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Becton Dickinson cd34
Kaplan-Meier survival analysis ( A ) Kaplan-Meier plot representing the overall survival for AML groups M1, M2, M3, M4, M5 and M7. ( B ) Kaplan-Meier plot representing the correlation between LSC genes <t>CD34</t> , TIM-3 , CLL-1 and BMI-1 expression and survival. CD34 ( p = 0.0022), TIM-3 ( p = 0.0035), CLL-1 ( p = 0.0006), BMI-1 ( p
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1) Product Images from "Acute myeloid leukemia stem cell markers in prognosis and targeted therapy: potential impact of BMI-1, TIM-3 and CLL-1"

Article Title: Acute myeloid leukemia stem cell markers in prognosis and targeted therapy: potential impact of BMI-1, TIM-3 and CLL-1

Journal: Oncotarget

doi: 10.18632/oncotarget.11063

Kaplan-Meier survival analysis ( A ) Kaplan-Meier plot representing the overall survival for AML groups M1, M2, M3, M4, M5 and M7. ( B ) Kaplan-Meier plot representing the correlation between LSC genes CD34 , TIM-3 , CLL-1 and BMI-1 expression and survival. CD34 ( p = 0.0022), TIM-3 ( p = 0.0035), CLL-1 ( p = 0.0006), BMI-1 ( p
Figure Legend Snippet: Kaplan-Meier survival analysis ( A ) Kaplan-Meier plot representing the overall survival for AML groups M1, M2, M3, M4, M5 and M7. ( B ) Kaplan-Meier plot representing the correlation between LSC genes CD34 , TIM-3 , CLL-1 and BMI-1 expression and survival. CD34 ( p = 0.0022), TIM-3 ( p = 0.0035), CLL-1 ( p = 0.0006), BMI-1 ( p

Techniques Used: Expressing

Kasumi-1 cell line proliferation with antibodies against CD44, CD34, CLL-1, TIM-3 or BMI-1 small molecular inhibitor PTC-209, measured with MTT assay ( A ) Anti-CD44 (1.0 μg/100 μl), anti-CD34 (1.0 μg/100 μl), anti-CLL-1 (0.5 μg/100 μl), and anti-TIM-3 (0.5 μg/100 μl). ( B ) PTC-209 at concentrations ranging from 0.2 to 4 μM. Fold changes in cell proliferation are expressed as mean ± S.D., n = 3. One-way ANOVA was used followed by the Newman-Keuls post-test (*** p
Figure Legend Snippet: Kasumi-1 cell line proliferation with antibodies against CD44, CD34, CLL-1, TIM-3 or BMI-1 small molecular inhibitor PTC-209, measured with MTT assay ( A ) Anti-CD44 (1.0 μg/100 μl), anti-CD34 (1.0 μg/100 μl), anti-CLL-1 (0.5 μg/100 μl), and anti-TIM-3 (0.5 μg/100 μl). ( B ) PTC-209 at concentrations ranging from 0.2 to 4 μM. Fold changes in cell proliferation are expressed as mean ± S.D., n = 3. One-way ANOVA was used followed by the Newman-Keuls post-test (*** p

Techniques Used: MTT Assay

KG-1a cell line proliferation with antibodies against CD44, CD34, CLL-1, TIM-3 or BMI-1 small molecular inhibitor PTC-209, measured with MTT assay ( A ) Anti-CD44 (1.0 μg/100 μl), anti-CD34 (1.0 μg/100 μl), anti-CLL-1 (0.5 μg/100 μl), and anti-TIM-3 (0.5 μg/100 μl). ( B ) PTC-209 at concentrations ranging from 0.2 to 4 μM. Fold changes in cell proliferation are expressed as mean ± S.D., n = 3. One-way ANOVA was used followed by the Newman-Keuls post-test (*** p
Figure Legend Snippet: KG-1a cell line proliferation with antibodies against CD44, CD34, CLL-1, TIM-3 or BMI-1 small molecular inhibitor PTC-209, measured with MTT assay ( A ) Anti-CD44 (1.0 μg/100 μl), anti-CD34 (1.0 μg/100 μl), anti-CLL-1 (0.5 μg/100 μl), and anti-TIM-3 (0.5 μg/100 μl). ( B ) PTC-209 at concentrations ranging from 0.2 to 4 μM. Fold changes in cell proliferation are expressed as mean ± S.D., n = 3. One-way ANOVA was used followed by the Newman-Keuls post-test (*** p

Techniques Used: MTT Assay

Leukemic stem cell genes expression analysis by qRT-PCR in bone marrow samples from AML patients versus control subjects Gene expression of: ( A ) CD34 , ( B ) CLL-1 , ( C ) BMI-1 , and ( D ) TIM-3 . Fold changes in the respective gene expression are expressed as mean ± S.E.M., n = 10 control and 40 patients (M1 = 5, M2 = 7, M3 = 5, M4 = 16, M5 = 4, M7 = 3). One-way ANOVA was used followed by the Newman-Keuls post-test ( ***p
Figure Legend Snippet: Leukemic stem cell genes expression analysis by qRT-PCR in bone marrow samples from AML patients versus control subjects Gene expression of: ( A ) CD34 , ( B ) CLL-1 , ( C ) BMI-1 , and ( D ) TIM-3 . Fold changes in the respective gene expression are expressed as mean ± S.E.M., n = 10 control and 40 patients (M1 = 5, M2 = 7, M3 = 5, M4 = 16, M5 = 4, M7 = 3). One-way ANOVA was used followed by the Newman-Keuls post-test ( ***p

Techniques Used: Expressing, Quantitative RT-PCR

2) Product Images from "Mobilized Multipotent Hematopoietic Progenitors Stabilize and Expand Regulatory T Cells to Protect Against Autoimmune Encephalomyelitis"

Article Title: Mobilized Multipotent Hematopoietic Progenitors Stabilize and Expand Regulatory T Cells to Protect Against Autoimmune Encephalomyelitis

Journal: Frontiers in Immunology

doi: 10.3389/fimmu.2020.607175

Preparation and characterization of mobilized MPP. (A) Upon mobilization with G-CSF and Flt3L, spleen c-kit + cells were magnetically sorted, further stained with c-kit, Sca-1, CD11b and CD34 and cell-sorted as c-kit + Sca-1 + CD34 + CD11b −/low cells. (B) SLAM markers including CD150 and CD48 and Flt3 were used for characterization of mobilized cell sorted c-kit + Sca-1 + CD34 + CD11b −/low progenitors as 80% MPP3 (CD150 − ) and 20% MPP2 (CD150 + ). (C) The differentiation properties of mobilized MPP were assessed after 7 days of co-culture upon OP9 and OP9Δ4 stromal cells in the presence of SCF (1 ng/ml), IL-7 (8 ng/ml) and Flt3L (10 ng/ml). Cells were recovered and stained for FACS analysis with different lineage markers. Percentages of the different subsets resulting from MPP differentiation are indicated.
Figure Legend Snippet: Preparation and characterization of mobilized MPP. (A) Upon mobilization with G-CSF and Flt3L, spleen c-kit + cells were magnetically sorted, further stained with c-kit, Sca-1, CD11b and CD34 and cell-sorted as c-kit + Sca-1 + CD34 + CD11b −/low cells. (B) SLAM markers including CD150 and CD48 and Flt3 were used for characterization of mobilized cell sorted c-kit + Sca-1 + CD34 + CD11b −/low progenitors as 80% MPP3 (CD150 − ) and 20% MPP2 (CD150 + ). (C) The differentiation properties of mobilized MPP were assessed after 7 days of co-culture upon OP9 and OP9Δ4 stromal cells in the presence of SCF (1 ng/ml), IL-7 (8 ng/ml) and Flt3L (10 ng/ml). Cells were recovered and stained for FACS analysis with different lineage markers. Percentages of the different subsets resulting from MPP differentiation are indicated.

Techniques Used: Staining, Co-Culture Assay, FACS

3) Product Images from "Serum-mediated Activation of Bone Marrow–derived Mesenchymal Stem Cells in Ischemic Stroke Patients"

Article Title: Serum-mediated Activation of Bone Marrow–derived Mesenchymal Stem Cells in Ischemic Stroke Patients

Journal: Cell Transplantation

doi: 10.1177/0963689718755404

Evaluation of phenotypic characteristics of mesenchymal stem cells (MSCs). (A) Representative phase contrast images of human MSCs (hMSCs) expanded with the different serums. (B) Cumulative population doubling level of hMSCs cultured in Dulbecco’s modified Eagle’s medium with 10% fetal bovine serum (FBS), control serum (NS), and stroke patient serum (SS). (C) Fluorescent-activated cell sorting analysis of hMSCs cultured with different types of serum. Quantitative analysis of the percentages of cells expressing CD90, CD73 (positive markers), and CD34, CD45 (negative markers). The relative expression levels of both human vascular endothelial growth factor (D) and human fibroblast growth factor (F) were significantly increased in SS-hMSCs than FBS-hMSCs or NS-hMSCs. Human glial cell–derived neurotrophic factor (E) expression level was significantly lower in FBS-hMSCs than NS-hMSCs and SS-hMSCs. All data are presented as mean + SD (** P
Figure Legend Snippet: Evaluation of phenotypic characteristics of mesenchymal stem cells (MSCs). (A) Representative phase contrast images of human MSCs (hMSCs) expanded with the different serums. (B) Cumulative population doubling level of hMSCs cultured in Dulbecco’s modified Eagle’s medium with 10% fetal bovine serum (FBS), control serum (NS), and stroke patient serum (SS). (C) Fluorescent-activated cell sorting analysis of hMSCs cultured with different types of serum. Quantitative analysis of the percentages of cells expressing CD90, CD73 (positive markers), and CD34, CD45 (negative markers). The relative expression levels of both human vascular endothelial growth factor (D) and human fibroblast growth factor (F) were significantly increased in SS-hMSCs than FBS-hMSCs or NS-hMSCs. Human glial cell–derived neurotrophic factor (E) expression level was significantly lower in FBS-hMSCs than NS-hMSCs and SS-hMSCs. All data are presented as mean + SD (** P

Techniques Used: Cell Culture, Modification, FACS, Expressing, Derivative Assay

4) Product Images from "Zoledronic acid prevents the tumor-promoting effects of mesenchymal stem cells via MCP-1 dependent recruitment of macrophages"

Article Title: Zoledronic acid prevents the tumor-promoting effects of mesenchymal stem cells via MCP-1 dependent recruitment of macrophages

Journal: Oncotarget

doi:

The characterization of MSC A. Frozen sections of breast carcinoma tumor were immunostained with anti-CD29 and CD45. CD29 + and CD45 + stem cells (arrow) migrated into the tumor site. Nuclei were stained with DAPI (blue). Scale bars = 50 μm (left) and 5 μm (right) respectively. B. Quantitative analysis of cell marker expression by FACS. MSC express high levels of CD29, CD73, CD90 and CD105, but almost negative for CD34 and CD45.
Figure Legend Snippet: The characterization of MSC A. Frozen sections of breast carcinoma tumor were immunostained with anti-CD29 and CD45. CD29 + and CD45 + stem cells (arrow) migrated into the tumor site. Nuclei were stained with DAPI (blue). Scale bars = 50 μm (left) and 5 μm (right) respectively. B. Quantitative analysis of cell marker expression by FACS. MSC express high levels of CD29, CD73, CD90 and CD105, but almost negative for CD34 and CD45.

Techniques Used: Staining, Marker, Expressing, FACS

5) Product Images from "A novel method to derive amniotic fluid stem cells for therapeutic purposes"

Article Title: A novel method to derive amniotic fluid stem cells for therapeutic purposes

Journal: BMC Cell Biology

doi: 10.1186/1471-2121-11-79

Characteristics of hAFS cells obtained by the starter cell method . (A) Clonal hAFS cells at passage 18 show an Oct-4 specific signal after staining with FITC-conjugated secondary antibody against anti-Oct4a with fluorescent microscope at 20× magnification. The Oct-4 positive signal is exhibit in hAFS population as shown by merge of Oct-4a and Hoechst 33342 staining. (B) The hAFS cell line at passage 18 has expression of Oct-4a and HLA-ABC, but not HLA-DR, Nanog and Sox2 by cDNA analysis using RT-PCR. The expression of Nestin was observed in hAFS cell-derived neurons. C means negative control, while gDNA was used as positive control of PCR amplification. (C) The flow cytometry analysis shows expression of SSEA4, CD29, CD44, CD73, CD90, CD105 and CD133, but not CD34 or CD45 on the hAFS cell surface.
Figure Legend Snippet: Characteristics of hAFS cells obtained by the starter cell method . (A) Clonal hAFS cells at passage 18 show an Oct-4 specific signal after staining with FITC-conjugated secondary antibody against anti-Oct4a with fluorescent microscope at 20× magnification. The Oct-4 positive signal is exhibit in hAFS population as shown by merge of Oct-4a and Hoechst 33342 staining. (B) The hAFS cell line at passage 18 has expression of Oct-4a and HLA-ABC, but not HLA-DR, Nanog and Sox2 by cDNA analysis using RT-PCR. The expression of Nestin was observed in hAFS cell-derived neurons. C means negative control, while gDNA was used as positive control of PCR amplification. (C) The flow cytometry analysis shows expression of SSEA4, CD29, CD44, CD73, CD90, CD105 and CD133, but not CD34 or CD45 on the hAFS cell surface.

Techniques Used: Staining, Microscopy, Expressing, Reverse Transcription Polymerase Chain Reaction, Derivative Assay, Negative Control, Positive Control, Polymerase Chain Reaction, Amplification, Flow Cytometry, Cytometry

6) Product Images from "Knockdown of insulin-like growth factor 1 exerts a protective effect on hypoxic injury of aged BM-MSCs: role of autophagy"

Article Title: Knockdown of insulin-like growth factor 1 exerts a protective effect on hypoxic injury of aged BM-MSCs: role of autophagy

Journal: Stem Cell Research & Therapy

doi: 10.1186/s13287-018-1028-5

Characterization of young and aged BM-MSCs. Flow cytometric results show that young and aged BM-MSCs were consistently negative for CD31, CD34, and CD45, and positive for CD29, CD44, and CD90
Figure Legend Snippet: Characterization of young and aged BM-MSCs. Flow cytometric results show that young and aged BM-MSCs were consistently negative for CD31, CD34, and CD45, and positive for CD29, CD44, and CD90

Techniques Used: Flow Cytometry

7) Product Images from "Human T-Cell Leukemia Virus Type 1 Tax Oncoprotein Suppression of Multilineage Hematopoiesis of CD34+ Cells In Vitro"

Article Title: Human T-Cell Leukemia Virus Type 1 Tax Oncoprotein Suppression of Multilineage Hematopoiesis of CD34+ Cells In Vitro

Journal: Journal of Virology

doi: 10.1128/JVI.77.22.12152-12164.2003

FACS isolation of CD34 + cells infected with lentiviral vectors. Purified CD34 + cells (3 × 10 6 ) were infected with lentiviral vectors (MOI = 3) by centrifugation (4 h at 2,500 rpm) in a Beckman GPR centrifuge. Cells were incubated for 72 h in IMDM supplemented with 10% FBS and 30 μl of cytokine cocktail StemSpan CC100. Cells were incubated with a PE-conjugated human-specific MAb against the CD34 marker, and cell sorting was performed with a FACSVantage flow cytometer. An additional cell sample was incubated with a mouse immunoglobulin G γ isotype control antibody to set compensation and gates for FACS. The R2 gate in each panel represents the gate set to isolate CD34 + GFP + ). The percentage of CD34 + GFP + cells is displayed in the upper right panel. Mock-transduced CD34 + cells were isolated only on the basis of CD34 + expression and are represented by the R3 gate. LVs used in each transduction are as follows: GFP, HR′CMV-GFP; Tax1, HR′CMV-Tax1/GFP; Tax1(−), HR′CMV-Tax1(−)/GFP; Tax2, HR′CMV-Tax2/GFP; and Vpr, HR′CMV-Vpr/GFP. The purity of the sorted cell populations was not determined after isolation due to the limited numbers of cells recovered by FACS.
Figure Legend Snippet: FACS isolation of CD34 + cells infected with lentiviral vectors. Purified CD34 + cells (3 × 10 6 ) were infected with lentiviral vectors (MOI = 3) by centrifugation (4 h at 2,500 rpm) in a Beckman GPR centrifuge. Cells were incubated for 72 h in IMDM supplemented with 10% FBS and 30 μl of cytokine cocktail StemSpan CC100. Cells were incubated with a PE-conjugated human-specific MAb against the CD34 marker, and cell sorting was performed with a FACSVantage flow cytometer. An additional cell sample was incubated with a mouse immunoglobulin G γ isotype control antibody to set compensation and gates for FACS. The R2 gate in each panel represents the gate set to isolate CD34 + GFP + ). The percentage of CD34 + GFP + cells is displayed in the upper right panel. Mock-transduced CD34 + cells were isolated only on the basis of CD34 + expression and are represented by the R3 gate. LVs used in each transduction are as follows: GFP, HR′CMV-GFP; Tax1, HR′CMV-Tax1/GFP; Tax1(−), HR′CMV-Tax1(−)/GFP; Tax2, HR′CMV-Tax2/GFP; and Vpr, HR′CMV-Vpr/GFP. The purity of the sorted cell populations was not determined after isolation due to the limited numbers of cells recovered by FACS.

Techniques Used: FACS, Isolation, Infection, Purification, Centrifugation, Incubation, Marker, Flow Cytometry, Cytometry, Expressing, Transduction

Clonogenic colony-forming activity of lentiviral vector- transduced CD34 + cells. GFP + CD34 + cells were purified by FACS, and isolated cells (10 3 ). (A) Total number of myeloid, erythroid and pluripotential colonies per CD34 + GFP + cells (10 3 ) plated was determined at 14 days postplating. Purified CD34 + GFP + cell samples were plated in triplicate. Each column represents a separate sorting experiment. Colony-forming activities were assayed four times for each transduction, except for Tax1(−) and Tax2-transduced CD34 + cells, which were assayed three times (sorting experiments 2, 3, and 4). Transduction of CD34 + cells with HR′CMV-Vpr/GFP resulted in no colony formation in FACS experiments 1 and 4 and is indicated by an asterisk. (B) Relative distribution of clonogenic colonies. Colonies were analyzed by morphology and characterized as CFU-GM, BFU-E, or HPP-CFC. The average numbers of CFU-GM colonies that arose per 10 3 purified CD34 + GFP + cells plated were 38.8 (Mock), 24.4 (GFP), 7.3 (Tax1), 21.2 [Tax1(−)], and 21.4 (Tax2). The average numbers of BFU-E colonies arising per 10 3 CD34 + GFP+ cells plated were 14.9 (Mock), 9.0 (GFP), 2.8 (Tax1), 8.0 [Tax1(−)], and 8.3 (Tax2). The average numbers of CFU-HPP colonies arising per 10 3 CD34 + GFP + cells plated were 6.0 (Mock), 3.6 (GFP), 1.2 (Tax1), 3.2 [Tax1(−)], and 3.3 (Tax2). Statistical analysis was performed by ANOVA ( P
Figure Legend Snippet: Clonogenic colony-forming activity of lentiviral vector- transduced CD34 + cells. GFP + CD34 + cells were purified by FACS, and isolated cells (10 3 ). (A) Total number of myeloid, erythroid and pluripotential colonies per CD34 + GFP + cells (10 3 ) plated was determined at 14 days postplating. Purified CD34 + GFP + cell samples were plated in triplicate. Each column represents a separate sorting experiment. Colony-forming activities were assayed four times for each transduction, except for Tax1(−) and Tax2-transduced CD34 + cells, which were assayed three times (sorting experiments 2, 3, and 4). Transduction of CD34 + cells with HR′CMV-Vpr/GFP resulted in no colony formation in FACS experiments 1 and 4 and is indicated by an asterisk. (B) Relative distribution of clonogenic colonies. Colonies were analyzed by morphology and characterized as CFU-GM, BFU-E, or HPP-CFC. The average numbers of CFU-GM colonies that arose per 10 3 purified CD34 + GFP + cells plated were 38.8 (Mock), 24.4 (GFP), 7.3 (Tax1), 21.2 [Tax1(−)], and 21.4 (Tax2). The average numbers of BFU-E colonies arising per 10 3 CD34 + GFP+ cells plated were 14.9 (Mock), 9.0 (GFP), 2.8 (Tax1), 8.0 [Tax1(−)], and 8.3 (Tax2). The average numbers of CFU-HPP colonies arising per 10 3 CD34 + GFP + cells plated were 6.0 (Mock), 3.6 (GFP), 1.2 (Tax1), 3.2 [Tax1(−)], and 3.3 (Tax2). Statistical analysis was performed by ANOVA ( P

Techniques Used: Activity Assay, Plasmid Preparation, Purification, FACS, Isolation, Transduction

8) Product Images from "Cardiac Migration of Endogenous Mesenchymal Stromal Cells in Patients with Inflammatory Cardiomyopathy"

Article Title: Cardiac Migration of Endogenous Mesenchymal Stromal Cells in Patients with Inflammatory Cardiomyopathy

Journal: Mediators of Inflammation

doi: 10.1155/2015/308185

Immunostaining and quantification of cardiac MSC related to cardiac inflammation and expression of SDF-1 α mRNA. (a) Representative image of immunofluorescence of MSC in EMB. Serial sections were stained with (A) an overlay of α -sarcomeric actin (red), nuclear counterstained (DAPI, blue), anti-CD105 and anti-CD90 (red, and B), and anti-CD45 (green, and C) as a lymphocytes marker (the arrow image (A) highlights the MSC). (b) Quantitative analysis of CD45 − CD34 − CD105 + CD90 + -MSC in EMB of patients without ( n = 6) and with ( n = 23) cardiac inflammation, as defined by the number of LFA-positive cells (box plots are given as median and IQR; whiskers represent 95% CI). (c) Quantitative analysis of CD45 − CD34 − CD105 + CD90 + -MSC in EMB after dichotomisation with respect to SDF-1 α mRNA expression in their EMB (cut-off: median, box plots are given for median and IQR; whiskers represent 95% CI). (d) Correlation between the transcardiac gradient of circulating MSC and MSC in EMB. (e) Representative image of immunofluorescence of MSC and proliferation in EMB. The same serial sections as indicated above (3(a)) were stained with (A) anti-Ki67 (yellow) for proliferation, (B) overlay of CD105 (red), anti-CD45 (green), and Ki67 (yellow), and (C) overlay of α -sarcomeric actin (red), nuclei (DAPI, blue), CD105 (red), anti-CD45 (green), and Ki67 (yellow).
Figure Legend Snippet: Immunostaining and quantification of cardiac MSC related to cardiac inflammation and expression of SDF-1 α mRNA. (a) Representative image of immunofluorescence of MSC in EMB. Serial sections were stained with (A) an overlay of α -sarcomeric actin (red), nuclear counterstained (DAPI, blue), anti-CD105 and anti-CD90 (red, and B), and anti-CD45 (green, and C) as a lymphocytes marker (the arrow image (A) highlights the MSC). (b) Quantitative analysis of CD45 − CD34 − CD105 + CD90 + -MSC in EMB of patients without ( n = 6) and with ( n = 23) cardiac inflammation, as defined by the number of LFA-positive cells (box plots are given as median and IQR; whiskers represent 95% CI). (c) Quantitative analysis of CD45 − CD34 − CD105 + CD90 + -MSC in EMB after dichotomisation with respect to SDF-1 α mRNA expression in their EMB (cut-off: median, box plots are given for median and IQR; whiskers represent 95% CI). (d) Correlation between the transcardiac gradient of circulating MSC and MSC in EMB. (e) Representative image of immunofluorescence of MSC and proliferation in EMB. The same serial sections as indicated above (3(a)) were stained with (A) anti-Ki67 (yellow) for proliferation, (B) overlay of CD105 (red), anti-CD45 (green), and Ki67 (yellow), and (C) overlay of α -sarcomeric actin (red), nuclei (DAPI, blue), CD105 (red), anti-CD45 (green), and Ki67 (yellow).

Techniques Used: Immunostaining, Expressing, Immunofluorescence, Staining, Marker

Flow cytometry analysis strategy and quantification of transcardiac gradients of circulating MSC in CMi. (a) Cultivated MSC (bar indicates 200 μ m) isolated from bone marrow were stained with directly FITC-conjugated monoclonal antibodies against human CD45 and CD34, and anti-CD11b directly conjugated to AF488 (all from BD). After setting a morphological gate defined by forward/side scatter (FSC and SSC, R1), CD45 − CD34 − CD11b − cells (R2) defined as belonging to both R1 and R2 (first row) were stained with CD73-PE, CD106-APC, CD90-APC, CD29-APC, CD105-APC, or CD44-APC (second row). (b) Measurement of circulating MSC in patients with CMi. MNCs were isolated from peripheral blood using a Ficoll gradient. Circulating MSC were analysed by flow cytometry. First, a regional gate was defined to exclude debris and platelets defined by forward/side scatter (FSC and SSC, R1). The events in R1 are then displayed on a CD45CD34CD11b versus SSC dot plot and a second gate (R2) is produced to include the cluster of CD45 − CD34 − CD11b − cells. The right margin was fixed for all patients. A total of at least 100,000 events in R1 and R2 were counted. (c) Quantification of the number of circulating MSC sampled from the aortic root and simultaneously from the coronary sinus in patients with cardiac inflammation (box plots are given as median and IQR; whiskers represent 95% CI). The lines between the boxes represent numbers of CD45 − CD34 − CD11b − /100 MNC sampled from either the aorta or coronary sinus of the same individual.
Figure Legend Snippet: Flow cytometry analysis strategy and quantification of transcardiac gradients of circulating MSC in CMi. (a) Cultivated MSC (bar indicates 200 μ m) isolated from bone marrow were stained with directly FITC-conjugated monoclonal antibodies against human CD45 and CD34, and anti-CD11b directly conjugated to AF488 (all from BD). After setting a morphological gate defined by forward/side scatter (FSC and SSC, R1), CD45 − CD34 − CD11b − cells (R2) defined as belonging to both R1 and R2 (first row) were stained with CD73-PE, CD106-APC, CD90-APC, CD29-APC, CD105-APC, or CD44-APC (second row). (b) Measurement of circulating MSC in patients with CMi. MNCs were isolated from peripheral blood using a Ficoll gradient. Circulating MSC were analysed by flow cytometry. First, a regional gate was defined to exclude debris and platelets defined by forward/side scatter (FSC and SSC, R1). The events in R1 are then displayed on a CD45CD34CD11b versus SSC dot plot and a second gate (R2) is produced to include the cluster of CD45 − CD34 − CD11b − cells. The right margin was fixed for all patients. A total of at least 100,000 events in R1 and R2 were counted. (c) Quantification of the number of circulating MSC sampled from the aortic root and simultaneously from the coronary sinus in patients with cardiac inflammation (box plots are given as median and IQR; whiskers represent 95% CI). The lines between the boxes represent numbers of CD45 − CD34 − CD11b − /100 MNC sampled from either the aorta or coronary sinus of the same individual.

Techniques Used: Flow Cytometry, Cytometry, Isolation, Staining, Produced

9) Product Images from "Increased CD40+ fibrocytes in patients with idiopathic orbital inflammation"

Article Title: Increased CD40+ fibrocytes in patients with idiopathic orbital inflammation

Journal: Ophthalmic plastic and reconstructive surgery

doi: 10.1097/IOP.0000000000000243

Immunohistochemistry of orbital fat from three IOI patients stained with CD34 alpha SMA and CD31. Spindle-shaped CD34 + cells (A, D,and G), are present in a perivascular distribution with early fibrosis and mild chronic inflammation. α–SMA+ cells (B, E, and H) showed staining of only smooth muscle of blood vessels and myoepithelial cells in sequential tissue sections. CD31+ cell staining was limited to blood vessel endothelial cells(C, F, and I). All 400X of the same region.
Figure Legend Snippet: Immunohistochemistry of orbital fat from three IOI patients stained with CD34 alpha SMA and CD31. Spindle-shaped CD34 + cells (A, D,and G), are present in a perivascular distribution with early fibrosis and mild chronic inflammation. α–SMA+ cells (B, E, and H) showed staining of only smooth muscle of blood vessels and myoepithelial cells in sequential tissue sections. CD31+ cell staining was limited to blood vessel endothelial cells(C, F, and I). All 400X of the same region.

Techniques Used: Immunohistochemistry, Staining

Increased frequency of CD45+/ Col1 +/ CD34+ fibrocytes in peripheral blood from patients with IOI compared to healthy donors (IOI mean FI= 22.6 +/− 12.2, n= 7 vs Healthy mean FI= 2.5 +/− 3.6, n= 19, p=0.001). Data presented as fraction of monocytes (fibrocyte index, FI) co-expressing CD45, Col1 and CD34 calculated and analyzed by flow cytometry
Figure Legend Snippet: Increased frequency of CD45+/ Col1 +/ CD34+ fibrocytes in peripheral blood from patients with IOI compared to healthy donors (IOI mean FI= 22.6 +/− 12.2, n= 7 vs Healthy mean FI= 2.5 +/− 3.6, n= 19, p=0.001). Data presented as fraction of monocytes (fibrocyte index, FI) co-expressing CD45, Col1 and CD34 calculated and analyzed by flow cytometry

Techniques Used: Expressing, Flow Cytometry, Cytometry

Cultured fibrocytes from an IOI patient express CD34 (A), Col-1 (B), α–SMA (C), and CD40 (D) shown by immunofluorescence staining (left panel) and by flow cytometry (right panel). Spindle or stellate-shaped cellular morphology is consistently seen (insets). ISO (isotope) serves as a positive control.
Figure Legend Snippet: Cultured fibrocytes from an IOI patient express CD34 (A), Col-1 (B), α–SMA (C), and CD40 (D) shown by immunofluorescence staining (left panel) and by flow cytometry (right panel). Spindle or stellate-shaped cellular morphology is consistently seen (insets). ISO (isotope) serves as a positive control.

Techniques Used: Cell Culture, Immunofluorescence, Staining, Flow Cytometry, Cytometry, Positive Control

10) Product Images from "Mobilized Multipotent Hematopoietic Progenitors Stabilize and Expand Regulatory T Cells to Protect Against Autoimmune Encephalomyelitis"

Article Title: Mobilized Multipotent Hematopoietic Progenitors Stabilize and Expand Regulatory T Cells to Protect Against Autoimmune Encephalomyelitis

Journal: Frontiers in Immunology

doi: 10.3389/fimmu.2020.607175

Preparation and characterization of mobilized MPP. (A) Upon mobilization with G-CSF and Flt3L, spleen c-kit + cells were magnetically sorted, further stained with c-kit, Sca-1, CD11b and CD34 and cell-sorted as c-kit + Sca-1 + CD34 + CD11b −/low cells. (B) SLAM markers including CD150 and CD48 and Flt3 were used for characterization of mobilized cell sorted c-kit + Sca-1 + CD34 + CD11b −/low progenitors as 80% MPP3 (CD150 − ) and 20% MPP2 (CD150 + ). (C) The differentiation properties of mobilized MPP were assessed after 7 days of co-culture upon OP9 and OP9Δ4 stromal cells in the presence of SCF (1 ng/ml), IL-7 (8 ng/ml) and Flt3L (10 ng/ml). Cells were recovered and stained for FACS analysis with different lineage markers. Percentages of the different subsets resulting from MPP differentiation are indicated.
Figure Legend Snippet: Preparation and characterization of mobilized MPP. (A) Upon mobilization with G-CSF and Flt3L, spleen c-kit + cells were magnetically sorted, further stained with c-kit, Sca-1, CD11b and CD34 and cell-sorted as c-kit + Sca-1 + CD34 + CD11b −/low cells. (B) SLAM markers including CD150 and CD48 and Flt3 were used for characterization of mobilized cell sorted c-kit + Sca-1 + CD34 + CD11b −/low progenitors as 80% MPP3 (CD150 − ) and 20% MPP2 (CD150 + ). (C) The differentiation properties of mobilized MPP were assessed after 7 days of co-culture upon OP9 and OP9Δ4 stromal cells in the presence of SCF (1 ng/ml), IL-7 (8 ng/ml) and Flt3L (10 ng/ml). Cells were recovered and stained for FACS analysis with different lineage markers. Percentages of the different subsets resulting from MPP differentiation are indicated.

Techniques Used: Staining, Co-Culture Assay, FACS

11) Product Images from "A splicing factor switch controls hematopoietic lineage specification of pluripotent stem cells"

Article Title: A splicing factor switch controls hematopoietic lineage specification of pluripotent stem cells

Journal: EMBO Reports

doi: 10.15252/embr.202050535

Pervasive and dynamic alternative splicing occurs during human hematopoietic differentiation induced from hESCs Principal component analysis (PCA) of genes (left) and transcripts (right) in highly purified cells at various differentiation stages, including ESCs, APLNR + cells, CD31 + CD34 + cells, and CD43 + cells during hematopoietic differentiation induction from hESCs. Widespread occurrence ( > 85% of expressed genes) of alternative splicing in expressed genes (FPKM > 1 in at least one differentiation stage) at distinct differentiation stages during hematopoietic development. Average number of isoforms per gene at each differentiation stage. Analysis of isoform variants within each expressing gene at distinct differentiation stages. Number and frequency of five major splicing events at distinct differentiation stages, including mutually exclusive exon (MXE), alternative 5′ splicing (A5SS), alternative 3′ splicing (A3SS), intron retention (IR), and exon skipping (ES). The cutoff of splicing event of an expressed gene is 0.05
Figure Legend Snippet: Pervasive and dynamic alternative splicing occurs during human hematopoietic differentiation induced from hESCs Principal component analysis (PCA) of genes (left) and transcripts (right) in highly purified cells at various differentiation stages, including ESCs, APLNR + cells, CD31 + CD34 + cells, and CD43 + cells during hematopoietic differentiation induction from hESCs. Widespread occurrence ( > 85% of expressed genes) of alternative splicing in expressed genes (FPKM > 1 in at least one differentiation stage) at distinct differentiation stages during hematopoietic development. Average number of isoforms per gene at each differentiation stage. Analysis of isoform variants within each expressing gene at distinct differentiation stages. Number and frequency of five major splicing events at distinct differentiation stages, including mutually exclusive exon (MXE), alternative 5′ splicing (A5SS), alternative 3′ splicing (A3SS), intron retention (IR), and exon skipping (ES). The cutoff of splicing event of an expressed gene is 0.05

Techniques Used: Purification, Expressing

NUMB Expression and its splicing regulation Expression of NUMB and its family member NUMBLIKE ( NUMBL ) during human hematopoietic development by RNA‐Seq. Western blotting showing NUMB‐S overexpression upon DOX induction with anti‐FLAG antibody. The GAPDH gene was used as a loading control. Expression of NUMBL in day 2‐APLNR + cells, day 5‐DMSO–treated, and day 5‐PLB‐treated cells. The upper panel is a representative RT–PCR electropherogram showing the inclusion or exclusion of NUMB exon 9 in SRSF2 depleted cells on day 5 of differentiation. The bar graph showing the changes of exon 9 inclusion obtained from the RT–PCR electropherogram. RT–qPCR measuring the expression of NUMB‐S in SRSF2 depleted cells on day 5 of differentiation. The representative FACS plots of CD31 + CD34 + cells at day 5 of differentiation after treatment with DMSO and NOTCH inhibitor DAPT at various concentrations from day 2.5 to 5. Western blotting showing the expression of HES1 (detected by endogenous HES1 antibody as well as anti‐FLAG antibody) and SRSF2 in 293T cells. GADPH acts as a loading control. The splicing of NUMB exon 9 after HES1 overexpression. The top panel is a representative RT–PCR electropherogram showing the inclusion or exclusion of NUMB exon 9 without or with HES1 overexpression. The quantification is presented in the bottom bar graph. Data information: Results given are mean ± SD. P ‐values were determined by unpaired two‐tailed Student’s t ‐test in (D), (E), and (H). ns represents no significant difference. ** P
Figure Legend Snippet: NUMB Expression and its splicing regulation Expression of NUMB and its family member NUMBLIKE ( NUMBL ) during human hematopoietic development by RNA‐Seq. Western blotting showing NUMB‐S overexpression upon DOX induction with anti‐FLAG antibody. The GAPDH gene was used as a loading control. Expression of NUMBL in day 2‐APLNR + cells, day 5‐DMSO–treated, and day 5‐PLB‐treated cells. The upper panel is a representative RT–PCR electropherogram showing the inclusion or exclusion of NUMB exon 9 in SRSF2 depleted cells on day 5 of differentiation. The bar graph showing the changes of exon 9 inclusion obtained from the RT–PCR electropherogram. RT–qPCR measuring the expression of NUMB‐S in SRSF2 depleted cells on day 5 of differentiation. The representative FACS plots of CD31 + CD34 + cells at day 5 of differentiation after treatment with DMSO and NOTCH inhibitor DAPT at various concentrations from day 2.5 to 5. Western blotting showing the expression of HES1 (detected by endogenous HES1 antibody as well as anti‐FLAG antibody) and SRSF2 in 293T cells. GADPH acts as a loading control. The splicing of NUMB exon 9 after HES1 overexpression. The top panel is a representative RT–PCR electropherogram showing the inclusion or exclusion of NUMB exon 9 without or with HES1 overexpression. The quantification is presented in the bottom bar graph. Data information: Results given are mean ± SD. P ‐values were determined by unpaired two‐tailed Student’s t ‐test in (D), (E), and (H). ns represents no significant difference. ** P

Techniques Used: Expressing, RNA Sequencing Assay, Western Blot, Over Expression, Reverse Transcription Polymerase Chain Reaction, Quantitative RT-PCR, FACS, Two Tailed Test

Effects of long and short PLB treatment on hematopoietic differentiation The morphological alterations of cells treated without (DMSO control) or with various amount of PLB throughout the hematopoietic differentiation. Scale bar = 20 μm. Representative FACS plots illustrating the CD43 + HSPCs without or with PLB treatment at the indicated concentrations and treatment periods. Representative FACS plots showing the frequency of APLNR + cells on day 2 of differentiation and CD31 + CD34 + EPCs on day 5 without or with PLB treatment from day 0 to 2 at the indicated concentrations. The bar graph showing the percentage of APLNR + cells and CD31 + CD34 + EPCs of (C). Results given are mean ± SD. P ‐values were calculated by one‐way ANOVA followed by Dunnett’s test. ns represents no significant difference. All experiments were conducted for at least 3 biological replicates.
Figure Legend Snippet: Effects of long and short PLB treatment on hematopoietic differentiation The morphological alterations of cells treated without (DMSO control) or with various amount of PLB throughout the hematopoietic differentiation. Scale bar = 20 μm. Representative FACS plots illustrating the CD43 + HSPCs without or with PLB treatment at the indicated concentrations and treatment periods. Representative FACS plots showing the frequency of APLNR + cells on day 2 of differentiation and CD31 + CD34 + EPCs on day 5 without or with PLB treatment from day 0 to 2 at the indicated concentrations. The bar graph showing the percentage of APLNR + cells and CD31 + CD34 + EPCs of (C). Results given are mean ± SD. P ‐values were calculated by one‐way ANOVA followed by Dunnett’s test. ns represents no significant difference. All experiments were conducted for at least 3 biological replicates.

Techniques Used: FACS

Short PLB treatment exhibits minor cytotoxic effects Expression of LAS1L in day 2‐differentiated APLNR + cells and day 5‐differentiated CD31 + CD34 + cells by RNA‐Seq. The bottom panel showing the inclusion/exclusion of LAS1L exon 9 during hematopoietic differentiation by RT–PCR. The inclusion/exclusion of exon 9 of ATP5F1C and HDAC7 detecting by RT–PCR during hematopoietic differentiation. The top panel is a representative RT–PCR electropherogram showing the inclusion or exclusion of exon 9 in ATP5F1C (left) and HDAC7 (right) in cells at days 2 and 5 without or with PLB treatment at indicated concentrations, respectively. The quantification of PSI is presented in the bottom bar graph. P ‐values were calculated by one‐way followed by Dunnett’s test. The cellular morphology on days 3, 4, and 5 during hematopoietic differentiation after treatment with 1.25 or 2.5 nM PLB from day 2.5 to 5. Scale bar = 40 μm. Cellular apoptosis assessed using Annexin V and 7‐AAD at days 3 and 4 of differentiation by flow cytometry, with or without PLB treatment from day 2.5 to 5. The proportion of G0/G1, S, and G2/M cells at days 3 and 4 of differentiation, with or without PLB treatment from day 2.5 to 5, respectively. The cell cycle was determined using propidium iodide staining by flow cytometry. Immunofluorescent staining depicting low‐density lipoprotein (AcLDL) uptake from FACS‐sorted CD31 + cells without (DMSO) or with PLB (1.25 nM) treatment. CD144, LDL, and DAPI were stained (upper) by red, green, and blue, respectively. Scale bar = 40 μm. The bar graph showing the quantification of the LDL fluorescent intensity by the Volocity 3D image analysis software. The schematic illustrating the DOX‐inducible knockdown system with expression of SF3B1 shRNAs. The knockdown of SF3B1 was confirmed by RT–qPCR (left) and Western blotting (right) after inducing with DOX. The representative FACS plots of CD31 + CD34 + EPCs after SF3B1 depletion at day 5 of differentiation. The percentage of CD31 + CD34 + EPCs after SF3B1 depletion at day 5 of differentiation. Data information: Results given are mean ± SD. P ‐values were determined by Student’s t ‐test in (A), (E), (F), (H), (I), and (K). ns represents no significant difference. * P
Figure Legend Snippet: Short PLB treatment exhibits minor cytotoxic effects Expression of LAS1L in day 2‐differentiated APLNR + cells and day 5‐differentiated CD31 + CD34 + cells by RNA‐Seq. The bottom panel showing the inclusion/exclusion of LAS1L exon 9 during hematopoietic differentiation by RT–PCR. The inclusion/exclusion of exon 9 of ATP5F1C and HDAC7 detecting by RT–PCR during hematopoietic differentiation. The top panel is a representative RT–PCR electropherogram showing the inclusion or exclusion of exon 9 in ATP5F1C (left) and HDAC7 (right) in cells at days 2 and 5 without or with PLB treatment at indicated concentrations, respectively. The quantification of PSI is presented in the bottom bar graph. P ‐values were calculated by one‐way followed by Dunnett’s test. The cellular morphology on days 3, 4, and 5 during hematopoietic differentiation after treatment with 1.25 or 2.5 nM PLB from day 2.5 to 5. Scale bar = 40 μm. Cellular apoptosis assessed using Annexin V and 7‐AAD at days 3 and 4 of differentiation by flow cytometry, with or without PLB treatment from day 2.5 to 5. The proportion of G0/G1, S, and G2/M cells at days 3 and 4 of differentiation, with or without PLB treatment from day 2.5 to 5, respectively. The cell cycle was determined using propidium iodide staining by flow cytometry. Immunofluorescent staining depicting low‐density lipoprotein (AcLDL) uptake from FACS‐sorted CD31 + cells without (DMSO) or with PLB (1.25 nM) treatment. CD144, LDL, and DAPI were stained (upper) by red, green, and blue, respectively. Scale bar = 40 μm. The bar graph showing the quantification of the LDL fluorescent intensity by the Volocity 3D image analysis software. The schematic illustrating the DOX‐inducible knockdown system with expression of SF3B1 shRNAs. The knockdown of SF3B1 was confirmed by RT–qPCR (left) and Western blotting (right) after inducing with DOX. The representative FACS plots of CD31 + CD34 + EPCs after SF3B1 depletion at day 5 of differentiation. The percentage of CD31 + CD34 + EPCs after SF3B1 depletion at day 5 of differentiation. Data information: Results given are mean ± SD. P ‐values were determined by Student’s t ‐test in (A), (E), (F), (H), (I), and (K). ns represents no significant difference. * P

Techniques Used: Expressing, RNA Sequencing Assay, Reverse Transcription Polymerase Chain Reaction, Flow Cytometry, Staining, FACS, Software, Quantitative RT-PCR, Western Blot

The dynamic alternative splicing program during hESC hematopoietic differentiation Schematic representation of the strategies for fluorescence‐activated cell sorting (FACS) and transcriptomic analyses. During hematopoietic differentiation, human embryonic stem cells (hESCs, H1) on day 0, FACS‐purified lateral plate mesodermal APLNR + cells on day 2, purified CD31 + CD34 + endothelial progenitor cells (EPCs) on day 5, and purified CD43 + hematopoietic stem and progenitor cells (HSPCs) on day 8 were collected for RNA‐Seq, respectively. STAR Cufflinks, MISO, DESeq2, and rMATS were used to analyze the expression abundance of genes and transcripts, alternative splicing events, differentially expressed genes and transcripts, and differential splicing events, respectively. n = 3 technical replicates. Cumulative distribution curves of Log 2 (FPKM) of splicing factors ( n = 235) in hESCs APLNR + , CD31 + CD34 + , and CD43 + cells. The upper and lower dotted lines represent cumulative scores of 1 and 0, respectively. n = 3 technical replicates. The P ‐value was calculated by a two‐tailed Wilcoxon signed‐rank test. The heatmap illustrates the expression scaled by row of components within the major spliceosomal machinery ( n = 182) using unsupervised hierarchical clustering. The heatmap was scaled with Z‐Score using the log 2 (FPKM) expression of components within the major spliceosomal machinery. n = 3 technical replicates. The boxplots depict the expression of splicing factors in cluster 1 ( n = 115) and cluster 2 ( n = 67) identified in (C) at distinct differentiation stages. The central band indicates the median level of expression. Boxes present 25%‐75% of genes expression. The whiskers indicate the lowest and highest points within 1.5 × the interquartile. n = 3 technical replicates. P ‐values were calculated by a two‐tailed Wilcoxon signed‐rank test. The heatmap shows the dynamic expression of genes in the SF3A/3B complex (E) and SR family (F) at each indicated differentiation stage. Both heatmaps were scaled by row. The heatmaps were scaled with Z‐Score using the log 2 (FPKM) expression of indicated components. n = 3 technical replicates. The mRNA expression of splicing regulator SRSF2 and splicing factor PCBP2 during hematopoietic differentiation was measured by RT–qPCR. The ACTB gene was used as a control. Results given are mean ± standard deviation (SD). P ‐values were determined by an unpaired two‐tailed Student’s t ‐test. n ≥ 3 biological replicates. The number of differentially expressed transcripts at the isoform level (fold change (FC) > 2 FDR
Figure Legend Snippet: The dynamic alternative splicing program during hESC hematopoietic differentiation Schematic representation of the strategies for fluorescence‐activated cell sorting (FACS) and transcriptomic analyses. During hematopoietic differentiation, human embryonic stem cells (hESCs, H1) on day 0, FACS‐purified lateral plate mesodermal APLNR + cells on day 2, purified CD31 + CD34 + endothelial progenitor cells (EPCs) on day 5, and purified CD43 + hematopoietic stem and progenitor cells (HSPCs) on day 8 were collected for RNA‐Seq, respectively. STAR Cufflinks, MISO, DESeq2, and rMATS were used to analyze the expression abundance of genes and transcripts, alternative splicing events, differentially expressed genes and transcripts, and differential splicing events, respectively. n = 3 technical replicates. Cumulative distribution curves of Log 2 (FPKM) of splicing factors ( n = 235) in hESCs APLNR + , CD31 + CD34 + , and CD43 + cells. The upper and lower dotted lines represent cumulative scores of 1 and 0, respectively. n = 3 technical replicates. The P ‐value was calculated by a two‐tailed Wilcoxon signed‐rank test. The heatmap illustrates the expression scaled by row of components within the major spliceosomal machinery ( n = 182) using unsupervised hierarchical clustering. The heatmap was scaled with Z‐Score using the log 2 (FPKM) expression of components within the major spliceosomal machinery. n = 3 technical replicates. The boxplots depict the expression of splicing factors in cluster 1 ( n = 115) and cluster 2 ( n = 67) identified in (C) at distinct differentiation stages. The central band indicates the median level of expression. Boxes present 25%‐75% of genes expression. The whiskers indicate the lowest and highest points within 1.5 × the interquartile. n = 3 technical replicates. P ‐values were calculated by a two‐tailed Wilcoxon signed‐rank test. The heatmap shows the dynamic expression of genes in the SF3A/3B complex (E) and SR family (F) at each indicated differentiation stage. Both heatmaps were scaled by row. The heatmaps were scaled with Z‐Score using the log 2 (FPKM) expression of indicated components. n = 3 technical replicates. The mRNA expression of splicing regulator SRSF2 and splicing factor PCBP2 during hematopoietic differentiation was measured by RT–qPCR. The ACTB gene was used as a control. Results given are mean ± standard deviation (SD). P ‐values were determined by an unpaired two‐tailed Student’s t ‐test. n ≥ 3 biological replicates. The number of differentially expressed transcripts at the isoform level (fold change (FC) > 2 FDR

Techniques Used: Fluorescence, FACS, Purification, RNA Sequencing Assay, Expressing, Two Tailed Test, Quantitative RT-PCR, Standard Deviation

Effects of ectopic expression of constitutive splicing factors on human hematopoietic differentiation Volcano plot shows the differential splicing events regulated by PLB treated from day 2.5 to 5. The dotted line denotes FDR = 0.05. The scatter plot showing the correlation of splicing factors form Fig 3c (B) and all expressed genes (C) between day 2‐APLNR + cells and day 5‐PLB‐treated cells. r refers to the correlation coefficient. The protein–protein interaction network of 38 intersected splicing factors (Fig 3D ) (STRING: https://string‐db.org/ ). The mRNA expression of SF3A3 , SNRPD1, and SNRPE in day 2‐APLNR + cells, day 5‐DMSO–treated, and day 5‐PLB‐treated cells. N = 2 technical replicates. The upper panel illustrates the DOX‐inducible overexpression system. Western blotting confirmed the SRSF2 overexpression upon DOX induction with anti‐FLAG antibody. GAPDH was used as the loading control. RT–qPCR assay of the relative mRNA expression of SF3A3 , SNRPD1 , and SNRPE after overexpression upon DOX induction. The ACTB gene was used as a control. Data were normalized to the mRNA level of empty vector controls cells. Western blotting showing the protein expression of SF3A3, SNRPD1, and SNRPE after overexpression with anti‐FLAG antibody. The GAPDH gene was used as a control. Representative FACS plots of CD31 + CD34 + cells in SF3A3, SNRPD1, and SNRPE overexpressed (GFP + ) cells. Statistical analysis of the frequency of CD31 + CD34 + in SF3A3, SNRPD1, and SNRPE overexpressed (GFP + ) cells. Data information: P ‐values were calculated by one‐way ANOVA followed by Dunnett’s test. ns represents no significant difference. Results given are mean ± SD.. All experiments were conducted for at least 3 biological replicates unless stated otherwise Source data are available online for this figure.
Figure Legend Snippet: Effects of ectopic expression of constitutive splicing factors on human hematopoietic differentiation Volcano plot shows the differential splicing events regulated by PLB treated from day 2.5 to 5. The dotted line denotes FDR = 0.05. The scatter plot showing the correlation of splicing factors form Fig 3c (B) and all expressed genes (C) between day 2‐APLNR + cells and day 5‐PLB‐treated cells. r refers to the correlation coefficient. The protein–protein interaction network of 38 intersected splicing factors (Fig 3D ) (STRING: https://string‐db.org/ ). The mRNA expression of SF3A3 , SNRPD1, and SNRPE in day 2‐APLNR + cells, day 5‐DMSO–treated, and day 5‐PLB‐treated cells. N = 2 technical replicates. The upper panel illustrates the DOX‐inducible overexpression system. Western blotting confirmed the SRSF2 overexpression upon DOX induction with anti‐FLAG antibody. GAPDH was used as the loading control. RT–qPCR assay of the relative mRNA expression of SF3A3 , SNRPD1 , and SNRPE after overexpression upon DOX induction. The ACTB gene was used as a control. Data were normalized to the mRNA level of empty vector controls cells. Western blotting showing the protein expression of SF3A3, SNRPD1, and SNRPE after overexpression with anti‐FLAG antibody. The GAPDH gene was used as a control. Representative FACS plots of CD31 + CD34 + cells in SF3A3, SNRPD1, and SNRPE overexpressed (GFP + ) cells. Statistical analysis of the frequency of CD31 + CD34 + in SF3A3, SNRPD1, and SNRPE overexpressed (GFP + ) cells. Data information: P ‐values were calculated by one‐way ANOVA followed by Dunnett’s test. ns represents no significant difference. Results given are mean ± SD.. All experiments were conducted for at least 3 biological replicates unless stated otherwise Source data are available online for this figure.

Techniques Used: Expressing, Over Expression, Western Blot, Quantitative RT-PCR, Plasmid Preparation, FACS

Inhibition of splicing disrupts EPC and HEP generation The upper schematic illustrates the stage‐specific supplementation of splicing inhibitor PLB and on day 8 CD43 + HSPCs were examined by flow cytometry. The bottom bar graph showing the normalized frequency of CD43 + cells to DMSO control under distinct PLB treatment windows. P ‐values were calculated by one‐way followed by Dunnett’s test. The top panel is a representative RT–PCR electropherogram depicting the inclusion or exclusion LAS1L exon 9 in cells at day 2 as well as cells at day 5 without or with PLB treatment with indicated concentrations, respectively. The quantification of percent spliced‐in (PSI) was presented in the bottom bar graph. P ‐values were calculated by one‐way ANOVA followed by Dunnett’s test. The representative FACS plots show the generation of CD31 + CD34 + EPCs at day 5 of differentiation upon PLB treatment from day 2.5 to 5. The frequency of CD31 + CD34 + EPCs with PLB treatment in (C) was normalized to DMSO control. P ‐values were calculated by one‐way ANOVA followed by Dunnett’s test. Cell apoptotic level assessed using Annexin V and 7‐AAD at day 5 of differentiation by flow cytometry. The cells were treated with or without PLB from day 2.5 to 5. The proportion of G0/G1, S, and G2/M cells at day 5 of differentiation treated with or without PLB from day 2.5 to 5. The cell cycle was determined by flow cytometry with propidium iodide staining. The experimental schematic (upper panel). APLNR + cells on day 2 were FACS‐purified and treated with various dosages of PLB from day 2.5 to 5 during hematopoietic differentiation. On day 5, the generation of CD31 + CD34 + EPCs and CD31 + CD34 + CD73 − HEPs was assessed. The representative FACS plots showed the frequency of APLNR + cells, CD31 + CD34 + EPCs, and CD31 + CD34 + CD73 − HEPs without or with PLB treatment. The bar graphs show the percentage of CD31 + CD34 + EPCs and CD31 + CD34 + CD73 − HEPs without or with PLB treatment. Data information: Results given are mean ± SD. P ‐values were determined by an unpaired two‐tailed Student’s t ‐test in (E), (F), and (G). ns represents no significant difference, * P
Figure Legend Snippet: Inhibition of splicing disrupts EPC and HEP generation The upper schematic illustrates the stage‐specific supplementation of splicing inhibitor PLB and on day 8 CD43 + HSPCs were examined by flow cytometry. The bottom bar graph showing the normalized frequency of CD43 + cells to DMSO control under distinct PLB treatment windows. P ‐values were calculated by one‐way followed by Dunnett’s test. The top panel is a representative RT–PCR electropherogram depicting the inclusion or exclusion LAS1L exon 9 in cells at day 2 as well as cells at day 5 without or with PLB treatment with indicated concentrations, respectively. The quantification of percent spliced‐in (PSI) was presented in the bottom bar graph. P ‐values were calculated by one‐way ANOVA followed by Dunnett’s test. The representative FACS plots show the generation of CD31 + CD34 + EPCs at day 5 of differentiation upon PLB treatment from day 2.5 to 5. The frequency of CD31 + CD34 + EPCs with PLB treatment in (C) was normalized to DMSO control. P ‐values were calculated by one‐way ANOVA followed by Dunnett’s test. Cell apoptotic level assessed using Annexin V and 7‐AAD at day 5 of differentiation by flow cytometry. The cells were treated with or without PLB from day 2.5 to 5. The proportion of G0/G1, S, and G2/M cells at day 5 of differentiation treated with or without PLB from day 2.5 to 5. The cell cycle was determined by flow cytometry with propidium iodide staining. The experimental schematic (upper panel). APLNR + cells on day 2 were FACS‐purified and treated with various dosages of PLB from day 2.5 to 5 during hematopoietic differentiation. On day 5, the generation of CD31 + CD34 + EPCs and CD31 + CD34 + CD73 − HEPs was assessed. The representative FACS plots showed the frequency of APLNR + cells, CD31 + CD34 + EPCs, and CD31 + CD34 + CD73 − HEPs without or with PLB treatment. The bar graphs show the percentage of CD31 + CD34 + EPCs and CD31 + CD34 + CD73 − HEPs without or with PLB treatment. Data information: Results given are mean ± SD. P ‐values were determined by an unpaired two‐tailed Student’s t ‐test in (E), (F), and (G). ns represents no significant difference, * P

Techniques Used: Inhibition, Flow Cytometry, Reverse Transcription Polymerase Chain Reaction, FACS, Staining, Purification, Two Tailed Test

12) Product Images from "Human T-Cell Leukemia Virus Type 1 Tax Oncoprotein Suppression of Multilineage Hematopoiesis of CD34+ Cells In Vitro"

Article Title: Human T-Cell Leukemia Virus Type 1 Tax Oncoprotein Suppression of Multilineage Hematopoiesis of CD34+ Cells In Vitro

Journal: Journal of Virology

doi: 10.1128/JVI.77.22.12152-12164.2003

FACS isolation of CD34 + cells infected with lentiviral vectors. Purified CD34 + cells (3 × 10 6 ) were infected with lentiviral vectors (MOI = 3) by centrifugation (4 h at 2,500 rpm) in a Beckman GPR centrifuge. Cells were incubated for 72 h in IMDM supplemented with 10% FBS and 30 μl of cytokine cocktail StemSpan CC100. Cells were incubated with a PE-conjugated human-specific MAb against the CD34 marker, and cell sorting was performed with a FACSVantage flow cytometer. An additional cell sample was incubated with a mouse immunoglobulin G γ isotype control antibody to set compensation and gates for FACS. The R2 gate in each panel represents the gate set to isolate CD34 + GFP + ). The percentage of CD34 + GFP + cells is displayed in the upper right panel. Mock-transduced CD34 + cells were isolated only on the basis of CD34 + expression and are represented by the R3 gate. LVs used in each transduction are as follows: GFP, HR′CMV-GFP; Tax1, HR′CMV-Tax1/GFP; Tax1(−), HR′CMV-Tax1(−)/GFP; Tax2, HR′CMV-Tax2/GFP; and Vpr, HR′CMV-Vpr/GFP. The purity of the sorted cell populations was not determined after isolation due to the limited numbers of cells recovered by FACS.
Figure Legend Snippet: FACS isolation of CD34 + cells infected with lentiviral vectors. Purified CD34 + cells (3 × 10 6 ) were infected with lentiviral vectors (MOI = 3) by centrifugation (4 h at 2,500 rpm) in a Beckman GPR centrifuge. Cells were incubated for 72 h in IMDM supplemented with 10% FBS and 30 μl of cytokine cocktail StemSpan CC100. Cells were incubated with a PE-conjugated human-specific MAb against the CD34 marker, and cell sorting was performed with a FACSVantage flow cytometer. An additional cell sample was incubated with a mouse immunoglobulin G γ isotype control antibody to set compensation and gates for FACS. The R2 gate in each panel represents the gate set to isolate CD34 + GFP + ). The percentage of CD34 + GFP + cells is displayed in the upper right panel. Mock-transduced CD34 + cells were isolated only on the basis of CD34 + expression and are represented by the R3 gate. LVs used in each transduction are as follows: GFP, HR′CMV-GFP; Tax1, HR′CMV-Tax1/GFP; Tax1(−), HR′CMV-Tax1(−)/GFP; Tax2, HR′CMV-Tax2/GFP; and Vpr, HR′CMV-Vpr/GFP. The purity of the sorted cell populations was not determined after isolation due to the limited numbers of cells recovered by FACS.

Techniques Used: FACS, Isolation, Infection, Purification, Centrifugation, Incubation, Marker, Flow Cytometry, Cytometry, Expressing, Transduction

Clonogenic colony-forming activity of lentiviral vector- transduced CD34 + cells. GFP + CD34 + cells were purified by FACS, and isolated cells (10 3 ). (A) Total number of myeloid, erythroid and pluripotential colonies per CD34 + GFP + cells (10 3 ) plated was determined at 14 days postplating. Purified CD34 + GFP + cell samples were plated in triplicate. Each column represents a separate sorting experiment. Colony-forming activities were assayed four times for each transduction, except for Tax1(−) and Tax2-transduced CD34 + cells, which were assayed three times (sorting experiments 2, 3, and 4). Transduction of CD34 + cells with HR′CMV-Vpr/GFP resulted in no colony formation in FACS experiments 1 and 4 and is indicated by an asterisk. (B) Relative distribution of clonogenic colonies. Colonies were analyzed by morphology and characterized as CFU-GM, BFU-E, or HPP-CFC. The average numbers of CFU-GM colonies that arose per 10 3 purified CD34 + GFP + cells plated were 38.8 (Mock), 24.4 (GFP), 7.3 (Tax1), 21.2 [Tax1(−)], and 21.4 (Tax2). The average numbers of BFU-E colonies arising per 10 3 CD34 + GFP+ cells plated were 14.9 (Mock), 9.0 (GFP), 2.8 (Tax1), 8.0 [Tax1(−)], and 8.3 (Tax2). The average numbers of CFU-HPP colonies arising per 10 3 CD34 + GFP + cells plated were 6.0 (Mock), 3.6 (GFP), 1.2 (Tax1), 3.2 [Tax1(−)], and 3.3 (Tax2). Statistical analysis was performed by ANOVA ( P
Figure Legend Snippet: Clonogenic colony-forming activity of lentiviral vector- transduced CD34 + cells. GFP + CD34 + cells were purified by FACS, and isolated cells (10 3 ). (A) Total number of myeloid, erythroid and pluripotential colonies per CD34 + GFP + cells (10 3 ) plated was determined at 14 days postplating. Purified CD34 + GFP + cell samples were plated in triplicate. Each column represents a separate sorting experiment. Colony-forming activities were assayed four times for each transduction, except for Tax1(−) and Tax2-transduced CD34 + cells, which were assayed three times (sorting experiments 2, 3, and 4). Transduction of CD34 + cells with HR′CMV-Vpr/GFP resulted in no colony formation in FACS experiments 1 and 4 and is indicated by an asterisk. (B) Relative distribution of clonogenic colonies. Colonies were analyzed by morphology and characterized as CFU-GM, BFU-E, or HPP-CFC. The average numbers of CFU-GM colonies that arose per 10 3 purified CD34 + GFP + cells plated were 38.8 (Mock), 24.4 (GFP), 7.3 (Tax1), 21.2 [Tax1(−)], and 21.4 (Tax2). The average numbers of BFU-E colonies arising per 10 3 CD34 + GFP+ cells plated were 14.9 (Mock), 9.0 (GFP), 2.8 (Tax1), 8.0 [Tax1(−)], and 8.3 (Tax2). The average numbers of CFU-HPP colonies arising per 10 3 CD34 + GFP + cells plated were 6.0 (Mock), 3.6 (GFP), 1.2 (Tax1), 3.2 [Tax1(−)], and 3.3 (Tax2). Statistical analysis was performed by ANOVA ( P

Techniques Used: Activity Assay, Plasmid Preparation, Purification, FACS, Isolation, Transduction

13) Product Images from "A revised road map for the commitment of human cord blood CD34-negative hematopoietic stem cells"

Article Title: A revised road map for the commitment of human cord blood CD34-negative hematopoietic stem cells

Journal: Nature Communications

doi: 10.1038/s41467-018-04441-z

A comparison of the gene expression profiles of single-purified human CB-derived CD34 + and CD34 − and CD90 + HSCs by qRT-PCR. Multi-plex (79 genes) single-cell qRT-PCR using CD34 + and CD34 − HSCs and CD90 + HSCs was performed. a The 54 gene expression profiles in CD34 + and CD34 − HSCs and CD90 + HSC are depicted using a heatmap. These 54 genes were classified as HSC/HPC-, erythrocyte-, megakaryocyte-, myeloid cell-, lymphocyte- and epigenetics-related genes. b The expression of individual genes in CD34 + and CD34 − HSCs and CD90 + HSCs is depicted by violin plots. The HSC maintenance genes highly expressed in both CD34 + and CD34 − HSCs and CD90 + HSCs ( KIT , RUNK1 , TAL1 , BMI1 , DNMT3A , TGFBR1 and R2 ) are shown in the upper panel and highlighted by green color in a . The genes highly expressed in CD34 + HSCs ( IFITM1 , MPL , IKZF1 , ETV6 , ALDH1A1 and IGF1R ) are shown in the middle left panel and highlighted by pink color in a . The genes highly expressed in CD34 − HSCs ( EZH2 and MYB ) are shown in the middle right panel and highlighted by blue color in a . The gate names (R6 and R8) presented in this figure correspond to the same fractions in Fig. 1e, f . c Violin plots of the reference genes, including ACTB , GAPDH , PTPRC (CD45) and PGK-1 (left). Violin plots of the genes for CD34 and PROM1 (CD133) (middle). qRT-PCR of the VNN2 (GPI-80) mRNA expression in the 18Lin − CD34 + CD38 − CD133 + GPI-80 +/ − and 18Lin − CD34 − CD133 + GPI-80 +/ − cells, compared with the positive control (CB-derived neutrophils) and negative control (THP-1 cells) (right). n.d.: not detected
Figure Legend Snippet: A comparison of the gene expression profiles of single-purified human CB-derived CD34 + and CD34 − and CD90 + HSCs by qRT-PCR. Multi-plex (79 genes) single-cell qRT-PCR using CD34 + and CD34 − HSCs and CD90 + HSCs was performed. a The 54 gene expression profiles in CD34 + and CD34 − HSCs and CD90 + HSC are depicted using a heatmap. These 54 genes were classified as HSC/HPC-, erythrocyte-, megakaryocyte-, myeloid cell-, lymphocyte- and epigenetics-related genes. b The expression of individual genes in CD34 + and CD34 − HSCs and CD90 + HSCs is depicted by violin plots. The HSC maintenance genes highly expressed in both CD34 + and CD34 − HSCs and CD90 + HSCs ( KIT , RUNK1 , TAL1 , BMI1 , DNMT3A , TGFBR1 and R2 ) are shown in the upper panel and highlighted by green color in a . The genes highly expressed in CD34 + HSCs ( IFITM1 , MPL , IKZF1 , ETV6 , ALDH1A1 and IGF1R ) are shown in the middle left panel and highlighted by pink color in a . The genes highly expressed in CD34 − HSCs ( EZH2 and MYB ) are shown in the middle right panel and highlighted by blue color in a . The gate names (R6 and R8) presented in this figure correspond to the same fractions in Fig. 1e, f . c Violin plots of the reference genes, including ACTB , GAPDH , PTPRC (CD45) and PGK-1 (left). Violin plots of the genes for CD34 and PROM1 (CD133) (middle). qRT-PCR of the VNN2 (GPI-80) mRNA expression in the 18Lin − CD34 + CD38 − CD133 + GPI-80 +/ − and 18Lin − CD34 − CD133 + GPI-80 +/ − cells, compared with the positive control (CB-derived neutrophils) and negative control (THP-1 cells) (right). n.d.: not detected

Techniques Used: Expressing, Purification, Derivative Assay, Quantitative RT-PCR, Positive Control, Negative Control

A comparison of the gene expression profiles between CD34 + and CD34 − SRCs (HSCs) by a microarray. a The gene expression profiles of CD34 + and CD34 − SRCs (HSCs) were compared by a Gene Set Enrichment Analysis (GSEA) using the data from the microarray analysis. The gene sets enriched in CD34 − SRCs (blue bar) and those in CD34 + SRCs (red bar) with a Normalized Enrichment Score
Figure Legend Snippet: A comparison of the gene expression profiles between CD34 + and CD34 − SRCs (HSCs) by a microarray. a The gene expression profiles of CD34 + and CD34 − SRCs (HSCs) were compared by a Gene Set Enrichment Analysis (GSEA) using the data from the microarray analysis. The gene sets enriched in CD34 − SRCs (blue bar) and those in CD34 + SRCs (red bar) with a Normalized Enrichment Score

Techniques Used: Expressing, Microarray

The current and proposed models for human HSC hierarchy. a The current model 1 – 3 for the human HSC hierarchy. CD34 + HSCs as defined by a CD34 + CD38 − CD45RA − CD90 + CD49f + immunophenotype differentiate into MPPs, CMPs, MLPs, GMPs and MEPs. b The model 19 , 20 proposed based on our series of studies 18 , 21 , 22 , 25 , 33 – 36 , 60 , in which CD34 − HSCs are defined by a CD34 − CD38 low/ − CD45RA − FLT3 − CD110 − CD133 + GPI-80 + immunophenotype. CD34 − HSCs generate CD34 + HSCs in vitro 25 , 35 , 36 and in vivo 18 , 33 , suggesting that CD34 − HSCs reside at the apex of human HSC hierarchy. CD34 − HSCs, then, differentiate into MPPs, CMPs, GMPs and MEPs according to the current model 1 – 3 . Incorporating the present studies, a revised road map, which allows a commitment/differentiation pathway of CD34 − HSCs directly into MEPs (bypass route), is shown. MPP: multipotent progenitor, MLP: multilymphoid progenitor, CMP: common myeloid progenitor, GMP: granulocyte/macrophage progenitor, MEP: megakaryocyte/erythrocyte progenitor
Figure Legend Snippet: The current and proposed models for human HSC hierarchy. a The current model 1 – 3 for the human HSC hierarchy. CD34 + HSCs as defined by a CD34 + CD38 − CD45RA − CD90 + CD49f + immunophenotype differentiate into MPPs, CMPs, MLPs, GMPs and MEPs. b The model 19 , 20 proposed based on our series of studies 18 , 21 , 22 , 25 , 33 – 36 , 60 , in which CD34 − HSCs are defined by a CD34 − CD38 low/ − CD45RA − FLT3 − CD110 − CD133 + GPI-80 + immunophenotype. CD34 − HSCs generate CD34 + HSCs in vitro 25 , 35 , 36 and in vivo 18 , 33 , suggesting that CD34 − HSCs reside at the apex of human HSC hierarchy. CD34 − HSCs, then, differentiate into MPPs, CMPs, GMPs and MEPs according to the current model 1 – 3 . Incorporating the present studies, a revised road map, which allows a commitment/differentiation pathway of CD34 − HSCs directly into MEPs (bypass route), is shown. MPP: multipotent progenitor, MLP: multilymphoid progenitor, CMP: common myeloid progenitor, GMP: granulocyte/macrophage progenitor, MEP: megakaryocyte/erythrocyte progenitor

Techniques Used: In Vitro, In Vivo

Representative FACS profile and colony-forming capacity of highly purified CB-derived 18Lin - CD34 + CD38 - CD133 + GPI-80 +/ − and 18Lin - CD34 − CD133 + GPI-80 +/ − cells. A representative FACS profile is shown. a The forward scatter/side scatter (FSC/SSC) profile of immunomagnetically separated Lin − cells. The R1 gate was set on the blast-lymphocyte window. b The R2 gate was set on the 18Lin − living cells. c The R2 gated cells were subdivided into two fractions: 18Lin − CD45 + CD34 + (R3) and CD34 − (R4) cells, according to their expression of CD34. The definitions of CD34 +/ − cells are as follows: the CD34 + fraction contains cells expressing > 5% of the maximum BV421 fluorescence intensity (FI). The CD34 − level of FI was determined based on the Fluorescence Minus One controls. d The cells residing in the R3 gate were further subdivided into 18Lin − CD45 + CD34 + CD38 − (R5) cells. The CD38 − fraction contains cells expressing
Figure Legend Snippet: Representative FACS profile and colony-forming capacity of highly purified CB-derived 18Lin - CD34 + CD38 - CD133 + GPI-80 +/ − and 18Lin - CD34 − CD133 + GPI-80 +/ − cells. A representative FACS profile is shown. a The forward scatter/side scatter (FSC/SSC) profile of immunomagnetically separated Lin − cells. The R1 gate was set on the blast-lymphocyte window. b The R2 gate was set on the 18Lin − living cells. c The R2 gated cells were subdivided into two fractions: 18Lin − CD45 + CD34 + (R3) and CD34 − (R4) cells, according to their expression of CD34. The definitions of CD34 +/ − cells are as follows: the CD34 + fraction contains cells expressing > 5% of the maximum BV421 fluorescence intensity (FI). The CD34 − level of FI was determined based on the Fluorescence Minus One controls. d The cells residing in the R3 gate were further subdivided into 18Lin − CD45 + CD34 + CD38 − (R5) cells. The CD38 − fraction contains cells expressing

Techniques Used: FACS, Purification, Derivative Assay, Expressing, Fluorescence

SCID-repopulating cell activity and serial analyses of human cell repopulation of 18Lin − CD34 + CD38 − CD133 + GPI-80 + and 18Lin − CD34 − CD133 + GPI-80 + cells in primary and secondary NOG mice by IBMI. a A schematic illustration of primary and secondary transplantation of CD34 + and CD34 − SRCs is shown. PR, primary recipient; SR, secondary recipient. b The long-term repopulating potential of the CD34 + and CD34 − SRCs was determined by serially analyzing the kinetics of BM engraftment for 20 to 22 weeks in primary NOG mice that received transplants of 200 18Lin − CD34 + CD38 − CD133 + GPI-80 + cells (41 SRCs) ( n = 25) and 200 18Lin − CD34 − CD133 + GPI-80 + cells (25 SRCs) ( n = 23), respectively. All of the primary recipient mice were highly repopulated with human CD45 + cells. The human CD45 + cell repopulation rates of the mice that were transplanted with CD34 + SRCs at 12 and 18 weeks after transplantation were significantly higher than those of the mice that were transplanted with CD34 − SRCs. However, the repopulation rates of both mice were comparable at 20 to 22 weeks after transplantation (mean ± S.D., * p
Figure Legend Snippet: SCID-repopulating cell activity and serial analyses of human cell repopulation of 18Lin − CD34 + CD38 − CD133 + GPI-80 + and 18Lin − CD34 − CD133 + GPI-80 + cells in primary and secondary NOG mice by IBMI. a A schematic illustration of primary and secondary transplantation of CD34 + and CD34 − SRCs is shown. PR, primary recipient; SR, secondary recipient. b The long-term repopulating potential of the CD34 + and CD34 − SRCs was determined by serially analyzing the kinetics of BM engraftment for 20 to 22 weeks in primary NOG mice that received transplants of 200 18Lin − CD34 + CD38 − CD133 + GPI-80 + cells (41 SRCs) ( n = 25) and 200 18Lin − CD34 − CD133 + GPI-80 + cells (25 SRCs) ( n = 23), respectively. All of the primary recipient mice were highly repopulated with human CD45 + cells. The human CD45 + cell repopulation rates of the mice that were transplanted with CD34 + SRCs at 12 and 18 weeks after transplantation were significantly higher than those of the mice that were transplanted with CD34 − SRCs. However, the repopulation rates of both mice were comparable at 20 to 22 weeks after transplantation (mean ± S.D., * p

Techniques Used: Activity Assay, Mouse Assay, Transplantation Assay

Single-cell-based gene expression profiles of human CB-derived CD34 + and CD34 − HSCs. Using highly purified human CB-derived 18Lin − CD34 + CD38 − CD133 + GPI-80 + and 18Lin − CD34 − CD133 + GPI-80 + cells, we performed a single-cell-based gene expression analysis. The immunophenotypes of target cells, including controls, are presented in Supplementary Data 6 and the 79 target genes that play important roles in the pathway of HSC development/differentiation 63 are listed in Supplementary Data 7 . a A principal component analysis (PCA) revealed that the gene expression profiles in individual CD34 + ( n = 33) and CD34 − HSCs ( n = 23) were clearly different. The gene expression profiles of individual CD90 + HSCs ( n = 5) were similar to those of CD34 + HSCs but not to those of CD34 − HSCs. The dotted line represents the border region between the CD34 + and CD34 − HSCs calculated by a Fisher’s linear discriminant analysis. b An unsupervised hierarchical clustering analysis (Dendrogram) clearly showed two clusters, 1 and 2. Interestingly, all CD34 − HSCs belong to cluster 1. Three subgroups were detected in cluster 1. The left-most subgroup uniformly contained 10 CD34 − HSCs. The remaining 13 CD34 − HSCs were scattered between the other two subgroups mixed with CD34 + HSCs and CD90 + HSCs and MPPs. In contrast, some of the CD34 + HSCs and CD90 + HSCs, most of the MPP, and all other HPCs (CMP, GMP, MEP and MLP) belonged to cluster 2. These results demonstrated that the gene expression profiles of CD34 − HSC were unique and largely differed from those of other classes of CD34 + HSPCs. c A hierarchical clustering analysis of 62 genes (heatmap) detected 3 clusters ( a – c ), which are highlighted with yellow squares. Cluster (A) contained HSC/HPC-related genes, including RUNX1 , BMI1 , DNMT3a and SCL/TAL1 , in addition to CD34 and PROM1 (CD133). Cluster (B) contained HPC-related genes, including MYB , FOXO3A , SMAD4 , NFE2 and NOTCH1 . Cluster (C) contained mature cell-related genes, including those for various cytokine receptors ( CSF2RA , IL-6R , CSF1R , IGF2R and IL-3RA )
Figure Legend Snippet: Single-cell-based gene expression profiles of human CB-derived CD34 + and CD34 − HSCs. Using highly purified human CB-derived 18Lin − CD34 + CD38 − CD133 + GPI-80 + and 18Lin − CD34 − CD133 + GPI-80 + cells, we performed a single-cell-based gene expression analysis. The immunophenotypes of target cells, including controls, are presented in Supplementary Data 6 and the 79 target genes that play important roles in the pathway of HSC development/differentiation 63 are listed in Supplementary Data 7 . a A principal component analysis (PCA) revealed that the gene expression profiles in individual CD34 + ( n = 33) and CD34 − HSCs ( n = 23) were clearly different. The gene expression profiles of individual CD90 + HSCs ( n = 5) were similar to those of CD34 + HSCs but not to those of CD34 − HSCs. The dotted line represents the border region between the CD34 + and CD34 − HSCs calculated by a Fisher’s linear discriminant analysis. b An unsupervised hierarchical clustering analysis (Dendrogram) clearly showed two clusters, 1 and 2. Interestingly, all CD34 − HSCs belong to cluster 1. Three subgroups were detected in cluster 1. The left-most subgroup uniformly contained 10 CD34 − HSCs. The remaining 13 CD34 − HSCs were scattered between the other two subgroups mixed with CD34 + HSCs and CD90 + HSCs and MPPs. In contrast, some of the CD34 + HSCs and CD90 + HSCs, most of the MPP, and all other HPCs (CMP, GMP, MEP and MLP) belonged to cluster 2. These results demonstrated that the gene expression profiles of CD34 − HSC were unique and largely differed from those of other classes of CD34 + HSPCs. c A hierarchical clustering analysis of 62 genes (heatmap) detected 3 clusters ( a – c ), which are highlighted with yellow squares. Cluster (A) contained HSC/HPC-related genes, including RUNX1 , BMI1 , DNMT3a and SCL/TAL1 , in addition to CD34 and PROM1 (CD133). Cluster (B) contained HPC-related genes, including MYB , FOXO3A , SMAD4 , NFE2 and NOTCH1 . Cluster (C) contained mature cell-related genes, including those for various cytokine receptors ( CSF2RA , IL-6R , CSF1R , IGF2R and IL-3RA )

Techniques Used: Expressing, Derivative Assay, Purification

Human multi-lineage hematopoietic repopulation abilities of single CD34 + and CD34 − SRCs. Human multi-lineage hematopoietic repopulations in the primary recipient NSG mice that received a single 18Lin − CD34 + CD38 − CD133 + GPI-80 + (Mouse ID: 34 + NSG001) or b single 18Lin − CD34 − CD133 + GPI-80 + (Mouse ID: 34 − NSG027) cells were analyzed by 6-color FCM at 22 or 21 weeks after transplantation. The expression of CD19, CD33 CD34 (BM, PB and spleen), CD11b and CD14 (BM), CD41 (BM and spleen), CD235a (BM) and CD56 (spleen) in living human CD45 + cells was analyzed. The presence of CD41 + and CD235a + cells in BM was analyzed using a human CD45 +/ − cell gate (depicted by red dotted lines). The mouse ID numbers presented in the figure corresponded those listed in Supplementary Data 3a . Analyses of the BM (other bones), PB and spleens of the two representative mice that received either CD34 + SRC a or CD34 − SRC b revealed that both SRCs had an in vivo differentiation capacity comparable to that of CD34 + stem/progenitor cells, CD19 + B-lymphoids, CD33 + /CD11b + myeloids, CD14 + monocytes, CD235a + erythroid and CD41 + megakaryocytic lineages. CD56 + NK cells were detected in the spleen. These results confirmed that both CD34 + and CD34 − SRCs had definite multi-lineage differentiation potential. Detailed FCM data of all recipient mice that received single CD34 + and CD34 − SRCs are presented in Supplementary Data 3b
Figure Legend Snippet: Human multi-lineage hematopoietic repopulation abilities of single CD34 + and CD34 − SRCs. Human multi-lineage hematopoietic repopulations in the primary recipient NSG mice that received a single 18Lin − CD34 + CD38 − CD133 + GPI-80 + (Mouse ID: 34 + NSG001) or b single 18Lin − CD34 − CD133 + GPI-80 + (Mouse ID: 34 − NSG027) cells were analyzed by 6-color FCM at 22 or 21 weeks after transplantation. The expression of CD19, CD33 CD34 (BM, PB and spleen), CD11b and CD14 (BM), CD41 (BM and spleen), CD235a (BM) and CD56 (spleen) in living human CD45 + cells was analyzed. The presence of CD41 + and CD235a + cells in BM was analyzed using a human CD45 +/ − cell gate (depicted by red dotted lines). The mouse ID numbers presented in the figure corresponded those listed in Supplementary Data 3a . Analyses of the BM (other bones), PB and spleens of the two representative mice that received either CD34 + SRC a or CD34 − SRC b revealed that both SRCs had an in vivo differentiation capacity comparable to that of CD34 + stem/progenitor cells, CD19 + B-lymphoids, CD33 + /CD11b + myeloids, CD14 + monocytes, CD235a + erythroid and CD41 + megakaryocytic lineages. CD56 + NK cells were detected in the spleen. These results confirmed that both CD34 + and CD34 − SRCs had definite multi-lineage differentiation potential. Detailed FCM data of all recipient mice that received single CD34 + and CD34 − SRCs are presented in Supplementary Data 3b

Techniques Used: Mouse Assay, Transplantation Assay, Expressing, In Vivo

14) Product Images from "Pioneer factors govern super-enhancer dynamics in stem cell plasticity and lineage choice"

Article Title: Pioneer factors govern super-enhancer dynamics in stem cell plasticity and lineage choice

Journal: Nature

doi: 10.1038/nature14289

FACS purification strategy to isolate HFSCs and TACs a , FACS purification of WT HFSCs for ChIP-Seq according to established markers α6 hi and CD34 + 26 . Sca1 is used to remove basal epidermal cells. b , FACS purification of TACs from Krt14-H2B-GFP mice 29 . TACs are GFP low Sca1 - α6 low/- CD34 - . c , Epifluorescence of Krt14-driven H2B-GFP. HFSCs and epidermal cells are GFP hi , whereas TACs are GFP low . d , q-PCR to verify the FACS sorting strategy and measure enrichment of cell-type specific marker genes. Mean and standard deviation are shown ( n = 3). P -values from t -test: * P
Figure Legend Snippet: FACS purification strategy to isolate HFSCs and TACs a , FACS purification of WT HFSCs for ChIP-Seq according to established markers α6 hi and CD34 + 26 . Sca1 is used to remove basal epidermal cells. b , FACS purification of TACs from Krt14-H2B-GFP mice 29 . TACs are GFP low Sca1 - α6 low/- CD34 - . c , Epifluorescence of Krt14-driven H2B-GFP. HFSCs and epidermal cells are GFP hi , whereas TACs are GFP low . d , q-PCR to verify the FACS sorting strategy and measure enrichment of cell-type specific marker genes. Mean and standard deviation are shown ( n = 3). P -values from t -test: * P

Techniques Used: FACS, Purification, Chromatin Immunoprecipitation, Mouse Assay, Polymerase Chain Reaction, Marker, Standard Deviation

15) Product Images from "Human Adipose-Derived Mesenchymal Progenitor Cells Engraft into Rabbit Articular Cartilage"

Article Title: Human Adipose-Derived Mesenchymal Progenitor Cells Engraft into Rabbit Articular Cartilage

Journal: International Journal of Molecular Sciences

doi: 10.3390/ijms160612076

Phenotype of haMPCs. The haMPCs are positive for CD90, CD73, CD29, CD 49d and HLA-I, negative for CD45, CD14, CD34, HLA-DR and Actin.
Figure Legend Snippet: Phenotype of haMPCs. The haMPCs are positive for CD90, CD73, CD29, CD 49d and HLA-I, negative for CD45, CD14, CD34, HLA-DR and Actin.

Techniques Used:

16) Product Images from "Isolation and Characterization of Porcine Amniotic Fluid-Derived Multipotent Stem Cells"

Article Title: Isolation and Characterization of Porcine Amniotic Fluid-Derived Multipotent Stem Cells

Journal: PLoS ONE

doi: 10.1371/journal.pone.0019964

Determination of specific gene markers in pAF-MSCs. (A) The expression of cell surface antigens was examined by flow cytometry. The antibodies of CD34, CD117 and CD166 were labeled with PE, and CD45, CD44 and CD54 were labeled with FITC. (B) The mRNA expressions of CD90, CD117 and HLA-abc, but not CD45 were detectable in pAF-MSCs by semi-quantitative RT-PCR assay. The RNA samples were prepared from pAF-MSCs at 4 th , 8 th , 12 th and 16 th passages. The β-actin was used as internal control. (C) The pluripotent markers of ES cell were determined in pAF-MSCs (at 6 th passage) by immunofluorescence assay. The antibodies against Oct4, Nanog, SSEA1, SSEA4, Tra-1-60 and Tra-1-81 were conducted and the positive staining showed green fluorescence (FITC). The nuclei were stained by Hoechst 33342 (blue fluorescence).
Figure Legend Snippet: Determination of specific gene markers in pAF-MSCs. (A) The expression of cell surface antigens was examined by flow cytometry. The antibodies of CD34, CD117 and CD166 were labeled with PE, and CD45, CD44 and CD54 were labeled with FITC. (B) The mRNA expressions of CD90, CD117 and HLA-abc, but not CD45 were detectable in pAF-MSCs by semi-quantitative RT-PCR assay. The RNA samples were prepared from pAF-MSCs at 4 th , 8 th , 12 th and 16 th passages. The β-actin was used as internal control. (C) The pluripotent markers of ES cell were determined in pAF-MSCs (at 6 th passage) by immunofluorescence assay. The antibodies against Oct4, Nanog, SSEA1, SSEA4, Tra-1-60 and Tra-1-81 were conducted and the positive staining showed green fluorescence (FITC). The nuclei were stained by Hoechst 33342 (blue fluorescence).

Techniques Used: Expressing, Flow Cytometry, Cytometry, Labeling, Quantitative RT-PCR, Immunofluorescence, Staining, Fluorescence

17) Product Images from "Mesenchymal stem cells derived from breast cancer tissue promote the proliferation and migration of the MCF-7 cell line in vitro"

Article Title: Mesenchymal stem cells derived from breast cancer tissue promote the proliferation and migration of the MCF-7 cell line in vitro

Journal: Oncology Letters

doi: 10.3892/ol.2013.1619

Surface antigens of BC-MSCs. BC-MSCs were positive for CD13, CD29, CD44, CD71, CD105 and HLA-I, but negative for CD4, CD10, CD14, CD34, CD38 and HLA-DR. BC-MSCs, breast cancer mesenchymal stem cells.
Figure Legend Snippet: Surface antigens of BC-MSCs. BC-MSCs were positive for CD13, CD29, CD44, CD71, CD105 and HLA-I, but negative for CD4, CD10, CD14, CD34, CD38 and HLA-DR. BC-MSCs, breast cancer mesenchymal stem cells.

Techniques Used:

18) Product Images from "Rapamycin-Induced Autophagy Promotes the Chondrogenic Differentiation of Synovium-Derived Mesenchymal Stem Cells in the Temporomandibular Joint in Response to IL-1β"

Article Title: Rapamycin-Induced Autophagy Promotes the Chondrogenic Differentiation of Synovium-Derived Mesenchymal Stem Cells in the Temporomandibular Joint in Response to IL-1β

Journal: BioMed Research International

doi: 10.1155/2020/4035306

Characterization of SMSCs in the temporomandibular joint. (a) SMSCs tested positive for CD90, CD73, CD105, CD44, and negative for CD34, CD11b, CD45, HLA-DR. (b) Alizarin red staining of SMSCs cultured in osteogenic medium for 2 weeks, and the relative levels of RUNX2 and ALP mRNA in the control and osteogenic induction groups (scale bars = 100 μ m; ∗ indicates p
Figure Legend Snippet: Characterization of SMSCs in the temporomandibular joint. (a) SMSCs tested positive for CD90, CD73, CD105, CD44, and negative for CD34, CD11b, CD45, HLA-DR. (b) Alizarin red staining of SMSCs cultured in osteogenic medium for 2 weeks, and the relative levels of RUNX2 and ALP mRNA in the control and osteogenic induction groups (scale bars = 100 μ m; ∗ indicates p

Techniques Used: Staining, Cell Culture

19) Product Images from "Human T-Cell Leukemia Virus Type 1 Tax Oncoprotein Suppression of Multilineage Hematopoiesis of CD34+ Cells In Vitro"

Article Title: Human T-Cell Leukemia Virus Type 1 Tax Oncoprotein Suppression of Multilineage Hematopoiesis of CD34+ Cells In Vitro

Journal: Journal of Virology

doi: 10.1128/JVI.77.22.12152-12164.2003

FACS isolation of CD34 + cells infected with lentiviral vectors. Purified CD34 + cells (3 × 10 6 ) were infected with lentiviral vectors (MOI = 3) by centrifugation (4 h at 2,500 rpm) in a Beckman GPR centrifuge. Cells were incubated for 72 h in IMDM supplemented with 10% FBS and 30 μl of cytokine cocktail StemSpan CC100. Cells were incubated with a PE-conjugated human-specific MAb against the CD34 marker, and cell sorting was performed with a FACSVantage flow cytometer. An additional cell sample was incubated with a mouse immunoglobulin G γ isotype control antibody to set compensation and gates for FACS. The R2 gate in each panel represents the gate set to isolate CD34 + GFP + ). The percentage of CD34 + GFP + cells is displayed in the upper right panel. Mock-transduced CD34 + cells were isolated only on the basis of CD34 + expression and are represented by the R3 gate. LVs used in each transduction are as follows: GFP, HR′CMV-GFP; Tax1, HR′CMV-Tax1/GFP; Tax1(−), HR′CMV-Tax1(−)/GFP; Tax2, HR′CMV-Tax2/GFP; and Vpr, HR′CMV-Vpr/GFP. The purity of the sorted cell populations was not determined after isolation due to the limited numbers of cells recovered by FACS.
Figure Legend Snippet: FACS isolation of CD34 + cells infected with lentiviral vectors. Purified CD34 + cells (3 × 10 6 ) were infected with lentiviral vectors (MOI = 3) by centrifugation (4 h at 2,500 rpm) in a Beckman GPR centrifuge. Cells were incubated for 72 h in IMDM supplemented with 10% FBS and 30 μl of cytokine cocktail StemSpan CC100. Cells were incubated with a PE-conjugated human-specific MAb against the CD34 marker, and cell sorting was performed with a FACSVantage flow cytometer. An additional cell sample was incubated with a mouse immunoglobulin G γ isotype control antibody to set compensation and gates for FACS. The R2 gate in each panel represents the gate set to isolate CD34 + GFP + ). The percentage of CD34 + GFP + cells is displayed in the upper right panel. Mock-transduced CD34 + cells were isolated only on the basis of CD34 + expression and are represented by the R3 gate. LVs used in each transduction are as follows: GFP, HR′CMV-GFP; Tax1, HR′CMV-Tax1/GFP; Tax1(−), HR′CMV-Tax1(−)/GFP; Tax2, HR′CMV-Tax2/GFP; and Vpr, HR′CMV-Vpr/GFP. The purity of the sorted cell populations was not determined after isolation due to the limited numbers of cells recovered by FACS.

Techniques Used: FACS, Isolation, Infection, Purification, Centrifugation, Incubation, Marker, Flow Cytometry, Cytometry, Expressing, Transduction

Clonogenic colony-forming activity of lentiviral vector- transduced CD34 + cells. GFP + CD34 + cells were purified by FACS, and isolated cells (10 3 ). (A) Total number of myeloid, erythroid and pluripotential colonies per CD34 + GFP + cells (10 3 ) plated was determined at 14 days postplating. Purified CD34 + GFP + cell samples were plated in triplicate. Each column represents a separate sorting experiment. Colony-forming activities were assayed four times for each transduction, except for Tax1(−) and Tax2-transduced CD34 + cells, which were assayed three times (sorting experiments 2, 3, and 4). Transduction of CD34 + cells with HR′CMV-Vpr/GFP resulted in no colony formation in FACS experiments 1 and 4 and is indicated by an asterisk. (B) Relative distribution of clonogenic colonies. Colonies were analyzed by morphology and characterized as CFU-GM, BFU-E, or HPP-CFC. The average numbers of CFU-GM colonies that arose per 10 3 purified CD34 + GFP + cells plated were 38.8 (Mock), 24.4 (GFP), 7.3 (Tax1), 21.2 [Tax1(−)], and 21.4 (Tax2). The average numbers of BFU-E colonies arising per 10 3 CD34 + GFP+ cells plated were 14.9 (Mock), 9.0 (GFP), 2.8 (Tax1), 8.0 [Tax1(−)], and 8.3 (Tax2). The average numbers of CFU-HPP colonies arising per 10 3 CD34 + GFP + cells plated were 6.0 (Mock), 3.6 (GFP), 1.2 (Tax1), 3.2 [Tax1(−)], and 3.3 (Tax2). Statistical analysis was performed by ANOVA ( P
Figure Legend Snippet: Clonogenic colony-forming activity of lentiviral vector- transduced CD34 + cells. GFP + CD34 + cells were purified by FACS, and isolated cells (10 3 ). (A) Total number of myeloid, erythroid and pluripotential colonies per CD34 + GFP + cells (10 3 ) plated was determined at 14 days postplating. Purified CD34 + GFP + cell samples were plated in triplicate. Each column represents a separate sorting experiment. Colony-forming activities were assayed four times for each transduction, except for Tax1(−) and Tax2-transduced CD34 + cells, which were assayed three times (sorting experiments 2, 3, and 4). Transduction of CD34 + cells with HR′CMV-Vpr/GFP resulted in no colony formation in FACS experiments 1 and 4 and is indicated by an asterisk. (B) Relative distribution of clonogenic colonies. Colonies were analyzed by morphology and characterized as CFU-GM, BFU-E, or HPP-CFC. The average numbers of CFU-GM colonies that arose per 10 3 purified CD34 + GFP + cells plated were 38.8 (Mock), 24.4 (GFP), 7.3 (Tax1), 21.2 [Tax1(−)], and 21.4 (Tax2). The average numbers of BFU-E colonies arising per 10 3 CD34 + GFP+ cells plated were 14.9 (Mock), 9.0 (GFP), 2.8 (Tax1), 8.0 [Tax1(−)], and 8.3 (Tax2). The average numbers of CFU-HPP colonies arising per 10 3 CD34 + GFP + cells plated were 6.0 (Mock), 3.6 (GFP), 1.2 (Tax1), 3.2 [Tax1(−)], and 3.3 (Tax2). Statistical analysis was performed by ANOVA ( P

Techniques Used: Activity Assay, Plasmid Preparation, Purification, FACS, Isolation, Transduction

20) Product Images from "Optimization of ex vivo hematopoietic stem cell expansion in intermittent dynamic cultures"

Article Title: Optimization of ex vivo hematopoietic stem cell expansion in intermittent dynamic cultures

Journal: Biotechnology Letters

doi: 10.1007/s10529-010-0355-0

Immunophenotypes of expanded cells in static and dynamic cultures with freshly isolated CD34 + cells at day 9
Figure Legend Snippet: Immunophenotypes of expanded cells in static and dynamic cultures with freshly isolated CD34 + cells at day 9

Techniques Used: Isolation

Fold increase of TNCs ( a ) and CD34 + /CD38 − ( b ) cells in static and dynamic cultures with fresh isolated cells for 9 days. The concentrations of the inoculum and maintenance density were at 5 × 10 4 and 10 6 cells/ml, respectively
Figure Legend Snippet: Fold increase of TNCs ( a ) and CD34 + /CD38 − ( b ) cells in static and dynamic cultures with fresh isolated cells for 9 days. The concentrations of the inoculum and maintenance density were at 5 × 10 4 and 10 6 cells/ml, respectively

Techniques Used: Isolation

Immunophenotypes of expanded cells in static and dynamic cultures with thawed CD34 + cells at day 9. These cells were pre-cultured for 3 days in static condition before inoculation of the cells into shake-flasks
Figure Legend Snippet: Immunophenotypes of expanded cells in static and dynamic cultures with thawed CD34 + cells at day 9. These cells were pre-cultured for 3 days in static condition before inoculation of the cells into shake-flasks

Techniques Used: Cell Culture

21) Product Images from "Optimization of ex vivo hematopoietic stem cell expansion in intermittent dynamic cultures"

Article Title: Optimization of ex vivo hematopoietic stem cell expansion in intermittent dynamic cultures

Journal: Biotechnology Letters

doi: 10.1007/s10529-010-0355-0

Immunophenotypes of expanded cells in static and dynamic cultures with freshly isolated CD34 + cells at day 9
Figure Legend Snippet: Immunophenotypes of expanded cells in static and dynamic cultures with freshly isolated CD34 + cells at day 9

Techniques Used: Isolation

Fold increase of TNCs ( a ) and CD34 + /CD38 − ( b ) cells in static and dynamic cultures with fresh isolated cells for 9 days. The concentrations of the inoculum and maintenance density were at 5 × 10 4 and 10 6 cells/ml, respectively
Figure Legend Snippet: Fold increase of TNCs ( a ) and CD34 + /CD38 − ( b ) cells in static and dynamic cultures with fresh isolated cells for 9 days. The concentrations of the inoculum and maintenance density were at 5 × 10 4 and 10 6 cells/ml, respectively

Techniques Used: Isolation

Immunophenotypes of expanded cells in static and dynamic cultures with thawed CD34 + cells at day 9. These cells were pre-cultured for 3 days in static condition before inoculation of the cells into shake-flasks
Figure Legend Snippet: Immunophenotypes of expanded cells in static and dynamic cultures with thawed CD34 + cells at day 9. These cells were pre-cultured for 3 days in static condition before inoculation of the cells into shake-flasks

Techniques Used: Cell Culture

22) Product Images from "Rapid mobilization of murine and human hematopoietic stem and progenitor cells with AMD3100, a CXCR4 antagonist"

Article Title: Rapid mobilization of murine and human hematopoietic stem and progenitor cells with AMD3100, a CXCR4 antagonist

Journal: The Journal of Experimental Medicine

doi: 10.1084/jem.20041385

Influence of AMD3100, G-CSF, and the combination of G-CSF plus AMD3100 on mobilization of NOD-SCID SRCs from normal human volunteers (apheresis samples), surface expression of adhesion molecules and chemotaxis of CD34 + cells, and homing of mobilized murine Sca1 + Lin − cells. (A) SRCs per kg in apheresis samples from G-CSF– ( n = 3), G-CSF + AMD3100- (160 μg/kg; n = 3), and AMD3100- (240 μg/kg; n = 4) mobilized circulating blood. Each set of test samples was assayed simultaneously in limiting dilutions in conditioned NOD-SCID mice. For every sample, four different cell concentrations were used and four mice were transplanted with each cell concentration. Mice were assayed for chimerism 8 wk later; those that demonstrated > 0.2% chimerism (total CD45 + cells in BM) were considered to be positive. Percentage of negative mice were used to calculate SRC frequencies. (B, i) Expression of CD49d (VLA-4), CD49e (VLA-5), CD26L (L-selectin), and CXCR4 on mobilized CD34 + cells (mean ± 1SEM of percent positive cells of five to seven different G-CSF samples, three AMD3100 plus G-CSF samples, and four BM samples). (B, ii) Mean fluorescent intensity (MFI) of positive samples. Same number of samples evaluated as in B, i except G-CSF group has a different number ( n =7). (C) CD34 + cells isolated from G-CSF ( n = 6) and G-CSF plus AMD3100 ( n = 3) mobilized peripheral blood were assessed for chemotaxis to SDF-1/CXCL12 (100 ng/ml) and results expressed as percentage of migrated cells. Data for each sample were collected in duplicate. (D, i) Homing of CD45.2 + Sca1 + Lin − BM cells from C57Bl/6 mice into lethally irradiated CD45.1 + B6.BoyJ BM from samples mobilized with G-CSF (2 times/d for 4 d as in Fig. 2B ; n = 3), G-CSF + AMD3100 (5 mg/kg; n = 4), and AMD3100 (5 mg/kg; n = 3). Homing was assessed as in Materials and methods. (D, ii) Competitive repopulation of CD45.2 + BM cells recovered after homing shown in D, i. Results of chimerism are after 4 mo in 2° irradiated B6.BoyJ recipients. For A–D, significant differences compared with G-CSF: *P
Figure Legend Snippet: Influence of AMD3100, G-CSF, and the combination of G-CSF plus AMD3100 on mobilization of NOD-SCID SRCs from normal human volunteers (apheresis samples), surface expression of adhesion molecules and chemotaxis of CD34 + cells, and homing of mobilized murine Sca1 + Lin − cells. (A) SRCs per kg in apheresis samples from G-CSF– ( n = 3), G-CSF + AMD3100- (160 μg/kg; n = 3), and AMD3100- (240 μg/kg; n = 4) mobilized circulating blood. Each set of test samples was assayed simultaneously in limiting dilutions in conditioned NOD-SCID mice. For every sample, four different cell concentrations were used and four mice were transplanted with each cell concentration. Mice were assayed for chimerism 8 wk later; those that demonstrated > 0.2% chimerism (total CD45 + cells in BM) were considered to be positive. Percentage of negative mice were used to calculate SRC frequencies. (B, i) Expression of CD49d (VLA-4), CD49e (VLA-5), CD26L (L-selectin), and CXCR4 on mobilized CD34 + cells (mean ± 1SEM of percent positive cells of five to seven different G-CSF samples, three AMD3100 plus G-CSF samples, and four BM samples). (B, ii) Mean fluorescent intensity (MFI) of positive samples. Same number of samples evaluated as in B, i except G-CSF group has a different number ( n =7). (C) CD34 + cells isolated from G-CSF ( n = 6) and G-CSF plus AMD3100 ( n = 3) mobilized peripheral blood were assessed for chemotaxis to SDF-1/CXCL12 (100 ng/ml) and results expressed as percentage of migrated cells. Data for each sample were collected in duplicate. (D, i) Homing of CD45.2 + Sca1 + Lin − BM cells from C57Bl/6 mice into lethally irradiated CD45.1 + B6.BoyJ BM from samples mobilized with G-CSF (2 times/d for 4 d as in Fig. 2B ; n = 3), G-CSF + AMD3100 (5 mg/kg; n = 4), and AMD3100 (5 mg/kg; n = 3). Homing was assessed as in Materials and methods. (D, ii) Competitive repopulation of CD45.2 + BM cells recovered after homing shown in D, i. Results of chimerism are after 4 mo in 2° irradiated B6.BoyJ recipients. For A–D, significant differences compared with G-CSF: *P

Techniques Used: Expressing, Chemotaxis Assay, Mouse Assay, Concentration Assay, Isolation, Irradiation

23) Product Images from "Bone Marrow Osteoblast Damage by Chemotherapeutic Agents"

Article Title: Bone Marrow Osteoblast Damage by Chemotherapeutic Agents

Journal: PLoS ONE

doi: 10.1371/journal.pone.0030758

Melphalan or rTGF-β1 exposure diminished the ability of HOB to support CD34+ bone marrow cells. A ) To evaluate HOB support of CD34+ bone marrow cells, HOB cells were exposed to melphalan [25 µg/ml] or rTGF-β1 [10 ng/ml] for 24 hours. After the 24 hour exposure, the HOB were thoroughly rinsed and CD34+ cells (8.8×10 5 ) were added in co-culture. Recombinant IL-3 (100 ng/ml) was added in all groups. CD34+ cells were collected after co-culture for 48 hours and the samples were analyzed for the expression of cell surface markers. B ) HOB were exposed to melphalan [50 µg/ml] or rTGF-β1 [10 ng/ml] for 24 hours. The HOB layers were rinsed and CD34+ bone marrow cells (1.75×10 5 ) were added in co-culture for 48 hours. The CD34+ cells were collected from the HOB layers, viability determined, and 1×10 3 viable CD34+ bone marrow cells were added to methocult containing factors for myeloid lineage in triplicate. Colonies were counted and scored after 7 days in culture. *p
Figure Legend Snippet: Melphalan or rTGF-β1 exposure diminished the ability of HOB to support CD34+ bone marrow cells. A ) To evaluate HOB support of CD34+ bone marrow cells, HOB cells were exposed to melphalan [25 µg/ml] or rTGF-β1 [10 ng/ml] for 24 hours. After the 24 hour exposure, the HOB were thoroughly rinsed and CD34+ cells (8.8×10 5 ) were added in co-culture. Recombinant IL-3 (100 ng/ml) was added in all groups. CD34+ cells were collected after co-culture for 48 hours and the samples were analyzed for the expression of cell surface markers. B ) HOB were exposed to melphalan [50 µg/ml] or rTGF-β1 [10 ng/ml] for 24 hours. The HOB layers were rinsed and CD34+ bone marrow cells (1.75×10 5 ) were added in co-culture for 48 hours. The CD34+ cells were collected from the HOB layers, viability determined, and 1×10 3 viable CD34+ bone marrow cells were added to methocult containing factors for myeloid lineage in triplicate. Colonies were counted and scored after 7 days in culture. *p

Techniques Used: Co-Culture Assay, Recombinant, Expressing

24) Product Images from "A revised road map for the commitment of human cord blood CD34-negative hematopoietic stem cells"

Article Title: A revised road map for the commitment of human cord blood CD34-negative hematopoietic stem cells

Journal: Nature Communications

doi: 10.1038/s41467-018-04441-z

A comparison of the gene expression profiles of single-purified human CB-derived CD34 + and CD34 − and CD90 + HSCs by qRT-PCR. Multi-plex (79 genes) single-cell qRT-PCR using CD34 + and CD34 − HSCs and CD90 + HSCs was performed. a The 54 gene expression profiles in CD34 + and CD34 − HSCs and CD90 + HSC are depicted using a heatmap. These 54 genes were classified as HSC/HPC-, erythrocyte-, megakaryocyte-, myeloid cell-, lymphocyte- and epigenetics-related genes. b The expression of individual genes in CD34 + and CD34 − HSCs and CD90 + HSCs is depicted by violin plots. The HSC maintenance genes highly expressed in both CD34 + and CD34 − HSCs and CD90 + HSCs ( KIT , RUNK1 , TAL1 , BMI1 , DNMT3A , TGFBR1 and R2 ) are shown in the upper panel and highlighted by green color in a . The genes highly expressed in CD34 + HSCs ( IFITM1 , MPL , IKZF1 , ETV6 , ALDH1A1 and IGF1R ) are shown in the middle left panel and highlighted by pink color in a . The genes highly expressed in CD34 − HSCs ( EZH2 and MYB ) are shown in the middle right panel and highlighted by blue color in a . The gate names (R6 and R8) presented in this figure correspond to the same fractions in Fig. 1e, f . c Violin plots of the reference genes, including ACTB , GAPDH , PTPRC (CD45) and PGK-1 (left). Violin plots of the genes for CD34 and PROM1 (CD133) (middle). qRT-PCR of the VNN2 (GPI-80) mRNA expression in the 18Lin − CD34 + CD38 − CD133 + GPI-80 +/ − and 18Lin − CD34 − CD133 + GPI-80 +/ − cells, compared with the positive control (CB-derived neutrophils) and negative control (THP-1 cells) (right). n.d.: not detected
Figure Legend Snippet: A comparison of the gene expression profiles of single-purified human CB-derived CD34 + and CD34 − and CD90 + HSCs by qRT-PCR. Multi-plex (79 genes) single-cell qRT-PCR using CD34 + and CD34 − HSCs and CD90 + HSCs was performed. a The 54 gene expression profiles in CD34 + and CD34 − HSCs and CD90 + HSC are depicted using a heatmap. These 54 genes were classified as HSC/HPC-, erythrocyte-, megakaryocyte-, myeloid cell-, lymphocyte- and epigenetics-related genes. b The expression of individual genes in CD34 + and CD34 − HSCs and CD90 + HSCs is depicted by violin plots. The HSC maintenance genes highly expressed in both CD34 + and CD34 − HSCs and CD90 + HSCs ( KIT , RUNK1 , TAL1 , BMI1 , DNMT3A , TGFBR1 and R2 ) are shown in the upper panel and highlighted by green color in a . The genes highly expressed in CD34 + HSCs ( IFITM1 , MPL , IKZF1 , ETV6 , ALDH1A1 and IGF1R ) are shown in the middle left panel and highlighted by pink color in a . The genes highly expressed in CD34 − HSCs ( EZH2 and MYB ) are shown in the middle right panel and highlighted by blue color in a . The gate names (R6 and R8) presented in this figure correspond to the same fractions in Fig. 1e, f . c Violin plots of the reference genes, including ACTB , GAPDH , PTPRC (CD45) and PGK-1 (left). Violin plots of the genes for CD34 and PROM1 (CD133) (middle). qRT-PCR of the VNN2 (GPI-80) mRNA expression in the 18Lin − CD34 + CD38 − CD133 + GPI-80 +/ − and 18Lin − CD34 − CD133 + GPI-80 +/ − cells, compared with the positive control (CB-derived neutrophils) and negative control (THP-1 cells) (right). n.d.: not detected

Techniques Used: Expressing, Purification, Derivative Assay, Quantitative RT-PCR, Positive Control, Negative Control

A comparison of the gene expression profiles between CD34 + and CD34 − SRCs (HSCs) by a microarray. a The gene expression profiles of CD34 + and CD34 − SRCs (HSCs) were compared by a Gene Set Enrichment Analysis (GSEA) using the data from the microarray analysis. The gene sets enriched in CD34 − SRCs (blue bar) and those in CD34 + SRCs (red bar) with a Normalized Enrichment Score
Figure Legend Snippet: A comparison of the gene expression profiles between CD34 + and CD34 − SRCs (HSCs) by a microarray. a The gene expression profiles of CD34 + and CD34 − SRCs (HSCs) were compared by a Gene Set Enrichment Analysis (GSEA) using the data from the microarray analysis. The gene sets enriched in CD34 − SRCs (blue bar) and those in CD34 + SRCs (red bar) with a Normalized Enrichment Score

Techniques Used: Expressing, Microarray

The current and proposed models for human HSC hierarchy. a The current model 1 – 3 for the human HSC hierarchy. CD34 + HSCs as defined by a CD34 + CD38 − CD45RA − CD90 + CD49f + immunophenotype differentiate into MPPs, CMPs, MLPs, GMPs and MEPs. b The model 19 , 20 proposed based on our series of studies 18 , 21 , 22 , 25 , 33 – 36 , 60 , in which CD34 − HSCs are defined by a CD34 − CD38 low/ − CD45RA − FLT3 − CD110 − CD133 + GPI-80 + immunophenotype. CD34 − HSCs generate CD34 + HSCs in vitro 25 , 35 , 36 and in vivo 18 , 33 , suggesting that CD34 − HSCs reside at the apex of human HSC hierarchy. CD34 − HSCs, then, differentiate into MPPs, CMPs, GMPs and MEPs according to the current model 1 – 3 . Incorporating the present studies, a revised road map, which allows a commitment/differentiation pathway of CD34 − HSCs directly into MEPs (bypass route), is shown. MPP: multipotent progenitor, MLP: multilymphoid progenitor, CMP: common myeloid progenitor, GMP: granulocyte/macrophage progenitor, MEP: megakaryocyte/erythrocyte progenitor
Figure Legend Snippet: The current and proposed models for human HSC hierarchy. a The current model 1 – 3 for the human HSC hierarchy. CD34 + HSCs as defined by a CD34 + CD38 − CD45RA − CD90 + CD49f + immunophenotype differentiate into MPPs, CMPs, MLPs, GMPs and MEPs. b The model 19 , 20 proposed based on our series of studies 18 , 21 , 22 , 25 , 33 – 36 , 60 , in which CD34 − HSCs are defined by a CD34 − CD38 low/ − CD45RA − FLT3 − CD110 − CD133 + GPI-80 + immunophenotype. CD34 − HSCs generate CD34 + HSCs in vitro 25 , 35 , 36 and in vivo 18 , 33 , suggesting that CD34 − HSCs reside at the apex of human HSC hierarchy. CD34 − HSCs, then, differentiate into MPPs, CMPs, GMPs and MEPs according to the current model 1 – 3 . Incorporating the present studies, a revised road map, which allows a commitment/differentiation pathway of CD34 − HSCs directly into MEPs (bypass route), is shown. MPP: multipotent progenitor, MLP: multilymphoid progenitor, CMP: common myeloid progenitor, GMP: granulocyte/macrophage progenitor, MEP: megakaryocyte/erythrocyte progenitor

Techniques Used: In Vitro, In Vivo

Representative FACS profile and colony-forming capacity of highly purified CB-derived 18Lin - CD34 + CD38 - CD133 + GPI-80 +/ − and 18Lin - CD34 − CD133 + GPI-80 +/ − cells. A representative FACS profile is shown. a The forward scatter/side scatter (FSC/SSC) profile of immunomagnetically separated Lin − cells. The R1 gate was set on the blast-lymphocyte window. b The R2 gate was set on the 18Lin − living cells. c The R2 gated cells were subdivided into two fractions: 18Lin − CD45 + CD34 + (R3) and CD34 − (R4) cells, according to their expression of CD34. The definitions of CD34 +/ − cells are as follows: the CD34 + fraction contains cells expressing > 5% of the maximum BV421 fluorescence intensity (FI). The CD34 − level of FI was determined based on the Fluorescence Minus One controls. d The cells residing in the R3 gate were further subdivided into 18Lin − CD45 + CD34 + CD38 − (R5) cells. The CD38 − fraction contains cells expressing
Figure Legend Snippet: Representative FACS profile and colony-forming capacity of highly purified CB-derived 18Lin - CD34 + CD38 - CD133 + GPI-80 +/ − and 18Lin - CD34 − CD133 + GPI-80 +/ − cells. A representative FACS profile is shown. a The forward scatter/side scatter (FSC/SSC) profile of immunomagnetically separated Lin − cells. The R1 gate was set on the blast-lymphocyte window. b The R2 gate was set on the 18Lin − living cells. c The R2 gated cells were subdivided into two fractions: 18Lin − CD45 + CD34 + (R3) and CD34 − (R4) cells, according to their expression of CD34. The definitions of CD34 +/ − cells are as follows: the CD34 + fraction contains cells expressing > 5% of the maximum BV421 fluorescence intensity (FI). The CD34 − level of FI was determined based on the Fluorescence Minus One controls. d The cells residing in the R3 gate were further subdivided into 18Lin − CD45 + CD34 + CD38 − (R5) cells. The CD38 − fraction contains cells expressing

Techniques Used: FACS, Purification, Derivative Assay, Expressing, Fluorescence

SCID-repopulating cell activity and serial analyses of human cell repopulation of 18Lin − CD34 + CD38 − CD133 + GPI-80 + and 18Lin − CD34 − CD133 + GPI-80 + cells in primary and secondary NOG mice by IBMI. a A schematic illustration of primary and secondary transplantation of CD34 + and CD34 − SRCs is shown. PR, primary recipient; SR, secondary recipient. b The long-term repopulating potential of the CD34 + and CD34 − SRCs was determined by serially analyzing the kinetics of BM engraftment for 20 to 22 weeks in primary NOG mice that received transplants of 200 18Lin − CD34 + CD38 − CD133 + GPI-80 + cells (41 SRCs) ( n = 25) and 200 18Lin − CD34 − CD133 + GPI-80 + cells (25 SRCs) ( n = 23), respectively. All of the primary recipient mice were highly repopulated with human CD45 + cells. The human CD45 + cell repopulation rates of the mice that were transplanted with CD34 + SRCs at 12 and 18 weeks after transplantation were significantly higher than those of the mice that were transplanted with CD34 − SRCs. However, the repopulation rates of both mice were comparable at 20 to 22 weeks after transplantation (mean ± S.D., * p
Figure Legend Snippet: SCID-repopulating cell activity and serial analyses of human cell repopulation of 18Lin − CD34 + CD38 − CD133 + GPI-80 + and 18Lin − CD34 − CD133 + GPI-80 + cells in primary and secondary NOG mice by IBMI. a A schematic illustration of primary and secondary transplantation of CD34 + and CD34 − SRCs is shown. PR, primary recipient; SR, secondary recipient. b The long-term repopulating potential of the CD34 + and CD34 − SRCs was determined by serially analyzing the kinetics of BM engraftment for 20 to 22 weeks in primary NOG mice that received transplants of 200 18Lin − CD34 + CD38 − CD133 + GPI-80 + cells (41 SRCs) ( n = 25) and 200 18Lin − CD34 − CD133 + GPI-80 + cells (25 SRCs) ( n = 23), respectively. All of the primary recipient mice were highly repopulated with human CD45 + cells. The human CD45 + cell repopulation rates of the mice that were transplanted with CD34 + SRCs at 12 and 18 weeks after transplantation were significantly higher than those of the mice that were transplanted with CD34 − SRCs. However, the repopulation rates of both mice were comparable at 20 to 22 weeks after transplantation (mean ± S.D., * p

Techniques Used: Activity Assay, Mouse Assay, Transplantation Assay

Single-cell-based gene expression profiles of human CB-derived CD34 + and CD34 − HSCs. Using highly purified human CB-derived 18Lin − CD34 + CD38 − CD133 + GPI-80 + and 18Lin − CD34 − CD133 + GPI-80 + cells, we performed a single-cell-based gene expression analysis. The immunophenotypes of target cells, including controls, are presented in Supplementary Data 6 and the 79 target genes that play important roles in the pathway of HSC development/differentiation 63 are listed in Supplementary Data 7 . a A principal component analysis (PCA) revealed that the gene expression profiles in individual CD34 + ( n = 33) and CD34 − HSCs ( n = 23) were clearly different. The gene expression profiles of individual CD90 + HSCs ( n = 5) were similar to those of CD34 + HSCs but not to those of CD34 − HSCs. The dotted line represents the border region between the CD34 + and CD34 − HSCs calculated by a Fisher’s linear discriminant analysis. b An unsupervised hierarchical clustering analysis (Dendrogram) clearly showed two clusters, 1 and 2. Interestingly, all CD34 − HSCs belong to cluster 1. Three subgroups were detected in cluster 1. The left-most subgroup uniformly contained 10 CD34 − HSCs. The remaining 13 CD34 − HSCs were scattered between the other two subgroups mixed with CD34 + HSCs and CD90 + HSCs and MPPs. In contrast, some of the CD34 + HSCs and CD90 + HSCs, most of the MPP, and all other HPCs (CMP, GMP, MEP and MLP) belonged to cluster 2. These results demonstrated that the gene expression profiles of CD34 − HSC were unique and largely differed from those of other classes of CD34 + HSPCs. c A hierarchical clustering analysis of 62 genes (heatmap) detected 3 clusters ( a – c ), which are highlighted with yellow squares. Cluster (A) contained HSC/HPC-related genes, including RUNX1 , BMI1 , DNMT3a and SCL/TAL1 , in addition to CD34 and PROM1 (CD133). Cluster (B) contained HPC-related genes, including MYB , FOXO3A , SMAD4 , NFE2 and NOTCH1 . Cluster (C) contained mature cell-related genes, including those for various cytokine receptors ( CSF2RA , IL-6R , CSF1R , IGF2R and IL-3RA )
Figure Legend Snippet: Single-cell-based gene expression profiles of human CB-derived CD34 + and CD34 − HSCs. Using highly purified human CB-derived 18Lin − CD34 + CD38 − CD133 + GPI-80 + and 18Lin − CD34 − CD133 + GPI-80 + cells, we performed a single-cell-based gene expression analysis. The immunophenotypes of target cells, including controls, are presented in Supplementary Data 6 and the 79 target genes that play important roles in the pathway of HSC development/differentiation 63 are listed in Supplementary Data 7 . a A principal component analysis (PCA) revealed that the gene expression profiles in individual CD34 + ( n = 33) and CD34 − HSCs ( n = 23) were clearly different. The gene expression profiles of individual CD90 + HSCs ( n = 5) were similar to those of CD34 + HSCs but not to those of CD34 − HSCs. The dotted line represents the border region between the CD34 + and CD34 − HSCs calculated by a Fisher’s linear discriminant analysis. b An unsupervised hierarchical clustering analysis (Dendrogram) clearly showed two clusters, 1 and 2. Interestingly, all CD34 − HSCs belong to cluster 1. Three subgroups were detected in cluster 1. The left-most subgroup uniformly contained 10 CD34 − HSCs. The remaining 13 CD34 − HSCs were scattered between the other two subgroups mixed with CD34 + HSCs and CD90 + HSCs and MPPs. In contrast, some of the CD34 + HSCs and CD90 + HSCs, most of the MPP, and all other HPCs (CMP, GMP, MEP and MLP) belonged to cluster 2. These results demonstrated that the gene expression profiles of CD34 − HSC were unique and largely differed from those of other classes of CD34 + HSPCs. c A hierarchical clustering analysis of 62 genes (heatmap) detected 3 clusters ( a – c ), which are highlighted with yellow squares. Cluster (A) contained HSC/HPC-related genes, including RUNX1 , BMI1 , DNMT3a and SCL/TAL1 , in addition to CD34 and PROM1 (CD133). Cluster (B) contained HPC-related genes, including MYB , FOXO3A , SMAD4 , NFE2 and NOTCH1 . Cluster (C) contained mature cell-related genes, including those for various cytokine receptors ( CSF2RA , IL-6R , CSF1R , IGF2R and IL-3RA )

Techniques Used: Expressing, Derivative Assay, Purification

Human multi-lineage hematopoietic repopulation abilities of single CD34 + and CD34 − SRCs. Human multi-lineage hematopoietic repopulations in the primary recipient NSG mice that received a single 18Lin − CD34 + CD38 − CD133 + GPI-80 + (Mouse ID: 34 + NSG001) or b single 18Lin − CD34 − CD133 + GPI-80 + (Mouse ID: 34 − NSG027) cells were analyzed by 6-color FCM at 22 or 21 weeks after transplantation. The expression of CD19, CD33 CD34 (BM, PB and spleen), CD11b and CD14 (BM), CD41 (BM and spleen), CD235a (BM) and CD56 (spleen) in living human CD45 + cells was analyzed. The presence of CD41 + and CD235a + cells in BM was analyzed using a human CD45 +/ − cell gate (depicted by red dotted lines). The mouse ID numbers presented in the figure corresponded those listed in Supplementary Data 3a . Analyses of the BM (other bones), PB and spleens of the two representative mice that received either CD34 + SRC a or CD34 − SRC b revealed that both SRCs had an in vivo differentiation capacity comparable to that of CD34 + stem/progenitor cells, CD19 + B-lymphoids, CD33 + /CD11b + myeloids, CD14 + monocytes, CD235a + erythroid and CD41 + megakaryocytic lineages. CD56 + NK cells were detected in the spleen. These results confirmed that both CD34 + and CD34 − SRCs had definite multi-lineage differentiation potential. Detailed FCM data of all recipient mice that received single CD34 + and CD34 − SRCs are presented in Supplementary Data 3b
Figure Legend Snippet: Human multi-lineage hematopoietic repopulation abilities of single CD34 + and CD34 − SRCs. Human multi-lineage hematopoietic repopulations in the primary recipient NSG mice that received a single 18Lin − CD34 + CD38 − CD133 + GPI-80 + (Mouse ID: 34 + NSG001) or b single 18Lin − CD34 − CD133 + GPI-80 + (Mouse ID: 34 − NSG027) cells were analyzed by 6-color FCM at 22 or 21 weeks after transplantation. The expression of CD19, CD33 CD34 (BM, PB and spleen), CD11b and CD14 (BM), CD41 (BM and spleen), CD235a (BM) and CD56 (spleen) in living human CD45 + cells was analyzed. The presence of CD41 + and CD235a + cells in BM was analyzed using a human CD45 +/ − cell gate (depicted by red dotted lines). The mouse ID numbers presented in the figure corresponded those listed in Supplementary Data 3a . Analyses of the BM (other bones), PB and spleens of the two representative mice that received either CD34 + SRC a or CD34 − SRC b revealed that both SRCs had an in vivo differentiation capacity comparable to that of CD34 + stem/progenitor cells, CD19 + B-lymphoids, CD33 + /CD11b + myeloids, CD14 + monocytes, CD235a + erythroid and CD41 + megakaryocytic lineages. CD56 + NK cells were detected in the spleen. These results confirmed that both CD34 + and CD34 − SRCs had definite multi-lineage differentiation potential. Detailed FCM data of all recipient mice that received single CD34 + and CD34 − SRCs are presented in Supplementary Data 3b

Techniques Used: Mouse Assay, Transplantation Assay, Expressing, In Vivo

25) Product Images from "A Crucial Role of CXCL14 for Promoting Regulatory T Cells Activation in Stroke"

Article Title: A Crucial Role of CXCL14 for Promoting Regulatory T Cells Activation in Stroke

Journal: Theranostics

doi: 10.7150/thno.17558

CXCL14 Exerts Immunomodulation via Regulating Immature Dendritic Cell (iDC) and Regulatory T Cells (Treg). ( A ) In the representative 3-D image, some CD11c + CD34 + iDC were observed in the human stroke hemisphere (upper panel). Moreover, increased numbers of CD11c + CD34 + cells were found in the ischemic brain of CXCL14 +/+ mice compared to that of CXCL14 -/- mice (middle panel). Furthermore, significantly increased numbers of CD4 + CD25 + Foxp3 + Treg were found in the CXCL14 +/+ mice compared to those of CXCL14 -/- mice at 5 days after cerebral ischemia (lower panel). ( B ) ELISA showed a significant decrease in the levels of IL-10 and TGF-beta in brain homogenates of CXCL14 -/- mice compared with those of wild type mice. ( C ) Infarct volume was significantly increased in CXCL14 -/- mice compared to that of CXCL14 +/+ mice. Furthermore, infarct volume was larger in CD25-Ab-treated, CD11c-Ab-treated and IL-2-Ab-treated CXCL14 +/+ mice than in saline-treated CXCL14 +/+ mice. ( D ) Overexpression of CXCL14 significantly increased cell proliferation, as shown by trypan blue, which excluded viable cells and the BrdU labeling index. ( E ) The transwell migration assay showed that CXCL14 is highly attracted to CD11c + B220 - cells, but not to CD11c - B220 + cells or to CD11c - B220 - cells. In iDC migration profiles for either chemokine after culturing for 8 days, CD11c + iDC responded to CXCL14 and to CCL2 chemoattraction. ( F ) CD11c + iDC treated with CXCL14 could move across the membrane in a concentration-dependent manner (SDF-1α as a positive control) in the transwell migration assay. In contrast, CXCL14-induced iDC migratory activity could be neutralized by adding the CXCL14-Ab and SB-3CT. LV-PrP C -shRNA or LV-PECAM-1-shRNA transduction also inhibited the CXCL14-induced iDC trafficking. The mean ± SEM is shown. * P
Figure Legend Snippet: CXCL14 Exerts Immunomodulation via Regulating Immature Dendritic Cell (iDC) and Regulatory T Cells (Treg). ( A ) In the representative 3-D image, some CD11c + CD34 + iDC were observed in the human stroke hemisphere (upper panel). Moreover, increased numbers of CD11c + CD34 + cells were found in the ischemic brain of CXCL14 +/+ mice compared to that of CXCL14 -/- mice (middle panel). Furthermore, significantly increased numbers of CD4 + CD25 + Foxp3 + Treg were found in the CXCL14 +/+ mice compared to those of CXCL14 -/- mice at 5 days after cerebral ischemia (lower panel). ( B ) ELISA showed a significant decrease in the levels of IL-10 and TGF-beta in brain homogenates of CXCL14 -/- mice compared with those of wild type mice. ( C ) Infarct volume was significantly increased in CXCL14 -/- mice compared to that of CXCL14 +/+ mice. Furthermore, infarct volume was larger in CD25-Ab-treated, CD11c-Ab-treated and IL-2-Ab-treated CXCL14 +/+ mice than in saline-treated CXCL14 +/+ mice. ( D ) Overexpression of CXCL14 significantly increased cell proliferation, as shown by trypan blue, which excluded viable cells and the BrdU labeling index. ( E ) The transwell migration assay showed that CXCL14 is highly attracted to CD11c + B220 - cells, but not to CD11c - B220 + cells or to CD11c - B220 - cells. In iDC migration profiles for either chemokine after culturing for 8 days, CD11c + iDC responded to CXCL14 and to CCL2 chemoattraction. ( F ) CD11c + iDC treated with CXCL14 could move across the membrane in a concentration-dependent manner (SDF-1α as a positive control) in the transwell migration assay. In contrast, CXCL14-induced iDC migratory activity could be neutralized by adding the CXCL14-Ab and SB-3CT. LV-PrP C -shRNA or LV-PECAM-1-shRNA transduction also inhibited the CXCL14-induced iDC trafficking. The mean ± SEM is shown. * P

Techniques Used: Mouse Assay, Enzyme-linked Immunosorbent Assay, Over Expression, Labeling, Transwell Migration Assay, Migration, Concentration Assay, Positive Control, Activity Assay, shRNA, Transduction

26) Product Images from "In Vitro Expression of Cytokeratin 19 in Adipose-Derived Stem Cells Is Induced by Epidermal Growth Factor"

Article Title: In Vitro Expression of Cytokeratin 19 in Adipose-Derived Stem Cells Is Induced by Epidermal Growth Factor

Journal: Medical Science Monitor : International Medical Journal of Experimental and Clinical Research

doi: 10.12659/MSM.908647

Characterization of ADSCs by flow cytometry. ADSCs expressed the mesenchymal stem cell markers CD29, CD90, and CD105, but were primarily negative for the endothelial marker CD31, and the hematopoietic markers CD34 and CD45.
Figure Legend Snippet: Characterization of ADSCs by flow cytometry. ADSCs expressed the mesenchymal stem cell markers CD29, CD90, and CD105, but were primarily negative for the endothelial marker CD31, and the hematopoietic markers CD34 and CD45.

Techniques Used: Flow Cytometry, Cytometry, Marker

27) Product Images from "Bone marrow MSC from pediatric patients with B-ALL highly immunosuppress T-cell responses but do not compromise CD19-CAR T-cell activity"

Article Title: Bone marrow MSC from pediatric patients with B-ALL highly immunosuppress T-cell responses but do not compromise CD19-CAR T-cell activity

Journal: Journal for Immunotherapy of Cancer

doi: 10.1136/jitc-2020-001419

Characterization of BM-MSC from pediatric patients with B-ALL and age-matched HD. (A) Representative phase-contrast morphology of BM-MSC. (B) Immunophenotype of expanded BM-MSC at P3. Expression of CD105, CD90, CD73, CD13, nestin, CD45, CD34 and CD31 was analyzed by FACS. Shown are representative FACS histograms; gray histograms represent isotype-matched negative control mAb and blue histograms represent mAb-specific stained cells. (C) In vitro proliferation of BM-MSC calculated as population doubling over six passages. (D, E) Osteogenic and adipogenic differentiation capacity of BM-MSC. (D) Left panel, oil red-O staining indicative of adipogenic differentiation capacity. Right panel, Alizarin red staining and detection of AP alkaline phosphatase activity with NTB, indicative of osteogenic differentiation capacity. (E) Relative quantification of mRNA expression of the adipogenic transcription factors C/EBP-α and PPAR-γ and the osteogenic transcription factors BMP2 and RUNX2 by qRT-PCR. Data are shown as mean±SEM. *P
Figure Legend Snippet: Characterization of BM-MSC from pediatric patients with B-ALL and age-matched HD. (A) Representative phase-contrast morphology of BM-MSC. (B) Immunophenotype of expanded BM-MSC at P3. Expression of CD105, CD90, CD73, CD13, nestin, CD45, CD34 and CD31 was analyzed by FACS. Shown are representative FACS histograms; gray histograms represent isotype-matched negative control mAb and blue histograms represent mAb-specific stained cells. (C) In vitro proliferation of BM-MSC calculated as population doubling over six passages. (D, E) Osteogenic and adipogenic differentiation capacity of BM-MSC. (D) Left panel, oil red-O staining indicative of adipogenic differentiation capacity. Right panel, Alizarin red staining and detection of AP alkaline phosphatase activity with NTB, indicative of osteogenic differentiation capacity. (E) Relative quantification of mRNA expression of the adipogenic transcription factors C/EBP-α and PPAR-γ and the osteogenic transcription factors BMP2 and RUNX2 by qRT-PCR. Data are shown as mean±SEM. *P

Techniques Used: Expressing, FACS, Negative Control, Staining, In Vitro, Activity Assay, Quantitative RT-PCR

28) Product Images from "CD317 is over-expressed in B-cell chronic lymphocytic leukemia, but not B-cell acute lymphoblastic leukemia"

Article Title: CD317 is over-expressed in B-cell chronic lymphocytic leukemia, but not B-cell acute lymphoblastic leukemia

Journal: International Journal of Clinical and Experimental Pathology

doi:

Expression of CD317 on human normal bone marrow hematogones. Bone marrow specimens from 3 healthy individuals were stained with anti-CD317-PE or isotype-PE, and anti- CD34-FITC, anti-CD19-PerCP, anti-CD10-APC. The cells were analysed by flow cytometry.
Figure Legend Snippet: Expression of CD317 on human normal bone marrow hematogones. Bone marrow specimens from 3 healthy individuals were stained with anti-CD317-PE or isotype-PE, and anti- CD34-FITC, anti-CD19-PerCP, anti-CD10-APC. The cells were analysed by flow cytometry.

Techniques Used: Expressing, Staining, Flow Cytometry, Cytometry

29) Product Images from "Transplantation of placenta-derived mesenchymal stem cell-induced neural stem cells to treat spinal cord injury"

Article Title: Transplantation of placenta-derived mesenchymal stem cell-induced neural stem cells to treat spinal cord injury

Journal: Neural Regeneration Research

doi: 10.4103/1673-5374.147953

Identification of human placenta-derived mesenchymal stem cells. Flow cytometry analysis revealed that the cells were positive for CD90, CD105, and CD73 and negative for CD34, CD14, and CD45. These results demonstrate that the extracted and cultured cells were mesenchymal stem cells.
Figure Legend Snippet: Identification of human placenta-derived mesenchymal stem cells. Flow cytometry analysis revealed that the cells were positive for CD90, CD105, and CD73 and negative for CD34, CD14, and CD45. These results demonstrate that the extracted and cultured cells were mesenchymal stem cells.

Techniques Used: Derivative Assay, Flow Cytometry, Cytometry, Cell Culture

30) Product Images from "Isolation, Characterization, and Transplantation of Cardiac Endothelial Cells"

Article Title: Isolation, Characterization, and Transplantation of Cardiac Endothelial Cells

Journal: BioMed Research International

doi: 10.1155/2013/359412

Endothelial marker expression in early and late cultured cardiac endothelial cells. (a) q-RT-PCR of endothelial cell genes revealed a decreased expression of CD31, CD34, eNOS, VE-Cad, and vWF in late cultured EC (passage 6, black bar) compared to early cultured EC (passage 1, white bar). In contrast, expression of sFlt-1 was increased. (b) the table shows average Ct values of each endothelial genes expressed by sorted cardiac endothelial cells from atrium in passage 1 and 6. The experiment was run from three different samples in each passage represented as Sample 1, 2, and 3, respectively. Each sample was performed in triplicate per each target gene. The RNA from mouse whole heart was used as positive control. (c) the table represents efficiency ( E ) of primers that we used for q-RT-PCR. We calculated the percentage of primer efficiency by using the formula; E = [10 ∧ (−1/slope)] − 1 × 100 where E is the primer efficiency of the q-RT-PCR reaction and slope refers to the slope of the plot of Ct value versus the log of the input RNA amount. Our primer list demonstrates a slope between −4.19 and −3.42 which corresponds to an estimated efficiency between 73.4% and 95.9%, respectively [ 63 ]. (d) western blot for phosphorylated eNOS showed active eNOS in both early (passage 1 samples 1, 2, and 3) and late passaged EC (passage 6 samples 4, 5, and 6).
Figure Legend Snippet: Endothelial marker expression in early and late cultured cardiac endothelial cells. (a) q-RT-PCR of endothelial cell genes revealed a decreased expression of CD31, CD34, eNOS, VE-Cad, and vWF in late cultured EC (passage 6, black bar) compared to early cultured EC (passage 1, white bar). In contrast, expression of sFlt-1 was increased. (b) the table shows average Ct values of each endothelial genes expressed by sorted cardiac endothelial cells from atrium in passage 1 and 6. The experiment was run from three different samples in each passage represented as Sample 1, 2, and 3, respectively. Each sample was performed in triplicate per each target gene. The RNA from mouse whole heart was used as positive control. (c) the table represents efficiency ( E ) of primers that we used for q-RT-PCR. We calculated the percentage of primer efficiency by using the formula; E = [10 ∧ (−1/slope)] − 1 × 100 where E is the primer efficiency of the q-RT-PCR reaction and slope refers to the slope of the plot of Ct value versus the log of the input RNA amount. Our primer list demonstrates a slope between −4.19 and −3.42 which corresponds to an estimated efficiency between 73.4% and 95.9%, respectively [ 63 ]. (d) western blot for phosphorylated eNOS showed active eNOS in both early (passage 1 samples 1, 2, and 3) and late passaged EC (passage 6 samples 4, 5, and 6).

Techniques Used: Marker, Expressing, Cell Culture, Reverse Transcription Polymerase Chain Reaction, Positive Control, Western Blot

Flow-cytometry analysis of primary isolates of mouse cardiac endothelial cells. After mechanical disaggregation and enzymatic digestion, single cell suspension from murine whole heart (a), atrium (b), and ventricle (c) were incubated with combination of fluorescent-antibody to CD45, Sca-1, CD31, and CD34. Cells were first gated to include only small and low granulated cells from FSC-A and SSC-A dot plot. Hematopoietic cells identified by CD45+ were gated out. Sca-1+/CD31+ were identified as endothelial population. This population was further divided into CD34+ or CD34− endothelial cells.
Figure Legend Snippet: Flow-cytometry analysis of primary isolates of mouse cardiac endothelial cells. After mechanical disaggregation and enzymatic digestion, single cell suspension from murine whole heart (a), atrium (b), and ventricle (c) were incubated with combination of fluorescent-antibody to CD45, Sca-1, CD31, and CD34. Cells were first gated to include only small and low granulated cells from FSC-A and SSC-A dot plot. Hematopoietic cells identified by CD45+ were gated out. Sca-1+/CD31+ were identified as endothelial population. This population was further divided into CD34+ or CD34− endothelial cells.

Techniques Used: Flow Cytometry, Cytometry, Incubation

Freshly-sorted cardiac endothelial cells. Comparison of endothelial percentage within CD45− population among age (a) ( n = 28) and gender (b) ( n = 29). (c) The percentage of ECs within CD45− population between atrium and ventricle in 3-month-old mice ( n = 11). (d) q-RT-PCR levels for Flk-1 , soluble Flt-1, and VEGF-A of one male (M), one female (F), and freshly FACS-sorted ECs from atrium and ventricle normalized to GAPDH. (e) Representative RT-PCR analysis of freshly FACS-sorted ECs from atrium and ventricle (three females 15–24-month-old) showed expression of endothelial markers Flk-1, Flt-1, Tie-1, Tie-2, vWF, eNOS, VE-Cad, CD31, and CD34 while lacked cardiomyocyte marker NKx2.5 and endocardium marker GATA5. P values were calculated by ANOVA (a) or Student's t -test (b, c).
Figure Legend Snippet: Freshly-sorted cardiac endothelial cells. Comparison of endothelial percentage within CD45− population among age (a) ( n = 28) and gender (b) ( n = 29). (c) The percentage of ECs within CD45− population between atrium and ventricle in 3-month-old mice ( n = 11). (d) q-RT-PCR levels for Flk-1 , soluble Flt-1, and VEGF-A of one male (M), one female (F), and freshly FACS-sorted ECs from atrium and ventricle normalized to GAPDH. (e) Representative RT-PCR analysis of freshly FACS-sorted ECs from atrium and ventricle (three females 15–24-month-old) showed expression of endothelial markers Flk-1, Flt-1, Tie-1, Tie-2, vWF, eNOS, VE-Cad, CD31, and CD34 while lacked cardiomyocyte marker NKx2.5 and endocardium marker GATA5. P values were calculated by ANOVA (a) or Student's t -test (b, c).

Techniques Used: Mouse Assay, Reverse Transcription Polymerase Chain Reaction, FACS, Expressing, Marker

Murine cardiac endothelial cells. Heart tissue sections staining demonstrated (a–d) colocalization of CD31 and Sca-1, (e–h) CD31 and CD34, and (i–k) CD31 and vWF at endothelial lining layer. (l) Representative photograph of a heart section stained with secondary antibodies only (anti rat Alexa 488 and anti rabbit Alexa 594) and DAPI shows acceptable green and red background fluorescence. (m, n) eNOS and Caveolin-1 highlighted the endothelial layer. (o) alpha-SMA exclusively stained the smooth muscle cells of vessels without overlapping with CD31 which stained endothelial cell layer. (p) Staining of NG2 and Sca-1 showed Sca-1 staining in endothelium of coronary arteries and capillaries whereas NG2 is exclusively expressed by vascular smooth muscle cells. Photographs were taken with Zeiss Axiovert 200 using Axiovision v4.6.3 software. Scale bar = 100 μ m.
Figure Legend Snippet: Murine cardiac endothelial cells. Heart tissue sections staining demonstrated (a–d) colocalization of CD31 and Sca-1, (e–h) CD31 and CD34, and (i–k) CD31 and vWF at endothelial lining layer. (l) Representative photograph of a heart section stained with secondary antibodies only (anti rat Alexa 488 and anti rabbit Alexa 594) and DAPI shows acceptable green and red background fluorescence. (m, n) eNOS and Caveolin-1 highlighted the endothelial layer. (o) alpha-SMA exclusively stained the smooth muscle cells of vessels without overlapping with CD31 which stained endothelial cell layer. (p) Staining of NG2 and Sca-1 showed Sca-1 staining in endothelium of coronary arteries and capillaries whereas NG2 is exclusively expressed by vascular smooth muscle cells. Photographs were taken with Zeiss Axiovert 200 using Axiovision v4.6.3 software. Scale bar = 100 μ m.

Techniques Used: Staining, Fluorescence, Software

31) Product Images from "Essential role of FBXL5-mediated cellular iron homeostasis in maintenance of hematopoietic stem cells"

Article Title: Essential role of FBXL5-mediated cellular iron homeostasis in maintenance of hematopoietic stem cells

Journal: Nature Communications

doi: 10.1038/ncomms16114

Downregulation of FBXL5 expression is associated with human hematopoietic failure. ( a – c ) Microarray analysis of FBXL5 ( a ), TFR1 ( b ) and DMT1 ( c ) expression in Lin – CD34 + CD38 – CD90 + CD45RA – cells from healthy control (Ctrl) subjects ( n =11) or from MDS patients without deletion of chromosome 5q ( n =8). ( d , e ) Microarray analysis of FBXL5 ( d ) and TFR1 ( e ) expression in CD34 + BM mononuclear cells from healthy control subjects ( n =17) or from patients with refractory anaemia with ringed sideroblasts (RARS, n =48), refractory anaemia (RA, n =55), refractory anaemia with excess blasts 1 (RAEB1, n =37), or refractory anaemia with excess blasts 2 (RAEB2, n =43). Each point represents an individual donor, and horizontal lines indicate the mean. ( f ) Survival of MDS patients without deletion of chromosome 5q and with a high ( n =29) or low ( n =79) level of IRP2 expression in CD34 + hematopoietic progenitor cells. * P
Figure Legend Snippet: Downregulation of FBXL5 expression is associated with human hematopoietic failure. ( a – c ) Microarray analysis of FBXL5 ( a ), TFR1 ( b ) and DMT1 ( c ) expression in Lin – CD34 + CD38 – CD90 + CD45RA – cells from healthy control (Ctrl) subjects ( n =11) or from MDS patients without deletion of chromosome 5q ( n =8). ( d , e ) Microarray analysis of FBXL5 ( d ) and TFR1 ( e ) expression in CD34 + BM mononuclear cells from healthy control subjects ( n =17) or from patients with refractory anaemia with ringed sideroblasts (RARS, n =48), refractory anaemia (RA, n =55), refractory anaemia with excess blasts 1 (RAEB1, n =37), or refractory anaemia with excess blasts 2 (RAEB2, n =43). Each point represents an individual donor, and horizontal lines indicate the mean. ( f ) Survival of MDS patients without deletion of chromosome 5q and with a high ( n =29) or low ( n =79) level of IRP2 expression in CD34 + hematopoietic progenitor cells. * P

Techniques Used: Expressing, Microarray

32) Product Images from "Optimization of culture conditions for rapid clinical-scale expansion of human umbilical cord blood-derived mesenchymal stem cells"

Article Title: Optimization of culture conditions for rapid clinical-scale expansion of human umbilical cord blood-derived mesenchymal stem cells

Journal: Clinical and Translational Medicine

doi: 10.1186/s40169-017-0168-z

Effects of Ca 2+ /hypoxia on the stem cell phenotypes of hUCB-MSCs. a Immunophenotypic analysis of CD34, CD45, CD73, and CD105 expression in naïve or Ca 2+ /hypoxia-conditioned hUCB-MSCs was performed using flow cytometry. b After incubation in specialized
Figure Legend Snippet: Effects of Ca 2+ /hypoxia on the stem cell phenotypes of hUCB-MSCs. a Immunophenotypic analysis of CD34, CD45, CD73, and CD105 expression in naïve or Ca 2+ /hypoxia-conditioned hUCB-MSCs was performed using flow cytometry. b After incubation in specialized

Techniques Used: Expressing, Flow Cytometry, Cytometry, Incubation

33) Product Images from "Bone marrow mesenchymal stem cells differentiate into urothelial cells and the implications for reconstructing urinary bladder mucosa"

Article Title: Bone marrow mesenchymal stem cells differentiate into urothelial cells and the implications for reconstructing urinary bladder mucosa

Journal: Cytotechnology

doi: 10.1007/s10616-011-9376-3

Phenotypes of BMSCs as indicated by flow cytometry. BMSC markers CD29, CD44, CD105, and CD90. Hematopoietic progenitor markers CD34 and HLA-DR. Pan - leukocyte marker CD45. Differentiated marker CD71
Figure Legend Snippet: Phenotypes of BMSCs as indicated by flow cytometry. BMSC markers CD29, CD44, CD105, and CD90. Hematopoietic progenitor markers CD34 and HLA-DR. Pan - leukocyte marker CD45. Differentiated marker CD71

Techniques Used: Flow Cytometry, Cytometry, Marker

34) Product Images from "TLR9 ligation in pancreatic stellate cells promotes tumorigenesis"

Article Title: TLR9 ligation in pancreatic stellate cells promotes tumorigenesis

Journal: The Journal of Experimental Medicine

doi: 10.1084/jem.20142162

TLR9 is up-regulated during pancreatic oncogenesis in epithelial, inflammatory, and stromal cells . (A) Frozen sections from pancreata of 3-mo-old KC and KC;TLR9 −/− mice were stained for DAPI and TLR9 and visualized on a confocal microscope (63×; bar = 30 µm). Results were quantified based on 10 HPFs per slide. (B) 3- and 6-mo-old KC mice were analyzed by flow cytometry for pancreatic TLR9 expression on DCs, granulocytes, and macrophages. Mean fluorescence intensity (MFI) is indicated compared with respective isotype controls. Representative data and summary statistics from three mice per data point are shown. (C) 3-mo-old KC mice were analyzed by flow cytometry for pancreatic TLR9 expression on epithelial cells (CD45 − CD34 − CD133 + ) and cancer-associated fibroblasts (CD45 − CD34 − CD133 − PDGFR-α + ). Representative data and summary statistics from three mice per data point are shown. Mouse experiments were repeated more than five times with similar results. (D) TLR9 immunohistochemistry compared with isotype control was performed on normal human pancreata and pancreata from patients with PDAC (40×; bar = 60 µm). Representative images and quantitative data from four patients per group are shown. (E) Human PDAC and normal human pancreas specimens were stained using a mAb directed against HMGB1 (40×; bar = 60 µm). Representative images and quantitative data from four patients per group are shown (*, P
Figure Legend Snippet: TLR9 is up-regulated during pancreatic oncogenesis in epithelial, inflammatory, and stromal cells . (A) Frozen sections from pancreata of 3-mo-old KC and KC;TLR9 −/− mice were stained for DAPI and TLR9 and visualized on a confocal microscope (63×; bar = 30 µm). Results were quantified based on 10 HPFs per slide. (B) 3- and 6-mo-old KC mice were analyzed by flow cytometry for pancreatic TLR9 expression on DCs, granulocytes, and macrophages. Mean fluorescence intensity (MFI) is indicated compared with respective isotype controls. Representative data and summary statistics from three mice per data point are shown. (C) 3-mo-old KC mice were analyzed by flow cytometry for pancreatic TLR9 expression on epithelial cells (CD45 − CD34 − CD133 + ) and cancer-associated fibroblasts (CD45 − CD34 − CD133 − PDGFR-α + ). Representative data and summary statistics from three mice per data point are shown. Mouse experiments were repeated more than five times with similar results. (D) TLR9 immunohistochemistry compared with isotype control was performed on normal human pancreata and pancreata from patients with PDAC (40×; bar = 60 µm). Representative images and quantitative data from four patients per group are shown. (E) Human PDAC and normal human pancreas specimens were stained using a mAb directed against HMGB1 (40×; bar = 60 µm). Representative images and quantitative data from four patients per group are shown (*, P

Techniques Used: Mouse Assay, Staining, Microscopy, Flow Cytometry, Cytometry, Expressing, Fluorescence, Immunohistochemistry

35) Product Images from "Silencing Smad7 potentiates BMP2-induced chondrogenic differentiation and inhibits endochondral ossification in human synovial-derived mesenchymal stromal cells"

Article Title: Silencing Smad7 potentiates BMP2-induced chondrogenic differentiation and inhibits endochondral ossification in human synovial-derived mesenchymal stromal cells

Journal: Stem Cell Research & Therapy

doi: 10.1186/s13287-021-02202-2

Isolation and morphology of cultured hSMSCs; the proliferation potential and the identification of hSMSCs. a hSMSCs were migrated outwards from the freshly harvested synovium at 72 h; Morphology of the confluence hSMSCs was recorded at day 14; primary cultures (P0) and the fifth passage (P5) with spindle shapes on cell culture dish (original magnification × 40, scale bar =200 μm) b . Cell proliferation assessed by crystal violet staining assay. General observation of the stained cells was recorded at the indicated time-points. c The stained cells were dissolved for quantitatively OD reading at A590 nm. d Proliferation of P1 and P5 as determined by the CCK-8 method showed that no significant difference. e The MSC markers of hSMSCs at P1 by flow cytometry and hSMSCs were positive for the MSC markers CD73, CD90, CD105, and CD44, while weakly expressed the markers CD34, CD14, CD45, and HLA-DR. f The average mean fluorescence for all markers between three donor populations. g The potential for osteogenic, chondrogenic, and adipogenic differentiation of hSMSCs in vitro: osteogenic (g1.alkaline phosphatase staining and g2.Alzarin Red S staining), chondrogenic (g3.Alcian Blue staining and g4.COL 2 immunohistochemical staining), and adipogenic (g5.Oil Red O staining) (original magnification × 200, scale bar = 50 μm). h RT-qPCR assay was performed to determine that the expression of osteogenic chondrogenic and adipogenic differentiation relative factors, including RUNX2, Osterix, Sox9, COL2, and PPAR-γ. The data are shown as mean ± SD for three separate experiments. * P
Figure Legend Snippet: Isolation and morphology of cultured hSMSCs; the proliferation potential and the identification of hSMSCs. a hSMSCs were migrated outwards from the freshly harvested synovium at 72 h; Morphology of the confluence hSMSCs was recorded at day 14; primary cultures (P0) and the fifth passage (P5) with spindle shapes on cell culture dish (original magnification × 40, scale bar =200 μm) b . Cell proliferation assessed by crystal violet staining assay. General observation of the stained cells was recorded at the indicated time-points. c The stained cells were dissolved for quantitatively OD reading at A590 nm. d Proliferation of P1 and P5 as determined by the CCK-8 method showed that no significant difference. e The MSC markers of hSMSCs at P1 by flow cytometry and hSMSCs were positive for the MSC markers CD73, CD90, CD105, and CD44, while weakly expressed the markers CD34, CD14, CD45, and HLA-DR. f The average mean fluorescence for all markers between three donor populations. g The potential for osteogenic, chondrogenic, and adipogenic differentiation of hSMSCs in vitro: osteogenic (g1.alkaline phosphatase staining and g2.Alzarin Red S staining), chondrogenic (g3.Alcian Blue staining and g4.COL 2 immunohistochemical staining), and adipogenic (g5.Oil Red O staining) (original magnification × 200, scale bar = 50 μm). h RT-qPCR assay was performed to determine that the expression of osteogenic chondrogenic and adipogenic differentiation relative factors, including RUNX2, Osterix, Sox9, COL2, and PPAR-γ. The data are shown as mean ± SD for three separate experiments. * P

Techniques Used: Isolation, Cell Culture, Staining, CCK-8 Assay, Flow Cytometry, Fluorescence, In Vitro, Immunohistochemistry, Quantitative RT-PCR, Expressing

36) Product Images from "CD34 Expression by Hair Follicle Stem Cells Is Required for Skin Tumor Development in Mice"

Article Title: CD34 Expression by Hair Follicle Stem Cells Is Required for Skin Tumor Development in Mice

Journal:

doi: 10.1158/0008-5472.CAN-06-3128

CD34KO mouse skin lacks CD34 expression and appears normal histologically. Top, skin from 7-wk-old WT and CD34KO mice was fixed in 10% formalin and stained with H E; middle, single-cell suspensions of keratinocytes were stained with antibodies
Figure Legend Snippet: CD34KO mouse skin lacks CD34 expression and appears normal histologically. Top, skin from 7-wk-old WT and CD34KO mice was fixed in 10% formalin and stained with H E; middle, single-cell suspensions of keratinocytes were stained with antibodies

Techniques Used: Expressing, Mouse Assay, Staining

Localization of the hair follicle progenitor cell marker MTS24 in WT and CD34KO skin. To determine if lack of CD34 expression in hair follicles affected the localization of the hair follicle progenitor marker, untreated and TPA-treated CD34KO and WT skin
Figure Legend Snippet: Localization of the hair follicle progenitor cell marker MTS24 in WT and CD34KO skin. To determine if lack of CD34 expression in hair follicles affected the localization of the hair follicle progenitor marker, untreated and TPA-treated CD34KO and WT skin

Techniques Used: Marker, Expressing

37) Product Images from "High efficient isolation and systematic identification of human adipose-derived mesenchymal stem cells"

Article Title: High efficient isolation and systematic identification of human adipose-derived mesenchymal stem cells

Journal: Journal of Biomedical Science

doi: 10.1186/1423-0127-18-59

The hADSCs expressed a unique set of CD markers . (A) Flow cytometry analysis disclosed that the 3rd passage (P3) were positive for CD29, CD44, CD73, CD105 and CD166 with expression rates all up to 95%, but negative for CD31, CD34, CD45 and HLA-DR. (B) This immunophenotype was consistent among different passages. (C) Merged images from immunofluorescent staining of CD antigens (green) and propidium iodide (PI) staining of nuclei (red) demonstrated the same phenotype (Bars = 10 μm).
Figure Legend Snippet: The hADSCs expressed a unique set of CD markers . (A) Flow cytometry analysis disclosed that the 3rd passage (P3) were positive for CD29, CD44, CD73, CD105 and CD166 with expression rates all up to 95%, but negative for CD31, CD34, CD45 and HLA-DR. (B) This immunophenotype was consistent among different passages. (C) Merged images from immunofluorescent staining of CD antigens (green) and propidium iodide (PI) staining of nuclei (red) demonstrated the same phenotype (Bars = 10 μm).

Techniques Used: Flow Cytometry, Cytometry, Expressing, Staining

Immunocytochemical analysis and ultrastructure of hADSCs under endothelial differentiation . (A) The expression of endothelial-specific protein vascular endothelial growth factor receptor-2 (KDR), CD34 and CD31 were detected by diaminobenzidine staining of the secondary antibody (Bars = 50 μm). (B) Ultrastructural images showed clear specific endothelial granule, the Weibel-Palade body (arrow) (Bars = 200 nm).
Figure Legend Snippet: Immunocytochemical analysis and ultrastructure of hADSCs under endothelial differentiation . (A) The expression of endothelial-specific protein vascular endothelial growth factor receptor-2 (KDR), CD34 and CD31 were detected by diaminobenzidine staining of the secondary antibody (Bars = 50 μm). (B) Ultrastructural images showed clear specific endothelial granule, the Weibel-Palade body (arrow) (Bars = 200 nm).

Techniques Used: Expressing, Staining

38) Product Images from "Hyaluronic Acid Hydrogel Integrated with Mesenchymal Stem Cell-Secretome to Treat Endometrial Injury in a Rat Model of Asherman’s Syndrome"

Article Title: Hyaluronic Acid Hydrogel Integrated with Mesenchymal Stem Cell-Secretome to Treat Endometrial Injury in a Rat Model of Asherman’s Syndrome

Journal: Advanced healthcare materials

doi: 10.1002/adhm.201900411

Characterization of MSCs. Flow cytometry analysis of common MSC markers such as A) CD105 and B) CD90. MSCs are negative for C) CD31, D) CD34, and E) CD117. F) Summary of positive MSC cells with different markers.
Figure Legend Snippet: Characterization of MSCs. Flow cytometry analysis of common MSC markers such as A) CD105 and B) CD90. MSCs are negative for C) CD31, D) CD34, and E) CD117. F) Summary of positive MSC cells with different markers.

Techniques Used: Flow Cytometry

39) Product Images from "High frequency of concomitant mastocytosis in patients with acute myeloid leukemia exhibiting the transforming KIT mutation D816V †), High frequency of concomitant mastocytosis in patients with acute myeloid leukemia exhibiting the transforming KIT mutation D816V"

Article Title: High frequency of concomitant mastocytosis in patients with acute myeloid leukemia exhibiting the transforming KIT mutation D816V †), High frequency of concomitant mastocytosis in patients with acute myeloid leukemia exhibiting the transforming KIT mutation D816V

Journal: Molecular Oncology

doi: 10.1016/j.molonc.2010.04.008

Analysis of microdissected bone marrow cells for the presence of KIT D816V. (A–D) Bone marrow sections in a patient with AML and associated systemic mastocytosis (SM) were stained with an antibody against tryptase (for mast cell detection) (A,B) and an antibody against CD34 (for detection of AML blasts) (C,D). Images were taken before (A,C) and after (B,D) microdissection by lasercapturing. Microdissected cells after successful capturing are indicated by white spots. (E–H) Melting point analysis for the presence of KIT D816V in microdissected cells in two patients with SM and associated AML (SM‐AML). In each case, HMC‐1 cells were run in parallel as an internal positive control. In one patient (E,F), CD34+ AML blast cells did not exhibit KIT D816V (E) whereas mast cells (F) clearly expressed KIT D816V. In the second patient, both the AML blasts (G) and the mast cells (H) were found to carry the KIT D816V mutant.
Figure Legend Snippet: Analysis of microdissected bone marrow cells for the presence of KIT D816V. (A–D) Bone marrow sections in a patient with AML and associated systemic mastocytosis (SM) were stained with an antibody against tryptase (for mast cell detection) (A,B) and an antibody against CD34 (for detection of AML blasts) (C,D). Images were taken before (A,C) and after (B,D) microdissection by lasercapturing. Microdissected cells after successful capturing are indicated by white spots. (E–H) Melting point analysis for the presence of KIT D816V in microdissected cells in two patients with SM and associated AML (SM‐AML). In each case, HMC‐1 cells were run in parallel as an internal positive control. In one patient (E,F), CD34+ AML blast cells did not exhibit KIT D816V (E) whereas mast cells (F) clearly expressed KIT D816V. In the second patient, both the AML blasts (G) and the mast cells (H) were found to carry the KIT D816V mutant.

Techniques Used: Staining, Laser Capture Microdissection, Positive Control, Mutagenesis

40) Product Images from "Evidence of Mobilization of Pluripotent Stem Cells into Peripheral Blood of Patients with Myocardial Ischemia"

Article Title: Evidence of Mobilization of Pluripotent Stem Cells into Peripheral Blood of Patients with Myocardial Ischemia

Journal: Experimental hematology

doi: 10.1016/j.exphem.2010.08.003

Bar graphs showing the mRNA expression of pluripotent markers - Oct-4 and Nanog ( Panel A and Panel B , respectively), cardiac markers - Nkx2.5/Csx and GATA4 ( Panel C and Panel D , respectively) and endothelial antigen - vWF ( Panel E ) in sorted CD34+ cells
Figure Legend Snippet: Bar graphs showing the mRNA expression of pluripotent markers - Oct-4 and Nanog ( Panel A and Panel B , respectively), cardiac markers - Nkx2.5/Csx and GATA4 ( Panel C and Panel D , respectively) and endothelial antigen - vWF ( Panel E ) in sorted CD34+ cells

Techniques Used: Expressing

Related Articles

Isolation:

Article Title: Daily Ethanol Drinking Followed by an Abstinence Period Impairs Bone Marrow Niche and Mitochondrial Function of Hematopoietic Stem/Progenitor Cells in Rhesus Macaques
Article Snippet: The cell suspension was layered on a Ficoll density gradient followed by centrifugation for 30 min at 1500 rpm at room temperature. .. Mononuclear cells were collected from the Ficoll gradient interface and HSPCs cells were isolated using rhesus-specific antibodies to CD34 (BD Pharmingen, clone 563) and the Miltenyi Biotec (Bergisch Gladbach, Germany) anti-PE MACS magnetic bead system using supplied buffers and reagents. .. Specifically, 1–2×108 BM mononuclear cells derived from a single femur were resuspended in 1 ml wash buffer and mixed with 200 μl FcR blocking reagent (Miltenyi Biotec) and 100 μl PE-conjugated antibodies to CD34 (BD Pharmingen, clone 563).

Magnetic Cell Separation:

Article Title: Daily Ethanol Drinking Followed by an Abstinence Period Impairs Bone Marrow Niche and Mitochondrial Function of Hematopoietic Stem/Progenitor Cells in Rhesus Macaques
Article Snippet: The cell suspension was layered on a Ficoll density gradient followed by centrifugation for 30 min at 1500 rpm at room temperature. .. Mononuclear cells were collected from the Ficoll gradient interface and HSPCs cells were isolated using rhesus-specific antibodies to CD34 (BD Pharmingen, clone 563) and the Miltenyi Biotec (Bergisch Gladbach, Germany) anti-PE MACS magnetic bead system using supplied buffers and reagents. .. Specifically, 1–2×108 BM mononuclear cells derived from a single femur were resuspended in 1 ml wash buffer and mixed with 200 μl FcR blocking reagent (Miltenyi Biotec) and 100 μl PE-conjugated antibodies to CD34 (BD Pharmingen, clone 563).

Derivative Assay:

Article Title: Daily Ethanol Drinking Followed by an Abstinence Period Impairs Bone Marrow Niche and Mitochondrial Function of Hematopoietic Stem/Progenitor Cells in Rhesus Macaques
Article Snippet: Mononuclear cells were collected from the Ficoll gradient interface and HSPCs cells were isolated using rhesus-specific antibodies to CD34 (BD Pharmingen, clone 563) and the Miltenyi Biotec (Bergisch Gladbach, Germany) anti-PE MACS magnetic bead system using supplied buffers and reagents. .. Specifically, 1–2×108 BM mononuclear cells derived from a single femur were resuspended in 1 ml wash buffer and mixed with 200 μl FcR blocking reagent (Miltenyi Biotec) and 100 μl PE-conjugated antibodies to CD34 (BD Pharmingen, clone 563). .. Cells were incubated on a rocking platform for 20 min at 12–15°C, washed by centrifugation at 1000 rpm twice with 20 ml wash buffer, resuspended in 0.5–1 ml wash buffer supplied with 200 μl anti-PE MicroBeads (Miltenyi Biotec) and incubated for an additional 15 min.

Blocking Assay:

Article Title: Daily Ethanol Drinking Followed by an Abstinence Period Impairs Bone Marrow Niche and Mitochondrial Function of Hematopoietic Stem/Progenitor Cells in Rhesus Macaques
Article Snippet: Mononuclear cells were collected from the Ficoll gradient interface and HSPCs cells were isolated using rhesus-specific antibodies to CD34 (BD Pharmingen, clone 563) and the Miltenyi Biotec (Bergisch Gladbach, Germany) anti-PE MACS magnetic bead system using supplied buffers and reagents. .. Specifically, 1–2×108 BM mononuclear cells derived from a single femur were resuspended in 1 ml wash buffer and mixed with 200 μl FcR blocking reagent (Miltenyi Biotec) and 100 μl PE-conjugated antibodies to CD34 (BD Pharmingen, clone 563). .. Cells were incubated on a rocking platform for 20 min at 12–15°C, washed by centrifugation at 1000 rpm twice with 20 ml wash buffer, resuspended in 0.5–1 ml wash buffer supplied with 200 μl anti-PE MicroBeads (Miltenyi Biotec) and incubated for an additional 15 min.

Staining:

Article Title: A revised road map for the commitment of human cord blood CD34-negative hematopoietic stem cells
Article Snippet: The repopulation of human hematopoietic cells in the murine BM, PB, SP and Thy were determined by detecting the number of 7-AAD− cells positively stained with PB-conjugated anti-human CD45 mAb by six-color FCM (FACS CantoII). .. The cells were also stained with a PE-Cy7-conjugated anti-mouse CD45.1 mAb (Beckman Coulter); FITC- or BV510-conjugated anti-human CD3 (BM, Thy), CD19 (BM, PB, SP), CD11b (BM) and CD235a (BM) mAbs; PE-conjugated anti-human CD4 (Thy) (eBioscience), CD33 (BM, PB, SP), CD14 (BM) and CD41 (BM) mAbs (Beckman Coulter), and APC-conjugated anti-human CD8 (Thy) (eBioscience) and CD34 (BM, PB, SP) mAbs (BD Biosciences) for the detection of human stem/progenitor, B-lymphoid, T-lymphoid and myeloid/monocytic hematopoietic cells. .. For the precise analysis of the NK-cell development in the spleen, the cells were stained with an FITC-conjugated anti-human CD56 mAb (BD Biosciences).

Article Title: Functional phosphoproteomic analysis reveals cold-shock domain protein A to be a Bcr-Abl effector-regulating proliferation and transformation in chronic myeloid leukemia
Article Snippet: Following this, the column was washed four times with MACS buffer, removed from the magnet and the cells eluted with 2 ml MACS buffer. .. The purity of the CD34+ fraction was consistently above 96% as determined by flow cytometry (FACScalibur, Becton Dickinson, Oxford, UK) with anti-CD34 staining. .. Aliquots were immediately frozen for subsequent lysis for western blot analysis or cultured in leukemic cell growth media for proliferation assays.

Article Title: A revised road map for the commitment of human cord blood CD34-negative hematopoietic stem cells
Article Snippet: The repopulation of human hematopoietic cells in the murine BM, PB, SP and Thy were determined by detecting the number of 7-AAD− cells positively stained with PB-conjugated anti-human CD45 mAb by six-color FCM (FACS CantoII). .. The cells were also stained with a PE-Cy7-conjugated anti-mouse CD45.1 mAb (Beckman Coulter); FITC- or BV510-conjugated anti-human CD3 (BM, Thy), CD19 (BM, PB, SP), CD11b (BM) and CD235a (BM) mAbs; PE-conjugated anti-human CD4 (Thy) (eBioscience), CD33 (BM, PB, SP), CD14 (BM) and CD41 (BM, SP) mAbs (Beckman Coulter), and APC-conjugated anti-human CD8 (Thy) (eBioscience) and CD34 (BM, PB, SP) mAbs (BD Biosciences) for the detection of human stem/progenitor, B-lymphoid, T-lymphoid and myeloid/monocytic hematopoietic cells. .. For the precise analysis of the NK-cell development in the spleen, the cells were stained with an FITC-conjugated anti-human CD56 mAb (BD Biosciences).

Flow Cytometry:

Article Title: CD123 CAR T Cells for the Treatment of Myelodysplastic Syndrome
Article Snippet: For MDS-L cell line killing assays in vitro, CD123, CD3 and EGFRt expressions were evaluated by flow cytometry to determine the number of MDS-L cells and CAR T cells in coculture using following antibodies: anti-CD123-APC (BD Biosciences), anti-CD3-FITC (BioLegend), and anti-EGFRt-BV421 (Biolegend). .. For primary MDS cell killing assays, number of MDS stem cell and CAR T cells were assessed by flow cytometry with following antibodies: anti-CD34-PE/Cy7 (BD Biosciences), anti-CD38-PE-CF594 (BD Horizon), anti-CD123-APC (BD Biosciences), anti-CD3-FITC (BioLegend) and anti-EGFRt-BV421 (BioLegend). .. Viability of each cell population was determined by Live/Dead-APC/Cy7 (Invitrogen™ Thermo Fisher Scientific, MA).

Article Title: Functional phosphoproteomic analysis reveals cold-shock domain protein A to be a Bcr-Abl effector-regulating proliferation and transformation in chronic myeloid leukemia
Article Snippet: Following this, the column was washed four times with MACS buffer, removed from the magnet and the cells eluted with 2 ml MACS buffer. .. The purity of the CD34+ fraction was consistently above 96% as determined by flow cytometry (FACScalibur, Becton Dickinson, Oxford, UK) with anti-CD34 staining. .. Aliquots were immediately frozen for subsequent lysis for western blot analysis or cultured in leukemic cell growth media for proliferation assays.

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  • 86
    Becton Dickinson cd34
    FACS isolation of <t>CD34</t> + cells infected with lentiviral vectors. Purified CD34 + cells (3 × 10 6 ) were infected with lentiviral vectors (MOI = 3) by centrifugation (4 h at 2,500 rpm) in a Beckman GPR centrifuge. Cells were incubated for 72 h in IMDM supplemented with 10% FBS and 30 μl of cytokine cocktail StemSpan CC100. Cells were incubated with a PE-conjugated human-specific MAb against the CD34 marker, and cell sorting was performed with a FACSVantage flow cytometer. An additional cell sample was incubated with a mouse immunoglobulin G γ isotype control antibody to set compensation and gates for FACS. The R2 gate in each panel represents the gate set to isolate CD34 + GFP + ). The percentage of CD34 + GFP + cells is displayed in the upper right panel. Mock-transduced CD34 + cells were isolated only on the basis of CD34 + expression and are represented by the R3 gate. LVs used in each transduction are as follows: GFP, HR′CMV-GFP; Tax1, HR′CMV-Tax1/GFP; Tax1(−), HR′CMV-Tax1(−)/GFP; Tax2, HR′CMV-Tax2/GFP; and Vpr, HR′CMV-Vpr/GFP. The purity of the sorted cell populations was not determined after isolation due to the limited numbers of cells recovered by FACS.
    Cd34, supplied by Becton Dickinson, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Becton Dickinson cd34 cell purity
    The mean percentage of migration toward stromal cell-derived factor 1 (SDF-1) in different culture conditions. The chemotactic effect of SDF-1 on <t>CD34+</t> cells migration in the 6 different culture conditions after 4 hr. Results show mean percentage of migration from 3 independent experiments. Error bars represent SD. * P
    Cd34 Cell Purity, supplied by Becton Dickinson, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    FACS isolation of CD34 + cells infected with lentiviral vectors. Purified CD34 + cells (3 × 10 6 ) were infected with lentiviral vectors (MOI = 3) by centrifugation (4 h at 2,500 rpm) in a Beckman GPR centrifuge. Cells were incubated for 72 h in IMDM supplemented with 10% FBS and 30 μl of cytokine cocktail StemSpan CC100. Cells were incubated with a PE-conjugated human-specific MAb against the CD34 marker, and cell sorting was performed with a FACSVantage flow cytometer. An additional cell sample was incubated with a mouse immunoglobulin G γ isotype control antibody to set compensation and gates for FACS. The R2 gate in each panel represents the gate set to isolate CD34 + GFP + ). The percentage of CD34 + GFP + cells is displayed in the upper right panel. Mock-transduced CD34 + cells were isolated only on the basis of CD34 + expression and are represented by the R3 gate. LVs used in each transduction are as follows: GFP, HR′CMV-GFP; Tax1, HR′CMV-Tax1/GFP; Tax1(−), HR′CMV-Tax1(−)/GFP; Tax2, HR′CMV-Tax2/GFP; and Vpr, HR′CMV-Vpr/GFP. The purity of the sorted cell populations was not determined after isolation due to the limited numbers of cells recovered by FACS.

    Journal: Journal of Virology

    Article Title: Human T-Cell Leukemia Virus Type 1 Tax Oncoprotein Suppression of Multilineage Hematopoiesis of CD34+ Cells In Vitro

    doi: 10.1128/JVI.77.22.12152-12164.2003

    Figure Lengend Snippet: FACS isolation of CD34 + cells infected with lentiviral vectors. Purified CD34 + cells (3 × 10 6 ) were infected with lentiviral vectors (MOI = 3) by centrifugation (4 h at 2,500 rpm) in a Beckman GPR centrifuge. Cells were incubated for 72 h in IMDM supplemented with 10% FBS and 30 μl of cytokine cocktail StemSpan CC100. Cells were incubated with a PE-conjugated human-specific MAb against the CD34 marker, and cell sorting was performed with a FACSVantage flow cytometer. An additional cell sample was incubated with a mouse immunoglobulin G γ isotype control antibody to set compensation and gates for FACS. The R2 gate in each panel represents the gate set to isolate CD34 + GFP + ). The percentage of CD34 + GFP + cells is displayed in the upper right panel. Mock-transduced CD34 + cells were isolated only on the basis of CD34 + expression and are represented by the R3 gate. LVs used in each transduction are as follows: GFP, HR′CMV-GFP; Tax1, HR′CMV-Tax1/GFP; Tax1(−), HR′CMV-Tax1(−)/GFP; Tax2, HR′CMV-Tax2/GFP; and Vpr, HR′CMV-Vpr/GFP. The purity of the sorted cell populations was not determined after isolation due to the limited numbers of cells recovered by FACS.

    Article Snippet: CD34+ cells transduced with Tax1 demonstrated a two- to fivefold reduction in clonogenic colony-forming activity in vitro, in comparison with CD34+ cells transduced with LVs encoding Tax2, Tax1(−), or only GFP (Fig. ).

    Techniques: FACS, Isolation, Infection, Purification, Centrifugation, Incubation, Marker, Flow Cytometry, Cytometry, Expressing, Transduction

    Clonogenic colony-forming activity of lentiviral vector- transduced CD34 + cells. GFP + CD34 + cells were purified by FACS, and isolated cells (10 3 ). (A) Total number of myeloid, erythroid and pluripotential colonies per CD34 + GFP + cells (10 3 ) plated was determined at 14 days postplating. Purified CD34 + GFP + cell samples were plated in triplicate. Each column represents a separate sorting experiment. Colony-forming activities were assayed four times for each transduction, except for Tax1(−) and Tax2-transduced CD34 + cells, which were assayed three times (sorting experiments 2, 3, and 4). Transduction of CD34 + cells with HR′CMV-Vpr/GFP resulted in no colony formation in FACS experiments 1 and 4 and is indicated by an asterisk. (B) Relative distribution of clonogenic colonies. Colonies were analyzed by morphology and characterized as CFU-GM, BFU-E, or HPP-CFC. The average numbers of CFU-GM colonies that arose per 10 3 purified CD34 + GFP + cells plated were 38.8 (Mock), 24.4 (GFP), 7.3 (Tax1), 21.2 [Tax1(−)], and 21.4 (Tax2). The average numbers of BFU-E colonies arising per 10 3 CD34 + GFP+ cells plated were 14.9 (Mock), 9.0 (GFP), 2.8 (Tax1), 8.0 [Tax1(−)], and 8.3 (Tax2). The average numbers of CFU-HPP colonies arising per 10 3 CD34 + GFP + cells plated were 6.0 (Mock), 3.6 (GFP), 1.2 (Tax1), 3.2 [Tax1(−)], and 3.3 (Tax2). Statistical analysis was performed by ANOVA ( P

    Journal: Journal of Virology

    Article Title: Human T-Cell Leukemia Virus Type 1 Tax Oncoprotein Suppression of Multilineage Hematopoiesis of CD34+ Cells In Vitro

    doi: 10.1128/JVI.77.22.12152-12164.2003

    Figure Lengend Snippet: Clonogenic colony-forming activity of lentiviral vector- transduced CD34 + cells. GFP + CD34 + cells were purified by FACS, and isolated cells (10 3 ). (A) Total number of myeloid, erythroid and pluripotential colonies per CD34 + GFP + cells (10 3 ) plated was determined at 14 days postplating. Purified CD34 + GFP + cell samples were plated in triplicate. Each column represents a separate sorting experiment. Colony-forming activities were assayed four times for each transduction, except for Tax1(−) and Tax2-transduced CD34 + cells, which were assayed three times (sorting experiments 2, 3, and 4). Transduction of CD34 + cells with HR′CMV-Vpr/GFP resulted in no colony formation in FACS experiments 1 and 4 and is indicated by an asterisk. (B) Relative distribution of clonogenic colonies. Colonies were analyzed by morphology and characterized as CFU-GM, BFU-E, or HPP-CFC. The average numbers of CFU-GM colonies that arose per 10 3 purified CD34 + GFP + cells plated were 38.8 (Mock), 24.4 (GFP), 7.3 (Tax1), 21.2 [Tax1(−)], and 21.4 (Tax2). The average numbers of BFU-E colonies arising per 10 3 CD34 + GFP+ cells plated were 14.9 (Mock), 9.0 (GFP), 2.8 (Tax1), 8.0 [Tax1(−)], and 8.3 (Tax2). The average numbers of CFU-HPP colonies arising per 10 3 CD34 + GFP + cells plated were 6.0 (Mock), 3.6 (GFP), 1.2 (Tax1), 3.2 [Tax1(−)], and 3.3 (Tax2). Statistical analysis was performed by ANOVA ( P

    Article Snippet: CD34+ cells transduced with Tax1 demonstrated a two- to fivefold reduction in clonogenic colony-forming activity in vitro, in comparison with CD34+ cells transduced with LVs encoding Tax2, Tax1(−), or only GFP (Fig. ).

    Techniques: Activity Assay, Plasmid Preparation, Purification, FACS, Isolation, Transduction

    Preparation and characterization of mobilized MPP. (A) Upon mobilization with G-CSF and Flt3L, spleen c-kit + cells were magnetically sorted, further stained with c-kit, Sca-1, CD11b and CD34 and cell-sorted as c-kit + Sca-1 + CD34 + CD11b −/low cells. (B) SLAM markers including CD150 and CD48 and Flt3 were used for characterization of mobilized cell sorted c-kit + Sca-1 + CD34 + CD11b −/low progenitors as 80% MPP3 (CD150 − ) and 20% MPP2 (CD150 + ). (C) The differentiation properties of mobilized MPP were assessed after 7 days of co-culture upon OP9 and OP9Δ4 stromal cells in the presence of SCF (1 ng/ml), IL-7 (8 ng/ml) and Flt3L (10 ng/ml). Cells were recovered and stained for FACS analysis with different lineage markers. Percentages of the different subsets resulting from MPP differentiation are indicated.

    Journal: Frontiers in Immunology

    Article Title: Mobilized Multipotent Hematopoietic Progenitors Stabilize and Expand Regulatory T Cells to Protect Against Autoimmune Encephalomyelitis

    doi: 10.3389/fimmu.2020.607175

    Figure Lengend Snippet: Preparation and characterization of mobilized MPP. (A) Upon mobilization with G-CSF and Flt3L, spleen c-kit + cells were magnetically sorted, further stained with c-kit, Sca-1, CD11b and CD34 and cell-sorted as c-kit + Sca-1 + CD34 + CD11b −/low cells. (B) SLAM markers including CD150 and CD48 and Flt3 were used for characterization of mobilized cell sorted c-kit + Sca-1 + CD34 + CD11b −/low progenitors as 80% MPP3 (CD150 − ) and 20% MPP2 (CD150 + ). (C) The differentiation properties of mobilized MPP were assessed after 7 days of co-culture upon OP9 and OP9Δ4 stromal cells in the presence of SCF (1 ng/ml), IL-7 (8 ng/ml) and Flt3L (10 ng/ml). Cells were recovered and stained for FACS analysis with different lineage markers. Percentages of the different subsets resulting from MPP differentiation are indicated.

    Article Snippet: Total splenocytes were magnetically sorted for c-kit+ cells with an automated magnetic cell sorter (Robosep, StemCell Technologies, Vancouver, BC, Canada), further stained with the mAbs directed against CD34 (BD Biosciences, Le Pont de Claix, France), Sca-1 (anti-Ly6A/E) and CD11b (eBioscience, ThermoFisher Scientific, Illkirch, France), and electronically sorted into c-kithigh Sca-1high CD34+ CD11b-/low cells with the FACS Aria II cell sorter (BD Biosciences).

    Techniques: Staining, Co-Culture Assay, FACS

    Pervasive and dynamic alternative splicing occurs during human hematopoietic differentiation induced from hESCs Principal component analysis (PCA) of genes (left) and transcripts (right) in highly purified cells at various differentiation stages, including ESCs, APLNR + cells, CD31 + CD34 + cells, and CD43 + cells during hematopoietic differentiation induction from hESCs. Widespread occurrence ( > 85% of expressed genes) of alternative splicing in expressed genes (FPKM > 1 in at least one differentiation stage) at distinct differentiation stages during hematopoietic development. Average number of isoforms per gene at each differentiation stage. Analysis of isoform variants within each expressing gene at distinct differentiation stages. Number and frequency of five major splicing events at distinct differentiation stages, including mutually exclusive exon (MXE), alternative 5′ splicing (A5SS), alternative 3′ splicing (A3SS), intron retention (IR), and exon skipping (ES). The cutoff of splicing event of an expressed gene is 0.05

    Journal: EMBO Reports

    Article Title: A splicing factor switch controls hematopoietic lineage specification of pluripotent stem cells

    doi: 10.15252/embr.202050535

    Figure Lengend Snippet: Pervasive and dynamic alternative splicing occurs during human hematopoietic differentiation induced from hESCs Principal component analysis (PCA) of genes (left) and transcripts (right) in highly purified cells at various differentiation stages, including ESCs, APLNR + cells, CD31 + CD34 + cells, and CD43 + cells during hematopoietic differentiation induction from hESCs. Widespread occurrence ( > 85% of expressed genes) of alternative splicing in expressed genes (FPKM > 1 in at least one differentiation stage) at distinct differentiation stages during hematopoietic development. Average number of isoforms per gene at each differentiation stage. Analysis of isoform variants within each expressing gene at distinct differentiation stages. Number and frequency of five major splicing events at distinct differentiation stages, including mutually exclusive exon (MXE), alternative 5′ splicing (A5SS), alternative 3′ splicing (A3SS), intron retention (IR), and exon skipping (ES). The cutoff of splicing event of an expressed gene is 0.05

    Article Snippet: The primary antibodies used for flow cytometry were as follows: PE cyanine 7 Mouse IgG1 (cat# 25‐4714‐42, eBioscience), APC Mouse IgG3 (cat#1C007A, R & D), PE IgG1 (cat# 555719, BD Bioscience), CD31 (cat# 555446, BD Biosciences), CD34 (cat# 555824, BD Biosciences), h‐APJ (cat# FAB856A, R & D), CD73 (cat# 561258, BD Biosciences), and CD43 (cat# 560978, eBioscience).

    Techniques: Purification, Expressing

    NUMB Expression and its splicing regulation Expression of NUMB and its family member NUMBLIKE ( NUMBL ) during human hematopoietic development by RNA‐Seq. Western blotting showing NUMB‐S overexpression upon DOX induction with anti‐FLAG antibody. The GAPDH gene was used as a loading control. Expression of NUMBL in day 2‐APLNR + cells, day 5‐DMSO–treated, and day 5‐PLB‐treated cells. The upper panel is a representative RT–PCR electropherogram showing the inclusion or exclusion of NUMB exon 9 in SRSF2 depleted cells on day 5 of differentiation. The bar graph showing the changes of exon 9 inclusion obtained from the RT–PCR electropherogram. RT–qPCR measuring the expression of NUMB‐S in SRSF2 depleted cells on day 5 of differentiation. The representative FACS plots of CD31 + CD34 + cells at day 5 of differentiation after treatment with DMSO and NOTCH inhibitor DAPT at various concentrations from day 2.5 to 5. Western blotting showing the expression of HES1 (detected by endogenous HES1 antibody as well as anti‐FLAG antibody) and SRSF2 in 293T cells. GADPH acts as a loading control. The splicing of NUMB exon 9 after HES1 overexpression. The top panel is a representative RT–PCR electropherogram showing the inclusion or exclusion of NUMB exon 9 without or with HES1 overexpression. The quantification is presented in the bottom bar graph. Data information: Results given are mean ± SD. P ‐values were determined by unpaired two‐tailed Student’s t ‐test in (D), (E), and (H). ns represents no significant difference. ** P

    Journal: EMBO Reports

    Article Title: A splicing factor switch controls hematopoietic lineage specification of pluripotent stem cells

    doi: 10.15252/embr.202050535

    Figure Lengend Snippet: NUMB Expression and its splicing regulation Expression of NUMB and its family member NUMBLIKE ( NUMBL ) during human hematopoietic development by RNA‐Seq. Western blotting showing NUMB‐S overexpression upon DOX induction with anti‐FLAG antibody. The GAPDH gene was used as a loading control. Expression of NUMBL in day 2‐APLNR + cells, day 5‐DMSO–treated, and day 5‐PLB‐treated cells. The upper panel is a representative RT–PCR electropherogram showing the inclusion or exclusion of NUMB exon 9 in SRSF2 depleted cells on day 5 of differentiation. The bar graph showing the changes of exon 9 inclusion obtained from the RT–PCR electropherogram. RT–qPCR measuring the expression of NUMB‐S in SRSF2 depleted cells on day 5 of differentiation. The representative FACS plots of CD31 + CD34 + cells at day 5 of differentiation after treatment with DMSO and NOTCH inhibitor DAPT at various concentrations from day 2.5 to 5. Western blotting showing the expression of HES1 (detected by endogenous HES1 antibody as well as anti‐FLAG antibody) and SRSF2 in 293T cells. GADPH acts as a loading control. The splicing of NUMB exon 9 after HES1 overexpression. The top panel is a representative RT–PCR electropherogram showing the inclusion or exclusion of NUMB exon 9 without or with HES1 overexpression. The quantification is presented in the bottom bar graph. Data information: Results given are mean ± SD. P ‐values were determined by unpaired two‐tailed Student’s t ‐test in (D), (E), and (H). ns represents no significant difference. ** P

    Article Snippet: The primary antibodies used for flow cytometry were as follows: PE cyanine 7 Mouse IgG1 (cat# 25‐4714‐42, eBioscience), APC Mouse IgG3 (cat#1C007A, R & D), PE IgG1 (cat# 555719, BD Bioscience), CD31 (cat# 555446, BD Biosciences), CD34 (cat# 555824, BD Biosciences), h‐APJ (cat# FAB856A, R & D), CD73 (cat# 561258, BD Biosciences), and CD43 (cat# 560978, eBioscience).

    Techniques: Expressing, RNA Sequencing Assay, Western Blot, Over Expression, Reverse Transcription Polymerase Chain Reaction, Quantitative RT-PCR, FACS, Two Tailed Test

    Effects of long and short PLB treatment on hematopoietic differentiation The morphological alterations of cells treated without (DMSO control) or with various amount of PLB throughout the hematopoietic differentiation. Scale bar = 20 μm. Representative FACS plots illustrating the CD43 + HSPCs without or with PLB treatment at the indicated concentrations and treatment periods. Representative FACS plots showing the frequency of APLNR + cells on day 2 of differentiation and CD31 + CD34 + EPCs on day 5 without or with PLB treatment from day 0 to 2 at the indicated concentrations. The bar graph showing the percentage of APLNR + cells and CD31 + CD34 + EPCs of (C). Results given are mean ± SD. P ‐values were calculated by one‐way ANOVA followed by Dunnett’s test. ns represents no significant difference. All experiments were conducted for at least 3 biological replicates.

    Journal: EMBO Reports

    Article Title: A splicing factor switch controls hematopoietic lineage specification of pluripotent stem cells

    doi: 10.15252/embr.202050535

    Figure Lengend Snippet: Effects of long and short PLB treatment on hematopoietic differentiation The morphological alterations of cells treated without (DMSO control) or with various amount of PLB throughout the hematopoietic differentiation. Scale bar = 20 μm. Representative FACS plots illustrating the CD43 + HSPCs without or with PLB treatment at the indicated concentrations and treatment periods. Representative FACS plots showing the frequency of APLNR + cells on day 2 of differentiation and CD31 + CD34 + EPCs on day 5 without or with PLB treatment from day 0 to 2 at the indicated concentrations. The bar graph showing the percentage of APLNR + cells and CD31 + CD34 + EPCs of (C). Results given are mean ± SD. P ‐values were calculated by one‐way ANOVA followed by Dunnett’s test. ns represents no significant difference. All experiments were conducted for at least 3 biological replicates.

    Article Snippet: The primary antibodies used for flow cytometry were as follows: PE cyanine 7 Mouse IgG1 (cat# 25‐4714‐42, eBioscience), APC Mouse IgG3 (cat#1C007A, R & D), PE IgG1 (cat# 555719, BD Bioscience), CD31 (cat# 555446, BD Biosciences), CD34 (cat# 555824, BD Biosciences), h‐APJ (cat# FAB856A, R & D), CD73 (cat# 561258, BD Biosciences), and CD43 (cat# 560978, eBioscience).

    Techniques: FACS

    Short PLB treatment exhibits minor cytotoxic effects Expression of LAS1L in day 2‐differentiated APLNR + cells and day 5‐differentiated CD31 + CD34 + cells by RNA‐Seq. The bottom panel showing the inclusion/exclusion of LAS1L exon 9 during hematopoietic differentiation by RT–PCR. The inclusion/exclusion of exon 9 of ATP5F1C and HDAC7 detecting by RT–PCR during hematopoietic differentiation. The top panel is a representative RT–PCR electropherogram showing the inclusion or exclusion of exon 9 in ATP5F1C (left) and HDAC7 (right) in cells at days 2 and 5 without or with PLB treatment at indicated concentrations, respectively. The quantification of PSI is presented in the bottom bar graph. P ‐values were calculated by one‐way followed by Dunnett’s test. The cellular morphology on days 3, 4, and 5 during hematopoietic differentiation after treatment with 1.25 or 2.5 nM PLB from day 2.5 to 5. Scale bar = 40 μm. Cellular apoptosis assessed using Annexin V and 7‐AAD at days 3 and 4 of differentiation by flow cytometry, with or without PLB treatment from day 2.5 to 5. The proportion of G0/G1, S, and G2/M cells at days 3 and 4 of differentiation, with or without PLB treatment from day 2.5 to 5, respectively. The cell cycle was determined using propidium iodide staining by flow cytometry. Immunofluorescent staining depicting low‐density lipoprotein (AcLDL) uptake from FACS‐sorted CD31 + cells without (DMSO) or with PLB (1.25 nM) treatment. CD144, LDL, and DAPI were stained (upper) by red, green, and blue, respectively. Scale bar = 40 μm. The bar graph showing the quantification of the LDL fluorescent intensity by the Volocity 3D image analysis software. The schematic illustrating the DOX‐inducible knockdown system with expression of SF3B1 shRNAs. The knockdown of SF3B1 was confirmed by RT–qPCR (left) and Western blotting (right) after inducing with DOX. The representative FACS plots of CD31 + CD34 + EPCs after SF3B1 depletion at day 5 of differentiation. The percentage of CD31 + CD34 + EPCs after SF3B1 depletion at day 5 of differentiation. Data information: Results given are mean ± SD. P ‐values were determined by Student’s t ‐test in (A), (E), (F), (H), (I), and (K). ns represents no significant difference. * P

    Journal: EMBO Reports

    Article Title: A splicing factor switch controls hematopoietic lineage specification of pluripotent stem cells

    doi: 10.15252/embr.202050535

    Figure Lengend Snippet: Short PLB treatment exhibits minor cytotoxic effects Expression of LAS1L in day 2‐differentiated APLNR + cells and day 5‐differentiated CD31 + CD34 + cells by RNA‐Seq. The bottom panel showing the inclusion/exclusion of LAS1L exon 9 during hematopoietic differentiation by RT–PCR. The inclusion/exclusion of exon 9 of ATP5F1C and HDAC7 detecting by RT–PCR during hematopoietic differentiation. The top panel is a representative RT–PCR electropherogram showing the inclusion or exclusion of exon 9 in ATP5F1C (left) and HDAC7 (right) in cells at days 2 and 5 without or with PLB treatment at indicated concentrations, respectively. The quantification of PSI is presented in the bottom bar graph. P ‐values were calculated by one‐way followed by Dunnett’s test. The cellular morphology on days 3, 4, and 5 during hematopoietic differentiation after treatment with 1.25 or 2.5 nM PLB from day 2.5 to 5. Scale bar = 40 μm. Cellular apoptosis assessed using Annexin V and 7‐AAD at days 3 and 4 of differentiation by flow cytometry, with or without PLB treatment from day 2.5 to 5. The proportion of G0/G1, S, and G2/M cells at days 3 and 4 of differentiation, with or without PLB treatment from day 2.5 to 5, respectively. The cell cycle was determined using propidium iodide staining by flow cytometry. Immunofluorescent staining depicting low‐density lipoprotein (AcLDL) uptake from FACS‐sorted CD31 + cells without (DMSO) or with PLB (1.25 nM) treatment. CD144, LDL, and DAPI were stained (upper) by red, green, and blue, respectively. Scale bar = 40 μm. The bar graph showing the quantification of the LDL fluorescent intensity by the Volocity 3D image analysis software. The schematic illustrating the DOX‐inducible knockdown system with expression of SF3B1 shRNAs. The knockdown of SF3B1 was confirmed by RT–qPCR (left) and Western blotting (right) after inducing with DOX. The representative FACS plots of CD31 + CD34 + EPCs after SF3B1 depletion at day 5 of differentiation. The percentage of CD31 + CD34 + EPCs after SF3B1 depletion at day 5 of differentiation. Data information: Results given are mean ± SD. P ‐values were determined by Student’s t ‐test in (A), (E), (F), (H), (I), and (K). ns represents no significant difference. * P

    Article Snippet: The primary antibodies used for flow cytometry were as follows: PE cyanine 7 Mouse IgG1 (cat# 25‐4714‐42, eBioscience), APC Mouse IgG3 (cat#1C007A, R & D), PE IgG1 (cat# 555719, BD Bioscience), CD31 (cat# 555446, BD Biosciences), CD34 (cat# 555824, BD Biosciences), h‐APJ (cat# FAB856A, R & D), CD73 (cat# 561258, BD Biosciences), and CD43 (cat# 560978, eBioscience).

    Techniques: Expressing, RNA Sequencing Assay, Reverse Transcription Polymerase Chain Reaction, Flow Cytometry, Staining, FACS, Software, Quantitative RT-PCR, Western Blot

    The dynamic alternative splicing program during hESC hematopoietic differentiation Schematic representation of the strategies for fluorescence‐activated cell sorting (FACS) and transcriptomic analyses. During hematopoietic differentiation, human embryonic stem cells (hESCs, H1) on day 0, FACS‐purified lateral plate mesodermal APLNR + cells on day 2, purified CD31 + CD34 + endothelial progenitor cells (EPCs) on day 5, and purified CD43 + hematopoietic stem and progenitor cells (HSPCs) on day 8 were collected for RNA‐Seq, respectively. STAR Cufflinks, MISO, DESeq2, and rMATS were used to analyze the expression abundance of genes and transcripts, alternative splicing events, differentially expressed genes and transcripts, and differential splicing events, respectively. n = 3 technical replicates. Cumulative distribution curves of Log 2 (FPKM) of splicing factors ( n = 235) in hESCs APLNR + , CD31 + CD34 + , and CD43 + cells. The upper and lower dotted lines represent cumulative scores of 1 and 0, respectively. n = 3 technical replicates. The P ‐value was calculated by a two‐tailed Wilcoxon signed‐rank test. The heatmap illustrates the expression scaled by row of components within the major spliceosomal machinery ( n = 182) using unsupervised hierarchical clustering. The heatmap was scaled with Z‐Score using the log 2 (FPKM) expression of components within the major spliceosomal machinery. n = 3 technical replicates. The boxplots depict the expression of splicing factors in cluster 1 ( n = 115) and cluster 2 ( n = 67) identified in (C) at distinct differentiation stages. The central band indicates the median level of expression. Boxes present 25%‐75% of genes expression. The whiskers indicate the lowest and highest points within 1.5 × the interquartile. n = 3 technical replicates. P ‐values were calculated by a two‐tailed Wilcoxon signed‐rank test. The heatmap shows the dynamic expression of genes in the SF3A/3B complex (E) and SR family (F) at each indicated differentiation stage. Both heatmaps were scaled by row. The heatmaps were scaled with Z‐Score using the log 2 (FPKM) expression of indicated components. n = 3 technical replicates. The mRNA expression of splicing regulator SRSF2 and splicing factor PCBP2 during hematopoietic differentiation was measured by RT–qPCR. The ACTB gene was used as a control. Results given are mean ± standard deviation (SD). P ‐values were determined by an unpaired two‐tailed Student’s t ‐test. n ≥ 3 biological replicates. The number of differentially expressed transcripts at the isoform level (fold change (FC) > 2 FDR

    Journal: EMBO Reports

    Article Title: A splicing factor switch controls hematopoietic lineage specification of pluripotent stem cells

    doi: 10.15252/embr.202050535

    Figure Lengend Snippet: The dynamic alternative splicing program during hESC hematopoietic differentiation Schematic representation of the strategies for fluorescence‐activated cell sorting (FACS) and transcriptomic analyses. During hematopoietic differentiation, human embryonic stem cells (hESCs, H1) on day 0, FACS‐purified lateral plate mesodermal APLNR + cells on day 2, purified CD31 + CD34 + endothelial progenitor cells (EPCs) on day 5, and purified CD43 + hematopoietic stem and progenitor cells (HSPCs) on day 8 were collected for RNA‐Seq, respectively. STAR Cufflinks, MISO, DESeq2, and rMATS were used to analyze the expression abundance of genes and transcripts, alternative splicing events, differentially expressed genes and transcripts, and differential splicing events, respectively. n = 3 technical replicates. Cumulative distribution curves of Log 2 (FPKM) of splicing factors ( n = 235) in hESCs APLNR + , CD31 + CD34 + , and CD43 + cells. The upper and lower dotted lines represent cumulative scores of 1 and 0, respectively. n = 3 technical replicates. The P ‐value was calculated by a two‐tailed Wilcoxon signed‐rank test. The heatmap illustrates the expression scaled by row of components within the major spliceosomal machinery ( n = 182) using unsupervised hierarchical clustering. The heatmap was scaled with Z‐Score using the log 2 (FPKM) expression of components within the major spliceosomal machinery. n = 3 technical replicates. The boxplots depict the expression of splicing factors in cluster 1 ( n = 115) and cluster 2 ( n = 67) identified in (C) at distinct differentiation stages. The central band indicates the median level of expression. Boxes present 25%‐75% of genes expression. The whiskers indicate the lowest and highest points within 1.5 × the interquartile. n = 3 technical replicates. P ‐values were calculated by a two‐tailed Wilcoxon signed‐rank test. The heatmap shows the dynamic expression of genes in the SF3A/3B complex (E) and SR family (F) at each indicated differentiation stage. Both heatmaps were scaled by row. The heatmaps were scaled with Z‐Score using the log 2 (FPKM) expression of indicated components. n = 3 technical replicates. The mRNA expression of splicing regulator SRSF2 and splicing factor PCBP2 during hematopoietic differentiation was measured by RT–qPCR. The ACTB gene was used as a control. Results given are mean ± standard deviation (SD). P ‐values were determined by an unpaired two‐tailed Student’s t ‐test. n ≥ 3 biological replicates. The number of differentially expressed transcripts at the isoform level (fold change (FC) > 2 FDR

    Article Snippet: The primary antibodies used for flow cytometry were as follows: PE cyanine 7 Mouse IgG1 (cat# 25‐4714‐42, eBioscience), APC Mouse IgG3 (cat#1C007A, R & D), PE IgG1 (cat# 555719, BD Bioscience), CD31 (cat# 555446, BD Biosciences), CD34 (cat# 555824, BD Biosciences), h‐APJ (cat# FAB856A, R & D), CD73 (cat# 561258, BD Biosciences), and CD43 (cat# 560978, eBioscience).

    Techniques: Fluorescence, FACS, Purification, RNA Sequencing Assay, Expressing, Two Tailed Test, Quantitative RT-PCR, Standard Deviation

    Effects of ectopic expression of constitutive splicing factors on human hematopoietic differentiation Volcano plot shows the differential splicing events regulated by PLB treated from day 2.5 to 5. The dotted line denotes FDR = 0.05. The scatter plot showing the correlation of splicing factors form Fig 3c (B) and all expressed genes (C) between day 2‐APLNR + cells and day 5‐PLB‐treated cells. r refers to the correlation coefficient. The protein–protein interaction network of 38 intersected splicing factors (Fig 3D ) (STRING: https://string‐db.org/ ). The mRNA expression of SF3A3 , SNRPD1, and SNRPE in day 2‐APLNR + cells, day 5‐DMSO–treated, and day 5‐PLB‐treated cells. N = 2 technical replicates. The upper panel illustrates the DOX‐inducible overexpression system. Western blotting confirmed the SRSF2 overexpression upon DOX induction with anti‐FLAG antibody. GAPDH was used as the loading control. RT–qPCR assay of the relative mRNA expression of SF3A3 , SNRPD1 , and SNRPE after overexpression upon DOX induction. The ACTB gene was used as a control. Data were normalized to the mRNA level of empty vector controls cells. Western blotting showing the protein expression of SF3A3, SNRPD1, and SNRPE after overexpression with anti‐FLAG antibody. The GAPDH gene was used as a control. Representative FACS plots of CD31 + CD34 + cells in SF3A3, SNRPD1, and SNRPE overexpressed (GFP + ) cells. Statistical analysis of the frequency of CD31 + CD34 + in SF3A3, SNRPD1, and SNRPE overexpressed (GFP + ) cells. Data information: P ‐values were calculated by one‐way ANOVA followed by Dunnett’s test. ns represents no significant difference. Results given are mean ± SD.. All experiments were conducted for at least 3 biological replicates unless stated otherwise Source data are available online for this figure.

    Journal: EMBO Reports

    Article Title: A splicing factor switch controls hematopoietic lineage specification of pluripotent stem cells

    doi: 10.15252/embr.202050535

    Figure Lengend Snippet: Effects of ectopic expression of constitutive splicing factors on human hematopoietic differentiation Volcano plot shows the differential splicing events regulated by PLB treated from day 2.5 to 5. The dotted line denotes FDR = 0.05. The scatter plot showing the correlation of splicing factors form Fig 3c (B) and all expressed genes (C) between day 2‐APLNR + cells and day 5‐PLB‐treated cells. r refers to the correlation coefficient. The protein–protein interaction network of 38 intersected splicing factors (Fig 3D ) (STRING: https://string‐db.org/ ). The mRNA expression of SF3A3 , SNRPD1, and SNRPE in day 2‐APLNR + cells, day 5‐DMSO–treated, and day 5‐PLB‐treated cells. N = 2 technical replicates. The upper panel illustrates the DOX‐inducible overexpression system. Western blotting confirmed the SRSF2 overexpression upon DOX induction with anti‐FLAG antibody. GAPDH was used as the loading control. RT–qPCR assay of the relative mRNA expression of SF3A3 , SNRPD1 , and SNRPE after overexpression upon DOX induction. The ACTB gene was used as a control. Data were normalized to the mRNA level of empty vector controls cells. Western blotting showing the protein expression of SF3A3, SNRPD1, and SNRPE after overexpression with anti‐FLAG antibody. The GAPDH gene was used as a control. Representative FACS plots of CD31 + CD34 + cells in SF3A3, SNRPD1, and SNRPE overexpressed (GFP + ) cells. Statistical analysis of the frequency of CD31 + CD34 + in SF3A3, SNRPD1, and SNRPE overexpressed (GFP + ) cells. Data information: P ‐values were calculated by one‐way ANOVA followed by Dunnett’s test. ns represents no significant difference. Results given are mean ± SD.. All experiments were conducted for at least 3 biological replicates unless stated otherwise Source data are available online for this figure.

    Article Snippet: The primary antibodies used for flow cytometry were as follows: PE cyanine 7 Mouse IgG1 (cat# 25‐4714‐42, eBioscience), APC Mouse IgG3 (cat#1C007A, R & D), PE IgG1 (cat# 555719, BD Bioscience), CD31 (cat# 555446, BD Biosciences), CD34 (cat# 555824, BD Biosciences), h‐APJ (cat# FAB856A, R & D), CD73 (cat# 561258, BD Biosciences), and CD43 (cat# 560978, eBioscience).

    Techniques: Expressing, Over Expression, Western Blot, Quantitative RT-PCR, Plasmid Preparation, FACS

    Inhibition of splicing disrupts EPC and HEP generation The upper schematic illustrates the stage‐specific supplementation of splicing inhibitor PLB and on day 8 CD43 + HSPCs were examined by flow cytometry. The bottom bar graph showing the normalized frequency of CD43 + cells to DMSO control under distinct PLB treatment windows. P ‐values were calculated by one‐way followed by Dunnett’s test. The top panel is a representative RT–PCR electropherogram depicting the inclusion or exclusion LAS1L exon 9 in cells at day 2 as well as cells at day 5 without or with PLB treatment with indicated concentrations, respectively. The quantification of percent spliced‐in (PSI) was presented in the bottom bar graph. P ‐values were calculated by one‐way ANOVA followed by Dunnett’s test. The representative FACS plots show the generation of CD31 + CD34 + EPCs at day 5 of differentiation upon PLB treatment from day 2.5 to 5. The frequency of CD31 + CD34 + EPCs with PLB treatment in (C) was normalized to DMSO control. P ‐values were calculated by one‐way ANOVA followed by Dunnett’s test. Cell apoptotic level assessed using Annexin V and 7‐AAD at day 5 of differentiation by flow cytometry. The cells were treated with or without PLB from day 2.5 to 5. The proportion of G0/G1, S, and G2/M cells at day 5 of differentiation treated with or without PLB from day 2.5 to 5. The cell cycle was determined by flow cytometry with propidium iodide staining. The experimental schematic (upper panel). APLNR + cells on day 2 were FACS‐purified and treated with various dosages of PLB from day 2.5 to 5 during hematopoietic differentiation. On day 5, the generation of CD31 + CD34 + EPCs and CD31 + CD34 + CD73 − HEPs was assessed. The representative FACS plots showed the frequency of APLNR + cells, CD31 + CD34 + EPCs, and CD31 + CD34 + CD73 − HEPs without or with PLB treatment. The bar graphs show the percentage of CD31 + CD34 + EPCs and CD31 + CD34 + CD73 − HEPs without or with PLB treatment. Data information: Results given are mean ± SD. P ‐values were determined by an unpaired two‐tailed Student’s t ‐test in (E), (F), and (G). ns represents no significant difference, * P

    Journal: EMBO Reports

    Article Title: A splicing factor switch controls hematopoietic lineage specification of pluripotent stem cells

    doi: 10.15252/embr.202050535

    Figure Lengend Snippet: Inhibition of splicing disrupts EPC and HEP generation The upper schematic illustrates the stage‐specific supplementation of splicing inhibitor PLB and on day 8 CD43 + HSPCs were examined by flow cytometry. The bottom bar graph showing the normalized frequency of CD43 + cells to DMSO control under distinct PLB treatment windows. P ‐values were calculated by one‐way followed by Dunnett’s test. The top panel is a representative RT–PCR electropherogram depicting the inclusion or exclusion LAS1L exon 9 in cells at day 2 as well as cells at day 5 without or with PLB treatment with indicated concentrations, respectively. The quantification of percent spliced‐in (PSI) was presented in the bottom bar graph. P ‐values were calculated by one‐way ANOVA followed by Dunnett’s test. The representative FACS plots show the generation of CD31 + CD34 + EPCs at day 5 of differentiation upon PLB treatment from day 2.5 to 5. The frequency of CD31 + CD34 + EPCs with PLB treatment in (C) was normalized to DMSO control. P ‐values were calculated by one‐way ANOVA followed by Dunnett’s test. Cell apoptotic level assessed using Annexin V and 7‐AAD at day 5 of differentiation by flow cytometry. The cells were treated with or without PLB from day 2.5 to 5. The proportion of G0/G1, S, and G2/M cells at day 5 of differentiation treated with or without PLB from day 2.5 to 5. The cell cycle was determined by flow cytometry with propidium iodide staining. The experimental schematic (upper panel). APLNR + cells on day 2 were FACS‐purified and treated with various dosages of PLB from day 2.5 to 5 during hematopoietic differentiation. On day 5, the generation of CD31 + CD34 + EPCs and CD31 + CD34 + CD73 − HEPs was assessed. The representative FACS plots showed the frequency of APLNR + cells, CD31 + CD34 + EPCs, and CD31 + CD34 + CD73 − HEPs without or with PLB treatment. The bar graphs show the percentage of CD31 + CD34 + EPCs and CD31 + CD34 + CD73 − HEPs without or with PLB treatment. Data information: Results given are mean ± SD. P ‐values were determined by an unpaired two‐tailed Student’s t ‐test in (E), (F), and (G). ns represents no significant difference, * P

    Article Snippet: The primary antibodies used for flow cytometry were as follows: PE cyanine 7 Mouse IgG1 (cat# 25‐4714‐42, eBioscience), APC Mouse IgG3 (cat#1C007A, R & D), PE IgG1 (cat# 555719, BD Bioscience), CD31 (cat# 555446, BD Biosciences), CD34 (cat# 555824, BD Biosciences), h‐APJ (cat# FAB856A, R & D), CD73 (cat# 561258, BD Biosciences), and CD43 (cat# 560978, eBioscience).

    Techniques: Inhibition, Flow Cytometry, Reverse Transcription Polymerase Chain Reaction, FACS, Staining, Purification, Two Tailed Test

    The mean percentage of migration toward stromal cell-derived factor 1 (SDF-1) in different culture conditions. The chemotactic effect of SDF-1 on CD34+ cells migration in the 6 different culture conditions after 4 hr. Results show mean percentage of migration from 3 independent experiments. Error bars represent SD. * P

    Journal: Iranian Journal of Basic Medical Sciences

    Article Title: Mild hypoxia and human bone marrow mesenchymal stem cells synergistically enhance expansion and homing capacity of human cord blood CD34+ stem cells

    doi: 10.22038/IJBMS.2018.26820.6561

    Figure Lengend Snippet: The mean percentage of migration toward stromal cell-derived factor 1 (SDF-1) in different culture conditions. The chemotactic effect of SDF-1 on CD34+ cells migration in the 6 different culture conditions after 4 hr. Results show mean percentage of migration from 3 independent experiments. Error bars represent SD. * P

    Article Snippet: CD34+ cell purity was evaluated by flowcytometry analysis using FITC- human CD34 antibody (BD Pharmingen).

    Techniques: Migration, Derivative Assay

    Total nucleated cells count in different culture conditions. CD34+ cells cultured in 6 different groups. After 7 days, total number of nucleated cells was counted. Data represent mean±SD from 3 independent experiments. Error bars represent SD.* P

    Journal: Iranian Journal of Basic Medical Sciences

    Article Title: Mild hypoxia and human bone marrow mesenchymal stem cells synergistically enhance expansion and homing capacity of human cord blood CD34+ stem cells

    doi: 10.22038/IJBMS.2018.26820.6561

    Figure Lengend Snippet: Total nucleated cells count in different culture conditions. CD34+ cells cultured in 6 different groups. After 7 days, total number of nucleated cells was counted. Data represent mean±SD from 3 independent experiments. Error bars represent SD.* P

    Article Snippet: CD34+ cell purity was evaluated by flowcytometry analysis using FITC- human CD34 antibody (BD Pharmingen).

    Techniques: Cell Culture

    CD34+ cells fold change in different culture conditions; expanded CD34+ cells from 6 different culture conditions evaluated by flowcytometry. Data represent mean±SD from 3 independent experiments. Error bars represent SD. * P

    Journal: Iranian Journal of Basic Medical Sciences

    Article Title: Mild hypoxia and human bone marrow mesenchymal stem cells synergistically enhance expansion and homing capacity of human cord blood CD34+ stem cells

    doi: 10.22038/IJBMS.2018.26820.6561

    Figure Lengend Snippet: CD34+ cells fold change in different culture conditions; expanded CD34+ cells from 6 different culture conditions evaluated by flowcytometry. Data represent mean±SD from 3 independent experiments. Error bars represent SD. * P

    Article Snippet: CD34+ cell purity was evaluated by flowcytometry analysis using FITC- human CD34 antibody (BD Pharmingen).

    Techniques:

    Colony fold change in different culture conditions. Results of clonogenic assay of cord blood CD34+ cells in different culture conditions plated after 14 days. The experiments performed in duplicate in 3 independent experiments. Error bars represent SD.* P

    Journal: Iranian Journal of Basic Medical Sciences

    Article Title: Mild hypoxia and human bone marrow mesenchymal stem cells synergistically enhance expansion and homing capacity of human cord blood CD34+ stem cells

    doi: 10.22038/IJBMS.2018.26820.6561

    Figure Lengend Snippet: Colony fold change in different culture conditions. Results of clonogenic assay of cord blood CD34+ cells in different culture conditions plated after 14 days. The experiments performed in duplicate in 3 independent experiments. Error bars represent SD.* P

    Article Snippet: CD34+ cell purity was evaluated by flowcytometry analysis using FITC- human CD34 antibody (BD Pharmingen).

    Techniques: Clonogenic Assay