Journal: Cell Discovery

Article Title: Targeting JNK pathway promotes human hematopoietic stem cell expansion

doi: 10.1038/s41421-018-0072-8

Figure Lengend Snippet: a Experimental schematic for the evaluation of JNK inhibitors on HSC expansion. CD34 + cells were cultured with StemSpan SFEM II medium supplemented with SCF, TPO, FLT3L in the presence of JNK-related molecules for 7 days, then the total cell expansion and CD34 + CD45RA - cell frequency was determined. b Frequency of CD34 + CD45RA - cell subsets in 7-day cultures of CD34 + cells supplemented with two representative JNK inhibitors including JNK-IN-8 (2 μM) and SP600125 (5 μM) ( n = 3 experiments). c Chemical structure for JNK-IN-8 (hereafter called J8). d The increasing fold of CD34 + CD45RA - subset frequency compared to DMSO after 10-day culture of CD34 + cells with different concentration of J8 (This data was drawn by R). See also to Supplementary Table . All data shown as mean values ± SD. Statistical significance was assessed using unpaired t test, where *** p < 0.001. See also Supplementary Fig.

Article Snippet: Human CD34 + CB cells were isolated using CD34 MicroBead Kit (Miltenyi Biotec, Cat: 130-046-703) according to the manufacturer’s instructions.

Techniques: Cell Culture, Concentration Assay

Journal: Cell Discovery

Article Title: Targeting JNK pathway promotes human hematopoietic stem cell expansion

doi: 10.1038/s41421-018-0072-8

Figure Lengend Snippet: a Representative flow plots of phenotypically defined cell subsets after 10-day culture of 10,000 fresh CD34 + cells supplemented with DMSO or J8 (2 μM) ( n = 3 experiments). b Fold expansion of indicated phenotypically defined cell population after 10-day culture of 10,000 fresh CD34 + cells supplemented with DMSO or J8 (2 μM) ( n = 3 experiments). TCC, total cell count. c CFU number of progenies from 1,000 day 0 CD34 + cells after 10-day culture supplemented with DMSO or J8 (2 μM) ( n = 3 experiments). G, CFU-granulocyte; M, CFU-macrophage; GM, CFU-granulocyte and macrophage; CFU-E CFU-erythrocyte; BFU-E, erythroid burst-forming unit; GEMM, CFU-granulocyte, erythroid, macrophage, and megakaryocyte. All data shown as mean values ± SD. Statistical significance was assessed using unpaired t test, where * p < 0.05, ** p < 0.01, and **** p < 0.0001; ns, not significant. See also Supplementary Fig.

Article Snippet: Human CD34 + CB cells were isolated using CD34 MicroBead Kit (Miltenyi Biotec, Cat: 130-046-703) according to the manufacturer’s instructions.

Techniques: Cell Counting

Journal: Cell Discovery

Article Title: Targeting JNK pathway promotes human hematopoietic stem cell expansion

doi: 10.1038/s41421-018-0072-8

Figure Lengend Snippet: a Human CD45 + engraftment level in the PB of recipients transplanted with DMSO or J8-expanded 10,000 day 0 CD34 + cells on 5, 9, 12, 16 weeks posttransplantation ( n = 2 independent experiments). Statistics significance between DMSO and J8 group on 5, 9, 12, 16 weeks was assessed by multiple t test respectively, where * p < 0.05, ** p < 0.01. b Representative flow plots of engraftment of J8-expanded and DMSO-expanded CD34 + cells in mouse recipients’ PB 16 weeks after transplantation. Progenies of 10,000 day 0 CD34 + cells after 10-day culture with DMSO or J8 were transplanted per mouse. c HSC frequencies of fresh CD34 + cells, J8, and DMSO cultured cells determined by LDA, calculated by ELDA software. As for overall test for differences in stem cell frequencies between any of the groups, p = 0.0055. The frequency of more than 1% human chimerism (human CD45 compared with all CD45) in the BM was regarded as positive response. d HSC frequencies presented as 1/fresh CD34 + cells equivalent for each condition from (c), the required confidence interval was 95%. See Supplementary Table . All data shown as mean values ± SD. Statistical significance was assessed using unpaired t test, where * p < 0.05, and ** p < 0.01. See also Supplementary Fig.

Article Snippet: Human CD34 + CB cells were isolated using CD34 MicroBead Kit (Miltenyi Biotec, Cat: 130-046-703) according to the manufacturer’s instructions.

Techniques: Transplantation Assay, Cell Culture, Software

Journal: Cell Discovery

Article Title: Targeting JNK pathway promotes human hematopoietic stem cell expansion

doi: 10.1038/s41421-018-0072-8

Figure Lengend Snippet: a Secondary engraftment in mouse recipients’ PB 21 weeks after transplantation of 1 × 10 7 cells from BM of the primary recipients injected with DMSO or J8-expanded 10,000 day 0 CD34 + cells 21 weeks after transplantation. ( n = 10 mouse from two independent experiments per group, * p = 0.0418 by two-tailed unpaired t test.) b HSC frequency in secondary recipient of J8 or DMSO-expanded cells calculated by ELDA software. More than 1% human CD45 engraftment in the BM was regarded as positive. As for overall test for differences in stem cell frequencies between any of the groups, p = 0.0251. c HSC frequencies presented as 1/fresh CD34 + cells equivalent for each condition from (b), the required confidence interval was 95%. More than 1% human CD45 engraftment in the BM was regarded as positive. See also Supplementary Table . All data shown as mean values ± SD. Statistical significance was assessed using unpaired t test, where * p < 0.05

Article Snippet: Human CD34 + CB cells were isolated using CD34 MicroBead Kit (Miltenyi Biotec, Cat: 130-046-703) according to the manufacturer’s instructions.

Techniques: Transplantation Assay, Injection, Two Tailed Test, Software

Journal: Cell Discovery

Article Title: Targeting JNK pathway promotes human hematopoietic stem cell expansion

doi: 10.1038/s41421-018-0072-8

Figure Lengend Snippet: a Relative mRNA expression of indicated JNK-related genes on day 5, CD34 + cells cultured with DMSO or J8 ( n = 3 experiments). b Western blot analysis of inhibition of phosphorylated c-Jun for DMSO and J8-treated CD34 + cells following serum stimulation for 30 min. c Representative flow cytometry profiles of CD34 + cells 5 days after transduction with lentivirus expressing shRNAs targeting c-Jun or scrambled shRNAs ( n = 3 experiments). d Total cell number of CD34 + CD45RA - cell population in indicated cultured CD34 + cells on day 5 posttransfection ( n = 3 experiments). e CFU numbers of 5, 000 cells on day 5 after transduction with lentivirus expressing shRNAs targeting c-Jun or scrambled shRNA ( n = 3 experiments). G, CFU-granulocyte; M, CFU-macrophage; GM, CFU-granulocyte and macrophage; CFU-E, CFU-erythrocyte; BFU-E, erythroid burst-forming units; GEMM, CFU-granulocyte, erythroid, macrophage, and megakaryocyte. See also Table . Sh-ctrl, CD34 + cells transfected with scrambled shRNA; Sh-J1, CD34 + cells transfected with 1# c-Jun shRNA; Sh-J2, CD34 + cells transfected with 2# c-Jun shRNA. All data shown as mean values ± SD. Statistical significance was assessed using unpaired t- test, where * p < 0.05; ** p < 0.01, and *** p < 0.001; ND, not detected. See also Supplementary Fig.

Article Snippet: Human CD34 + CB cells were isolated using CD34 MicroBead Kit (Miltenyi Biotec, Cat: 130-046-703) according to the manufacturer’s instructions.

Techniques: Expressing, Cell Culture, Western Blot, Inhibition, Flow Cytometry, Transduction, shRNA, Transfection

Journal: The Journal of Clinical Investigation

Article Title: Relieving DYRK1A repression of MKL1 confers an adult-like phenotype to human infantile megakaryocytes

doi: 10.1172/JCI154839

Figure Lengend Snippet: ( A – D ) Umbilical cord blood CD34 + cells were cultured in Mk medium with or without 5 μM Dyrk inhibitor harmine for 6 days. Cells were analyzed either by flow cytometry after costaining with FITC-anti-CD41 and PI, or by microscopy of cytospins. ( A ) Mk polyploidization (PI staining). ( B ) Morphology of cytospins subjected to Wright stain and light microscopy, original magnification, ×200; scale bar: 20 μm. ( C ) Mk size (FSC). ( D ) Mk complexity/granulation (SSC). Graphs for A – D , mean ± SEM for 3 independent experiments, Student’s t test. ( E – H ) Cells cultured and analyzed as in A but with or without 5 μM selective Dyrk1a inhibitor EHT 1610. ( E ) Mk polyploidization (PI). ( F ) Morphology in cytospins as in B . ( G ) Mk size (FSC). ( H ) Mk complexity/granulation (SSC). Graphs for E – H , mean ± SEM for 3 independent experiments. *P < 0.05; **P < 0.01; ***P < 0.005, Student’s t test.

Article Snippet: For prolonged expansion of undifferentiated human multipotent progenitors, CB CD34 + cells underwent 8 days of culture in prestimulation medium supplemented with 1 μM StemRegenin 1 (SR1, Selleckchem).

Techniques: Cell Culture, Flow Cytometry, Microscopy, Staining, Wright Stain, Light Microscopy

Journal: The Journal of Clinical Investigation

Article Title: Relieving DYRK1A repression of MKL1 confers an adult-like phenotype to human infantile megakaryocytes

doi: 10.1172/JCI154839

Figure Lengend Snippet: ( A and B ) In vitro platelet release assay. Cord blood CD34 + cells were cultured for up to 13 days in Mk medium with or without 5 μM harmine or 2.5 μM EHT 1610. Culture supernatants underwent flow cytometry after labeling with APC-anti-CD41 and thiazole orange (TO). Gating was based on size and CD41 + /TO + characteristics of normal donor platelets. Graphs show mean platelet numbers ± SEM for 4 independent experiments. *P < 0.05; ***P < 0.005, Student’s t test. ( C ) In vivo platelet release. Cord blood CD34 + progenitors were cultured for 11 days in Mk medium with or without 2.5 μM harmine. 4 × 10 6 cells were transplanted per irradiated NSG mouse. Peripheral blood samples were then evaluated for the presence of human platelets by flow cytometry with a human-specific CD41 antibody. The graph shows circulating human platelet count ± SEM, n = 4–6/group. *P < 0.05; ** *P < 0.005, 1-way ANOVA with Tukey’s post hoc test. ( D ) Lung entrapment of human mKs. Lung tissue was evaluated for the presence of human mKs by flow cytometry with a human-specific CD41 antibody. The graph shows the percent of cells expressing human CD41 ± SEM, n = 4–6/group, 1-way ANOVA with Tukey’s post hoc test.

Article Snippet: For prolonged expansion of undifferentiated human multipotent progenitors, CB CD34 + cells underwent 8 days of culture in prestimulation medium supplemented with 1 μM StemRegenin 1 (SR1, Selleckchem).

Techniques: In Vitro, Release Assay, Cell Culture, Flow Cytometry, Labeling, In Vivo, Irradiation, Expressing

Journal: The Journal of Clinical Investigation

Article Title: Relieving DYRK1A repression of MKL1 confers an adult-like phenotype to human infantile megakaryocytes

doi: 10.1172/JCI154839

Figure Lengend Snippet: ( A ) Effects of Dyrk1 inhibition on targets of MKL1 and P-TEFb. Cord blood CD34 + cells cultured for 6 days in Mk medium with or without 5 μM inhibitors underwent immunoblot (IB) of whole cell lysates. Arrows indicate Filamin A isoforms. ( B ) Tubulin-normalized densitometry signals from IBs as in ( A ). Graph shows mean fold changes with inhibitors ± SEM for 3 independent experiments. *P < 0.05; **P < 0.01, 1-way ANOVA with Tukey’s post hoc test. ( C ) Transcriptomic effects in human mK precursors of ontogenic stage (adult versus neonatal) and Dyrk inhibition (neonatal with or without inhibitors). CD34 + cells cultured for 4 days in Mk medium underwent purification of CD61 + cells followed by RNA-Seq. Overlapping genes (Sh) with hypergeometric P values are shown; n = 3 independent experiments. See for gene lists. ( D – F ) MKL1 requirement for morphogenesis enhancement. Marrow progenitors from indicated strains cultured for 3 days in murine Mk medium with or without 5 μM harmine underwent flow cytometry after costaining with FITC-anti-CD41 and PI. ( D ) Mk polyploidization (PI). ( E ) Graph shows relative percent Mk ≥ 8N ± SEM, n = 4/group. ***P < 0.005, 2-way ANOVA. ( F) Mk size (FSC). Graph shows mean ± SEM, n = 4/group. *P < 0.05, 2-way ANOVA. ( G ) MKL1 localization. Human CD34 + progenitors cultured 24 hours in Mk medium with or without 5 μM inhibitors underwent immunofluorescent staining (IF) and confocal microscopy (Zeiss LSM700, original magnification, ×630; scale bar: 10 μm). ( H ) Graph shows mean ratio nuclear/cytoplasmic MKL1 signal ± SEM for 3 independent experiments. *P < 0.05, 1-way ANOVA with Tukey’s post hoc test.

Article Snippet: For prolonged expansion of undifferentiated human multipotent progenitors, CB CD34 + cells underwent 8 days of culture in prestimulation medium supplemented with 1 μM StemRegenin 1 (SR1, Selleckchem).

Techniques: Inhibition, Cell Culture, Western Blot, Purification, RNA Sequencing, Flow Cytometry, Staining, Confocal Microscopy

Journal: The Journal of Clinical Investigation

Article Title: Relieving DYRK1A repression of MKL1 confers an adult-like phenotype to human infantile megakaryocytes

doi: 10.1172/JCI154839

Figure Lengend Snippet: ( A ) Induction of F-actin. Human CD34 + progenitors cultured 24 hours in mK medium with or without 5 μM inhibitors underwent staining with indicated fluorescent dyes followed by confocal microscopy original magnification, ×630; scale bar: 10 μm. ( B ) Graphs show mean fluorescent intensity (MFI) of Phalloidin Alexa 594 signals ± SEM for 3 independent experiments as in A . *P < 0.05, Student’s t test. ( C ) Induction of Ablim2. Progenitors cultured 5 days as in A underwent IB of whole cell lysates. Arrows show Ablim2 isoforms. ( D ) Tubulin-normalized densitometry signals from IBs as in ( C ). Graph shows mean fold changes with inhibitors ± SEM for 3 independent experiments. *P < 0.05, 1-way ANOVA with Tukey’s post hoc test.

Article Snippet: For prolonged expansion of undifferentiated human multipotent progenitors, CB CD34 + cells underwent 8 days of culture in prestimulation medium supplemented with 1 μM StemRegenin 1 (SR1, Selleckchem).

Techniques: Cell Culture, Staining, Confocal Microscopy

Journal: The Journal of Clinical Investigation

Article Title: Relieving DYRK1A repression of MKL1 confers an adult-like phenotype to human infantile megakaryocytes

doi: 10.1172/JCI154839

Figure Lengend Snippet: ( A ) Effect of Ablim2 deficiency on polyploidization response. Neonatal CD34 + cells transduced with control or ABLIM2 targeting lentiviral shRNA constructs were cultured for 5 days in Mk medium with or without 5 μM inhibitors followed by flow cytometry as in A. ( B and C ) Graphs show mean percent Mk ≥ 8N ± SEM for 3 independent experiments. *P < 0.05; ***P < 0.005, 2-way ANOVA comparing fold induction. ( D – F ) MKL1 localization. Cells transduced as in ( A ) underwent 24 hours in Mk medium with or without 5 μM inhibitors followed by IF staining and confocal microscopy. Original magnification, ×630; scale bar: 10 μm. ( F ) Graph shows mean ratio nuclear to cytoplasmic MKL1 signal ± SEM for 3 independent experiments. *P < 0.05; *** P < 0.005, 2-way ANOVA comparing fold change. ( G – I ) Induction of F-Actin. Cells as in D underwent staining with fluorescent dyes and confocal microscopy. Original magnification, ×630; scale bar: 10 μm. ( I ) Graph shows MFI of Phalloidin Alexa 594 signal ± SEM for 3 independent experiments. *P < 0.05, 2-way ANOVA comparing fold change. ( J ) Diagram of pathways influencing MKL1 in its programming of the fetal-adult mK transition. positive transcription elongation factor (P-TEFb) signaling determines levels of MKL1, and actin cytoskeleton determines function. Diagram shows the influence exerted on MKL1/SRF by Dyrk1 destabilization of the F-actin-binding factors Ablim2/STARS.

Article Snippet: For prolonged expansion of undifferentiated human multipotent progenitors, CB CD34 + cells underwent 8 days of culture in prestimulation medium supplemented with 1 μM StemRegenin 1 (SR1, Selleckchem).

Techniques: Transduction, Control, shRNA, Construct, Cell Culture, Flow Cytometry, Staining, Confocal Microscopy, Binding Assay

Journal: Experimental Animals

Article Title: A Long-term Follow-up Study on the Engraftment of Human Hematopoietic Stem Cells in Sheep

doi: 10.1538/expanim.63.475

Figure Lengend Snippet: Long-term engraftment in sheep after in-utero transplantation of human hematopoietic stem cells

Article Snippet: Human CB CD34 + cells, used as hematopoietic stem cells (HSCs), were isolated by immunomagnetic separation using an anti-human CD34 microbeads kit (Miltenyi Biotech, Auburn, CA, USA) according to the manufacturer’s instructions.

Techniques: Transplantation Assay, In Utero

Journal: Experimental Animals

Article Title: A Long-term Follow-up Study on the Engraftment of Human Hematopoietic Stem Cells in Sheep

doi: 10.1538/expanim.63.475

Figure Lengend Snippet: In vitro CFU assay for validating the viability and functionality of transplanted CD34 + cells. In order to assess the viability and functionality of human cord blood (CB) CD34 + cells in the three protocols, colony forming-unit (CFU) assays of each CB CD34 + sample were conducted. Results are shown as mean ± standard deviation. Statistical significance was determined by ANOVA test. No significant difference was observed among the three protocols.

Article Snippet: Human CB CD34 + cells, used as hematopoietic stem cells (HSCs), were isolated by immunomagnetic separation using an anti-human CD34 microbeads kit (Miltenyi Biotech, Auburn, CA, USA) according to the manufacturer’s instructions.

Techniques: In Vitro, Colony-forming Unit Assay, Standard Deviation

Journal: Experimental Animals

Article Title: A Long-term Follow-up Study on the Engraftment of Human Hematopoietic Stem Cells in Sheep

doi: 10.1538/expanim.63.475

Figure Lengend Snippet: Long-term follow-up of human HSC engraftment after IUT in sheep. Human cord blood (CB) CD34 + cells were transduced with HOXB4 and transplanted into intact (non-conditioned) fetal sheep ( HOXB4 protocol, ■). Intact (non-transduced) human CB CD34 + cells were transplanted into fetal sheep conditioned with busulfan (BU) (BU protocol, ●). In addition, intact human CB CD34 + cells were transplanted into intact fetal sheep (non-treatment protocol, ▲ ). The scatter plots show percentages of human colony-forming units (CFUs) at the indicated months after in utero transplantation (IUT). The percentage of human CFUs was calculated by dividing the number of CFUs expressing the human β2-microglobulin gene by the total number of CFUs being analyzed in the bone marrow.

Article Snippet: Human CB CD34 + cells, used as hematopoietic stem cells (HSCs), were isolated by immunomagnetic separation using an anti-human CD34 microbeads kit (Miltenyi Biotech, Auburn, CA, USA) according to the manufacturer’s instructions.

Techniques: Transduction, In Utero, Transplantation Assay, Expressing