cd34 cell isolation Search Results


human cd34 cd38 isolation kit  (Miltenyi Biotec)
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Miltenyi Biotec human cd34 cd38 isolation kit
( A – D ) Irradiated NSG mice were transplanted with MPB <t>CD34</t> + HSPC that were transduced with a GFP-encoding LV vector (gene transfer 63%–66%) either i.v. or i.b.m. at the indicated cell doses. Human cell engraftment was analyzed by determining the percentage of human CD45 + (hCD45 + ) cells and GM hCD45 + (hCD45 + GFP + ) cells in the BM obtained via BM aspirate at 12 weeks to assess short-term engraftment ( A and B ) and from pooled BM from femurs, tibias, and iliac crests at 24 weeks to determine long-term human engraftment ( C and D ). Bars represent median engraftment ± 95% CI. Each symbol represents an individual mouse; n = 18–20 mice per cell dose per transplant method. Mice were transplanted using 4 unique MPB donors in 4 experiments; statistical analysis was performed using 1-way ANOVA comparing i.v. and i.b.m. groups at each cell dose with correction for multiple comparisons. ( E – H ) Lineage output of the human xenograft. BM was stained with antibodies specific for T cell lineage (hCD3), B cell lineage (hCD19), myeloid lineage (hCD33), and HSPC <t>(hCD34)</t> to determine the lineage output of the transplanted total HSPC ( E and G ) and GM HSPC ( F and H ) at 12 weeks and 24 weeks after transplant. Statistical analysis was performed using 2-way ANOVA. * P < 0.05, ** P < 0.01.
Human Cd34 Cd38 Isolation Kit, supplied by Miltenyi Biotec, used in various techniques. Bioz Stars score: 95/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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( A – D ) Irradiated NSG mice were transplanted with MPB CD34 + HSPC that were transduced with a GFP-encoding LV vector (gene transfer 63%–66%) either i.v. or i.b.m. at the indicated cell doses. Human cell engraftment was analyzed by determining the percentage of human CD45 + (hCD45 + ) cells and GM hCD45 + (hCD45 + GFP + ) cells in the BM obtained via BM aspirate at 12 weeks to assess short-term engraftment ( A and B ) and from pooled BM from femurs, tibias, and iliac crests at 24 weeks to determine long-term human engraftment ( C and D ). Bars represent median engraftment ± 95% CI. Each symbol represents an individual mouse; n = 18–20 mice per cell dose per transplant method. Mice were transplanted using 4 unique MPB donors in 4 experiments; statistical analysis was performed using 1-way ANOVA comparing i.v. and i.b.m. groups at each cell dose with correction for multiple comparisons. ( E – H ) Lineage output of the human xenograft. BM was stained with antibodies specific for T cell lineage (hCD3), B cell lineage (hCD19), myeloid lineage (hCD33), and HSPC (hCD34) to determine the lineage output of the transplanted total HSPC ( E and G ) and GM HSPC ( F and H ) at 12 weeks and 24 weeks after transplant. Statistical analysis was performed using 2-way ANOVA. * P < 0.05, ** P < 0.01.

Journal: JCI Insight

Article Title: Differential CXCR4 expression on hematopoietic progenitor cells versus stem cells directs homing and engraftment

doi: 10.1172/jci.insight.151847

Figure Lengend Snippet: ( A – D ) Irradiated NSG mice were transplanted with MPB CD34 + HSPC that were transduced with a GFP-encoding LV vector (gene transfer 63%–66%) either i.v. or i.b.m. at the indicated cell doses. Human cell engraftment was analyzed by determining the percentage of human CD45 + (hCD45 + ) cells and GM hCD45 + (hCD45 + GFP + ) cells in the BM obtained via BM aspirate at 12 weeks to assess short-term engraftment ( A and B ) and from pooled BM from femurs, tibias, and iliac crests at 24 weeks to determine long-term human engraftment ( C and D ). Bars represent median engraftment ± 95% CI. Each symbol represents an individual mouse; n = 18–20 mice per cell dose per transplant method. Mice were transplanted using 4 unique MPB donors in 4 experiments; statistical analysis was performed using 1-way ANOVA comparing i.v. and i.b.m. groups at each cell dose with correction for multiple comparisons. ( E – H ) Lineage output of the human xenograft. BM was stained with antibodies specific for T cell lineage (hCD3), B cell lineage (hCD19), myeloid lineage (hCD33), and HSPC (hCD34) to determine the lineage output of the transplanted total HSPC ( E and G ) and GM HSPC ( F and H ) at 12 weeks and 24 weeks after transplant. Statistical analysis was performed using 2-way ANOVA. * P < 0.05, ** P < 0.01.

Article Snippet: CD34 + CD38 – cells were isolated from G-CSF MPB by magnetic selection with human CD34 + CD38 – Isolation Kit (Miltenyi Biotec, 130-114-822) and cryopreserved.

Techniques: Irradiation, Transduction, Plasmid Preparation, Staining

( A ) MPB CD34 + HSPC were injected i.v., i.b.m. using a standard syringe, or slow i.b.m. using a Hamilton syringe over 1 minute, and homing of CD34 + cells to the BM was analyzed 20–22 hours later. To determine if mechanical pressure in the injected femur alters homing, irradiated CD34 – cells were injected i.b.m., and CD34 + cells were injected i.v. into the same animal. Homing was determined in the injected femurs (IF) and non-IF with i.b.m. injections, and in both femurs (BF) combined with i.v. injection. Symbols represent individual animals; n = 3 mice per group; statistical analysis was performed using 2-way ANOVA. ( B and C ) For i.b.m. injections, BM from the IF and non-IF was analyzed for expression of CXCL12 and VCAM1 on mouse stromal cells (hCD45 – PKH-26 – , mouse lineage – mCD45 – mCD51 + cells). n = 5 mice per group; data were normalized to i.v. injected group; statistical analysis was performed by Mann-Whitney U test. ( D and E ) CXCR4 and VLA4 homing receptor expression of CD34 + HSPC, CD34 + CD38 + HPC, and CD34 + CD38 – CD90 + HSC-enriched populations. Data were normalized to CXCR4 and VLA4 expression on CD34 + HSPC. Symbols represent unique MPB donors; n = 5 per group; statistical analysis was performed using ANOVA. ( F ) Adhesion of CD34 + HSPC, CD34 + CD38 + HPC, and CD34 + CD38 – CD90 + HSC to CXCL12. Symbols represent unique MPB donors; n = 5 per group; statistical analysis was performed using ANOVA. ( G ) Binding of CD34 + HSPC, CD34 + CD38 + HPC, and CD34 + CD38 – CD90 + HSC to the VLA4 affinity detection ligand LDV-FITC. Data were normalized to LDV-FITC expression in CD34 + HSPC. Symbols represent unique MPB donors; n = 3 per group; statistical analysis was performed using 1-way ANOVA. * P < 0.05, ** P < 0.01, *** P < 0.001. In B – E and G , data were normalized to the indicated group, to account for variability in MFI between different experiments/donors.

Journal: JCI Insight

Article Title: Differential CXCR4 expression on hematopoietic progenitor cells versus stem cells directs homing and engraftment

doi: 10.1172/jci.insight.151847

Figure Lengend Snippet: ( A ) MPB CD34 + HSPC were injected i.v., i.b.m. using a standard syringe, or slow i.b.m. using a Hamilton syringe over 1 minute, and homing of CD34 + cells to the BM was analyzed 20–22 hours later. To determine if mechanical pressure in the injected femur alters homing, irradiated CD34 – cells were injected i.b.m., and CD34 + cells were injected i.v. into the same animal. Homing was determined in the injected femurs (IF) and non-IF with i.b.m. injections, and in both femurs (BF) combined with i.v. injection. Symbols represent individual animals; n = 3 mice per group; statistical analysis was performed using 2-way ANOVA. ( B and C ) For i.b.m. injections, BM from the IF and non-IF was analyzed for expression of CXCL12 and VCAM1 on mouse stromal cells (hCD45 – PKH-26 – , mouse lineage – mCD45 – mCD51 + cells). n = 5 mice per group; data were normalized to i.v. injected group; statistical analysis was performed by Mann-Whitney U test. ( D and E ) CXCR4 and VLA4 homing receptor expression of CD34 + HSPC, CD34 + CD38 + HPC, and CD34 + CD38 – CD90 + HSC-enriched populations. Data were normalized to CXCR4 and VLA4 expression on CD34 + HSPC. Symbols represent unique MPB donors; n = 5 per group; statistical analysis was performed using ANOVA. ( F ) Adhesion of CD34 + HSPC, CD34 + CD38 + HPC, and CD34 + CD38 – CD90 + HSC to CXCL12. Symbols represent unique MPB donors; n = 5 per group; statistical analysis was performed using ANOVA. ( G ) Binding of CD34 + HSPC, CD34 + CD38 + HPC, and CD34 + CD38 – CD90 + HSC to the VLA4 affinity detection ligand LDV-FITC. Data were normalized to LDV-FITC expression in CD34 + HSPC. Symbols represent unique MPB donors; n = 3 per group; statistical analysis was performed using 1-way ANOVA. * P < 0.05, ** P < 0.01, *** P < 0.001. In B – E and G , data were normalized to the indicated group, to account for variability in MFI between different experiments/donors.

Article Snippet: CD34 + CD38 – cells were isolated from G-CSF MPB by magnetic selection with human CD34 + CD38 – Isolation Kit (Miltenyi Biotec, 130-114-822) and cryopreserved.

Techniques: Injection, Irradiation, Expressing, MANN-WHITNEY, Binding Assay

( A – C ) High CXCR4 expression on HPC mediates their preferential homing locally with i.b.m. transplant; CD34 + HSPC were transduced with a BFP LV vector, and the CD34 + CD38 + BFP + HPC were sorted by flow cytometry 72 hours after gene transfer. HPC were blocked with AMD3100 (CXCR4 antagonist) or BIO5192 (VLA4 antagonist) before i.v. or i.b.m. delivery ( A ). Homing of hCD34 + cells was analyzed in the injected femur (IF) and non-IF for i.b.m. injected mice and in both femurs combined in i.v. injected mice. Data were normalized to i.v. injected control CD34 + CD38 + cells, and the fold increase is indicated within the bar ( B and C ). n = 5–6 mice per group; statistical analysis was performed by 1-way ANOVA. ( D – F ) Induction of high CXCR4 expression on CD34 + CD38 – HSC enriched population via gene transfer confers them with a homing advantage despite higher abundance of CD34 + CD38 + HPC. CD34 + HSPC were transduced with a BFP LV vector or a BFP-CXCR4 LV vector and sorted for transduced CD34 + CD38 – BFP + cells and untransduced CD34 + CD38 + BFP – HPC at 72 hours. Transduced CD34 + CD38 – cells were mixed with untransduced CD34 + CD38 + cells and transplanted i.v. or i.b.m., and homing of CD34 + CD38 – BFP + cells into BM was analyzed at 20–22 hours ( D ). CXCR4 expression on injected cells at time of transplant is shown and the MFI on injected cells at time of transplant was as follows: CD34 + CD38 – control, 16,930; CD34 + CD38 + control, 35,098; and CD34 + CD38 – CXCR4 transduced, 37,453. ( E ). Homing in BM is shown ( F ). n = 3–10 mice per experimental arm; statistical analysis was performed using 1-way ANOVA. * P < 0.05.

Journal: JCI Insight

Article Title: Differential CXCR4 expression on hematopoietic progenitor cells versus stem cells directs homing and engraftment

doi: 10.1172/jci.insight.151847

Figure Lengend Snippet: ( A – C ) High CXCR4 expression on HPC mediates their preferential homing locally with i.b.m. transplant; CD34 + HSPC were transduced with a BFP LV vector, and the CD34 + CD38 + BFP + HPC were sorted by flow cytometry 72 hours after gene transfer. HPC were blocked with AMD3100 (CXCR4 antagonist) or BIO5192 (VLA4 antagonist) before i.v. or i.b.m. delivery ( A ). Homing of hCD34 + cells was analyzed in the injected femur (IF) and non-IF for i.b.m. injected mice and in both femurs combined in i.v. injected mice. Data were normalized to i.v. injected control CD34 + CD38 + cells, and the fold increase is indicated within the bar ( B and C ). n = 5–6 mice per group; statistical analysis was performed by 1-way ANOVA. ( D – F ) Induction of high CXCR4 expression on CD34 + CD38 – HSC enriched population via gene transfer confers them with a homing advantage despite higher abundance of CD34 + CD38 + HPC. CD34 + HSPC were transduced with a BFP LV vector or a BFP-CXCR4 LV vector and sorted for transduced CD34 + CD38 – BFP + cells and untransduced CD34 + CD38 + BFP – HPC at 72 hours. Transduced CD34 + CD38 – cells were mixed with untransduced CD34 + CD38 + cells and transplanted i.v. or i.b.m., and homing of CD34 + CD38 – BFP + cells into BM was analyzed at 20–22 hours ( D ). CXCR4 expression on injected cells at time of transplant is shown and the MFI on injected cells at time of transplant was as follows: CD34 + CD38 – control, 16,930; CD34 + CD38 + control, 35,098; and CD34 + CD38 – CXCR4 transduced, 37,453. ( E ). Homing in BM is shown ( F ). n = 3–10 mice per experimental arm; statistical analysis was performed using 1-way ANOVA. * P < 0.05.

Article Snippet: CD34 + CD38 – cells were isolated from G-CSF MPB by magnetic selection with human CD34 + CD38 – Isolation Kit (Miltenyi Biotec, 130-114-822) and cryopreserved.

Techniques: Expressing, Transduction, Plasmid Preparation, Flow Cytometry, Injection, Control

CD34 + CD38 – HSC-enriched cells were obtained using immunomagnetic sorting, transduced with a GFP LV vector (gene transfer efficiency was 69%), and transplanted into NSG mice i.v. or i.b.m. in 2 limiting dilution doses. ( A and B ) Long-term (24 week) engraftment of total (hCD45 + ) and GM human (hCD45 + GFP + ) cells is shown. Symbols represent individual animals; n = 3–4 mice per treatment condition; statistical analysis was performed by comparing the modes of delivery comparing the different cell doses using 1-way ANOVA. * P < 0.05. ( C and D ) Lineage output at 24 weeks shows that the long-term human graft was multilineage, composed of B, T, and myeloid cells and CD34 + HSPC. Statistical analysis was performed using 2-way ANOVA.

Journal: JCI Insight

Article Title: Differential CXCR4 expression on hematopoietic progenitor cells versus stem cells directs homing and engraftment

doi: 10.1172/jci.insight.151847

Figure Lengend Snippet: CD34 + CD38 – HSC-enriched cells were obtained using immunomagnetic sorting, transduced with a GFP LV vector (gene transfer efficiency was 69%), and transplanted into NSG mice i.v. or i.b.m. in 2 limiting dilution doses. ( A and B ) Long-term (24 week) engraftment of total (hCD45 + ) and GM human (hCD45 + GFP + ) cells is shown. Symbols represent individual animals; n = 3–4 mice per treatment condition; statistical analysis was performed by comparing the modes of delivery comparing the different cell doses using 1-way ANOVA. * P < 0.05. ( C and D ) Lineage output at 24 weeks shows that the long-term human graft was multilineage, composed of B, T, and myeloid cells and CD34 + HSPC. Statistical analysis was performed using 2-way ANOVA.

Article Snippet: CD34 + CD38 – cells were isolated from G-CSF MPB by magnetic selection with human CD34 + CD38 – Isolation Kit (Miltenyi Biotec, 130-114-822) and cryopreserved.

Techniques: Transduction, Plasmid Preparation

( A ) Construct design. The aa sequence of VPR and its mutant version generated is shown. VPR, a small HIV-1 accessory protein, is carried in viral particles bound to gag via residues in its central region that fold into 3 α-helices. The C-terminal domain has 6 arginine residues that potentiate nuclear localization, G 2 M arrest, and apoptosis. S79 phosphorylation is important for cell cycle arrest. We truncated VPR at the 78 aa and mutated VPR R77Q . W54 and Q65 interacts with DNA damage response (DDR) proteins via UNG2 and DCAF and were mutated to VPR W54R and VPR Q65R . The triple mutated and truncated VPR (VPR MT ) would lack pathogenicity (residues known to be associated with VPR toxicity are highlighted in red) but allow binding to gag. VPR MT was fused to CXCR4 cDNA via the HIV-1 protease cleavage site (PCS) to generate VPR MT -CXCR4. ( B ) K562 cells were transduced with LV CXCR4 vector-like particles (VLP; empty vector particles lacking the vector genome) at increasing particle concentration and analyzed for CXCR4 expression using flow cytometry. Mean fluorescence intensity (MFI) of CXCR4 is listed against volume of VLP added. ( C and D ) A GFP-encoding LV was either packaged using standard packaging plasmids (LV, black) or packaged with VPR MT -CXCR4 plasmid in addition in order to package the CXCR protein attached to the LV capsid (LV CXCR4 , red). CXCR4 expression on MPB CD34 + cells transduced with LV CXCR4 vector compared with cells transduced with the control LV vector 24 hours following gene transfer is shown. n = 4; statistical analysis was performed by Mann-Whitney U test. ( E ) The time course of CXCR4 expression in LV CXCR4 HSPC, normalized to that of control LV HSPC, is shown, with expression peaking at 24 hours that returns to baseline by 72 hours. Symbols represent individual MPB donors; n = 3.

Journal: JCI Insight

Article Title: Differential CXCR4 expression on hematopoietic progenitor cells versus stem cells directs homing and engraftment

doi: 10.1172/jci.insight.151847

Figure Lengend Snippet: ( A ) Construct design. The aa sequence of VPR and its mutant version generated is shown. VPR, a small HIV-1 accessory protein, is carried in viral particles bound to gag via residues in its central region that fold into 3 α-helices. The C-terminal domain has 6 arginine residues that potentiate nuclear localization, G 2 M arrest, and apoptosis. S79 phosphorylation is important for cell cycle arrest. We truncated VPR at the 78 aa and mutated VPR R77Q . W54 and Q65 interacts with DNA damage response (DDR) proteins via UNG2 and DCAF and were mutated to VPR W54R and VPR Q65R . The triple mutated and truncated VPR (VPR MT ) would lack pathogenicity (residues known to be associated with VPR toxicity are highlighted in red) but allow binding to gag. VPR MT was fused to CXCR4 cDNA via the HIV-1 protease cleavage site (PCS) to generate VPR MT -CXCR4. ( B ) K562 cells were transduced with LV CXCR4 vector-like particles (VLP; empty vector particles lacking the vector genome) at increasing particle concentration and analyzed for CXCR4 expression using flow cytometry. Mean fluorescence intensity (MFI) of CXCR4 is listed against volume of VLP added. ( C and D ) A GFP-encoding LV was either packaged using standard packaging plasmids (LV, black) or packaged with VPR MT -CXCR4 plasmid in addition in order to package the CXCR protein attached to the LV capsid (LV CXCR4 , red). CXCR4 expression on MPB CD34 + cells transduced with LV CXCR4 vector compared with cells transduced with the control LV vector 24 hours following gene transfer is shown. n = 4; statistical analysis was performed by Mann-Whitney U test. ( E ) The time course of CXCR4 expression in LV CXCR4 HSPC, normalized to that of control LV HSPC, is shown, with expression peaking at 24 hours that returns to baseline by 72 hours. Symbols represent individual MPB donors; n = 3.

Article Snippet: CD34 + CD38 – cells were isolated from G-CSF MPB by magnetic selection with human CD34 + CD38 – Isolation Kit (Miltenyi Biotec, 130-114-822) and cryopreserved.

Techniques: Construct, Sequencing, Mutagenesis, Generated, Phospho-proteomics, Binding Assay, Transduction, Plasmid Preparation, Concentration Assay, Expressing, Flow Cytometry, Fluorescence, Control, MANN-WHITNEY

Experimental schema for assessment of homing and engraftment of CD34 + CD38 – cells transduced with GFP LV or GFP LV CXCR4 that were transplanted into NSG mice either via i.v. or i.b.m. delivery. ( A ) The number of animals used for the homing experiment and engraftment experiments is indicated under each experimental arm. ( B ) Homing of CD34 + CD38 – cells in the BM. Data were normalized to i.v. injected control CD34 + CD38 – cells, and the fold increase is indicated within the bar. Symbols represent individual mice; statistical analysis was performed using ANOVA. ( C and D ) Long-term engraftment of CD34 + CD38 – cells was assessed by determining the percentage of human CD45 + GFP – cells ( C ) and human CD45 + GFP + ( D ) cells 24 weeks following transplant. Data were normalized to i.v. transplanted GFP LV transduced CD34 + CD38 – cells, and the fold increase is indicated within the bar. Gene transfer was 60% with GFP LV and 52% with GFP LV CXCR4 . Symbols represent individual mice; statistical analysis was performed by 1-way ANOVA. * P < 0.05, ** P < 0.01.

Journal: JCI Insight

Article Title: Differential CXCR4 expression on hematopoietic progenitor cells versus stem cells directs homing and engraftment

doi: 10.1172/jci.insight.151847

Figure Lengend Snippet: Experimental schema for assessment of homing and engraftment of CD34 + CD38 – cells transduced with GFP LV or GFP LV CXCR4 that were transplanted into NSG mice either via i.v. or i.b.m. delivery. ( A ) The number of animals used for the homing experiment and engraftment experiments is indicated under each experimental arm. ( B ) Homing of CD34 + CD38 – cells in the BM. Data were normalized to i.v. injected control CD34 + CD38 – cells, and the fold increase is indicated within the bar. Symbols represent individual mice; statistical analysis was performed using ANOVA. ( C and D ) Long-term engraftment of CD34 + CD38 – cells was assessed by determining the percentage of human CD45 + GFP – cells ( C ) and human CD45 + GFP + ( D ) cells 24 weeks following transplant. Data were normalized to i.v. transplanted GFP LV transduced CD34 + CD38 – cells, and the fold increase is indicated within the bar. Gene transfer was 60% with GFP LV and 52% with GFP LV CXCR4 . Symbols represent individual mice; statistical analysis was performed by 1-way ANOVA. * P < 0.05, ** P < 0.01.

Article Snippet: CD34 + CD38 – cells were isolated from G-CSF MPB by magnetic selection with human CD34 + CD38 – Isolation Kit (Miltenyi Biotec, 130-114-822) and cryopreserved.

Techniques: Transduction, Injection, Control