Human CD34+ Cells Rapidly Mobilized by AMD3100 Repopulate NOD/SCID Mice with Equivalent Efficiency as CD34+ Cells Mobilized by G-CSF.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 1186-1186
Author(s):  
David A. Hess ◽  
Jesper Bonde ◽  
Timothy P. Craft ◽  
Louisa Wirthlin ◽  
John F. DiPersio ◽  
...  
Keyword(s):  
Mononuclear Cells ◽  
Paired Comparison ◽  
Drug Clearance ◽  
Scid Mice ◽  
Lymphoid Cells ◽  
Additional Patient ◽  
Cxcr4 Antagonist ◽  
Post Transplantation ◽  
Hematopoietic Stem ◽  
Cd34 Cells

Abstract Interactions between the chemokine receptor CXCR4 and its ligand, stromal derived factor-1, regulate hematopoietic stem cell migration. The CXCR4 antagonist, AMD3100, has recently been shown to rapidly mobilize primitive hematopoietic cells. However, the functional properties of stem and progenitor cells mobilized with this agent are not well characterized. Thus, we directly compared the NOD/SCID repopulating function of CD34+ cells rapidly mobilized (4 hours) by AMD3100 versus CD34+ cells mobilized by 5 days of G-CSF treatment. Healthy, matched sibling donors were leukapheresed after a single injection of 240ug/kg AMD3100, and produced an enrichment of circulating CD34+ cells to 0.6% of the total mononuclear cells (MNC). After 2 weeks of drug clearance, the same donor was mobilized with G-CSF (0.4% CD34+ cells), allowing a paired comparison of the repopulating function of cells mobilized by these two regimens. Total MNC, CD34+ cells (>95% purity), and lineage depleted (Lin−) cells (48–55% CD34+) were isolated and transplanted into NOD/SCID mice at various doses. Injection of 106–107 (approximately 5x103–5x104 CD34+ cells) AMD3100-mobilized MNC resulted in bone marrow (BM) engraftment in 6 of 10 mice, whereas equal doses of G-CSF mobilized MNC engrafted 3 of 10 mice. Higher cell doses (2x107 MNC, approximately 1x105 CD34+ cells) consistently produced engraftment in the BM, spleen, and peripheral blood of all mice, with higher levels of engraftment with AMD3100-mobilized cells (1.8±0.5%) compared to G-CSF-mobilized cells (0.4±0.05%, p<0.05). Similar analyses performed using purified CD34+ cells revealed similar engraftment frequencies for both leukapheresis products. Transplantation of 5x104–105 AMD3100-mobilized CD34+ cells engrafted 4 of 7 mice and G-CSF-mobilized CD34+ cells engrafted 5 of 10 mice. However, transplantation of 5x105 AMD3100-mobilized CD34+ cells consistently resulted in higher engraftment levels compared to G-CSF-mobilized CD34+ cells (3.4±1.1% versus 0.8±0.4% human cells, p<0.05). Multilineage hematopoietic differentiation of transplanted CD34+ cells was similar for AMD3100 and G-CSF-mobilized CD34+ cells, with production of myeloid cells (CD33+, CD14−CD13+CD66abce+/−), monocytes (CD14+), immature B-lymphoid cells (CD19+/−CD20+/−), and primitive repopulating (CD34+CD133+CD38−) cells 7–8 weeks post-transplantation. Similarly, AMD3100 and G-CSF-mobilized Lin− cells produced consistent engraftment in all transplanted mice (n=20), and demonstrated equivalent engraftment levels and multilineage differentiation when directly comparing AMD3100 versus G-CSF-mobilization. Ongoing analysis of additional patient samples will allow direct comparison of these repopulating cells by limiting dilution analysis using Poisson statistics. These preliminary studies indicate that human AMD3100-mobilized CD34+ cells possess at least equivalent repopulating capacity compared to G-CSF mobilized cells, and therefore represent a more rapidly obtainable source of hematopoietic stem cells for clinical transplantation.

Human CD34+Cells Mobilized by AMD3100 Demonstrate Enhanced NOD/SCID Repopulating Function Compared to CD34+ Cells Mobilized by Granulocyte Colony Stimulating Factor.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 1962-1962 ◽  
Author(s):  
David A. Hess ◽  
Louisa Wirthlin ◽  
Timothy P. Craft ◽  
Jesper Bonde ◽  
Ryan W. Lahey ◽  
...  
Keyword(s):  
Progenitor Cells ◽  
Dna Sequences ◽  
Peripheral Blood ◽  
Paired Comparison ◽  
Fold Increase ◽  
Human Cells ◽  
Lymphoid Cells ◽  
Post Transplantation ◽  
Hematopoietic Stem ◽  
Cd34 Cells

Abstract Interactions between stromal derived factor-1 (SDF-1 or CXCL12), and its receptor CXCR4 regulate hematopoietic stem and progenitor cell retention in the bone marrow. AMD3100, a bicyclam molecule that selectively blocks the interaction between CXCL12 and CXCR4, has recently been used in clinical trials to rapidly mobilize hematopoietic progenitor cells. However, the functional properties of human stem and progenitor cells mobilized with this agent are not well characterized. Here, we directly compared the NOD/SCID repopulating function of CD34+ cells rapidly mobilized (4 hours) by AMD3100 versus CD34+ cells mobilized after 5 days of G-CSF treatment. A total of 7 HLA-matched sibling donors were leukapheresed after a single injection of 240ug/kg AMD3100. After 1 week of drug clearance, the same donor was mobilized with G-CSF, allowing a paired comparison of the repopulating function of cells mobilized by the two agents. Total CD34+ cells mobilized by AMD3100 treatment averaged 1.2±0.4x106 CD34+ cells/kg (range 0.4–2.1x106 CD34+ cells/kg), as compared to G-CSF treatment at 3.2±0.9x106 CD34+ cells/kg (range 1.7–5.7 x106 CD34+ cells/kg). Leukapheresis total mononuclear cell (MNC) fraction or purified CD34+ cells (>90% purity), were isolated and transplanted into sublethally irradiated NOD/SCID mice at varying doses. BM, spleen, and peripheral blood of mice were harvested 7–8 weeks post-transplantation and analyzed by flow cytometry for the presence or absence of engrafting human cells. Low frequency human engraftment events (<0.2% human cells) were confirmed by PCR for P17H8 alpha-satellite human DNA sequences. Injection of 1–40x106 MNC or 0.5–5x105 CD34+ cells produced consistent human engraftment and allowed limiting dilution analysis using Poisson statistics to be performed on paired samples of AMD3100 and G-CSF leukapheresis products from 3 individual patients. The calculated frequencies of NOD/SCID repopulating cells (SRC) were 1 SRC in 11.5x106 AMD3100-mobilized MNC (n=50) compared to 1 SRC in 44.8x106 G-CSF-mobilized MNC (n=55). For purified CD34+ populations, the overall frequency of repopulating cells was 1 SRC in 1.0x105 AMD3100-mobilized CDC34+ cells (n=53) compared to 1 SRC in 3.1x105 G-CSF-mobilized CD34+ cells (n=45). These data correspond to a 3–4-fold increase in overall repopulating function demonstrated by AMD3100 mobilized cells. Multilineage hematopoietic differentiation of transplanted CD34+ cells was similar for AMD3100 and G-CSF-mobilized CD34+ cells, with equivalent production of myelo-monocytic cells (CD33+CD14+), immature B-lymphoid cells (CD19+CD20+), and primitive repopulating (CD34+CD133+CD38−) cells 7–8 weeks post-transplantation. These studies indicate that human AMD3100-mobilized MNC and purified CD34+ cells possess enhanced repopulating capacity, as compared to G-CSF mobilized counterparts from the same donor. Thus, AMD3100 mobilized peripheral blood represents a rapidly obtained and highly functional source of repopulating hematopoietic stem cells for clinical transplantation procedures.

Surface fucosylation of human cord blood cells augments binding to P-selectin and E-selectin and enhances engraftment in bone marrow

Blood ◽  
2004 ◽  
Vol 104 (10) ◽  
pp. 3091-3096 ◽  
Author(s):  
Lijun Xia ◽  
J. Michael McDaniel ◽  
Tadayuki Yago ◽  
Andrea Doeden ◽  
Rodger P. McEver
Keyword(s):  
Bone Marrow ◽  
Cord Blood ◽  
Mononuclear Cells ◽  
Scid Mice ◽  
Sialyl Lewis X ◽  
Human Umbilical Cord Blood ◽  
Human Umbilical Cord ◽  
Hematopoietic Stem ◽  
Similar Treatment ◽  
Cd34 Cells

Abstract Murine hematopoietic stem and progenitor cells (HSPCs) home to bone marrow in part by rolling on P-selectin and E-selectin expressed on endothelial cells. Human adult CD34+ cells, which are enriched in HSPCs, roll on endothelial selectins in bone marrow vessels of nonobese diabetic/severe combined immune deficiency (NOD/SCID) mice. Many human umbilical cord blood (CB) CD34+ cells do not roll in these vessels, in part because of an uncharacterized defect in binding to P-selectin. Selectin ligands must be α1-3 fucosylated to form glycan determinants such as sialyl Lewis x (sLex). We found that inadequate α1-3 fucosylation of CB CD34+ cells, particularly CD34+CD38–/low cells that are highly enriched in HSPCs, caused them to bind poorly to E-selectin as well as to P-selectin. Treatment of CB CD34+ cells with guanosine diphosphate (GDP) fucose and exogenous α1-3 fucosyltransferase VI increased cell-surface sLex determinants, augmented binding to fluid-phase P- and E-selectin, and improved cell rolling on P- and E-selectin under flow. Similar treatment of CB mononuclear cells enhanced engraftment of human hematopoietic cells in bone marrows of irradiated NOD/SCID mice. These observations suggest that α1-3 fucosylation of CB cells might be a simple and effective method to improve hematopoietic cell homing to and engraftment in bone marrows of patients receiving CB transplants.

CUL-4A Short Hairpin RNA (shRNA) Impairs Normal Hematopoiesis of Human Cord Blood CD34+ Cells In Vitro and In Vivo.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 2270-2270
Author(s):  
Jae Hung Shieh ◽  
Jianxuan Zhang ◽  
Xiaoling Lu ◽  
Pengbo Zhou ◽  
Malcolm A.S. Moore
Keyword(s):  
Cord Blood ◽  
Lentiviral Vector ◽  
Scid Mice ◽  
Lymphoid Cells ◽  
Mouse Bone Marrow ◽  
Post Transplantation ◽  
Cd34 Cells ◽  
B Lymphoid

Abstract The CUL-4A ubiquitination machinery regulates the stability of HOXA9 by promoting its ubiquinitation and proteasome-dependent degradation. Our previous study showed the perturbation of CUL-4A biosysthesis in 32D cells by RNA-mediated interference increased an accumulation of HOXA9, and impaired G-CSF-induced terminal differentiation to granulocytes. HOXA9 is preferentially expressed in primitive hematopoietic cells and downregulates with differentiation. In this study, a lentiviral vector expressed both CUL-4A shRNA and GFP was constructed and was used to transduce cord blood (CB) CD34+ cells (GFP-shCUL-4A-CD34+ cells). The ability of GFP-shCUL-4A-CD34+ cells to form CFC colonies, to differentiate into myeloid, erythroid, B-lymphoid and T-lymphoid cells in vitro, and to engraft in irradiated NOD/SCID mice was evaluated. Our results showed that knockdown of CUL-4A m-RNA dramatically inhibited hematopoisis both in vivo and in vitro. In the CFC assay, FACS-sorted GFPbright-shCUL-4A-CD34+ cell formed fewer colonies (5%) that were smaller size (&lt;500 cells/colonies) relative to progeny of GFPbright-CD34+ cells. In contrast, FACS-sorted GFPdim-shCUL-4A-CD34+ cells produced only 50% less colonies than the control GFPdim-CD34+ cells. However, GFPdim-shCUL-4A-CD34+ cells had 30% more colonies with &lt;500 cells/colony but 82% less colonies with &gt;500 cells/colony comparing to that of GFPdim-CD34+ cells. After transplanting the GFP-shCUL-4A-CD34+ cells into irradiated NOD/SCID mice for 6 weeks, GFP-shCUL-4A-CD45+ cells decreased more than 80% in the mouse bone marrow. These human GFP-shCUL-4A CD45 + cells were capable of forming GFP+-CFC but with &gt;80% reduction in their potency. No reduction in % of GFP-CD45+ cells and GFP+-CFC was observed in the GFP-CD34+ cells group pre- and post-transplantation. The in vitro culture systems showed that knockdown of CUL-4A mRNA in CB CD34+ cells resulted in the developmental impairment in monocytic, megakaryocytic, erythroid, B lymphoid and T-lymphoid lineages. This impairment is a time- and shCUL-4A dose-dependent process. Further study using cell cycle analysis indicated that transduction of CUL-4A shRNA to HL-60 or CD34+ cells induced apoptosis. Our results, for the first time, demonstrate an essential role of the CUL-4A ubiquitin ligase in maintaining cell survival to ensure proper differentiation of normal human stem and progenitor cells into different hematopoietic lineages both in vivo and in vitro.

Hoechst dye efflux reveals a novel CD7+CD34− lymphoid progenitor in human umbilical cord blood

Blood ◽  
2000 ◽  
Vol 96 (6) ◽  
pp. 2125-2133 ◽  
Author(s):  
Robert W. Storms ◽  
Margaret A. Goodell ◽  
Alan Fisher ◽  
Richard C. Mulligan ◽  
Clay Smith
Keyword(s):  
Stem Cells ◽  
Cord Blood ◽  
Umbilical Cord ◽  
Umbilical Cord Blood ◽  
Large Population ◽  
Lymphoid Cells ◽  
Human Umbilical Cord Blood ◽  
Human Umbilical Cord ◽  
Hematopoietic Stem ◽  
Cd34 Cells

Abstract A novel Hoechst 33342 dye efflux assay was recently developed that identifies a population of hematopoietic cells termed side population (SP) cells. In the bone marrow of multiple species, including mice and primates, the SP is composed primarily of CD34−cells, yet has many of the functional properties of hematopoietic stem cells (HSCs). This report characterizes SP cells from human umbilical cord blood (UCB). The SP in unfractionated UCB was enriched for CD34+ cells but also contained a large population of CD34− cells, many of which were mature lymphocytes. SP cells isolated from UCB that had been depleted of lineage-committed cells (Lin− UCB) contained CD34+ and CD34− cells in approximately equivalent proportions. Similar to previous descriptions of human HSCs, the CD34+Lin− SP cells were CD38dimHLA-DRdimThy-1dimCD45RA−CD71−and were enriched for myelo-erythroid precursors. In contrast, the CD34−Lin− SP cells were CD38−HLA-DR−Thy-1−CD71−and failed to generate myelo-erythroid progeny in vitro. The majority of these cells were CD7+CD11b+CD45RA+, as might be expected of early lymphoid cells, but did not express other lymphoid markers. The CD7+CD34−Lin− UCB SP cells did not proliferate in simple suspension cultures but did differentiate into natural killer cells when cultured on stroma with various cytokines. In conclusion, the human Lin− UCB SP contains both CD34+ multipotential stem cells and a novel CD7+CD34−Lin− lymphoid progenitor. This observation adds to the growing body of evidence that CD34− progenitors exist in humans.

scholarly journals In Vitro and In Vivo Evidence That Ex Vivo Cytokine Priming of Donor Marrow Cells May Ameliorate Posttransplant Thrombocytopenia

Blood ◽  
1998 ◽  
Vol 91 (1) ◽  
pp. 353-359 ◽  
Author(s):  
Mariusz Z. Ratajczak ◽  
Janina Ratajczak ◽  
Boguslaw Machalinski ◽  
Rosemarie Mick ◽  
Alan M. Gewirtz
Keyword(s):  
Mononuclear Cells ◽  
Recovery Period ◽  
Hematopoietic Stem ◽  
Platelet Recovery ◽  
Serum Free ◽  
Cd34 Cells ◽  
In Vivo Animal Model ◽  
Marrow Cells

AbstractThrombocytopenia is typically observed in patients undergoing hematopoietic stem cell transplantation. We hypothesized that delayed platelet count recovery might be ameliorated by increasing the number of megakaryocyte colony- forming units (CFU-Meg) in the hematopoietic cell graft. To test this hypothesis, we evaluated cytokine combinations and culture medium potentially useful for expanding CFU-Meg in vitro. We then examined the ability of expanded cells to accelerate platelet recovery in an animal transplant model. Depending on the cytokine combination used, we found that culturing marrow CD34+cells for 7 to 10 days in serum-free cultures was able to expand CFU-Meg ∼40 to 80 times over input number. Shorter incubation periods were also found to be effective and when CD34+ cells were exposed to thrombopoietin (TPO), kit ligand (KL), interleukin-1α (IL-1α), and IL-3 in serum-free cultures for as few as 48 hours, the number of assayable CFU-Meg was still increased ∼threefold over input number. Of interest, cytokine primed marrow cells were also found to form colonies in vitro more quickly than unprimed cells. The potential clinical utility of this short-term expansion strategy was subsequently tested in an in vivo animal model. Lethally irradiated Balb-C mice were transplanted with previously frozen syngeneic marrow mononuclear cells (106/mouse), one tenth of which (105) had been primed with [TPO, KL, IL-1a, and IL-3] under serum-free conditions for 36 hours before cryopreservation. Mice receiving the primed frozen marrow cells recovered their platelet and neutrophil counts 3 to 5 days earlier than mice transplanted with unprimed cells. Mice which received marrow cells that had been primed after thawing but before transplantation had similar recovery kinetics. We conclude that pretransplant priming of hematopoietic cells leads to faster recovery of all hematopoietic lineages. Equally important, donor cell priming before transplant may represent a highly cost-effective alternative to constant administration of cytokines during the posttransplant recovery period.

scholarly journals AMD3100 mobilizes hematopoietic stem cells with long-term repopulating capacity in nonhuman primates

Blood ◽  
2006 ◽  
Vol 107 (9) ◽  
pp. 3772-3778 ◽  
Author(s):  
André Larochelle ◽  
Allen Krouse ◽  
Mark Metzger ◽  
Donald Orlic ◽  
Robert E. Donahue ◽  
...  
Keyword(s):  
Stem Cells ◽  
Stem Cell ◽  
Hematopoietic Stem Cells ◽  
Genetic Manipulation ◽  
Retroviral Vectors ◽  
Lymphoid Cells ◽  
Alternative Source ◽  
Hematopoietic Stem ◽  
Cd34 Cells

AMD3100, a bicyclam antagonist of the chemokine receptor CXCR4, has been shown to induce rapid mobilization of CD34+ hematopoietic cells in mice, dogs, and humans, offering an alternative to G-CSF mobilization of peripheral-blood hematopoietic stem cells. In this study, AMD3100-mobilized CD34+ cells were phenotypically analyzed, marked with NeoR-containing retroviral vectors, and subsequently transplanted into myeloablated rhesus macaques. We show engraftment of transduced AMD3100-mobilized CD34+ cells with NeoR gene marked myeloid and lymphoid cells up to 32 months after transplantation, demonstrating the ability of AMD3100 to mobilize true long-term repopulating hematopoietic stem cells. More AMD3100-mobilized CD34+ cells are in the G1 phase of the cell cycle and more cells express CXCR4 and VLA-4 compared with G-CSF-mobilized CD34+ cells. In vivo gene marking levels obtained with AMD3100-mobilized CD34+ cells were better than those obtained using CD34+ cells mobilized with G-CSF alone. Overall, these results indicate that AMD3100 mobilizes a population of hematopoietic stem cells with intrinsic characteristics different from those of hematopoietic stem cells mobilized with G-CSF, suggesting fundamental differences in the mechanism of AMD3100-mediated and G-CSF-mediated hematopoietic stem cell mobilization. Thus, AMD3100-mobilized CD34+ cells represent an alternative source of hematopoietic stem cells for clinical stem cell transplantation and genetic manipulation with integrating retroviral vectors.

Durable and Robust Fetal Globin Induction without Anemia in Rhesus Monkeys Following Autologous Hematopoietic Stem Cell Transplant with BCL11A Erythroid Enhancer Editing

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 4632-4632 ◽  
Author(s):  
Selami Demirci ◽  
Jing Zeng ◽  
Yuxuan Wu ◽  
Naoya Uchida ◽  
Jackson Gamer ◽  
...  
Keyword(s):  
Stem Cell ◽  
Hematopoietic Stem Cell ◽  
Mononuclear Cells ◽  
Hematologic Toxicity ◽  
Cell Transplant ◽  
Post Transplantation ◽  
Hematopoietic Stem ◽  
Significant Difference ◽  
Hbf Induction

Elevated fetal hemoglobin (HbF, α2γ2) levels are clinically beneficial for patients with β-hemoglobinopathies. Editing of the erythroid-specific BCL11A enhancer induces HbF, inhibiting sickling and restoring globin chain balance in erythroid cells derived from hematopoietic stem and progenitor cells (HSPCs) from SCD and β-thalassemia patients respectively, without detectable genotoxicity or adverse effects on hematopoietic stem cell (HSC) function (Wu Y, Nat Med, 2019). Here, we sought to evaluate engraftment and HbF induction potential of erythroid-specific BCL11A enhancer edited CD34+ HSPCs in a non-human primate transplantation model in which hemoglobin switching is conserved. We targeted the erythroid-specific +58 DNAse I hypersensitive site of BCL11A, which has identical human and rhesus sequences at the spacer and protospacer adjacent motif (PAM) of the potent #1617 sgRNA. Ribonucleoprotein complex (RNP) composed of 3x-NLS SpCas9 protein and either BCL11A enhancer targeting (#1617) or AAVS1 targeting sgRNA was electroporated into rhesus CD34+ HSPCs (n=3). Following erythroid differentiation, substantial γ-globin expression (54-77%, p<0.01) was observed in BCL11A edited cells (81-85% indels) as compared to 19-25% and 15-24% for non-electroporated and AAVS1 edited cells, respectively, with no significant difference in red blood cell (RBC) enucleation efficiency (44-47%) among groups. We tested BCL11A enhancer editing with autologous HSC transplant in two cohorts, with two macaques per cohort. For cohort 1, we performed competitive engraftment of BCL11A enhancer and AAVS1 edited HSPCs to test long-term reconstitution. For cohort 2, we evaluated BCL11A enhancer editing alone to evaluate HbF induction and hematopoietic reconstitution. For each cohort, purified CD34+ HSPCs were electroporated with RNP one day after G-CSF and plerixafor mobilization and cultured for two days prior to cryopreservation. HSPCs were thawed and infused following 2×5 Gy total body irradiation. For cohort 1 (n=2, ZL25 and ZL22, 1.34-1.39×106 CD34+ HSPCs/kg), we observed reduced indel frequencies (8-41%) at early post-infusion time points compared to cell products (18-49%), suggesting indels in unfractionated HSPCs may overestimate those in engrafting cells and/or hematopoietic ablation was incomplete. From weeks 6 to 83, stable indel frequencies were detected in both BCL11A (~3-18%) and AAVS1 (~10-45%), suggesting no selective advantage for BCL11A enhancer edited, AAVS1 edited, or non-edited HSCs. For cohort 2 (BCL11A enhancer editing alone (n=2, ZM17 and ZM26, 1.78-6.06×106 CD34+ cells/kg), cell products showed improved editing with ~95% indels and ~65-78% γ-globin protein after in vitro erythroid culture. Animals engrafted with typical kinetics and displayed stable indel ratios up to 28 weeks post-transplantation. A significant correlation was detected between γ-globin level and indel frequency comparing all 4 transplanted animals and unedited controls (R2=0.76, p<0.01). In both edited and unedited animals γ-globin levels peaked in the first two months after transplantation and subsequently declined and plateaued. In ZM17 (~70% BCL11A enhancer indels at ~24 weeks), ~12% γ-globin was observed in peripheral blood (PB) at last measurement (compared to 0.5% γ-globin in RBC prior to transplant). In the same animal, editing ranged from 78-81% across all PB and bone marrow (BM) lineages (excluding CD3+ T-cells with 63% indels), including B-lymphoid, myeloid, erythroid, and HSPCs (in particular including 78% indels in CD71+ CD45- erythroblasts). Hemoglobin, hematocrit, and reticulocyte counts and peripheral smear appearance were all normal, suggesting no erythroid toxicity. Colony-forming ability of BM-derived mononuclear cells was similar in edited and control animals. In summary, we evaluated the clinical potential of autologous BCL11A erythroid enhancer editing in rhesus macaques. BCL11A enhancer edited HSCs can persist for at least 83 weeks post-transplant and provide therapeutic levels of HbF in peripheral RBCs without anemia or other apparent hematologic toxicity. Furthermore, these results emphasize input CD34+ HSPC dose and conditioning intensity as critical variables that influence gene editing following autologous HSCT. Overall, these findings support BCL11A erythroid enhancer genome editing as a promising strategy for therapeutic HbF induction. Disclosures Weiss: GlaxoSmithKline: Consultancy; Cellarity INC: Consultancy; Esperian: Consultancy; Beam Therapeutics: Consultancy; Rubius INC: Consultancy.

Role of Osteoclasts in Regulation of Human Hematopoietic Stem Cell Niche and Fate.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 4253-4253
Author(s):  
Shmuel Yaccoby ◽  
Kenichiro Yata ◽  
Yun Ge ◽  
Bart Barlogie ◽  
Joshua Epstein ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Cord Blood ◽  
Scid Mice ◽  
Common Mechanism ◽  
Hematopoietic Stem ◽  
Variable Expression ◽  
Cd34 Cells ◽  
Rabbit Bone

Abstract Recent studies indicate that osteoblasts play an important role in maintaining hematopoietic stem cells (HSCs) niche in the bone marrow microenvironment. The aim of study was to test the effect of osteoclasts on the fate of HSCs in a long term co-culture assay. To generate osteoclasts, peripheral blood mononuclear cells from mobilized donors were cultured for 6–10 days in αMEM media supplemented with 10% FCS, M-CSF and RANKL. After removal of non-adherent cells, the cultures contained 95% multinucleated osteoclasts and their precursors. These osteoclasts expressed TRAP and formed resorption pits on bone slices (Yaccoby et al., Cancer Res., 2004). CD34+ cells were purified from donor PBSCs and cord blood using immunomagnetic beads separation (&gt;95% purity). Adult and cord blood HSCs were co-cultured with osteoclasts for up to 3 and 10 months, respectively, in media lacking any cytokines. Because osteoclasts do not survive long without M-CSF and RANKL, the HSCs were transferred to fresh osteoclast cultures every 6–10 days. Unlike their tight adherence to stromal cells, HSCs did not adhere to the osteoclasts and were easily recovered from co-cultures by gentle pipetting. Following 1 to 3 weeks of co-culture, committed HSCs rapidly differentiated into various hematopoietic cell lineage, followed by phagocytosis of terminal differentiated hematopoietic cells by the osteoclasts. The remaining HSCs were highly viable (&gt;90% by trypan blue exclusion) and gradually lost their CD34 expression, so that the cultures contained subpopulations of HSCs expressing CD34−/lowCD38+ and CD34−/lowCD38−. Quantitive real time RT-PCR (qRT-PCR) revealed loss of expression of CD34 and reduced expression of CD45 by HSCs co-cultured with osteoclasts longer than 6 weeks. Variable expression of CD34 on HSCs was previously reported in murine but not human HSCs (Tajima et al., Blood, 2001). The co-cultured HSCs showed reduced capacity of generating in vitro hematopoietic colonies, and did not differentiate into osteoclasts upon stimulation with M-CSF and RANKL. We next tested the long term engraftment of these co-cultured HSCs in 2 animal models. In the first model, cord blood and adult HSCs from 2 donors recovered after &gt;6 weeks in co-culture were injected I.V. into irradiated NOD/SCID mice. In the second novel model, co-cultured cord blood and adult HSCs from 2 donors were injected directly into rabbit bones implanted subcutaneously in SCID mice (SCID-rab model), 6–8 weeks after rabbit bone implantation. After 2–4 months, 10%±3% human CD45-expressing cells were identified in the NOD/SCID mice femora and 8%±4% in the SCID-rab mice rabbit bone. Our study suggests that osteoclasts promote rapid differentiation of committed HSCs and induce conversion of CD34+ cells to CD34− stem cells with self renewal potential. Intriguingly, long term co-culture of primary CD138-selected myeloma plasma cells (n=16) with osteoclasts resulted in dedifferentiation of tumor cells from a mature CD45− phenotype to an immature, CD45-expressing cells, suggesting a common mechanism of osteoclast-induced HSC and myeloma cell plasticity. This indicates that osteoclasts are important bone marrow component regulating human HSC niche, plasticity and fate.

Hematopoietic Origin of Fibroblasts and Their Precursors.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 672-672 ◽  
Author(s):  
Yasuhiro Ebihara ◽  
Masahiro Masuya ◽  
Russell Owens ◽  
Su Yang ◽  
Richard P. Visconti ◽  
...  
Keyword(s):  
Stem Cells ◽  
Mononuclear Cells ◽  
Fluorescent Protein ◽  
Mesangial Cells ◽  
Pcr Analysis ◽  
Glomerular Mesangial Cells ◽  
General Belief ◽  
Hematopoietic Stem ◽  
Steel Factor ◽  
Cd34 Cells

Abstract Recent studies indicate that hematopoietic stem cells (HSCs) are capable of reconstituting a number of non-hematopoietic organs and tissues. To define the potential of the HSC precisely, we initiated transplantation studies of a clonal population of cells derived from a single HSC. We used the bone marrow (BM) of transgenic enhanced green fluorescent protein (EGFP) mice as the source of donor HSCs. We demonstrated the HSC origin of glomerular mesangial cells (Masuya et al, Blood 101: 2215, 2003) and brain microglial cells and pericyte-like perivascular cells (Hess et al, Exp Neurol 186: 134, 2004). These observations and the fact that glomerular mesangial cells and pericytes are considered myofibroblasts suggested that fibroblasts are also derived from HSCs. In this abstract, we present evidence for HSC origin of fibroblasts and their precursors, BM fibroblast colony-forming units (CFU-F) and peripheral blood (PB) fibrocytes. Lin−Sca-1+c-Kit+CD34− cells from the BM of adult EGFP mice were individually sorted into 96 well Corning plates and cultured for 7 days in the presence of Steel factor and IL-11 or Steel factor and G-CSF. Viable clones consisting of fewer than 20 cells were individually transplanted into lethally irradiated mice. EGFP+ mononuclear cells were sorted from the BM cells of recipients showing high-level, multilineage hematopoietic reconstitution and assayed for CFU-F in Retronectin-treated tissue culture plates. Colonies consisting of more than 50 adherent cells were all EGFP+. The majority of the cells comprising the colonies were fibroblast-like, exhibited spindle-shaped or polygonal cytoplasm and had clear, ovoid nuclei. Flow cytometric analyses revealed that these cells expressed collagen-1 and discoidin domain receptor 2 (DDR2) and exhibited a decreased intensity of CD45. RT-PCR analysis of these cells revealed the presence of mRNA for procollagen 1 alpha-1, vimentin, fibronectin and DDR2. Next we analyzed the PB for donor origin fibrocytes, a fibroblast-like cell type that expresses both fibroblastic and hematopoietic phenotypes in culture. When nucleated PB cells from clonally engrafted mice were cultured on fibronectin-coated dishes, proliferation of EGFP+ fibroblast-like cells was detected. Only one-third of the EGFP+ cells expressed CD45 and most of the EGFP+ cells expressed both collagen-I and DDR2. Similar results were obtained with EGFP+ cells from mice transplanted with 100 uncultured Lin−Sca-1+c-Kit+CD34− cells or 1x106 BM nucleated cells. These studies excluded the effects of short-term culture on HSC differentiation and established the HSC origin of CFU-F and fibrocytes. Classic studies of CFU-F by Friedenstein and others have led to the general belief that mesenchymal stem cells (MSCs), rather than HSCs, generate a number of tissues including adipocytes, osteoblasts, chondrocytes, myocytes and vascular endothelial cells. Here we unequivocally demonstrated that CFU-F are of HSC origin, warranting a re-evaluation of the relationship between HSCs and MSCs.

Use of Chromatin Modifying Agents for Ex Vivo Expansion of Human Umbilical Cord Blood Stem Cells.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 4208-4208
Author(s):  
Hiroto Araki ◽  
Nadim Mahmud ◽  
Mohammed Milhem ◽  
Mingjiang Xu ◽  
Ronald Hoffman
Keyword(s):  
Stem Cells ◽  
Cord Blood ◽  
Functional Properties ◽  
Ex Vivo ◽  
Fold Increase ◽  
Scid Mice ◽  
Human Umbilical Cord ◽  
Self Renewal ◽  
Hematopoietic Stem ◽  
Cd34 Cells

Abstract The fixed number of hematopoietic stem cells (HSCs) within a single cord blood (CB) unit has limited the use of CB grafts for allogeneic transplantation in adults. Efforts to promote self-renewal and expansion of HSCs have been met with limited success. Using presently available ex-vivo culture techniques HSCs lose their functional properties in proportion to the number of cellular divisions they have undergone. We hypothesized that chromatin modifying agents, 5-aza-2′-deoxycytidine (5azaD) and histone deacetylase inhibitor, trichostatin A (TSA) could reactivate pivotal genes required for retaining the functional properties of dividing HSC. We have demonstrated previously that the fate of human bone marrow CD34+ cells could be altered by the addition of 5azaD/TSA (Milhem et al. Blood.2004;103:4102). In our current studies we hypothesized that in vitro exposure of CB CD34+ cells to chromatin modifying agents might lead to optimal HSC expansion to permit transplantation of adults. A 12.5-fold expansion was observed in the 5azaD/TSA treated CD34+CD90+ cell cultures containing SCF, thrombopoietin and FLT3 ligand (cytokines) in comparison to the input cell number. Despite 9 days of culture, 35.4% ± 5.8% (n = 10) of the total cells in the cultures exposed to chromatin modifying agents were CD34+CD90+ as compared to 1.40 % ± 0.32% in the culture containing cytokines alone. The 12.5-fold expansion of CD34+CD90+ cells was associated with a 9.8-fold increase in the numbers of CFU-mix and 11.5-fold expansion of cobblestone area-forming cells (CAFC). The frequency of SCID repopulating cells (SRC) was 1 in 26,537 in primary CB CD34+CD90+ cells but was increased to 1 in 2,745 CD34+CD90+ cells following 9 days of culture in the presence of 5azaD/TSA resulting in a 9.6-fold expansion of the absolute number of SRC. In contrast, the cultures lacking 5azaD/TSA had a net loss of both CFC/CAFC as well as SRC. The expansion of cells maintaining CD34+CD90+ phenotype was not due to the retention of a quiescent population of cells since all of the CD34+CD90+ cells in the culture had undergone cellular division as demonstrated by labeling with a cytoplasmic dye. CD34+CD90+ cells that had undergone 5–10 cellular divisions in the presence of 5azaD/TSA but not in the absence still retained the ability to repopulate NOD/SCID mice. 5azaD/TSA treated CD34+CD90+ cells, but not CD34+CD90- cells were responsible for in vivo hematopoietic repopulation of NOD/SCID assay, suggesting a strong association between CD34+CD90+ phenotype and their ability to repopulate NOD/SCID mice. We next assessed the effect of 5azaD/TSA treatment on the expression of HOXB4, a transcription factor which has been implicated in HSC self-renewal. A significantly higher level of HOXB4 protein was detected by western blot analysis after 9 days of culture in the cells treated with 5azaD/TSA as compared to cells exposed to cytokines alone. The almost 10-fold increase in SRC achieved using the chromatin modifying agents should be sufficient to increase the numbers of engraftable HSC within a single human CB unit so as to permit these expanded grafts to be routinely used for transplanting adult recipients.

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