Short Pulse of a Small Peptide Agonist of Stromal Cell-Derived Factor - 1 (SDF-1) Enhances the Engraftment of Expanded Human Cord Blood Hematopoietic Progenitor Cells in NOD/SCID Mice.

Abstract SDF-1 is the ligand to the chemokine receptor CXCR-4. A small synthetic peptide agonist of SDF-1 (CTCE-0214) has been shown to expand human cord blood hematopoietic stem and progenitor cells. In this study, we investigated whether a brief exposure of expanded cord blood hematopoietic cells to CTCE-0214 can improve engraftment of the cells into NOD/SCID mice. Published in vivo studies demonstrated that the administration of CTCE-0214 to transplanted NOD/SCID mice mobilized human colony forming cells (CFC) and enhanced human thrombopoiesis (Exp Hematol 32, 300, 2004). Our earlier study showed that CTCE-0214 added to single factors of thrombopoietin (TPO), stem cell factor (SCF), or Flt-3 ligand (F3L) synergistically increased the survival of enriched cord blood CD34+ cells (Blood 102, 960a, 2003). In this study, we further investigated the effects of CTCE-0214 on the ex vivo expansion of CD34+ cells to multi-lineage progenitors and the homing and engraftment capacity of expanded human progenitor cells after a short in vitro exposure to the peptide prior to infusion into NOD/SCID mice. Enriched CD34+ cells (MACS) derived from cord blood were cultured for 8 days in serum-free medium QBSF-60 containing TPO (50 ng/ml), SCF (50 ng/nl) and F3L (80 ng/ml) (TSF), with or without CTCE-0214 (0.01 ng/ml) (TSF+CTCE-0214) added at day 4. Progenitor cells expanded for 8 days in the absence of CTCE-0214 were pulsed with the peptide (100 ng/ml) for 4 hours (TSFpCTCE-0214). Results are summarized in Table. CTCE-0214 significantly (N=30, p≤0.05, paired t-test) increased the fold expansion of total nucleated cells (TNC), CD34+ cells, CD34+CD38- cells, CFU-GM, CFU-E, and CFU-MK (total CFC). Expanded progenitor cells (with and without CTCE-0214) were then infused into irradiated NOD/SCID mice. After 6 weeks, enhanced engraftments of human CD45+ cells (p≤0.05, N=21) were demonstrated in the bone marrow (BM) of mice that received cells cultured in TSF+CTCE-0214. Interestingly, a short pulse of cells expanded in TSF to CTCE-0214 for 4 hours also significantly increased the NOD/SCID engraftment (N=18), although no major changes to the in vitro read-out parameters were observed. The mechanism could be associated with the increased homing capacity of progenitor cells after pulsing with CTCE-0214. In conclusion, our results showed that CTCE-0214 enhances the proliferation of early progenitor cells in culture and exposure to the peptide can enhance the engraftment potential of expanded cells in NOD/SCID mice. The SDF-1 peptide agonist could be developed for application to hematopoietic stem cell transplantation and ex vivo expansion. NOD/SCID Engraftment of Expanded Cord Blood Stem Cells TSF TSF+CTCE-0214 TSFpCTCE-0214 *Fold expansion (mean±SE); **% human CD45+ cells in BM of mice TNC* 84.6±10.4 123.5±15.3 88.5±11.2 CD34+* 8.5±1.3 14.1±2.1 9.6±1.6 CD34+CD38−* 24.6±4.8 48.7±8.6 27.5±5.3 Total CFC* 46.9±6.5 87.9±10.7 50.6±6.4 NOD/SCID** 2.8±0.9 6.7±2.5 8.3±4.0

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Activation of OCT4 Enhances Ex Vivo Expansion of Phenotypically Defined and Functionally Engraftable Human Cord Blood Hematopoietic Stem and Progenitor Cells By Regulating HOXB4 Expression

Blood ◽

10.1182/blood.v124.21.4332.4332 ◽

2014 ◽

Vol 124 (21) ◽

pp. 4332-4332

Author(s):

Xinxin Huang ◽

Scott Cooper ◽

Hal E. Broxmeyer

Keyword(s):

Cord Blood ◽

Progenitor Cells ◽

Ex Vivo ◽

Fold Increase ◽

Lentiviral Vectors ◽

Ex Vivo Expansion ◽

Transcriptional Factor ◽

Human Cord Blood ◽

Hematopoietic Stem ◽

Cd34 Cells

Abstract Allogeneic hematopoietic cell transplantation (HCT) is well established as a clinical means to treat patients with hematologic disorders and cancer. Human cord blood (CB) is a viable source of hematopoietic stem cells (HSC) for transplantation. However, numbers of nucleated cells retrieved, as well as limited numbers of HSC/progenitor cells (HPC) present, during collection may be problematic for treatment of adult patients with single CB HCT. One means to address the problem of limiting numbers of HSC/HPC is to ex vivo expand these cells for potential clinical use. While progress has been made in this endeavor, there is still a clinically relevant need for additional means to ex vivo expansion of human HSC and HPC. OCT4, a transcriptional factor, plays an essential role in pluripotency and somatic cell reprogramming, however, the functions of OCT4 in HSC are largely unexplored. We hypothesized that OCT4 is involved in HSC function and expansion, and thus we first evaluated the effects of OAC1 (Oct4-activating compound 1) on ex vivo culture of CB CD34+ cells in the presence of a cocktail of cytokines (SCF, TPO and Flt3L) known to ex vivo expand human HSC. We found that CB CD34+ cells treated with OAC1 for 4 days showed a significant increase (2.8 fold increase, p<0.01) above that of cytokine cocktail in the numbers of rigorously defined HSC by phenotype (Lin-CD34+CD38-CD45RA-CD90+CD49f+) and in vivo repopulating capacity in both primary (3.1 fold increase, p<0.01) and secondary (1.9 fold increase, p<0.01) recipient NSG mice. OAC1 also significantly increased numbers of granulocyte/macrophage (CFU-GM, 2.7 fold increase, p<0.01), erythroid (BFU-E, 2.2 fold increase, p<0.01), and granulocyte, erythroid, macrophage, megakaryocyte (CFU-GEMM, 2.6 fold increase, p<0.01) progenitors above that of cytokine combinations as determined by colony assays. To further confirm the role of OCT4 in human HSC, we performed OCT4 overexpression in CB CD34+ cells using lentiviral vectors and found that overexpression of OCT4 also resulted in significant increase (2.6 fold increase, p<0.01) in the number of phenotypic HSC compared to control vectors. Together, our data indicate that activation of OCT4 by OAC1 or lentiviral vectors enhances ex vivo expansion of cytokine stimulated human CB HSC. HOXB4 is a homeobox transcriptional factor that appears to be an essential regulator of HSC self-renewal. Overexpression of HOXB4 results in high-level ex vivo HSC expansion. It is reported that OCT4 can bind to the promoter region of HOXB4 at the site of 2952 bp from the transcription start point. We hypothesized that activation of OCT4 might work through upregulation of HOXB4 expression to ex vivo expand HSC. We observed that the expression of HOXB4 was largely increased (2.3 fold increase, p<0.01) after culture of CB CD34+ cells with OAC1 compared to vehicle control. siRNA mediated inhibition of OCT4 resulted in the marked reduction of HOXB4 expression (p<0.01) in OAC1-treated cells indicating that OAC1 treatment lead to OCT4-mediated upregulation of HOXB4 expression in HSC. Consistently, siRNA-mediated knockdown of HOXB4 expression led to a significant reduction in the number of Lin-CD34+CD38-CD45RA-CD90+CD49f+ HSC in OAC1-treated cells (p<0.05), suggesting HOXB4 is essential for the generation of primitive HSC in OAC1-treated cells. Our study has identified the OCT4-HOXB4 axis in ex vivo expansion of human CB HSC and sheds light on the potential clinical application of using OAC1 treatment to enhance ex vivo expansion of cytokine stimulated human HSC. Disclosures Broxmeyer: CordUse: Membership on an entity's Board of Directors or advisory committees.

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Different Histone Deacetylase Inhibitors Affect Distinct Cellular Targets within the Hierarchy of Hematopoietic Stem / Progenitor Cells.

Blood ◽

10.1182/blood.v106.11.1721.1721 ◽

2005 ◽

Vol 106 (11) ◽

pp. 1721-1721

Author(s):

Hiroto Araki ◽

Ronald Hoffman ◽

Nadim Mahmud

Keyword(s):

Progenitor Cells ◽

Histone Deacetylase ◽

Hdac Inhibitor ◽

Histone Deacetylase Inhibitors ◽

Trichostatin A ◽

Scid Mice ◽

Human Cord Blood ◽

Hematopoietic Stem ◽

Cd34 Cells

Abstract Recently several laboratories have examined the in vitro effects of chromatin modifying agents on hematopoiesis. We have previously reported that the sequential addition of a hypomethylating agent, 5-aza-2′-deoxyctidine (5azaD) and a histone deacetylase (HDAC) inhibitor, trichostatin A (TSA) to cultures of human cord blood (CB) CD34+ cells containing SCF, thrombopoietin and FLT-3 ligand (cytokines) resulted in a 10-fold expansion of SCID repopulating cells (SRC) (Araki et al. Blood2004: 104:881a). Recently others have shown that another HDAC inhibitor, valproic acid (VA) resulted in an expansion of CB CD34+ cells and murine hematopoietic stem / progenitor cells (HSPC) (DeFelice et al. Cancer Res.2005:65:1505, Beg et al. Cancer Res.2005:65:2537). In our current studies we have compared the efficacy of VA, TSA or 5azaD as single agents or in combination to promote the expansion of CB HSPC in vitro. The frequency and fold expansion of colony forming cells (CFC), cobblestone area-forming cells (CAFC) as well as SRC generated from CB CD34+ cells after 9 days of culture were examined. The addition of cytokines alone result in a 1.5-fold expansion of CD34+CD90+ cells. By contrast the addition of cytokines with VA led to a 65-fold expansion of the numbers of CD34+CD90+ cells as compared to a 1.3-fold, 5.6-fold, 4.2-fold or 12.5-fold expansion of CD34+CD90+ cells in cultures receiving cytokines with 5azaD, TSA or 5azaD/VA or 5azaD/TSA respectively. In vitro biological assays (CFC, CAFC) were performed to determine the correlation between CD34+CD90+ cell expansion and function. Cultures receiving cytokines alone or cytokines with VA had the greatest degree of expansion of CFC (14.4 and 18.6-fold respectively). By contrast cultures receiving cytokines alone contained only 70% of the numbers of CAFC as did the primary CB CD34+ cells. Cultures receiving VA or 5azaD/TSA had the greatest degree of expansion of CAFC numbers (9.6-fold and 11.5-fold respectively). The marrow repopulating potential of these various expanded cell populations were then assayed by transplanting them into NOD/SCID mice. CD34+ cells from cultures receiving cytokines alone or cytokines with 5azaD/VA were devoid of human hematopoietic cell chimerism. By contrast, all NOD/SCID mice receiving grafts from cultures treated with cytokines with 5azaD/TSA had evidence of human multilineage hematopoietic engraftment (7.5% ± 3.7%). Cells from cultures treated with cytokines with VA are capable of engraftment in 2 out of 6 mice with a barely detectable level of human cell chimerism (0.11%, 0.14%). We then assessed using western blot analysis whether the chromatin modifying agents might alter HSC function by upregulating HOXB4 protein levels. HOXB4 protein was detectable in cells cultured in the presence of cytokines with VA, cytokines with 5azaD/VA, cytokines with 5azaD/TSA but only cells treated with cytokines with 5azaD/TSA contained readily assayable SRC. These studies suggest that treatment with different chromatin modifying agents are capable of altering the differentiation program of distinct populations of HSPC. Some treatments (VA, 5azaD/VA) primarily affect CFC and CAFC but not SRC. While 5azaD/TSA targets CAFC and SRC but not CFC. In addition, although HOXB4 may participate in HSC self-renewal, additional genes are likely altered following 5azaD/TSA treatment which are required for the maintenance of SRC potential.

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Mesenchymal Stem Cell-Derived Microvesicles Support Ex Vivo Expansion of Cord Blood-Derived CD34+Cells

Stem Cells International ◽

10.1155/2016/6493241 ◽

2016 ◽

Vol 2016 ◽

pp. 1-13 ◽

Cited By ~ 19

Author(s):

Hui Xie ◽

Li Sun ◽

Liming Zhang ◽

Teng Liu ◽

Li Chen ◽

...

Keyword(s):

Cord Blood ◽

Progenitor Cells ◽

Mononuclear Cells ◽

Ex Vivo ◽

Ex Vivo Expansion ◽

Hematopoietic Stem ◽

Promising Therapeutic Approach ◽

Cd34 Cells ◽

Stem And Progenitor Cells

Mesenchymal stem cells (MSCs) are known to support the characteristic properties of hematopoietic stem and progenitor cells (HSPCs) in the bone marrow hematopoietic microenvironment. MSCs are used in coculture systems as a feeder layer for the ex vivo expansion of umbilical cord blood (CB) to increase the relatively low number of HSPCs in CB. Findings increasingly suggest that MSC-derived microvesicles (MSC-MVs) play an important role in the biological functions of their parent cells. We speculate that MSC-MVs may recapitulate the hematopoiesis-supporting effects of their parent cells. In the current study, we found MSC-MVs containing microRNAs that are involved in the regulation of hematopoiesis. We also demonstrated that MSC-MVs could improve the expansion of CB-derived mononuclear cells and CD34+cells and generate a greater number of primitive progenitor cells in vitro. Additionally, when MSC-MVs were added to the CB-MSC coculture system, they could improve the hematopoiesis-supporting effects of MSCs. These findings highlight the role of MSC-MVs in the ex vivo expansion of CB, which may offer a promising therapeutic approach in CB transplantation.

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The BET inhibitor CPI203 promotes ex vivo expansion of cord blood long-term repopulating HSCs and megakaryocytes

Blood ◽

10.1182/blood.2020005357 ◽

2020 ◽

Vol 136 (21) ◽

pp. 2410-2415 ◽

Cited By ~ 1

Author(s):

Peng Hua ◽

Joanna Hester ◽

George Adigbli ◽

Rong Li ◽

Bethan Psaila ◽

...

Keyword(s):

Cord Blood ◽

Progenitor Cells ◽

Ex Vivo ◽

Ex Vivo Expansion ◽

Human Cord Blood ◽

Hematopoietic Stem ◽

Bet Inhibitor ◽

Functional Studies

Abstract Although cytokine-mediated expansion of human hematopoietic stem cells (HSCs) can result in high yields of hematopoietic progenitor cells, this generally occurs at the expense of reduced bone marrow HSC repopulating ability, thereby limiting potential therapeutic applications. Because bromodomain-containing proteins (BCPs) have been demonstrated to regulate mouse HSC self-renewal and stemness, we screened small molecules targeting various BCPs as potential agents for ex vivo expansion of human HSCs. Of 10 compounds tested, only the bromodomain and extra-terminal motif inhibitor CPI203 enhanced the expansion of human cord blood HSCs without losing cell viability in vitro. The expanded cells also demonstrated improved engraftment and repopulation in serial transplantation assays. Transcriptomic and functional studies showed that the expansion of long-term repopulating HSCs was accompanied by synchronized expansion and maturation of megakaryocytes consistent with CPI203-mediated reprogramming of cord blood hematopoietic stem and progenitor cells. This approach may therefore prove beneficial for ex vivo gene editing, for enhanced platelet production, and for the improved usage of cord blood for transplantation research and therapy.

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Ex vivo treatment of proliferating human cord blood stem cells with stroma-derived factor–1 enhances their ability to engraft NOD/SCID mice

Blood ◽

10.1182/blood.v99.9.3454 ◽

2002 ◽

Vol 99 (9) ◽

pp. 3454-3457 ◽

Cited By ~ 51

Author(s):

Hanno Glimm ◽

Patrick Tang ◽

Ian Clark-Lewis ◽

Christof von Kalle ◽

Connie Eaves

Keyword(s):

Stem Cells ◽

Cord Blood ◽

Transforming Growth Factor ◽

Ex Vivo ◽

Severe Combined Immunodeficiency ◽

Human Cord Blood ◽

Hematopoietic Stem ◽

Steel Factor ◽

Tgf Β1

Abstract Ex vivo proliferation of hematopoietic stem cells (HSCs) is important for cellular and gene therapy but is limited by the observation that HSCs do not engraft as they transit S/G2/M. Recently identified candidate inhibitors of human HSC cycling are transforming growth factor-β1(TGF-β1) and stroma-derived factor–1 (SDF-1). To determine the ability of these factors to alter the transplantability of human HSCs proliferating in vitro, lin− cord blood cells were first cultured for 96 hours in serum-free medium containing Flt3 ligand, Steel factor, interleukin-3, interleukin-6, and granulocyte colony-stimulating factor. These cells were then transferred to medium containing Steel factor and thrombopoietin with or without SDF-1 and/or TGF-β1 for 48 hours. Exposure to SDF-1 but not TGF-β1 significantly increased (> 2-fold) the recovery of HSCs able to repopulate nonobese diabetic/severe combined immunodeficiency mice. These results suggest new strategies for improving the engraftment activity of HSCs stimulated to proliferate ex vivo.

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Ex-Vivo Generation and Expansion of Both Lymphoid and Myeloid Lineages from Human Cord Blood (CB) HSC Using a Serum-Free Human Mesenchymal Stem Cell Based Culture System.

Blood ◽

10.1182/blood.v104.11.2888.2888 ◽

2004 ◽

Vol 104 (11) ◽

pp. 2888-2888

Author(s):

Ana Frias ◽

Christopher D. Porada ◽

Kirsten B. Crapnell ◽

Joaquim M.S. Cabral ◽

Esmail D. Zanjani ◽

...

Keyword(s):

Stem Cell ◽

Cord Blood ◽

Culture System ◽

Ex Vivo ◽

Cell Number ◽

Human Cord Blood ◽

Serum Free ◽

Gm Csf ◽

Cd34 Cells

Abstract The in vitro culture of a hematopoietic stem cell (HSC) graft with either media containing animal-derived components or a feeder layer with ill-defined pathogenic potential such as xenogeneic cell lines or cells modified by viral transformation poses risks that concern scientists and regulatory agencies. In the present studies, we avoided these risks by evaluating the ability of a human stromal-based serum free culture system (hu-ST) to support the ex-vivo expansion/maintenance of human CB HSC. CB CD34+ enriched cells were cultured in serum free medium in the presence of hu-ST with SCF, bFGF, LIF and Flt-3, and the cultures were analyzed for expansion, phenotype and clonogenic ability. We have previously reported the ability of this culture system to allow the successful expansion/maintenance of HSC along the myeloid pathway. In the present study, we investigated whether we could further develop this culture system to simultaneously expand myelopoiesis and lymphopoiesis in vitro. To this end, cord blood CD34+ cells were cultured for a total of 28 days and analyzed every 3 days for expansion and phenotype. There was a progressive increase in CD34 cell number with time in culture. The differentiative profile was primarily shifted towards the myeloid lineage with the presence of CD33, CD15, and CD14. However, a significant number of CD7+ cells were also generated. At week 2 of culture, we observed that 30% of the cells in the culture were CD7 positive. These CD7+CD2-CD3-CD5-CD56-CD16-CD34- cells were then sorted and either plated on top of new irradiated hu-ST layers in the presence of SCF, FLT-3, IL-7, IL-2, and IL-15, or cultured with IL-4, GM-CSF, and FLT-3 in the absence of stroma. Both of these cultures were maintained for an additional 2 weeks. In both sets of cultures, further expansion in the total cell number occurred with the time in culture, and by the end of the week 2, we observed that 25.3±4.18% of the cells had become CD56+ CD3-, a phenotype consistent with that of NK cells. Furthermore, cytotoxicity assays were performed and showed cytotoxic activity that increased in an E:T ratio-dependent fashion. 38.6% of the CD7+ cells grown in the presence of IL-4, GM-CSF, and FLT-3 became CD123+CD11c-, a phenotype characteristic of nonactivated dendritic cells, while 7.3–12.1% adopted an activitated dendritic cell phenotype CD83+CD1a+. In summary, we developed an in vitro culture system that reproducibly allows the effective ex vivo expansion of human cord blood HSCs while maintaining the capability of generating both myeloid and lymphoid hematopoiesis in vitro.

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Attenuation of Surface CXCR4 Down-Regulation in Human Cord Blood HSC during Ex Vivo Expansion Using the HUBEC Co-Culture System.

Blood ◽

10.1182/blood.v106.11.1068.1068 ◽

2005 ◽

Vol 106 (11) ◽

pp. 1068-1068

Author(s):

Naoko Takebe ◽

Thomas MacVittie ◽

Xiangfei Cheng ◽

Ann M. Farese ◽

Emily Welty ◽

...

Keyword(s):

Cord Blood ◽

Culture System ◽

Ex Vivo ◽

Ex Vivo Expansion ◽

Receptor Internalization ◽

Down Regulation ◽

Human Cord Blood ◽

Hematopoietic Stem ◽

Gm Csf

Abstract Down-modulation of surface CXCR4, a G-protein-coupled receptor, in hematopoietic stem cells (HSCs) undergoing ex vivo expansion culturing is considered to be one of the major causes of marrow reconstitution failure, possibly due to an HSC homing defect. Recently, it has been reported that severe combined immunodeficiency (SCID)-repopulating cells (SRC) were expanded from the CD34-enriched human adult bone marrow (ABM) or cord blood (CB) hematopoietic stem cells (HSC) using a human brain endothelial cell (HUBEC) co-culture system. We found that primitive cord blood cells expressing surface CXCR4 (82+5%) lost this capability significantly during 7 days of ex vivo expansion in the HUBEC co-culture containing the cytokines stem cell factor (SCF), flt-3, interleukin (IL)-6, IL-3, and granulocyte macrophage colony stimulating factor (GM-CSF). Expression levels of other surface proteins relevant to HSC homing, such as CD49d, CD95, CD26, or CD11a, were not down-modulated. We hypothesized that CXCR4 down-regulation was caused by a receptor internalization and tested several methods to reverse CXCR4 internalization back to the surface, such as elimination of GM-CSF in the culture media, performing a non-contact culture using the transwell, or adding either 0.3Mor 0.4M sucrose, or 25μg/ml chlorpromazine (CPZ), 24 hours prior to the analysis. CPZ and sucrose are known inhibitors of the cytokine-induced endocytosis of CXCR4 in neutrophils (Bruhl H. et al. Eur J Immunol 2003). Interestingly, 0.4M sucrose showed approximately a 2-fold increase of surface CXCR4 expression on CB CD34+ cells by flow cytometry analysis. CPZ and 0.3M sucrose showed a moderate increase expression of CXCR4. Using a transwell HUBEC co-culture system, CXCR4 surface expression on CD34+ cells was down-regulated during the ex vivo culture. In vitro HSC migration test showed 3.1-fold increase in migration compared to the control after incubation of HSC with 0.1M sucrose for 16 hours prior to the in vitro migration study. Eliminating GM-CSF from the cytokine cocktail or adding MG132 increased migration 1.36- and 1.2-fold compared to the control. We are currently performing an in vivo homing assay using nonobese diabetic (NOD)-SCID mice. In conclusion, the HUBEC ex vivo culture system down-regulates surface CXCR4 in human cord blood HSC. The mechanism of CXCR4 surface down regulation may be receptor internalization by cytokines. Sucrose may be useful in attenuation of CXCR4 surface expression in CD34+ HSC by inhibition of receptor internalization via clathrin-coated pits.

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

Blood ◽

10.1182/blood.v106.11.4208.4208 ◽

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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Ex-Vivo Expansion of Human Cord Blood CD34+ Cells by Overexpression of Bmi-1.

Blood ◽

10.1182/blood.v108.11.1329.1329 ◽

2006 ◽

Vol 108 (11) ◽

pp. 1329-1329

Author(s):

Aleksandra Rizo ◽

Edo Vellenga ◽

Gerald de Haan ◽

Jan Jacob Schuringa

Keyword(s):

Cord Blood ◽

Cell Expansion ◽

Cultured Cells ◽

Ex Vivo ◽

Human Cord Blood ◽

Self Renewal ◽

Hematopoietic Stem ◽

Cd34 Cells ◽

Cord Blood Cd34

Abstract Hematopoietic stem cells (HSCs) are able to self-renew and differentiate into cells of all hematopoietic lineages. Because of this unique property, they are used for HSC transplantations and could serve as a potential source of cells for future gene therapy. However, the difficulty to expand or even maintain HSCs ex vivo has been a major limitation for their clinical applications. Here, we report that overexpression of the Polycomb group gene Bmi-1 in human cord blood-derived HSCs can potentially overcome this limitation as stem/progenitor cells could be maintained in liquid culture conditions for over 16 weeks. In mouse studies, it has been reported that increased expression of Bmi-1 promotes HSC self-renewal, while loss-of-function analysis revealed that Bmi-1 is implicated in maintenance of the hematopoietic stem cells (HSC). In a clinically more relevant model, using human cord blood CD34+ cells, we have established a long-term ex-vivo expansion method by stable overexpression of the Bmi-1 gene. Bmi-1-transduced cells proliferated in liquid cultures supplemented with 20% serum, SCF, TPO, Flt3 ligand, IL3 and IL6 for more than 4 months, with a cumulative cell expansion of more then 2×105-fold. The cells remained cytokine-dependent, while about 4% continued to express CD34 for over 20 weeks of culture. The cultured cells retained their progenitor activity throughout the long-term expansion protocol. The colony-forming units (CFUs) were present at a frequency of ~ 30 colonies per 10 000 cells 16 weeks after culture and consisted of CFU-GM, BFU-E and high numbers of CFU-GEMM type progenitors. After plating the transduced cells in co-cultures with the stromal cell line MS5, Bmi-1 cells showed a proliferative advantage as compared to control cells, with a cumulative cell expansion of 44,9 fold. The non-adherent cells from the co-cultures gave rise to higher numbers of colonies of all types (~70 colonies/10.000 cells) after 4 weeks of co-culture. The LTC-IC frequencies were 5-fold higher in the Bmi-1-transduced cells compared to control cells (1/361 v.s. 1/2077, respectively). Further studies will be focused on in-vivo transplantation of the long-term cultured cells in NOD/SCID mice to test their repopulating capacity. In conclusion, our data implicate Bmi-1 as an important modulator of human HSC self-renewal and suggest that it can be a potential target for therapeutic manipulation of human HSCs.

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Enforced Expression of GATA-2 Confers Quiescence on Human Hematopoietic Stem and Progenitor Cells In Vitro and In Vivo.

Blood ◽

10.1182/blood.v108.11.83.83 ◽

2006 ◽

Vol 108 (11) ◽

pp. 83-83

Author(s):

Alex J. Tipping ◽

Cristina Pina ◽

Anders Castor ◽

Ann Atzberger ◽

Dengli Hong ◽

...

Keyword(s):

Cell Cycle ◽

Progenitor Cells ◽

Zinc Finger ◽

Human Cord Blood ◽

Hematopoietic Stem ◽

Cd34 Cells ◽

Stem And Progenitor Cells ◽

Colony Number

Abstract Hematopoietic stem cells (HSCs) in adults are largely quiescent, periodically entering and exiting cell cycle to replenish the progenitor pool or to self-renew, without exhausting their number. Expression profiling of quiescent HSCs in our and other laboratories suggests that high expression of the zinc finger transcription factor GATA-2 correlates with quiescence. We show here that TGFβ1-induced quiescence of wild-type human cord blood CD34+ cells in vitro correlated with induction of endogenous GATA-2 expression. To directly test if GATA-2 has a causative role in HSC quiescence we constitutively expressed GATA-2 in human cord blood stem and progenitor cells using lentiviral vectors, and assessed the functional output from these cells. In both CD34+ and CD34+ CD38− populations, enforced GATA-2 expression conferred increased quiescence as assessed by Hoechst/Pyronin Y staining. CD34+ cells with enforced GATA-2 expression showed reductions in both colony number and size when assessed in multipotential CFC assays. In CFC assays conducted with more primitive CD34+ CD38− cells, colony number and size were also reduced, with myeloid and mixed colony number more reduced than erythroid colonies. Reduced CFC activity was not due to increased apoptosis, as judged by Annexin V staining of GATA-2-transduced CD34+ or CD34+ CD38− cells. To the contrary, in vitro cultures from GATA-2-transduced CD34+ CD38− cells showed increased protection from apoptosis. In vitro, proliferation of CD34+ CD38− cells was severely impaired by constitutive expression of GATA-2. Real-time PCR analysis showed no upregulation of classic cell cycle inhibitors such as p21, p57 or p16INK4A. However GATA-2 expression did cause repression of cyclin D3, EGR2, E2F4, ANGPT1 and C/EBPα. In stem cell assays, CD34+ CD38− cells constitutively expressing GATA-2 showed little or no LTC-IC activity. In xenografted NOD/SCID mice, transduced CD34+ CD38−cells expressing high levels of GATA-2 did not contribute to hematopoiesis, although cells expressing lower levels of GATA-2 did. This threshold effect is presumably due to DNA binding by GATA-2, as a zinc-finger deletion variant of GATA-2 shows contribution to hematopoiesis from cells irrespective of expression level. These NOD/SCID data suggest that levels of GATA-2 may play a part in the in vivo control of stem and progenitor cell proliferation. Taken together, our data demonstrate that GATA-2 enforces a transcriptional program on stem and progenitor cells which suppresses their responses to proliferative stimuli with the result that they remain quiescent in vitro and in vivo.

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