Decellularized Wharton jelly matrix: a biomimetic scaffold for ex vivo hematopoietic stem cell culture

Abstract Hematopoietic stem progenitor cells (HSPCs) reside in the bone marrow (BM) hematopoietic “niche,” a special 3-dimensional (3D) microenvironment that regulates HSPC self-renewal and multipotency. In this study, we evaluated a novel 3D in vitro culture system that uses components of the BM hematopoietic niche to expand umbilical cord blood (UCB) CD34+ cells. We developed this model using decellularized Wharton jelly matrix (DWJM) as an extracellular matrix (ECM) scaffold and human BM mesenchymal stromal cells (MSCs) as supporting niche cells. To assess the efficacy of this model in expanding CD34+ cells, we analyzed UCB CD34+ cells, following culture in DWJM, for proliferation, viability, self-renewal, multilineage differentiation, and transmigration capability. We found that DWJM significantly expanded UCB HSPC subset. It promoted UCB CD34+ cell quiescence, while maintaining their viability, differentiation potential with megakaryocytic differentiation bias, and clonogenic capacity. DWJM induced an increase in the frequency of c-kit+ cells, a population with enhanced self-renewal ability, and in CXCR4 expression in CD34+ cells, which enhanced their transmigration capability. The presence of BM MSCs in DWJM, however, impaired UCB CD34+ cell transmigration and suppressed CXCR4 expression. Transcriptome analysis indicated that DWJM upregulates a set of genes that are specifically involved in megakaryocytic differentiation, cell mobility, and BM homing. Collectively, our results indicate that the DWJM-based 3D culture system is a novel in vitro model that supports the proliferation of UCB CD34+ cells with enhanced transmigration potential, while maintaining their differentiation potential. Our findings shed light on the interplay between DWJM and BM MSCs in supporting the ex vivo culture of human UCB CD34+ cells for use in clinical transplantation.

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Maintaining the self-renewal and differentiation potential of human CD34+ hematopoietic cells using a single genetic element

Blood ◽

10.1182/blood-2003-05-1762 ◽

2003 ◽

Vol 102 (13) ◽

pp. 4369-4376 ◽

Cited By ~ 87

Author(s):

James C. Mulloy ◽

Jorg Cammenga ◽

Francisco J. Berguido ◽

Kaida Wu ◽

Ping Zhou ◽

...

Keyword(s):

Progenitor Cells ◽

Ex Vivo ◽

Severe Combined Immunodeficiency ◽

Hematopoietic Cells ◽

The Self ◽

Differentiation Potential ◽

Self Renewal ◽

Hematopoietic Stem ◽

Human Cd34

AbstractHematopoiesis is a complex process involving hematopoietic stem cell (HSC) self-renewal and lineage commitment decisions that must continue throughout life. Establishing a reproducible technique that allows for the long-term ex vivo expansion of human HSCs and maintains self-renewal and multipotential differentiation will allow us to better understand these processes, and we report the ability of the leukemia-associated AML1-ETO fusion protein to establish such a system. AML1-ETO-transduced human CD34+ hematopoietic cells routinely proliferate in liquid culture for more than 7 months, remain cytokine dependent for survival and proliferation, and demonstrate self-renewal of immature cells that retain both lymphoid and myeloid potential in vitro. These cells continue to express the CD34 cell surface marker and have ongoing telomerase activity with maintenance of telomere ends, however they do not cause leukemia in nonobese diabetic-severe combined immunodeficiency (NOD/SCID) mice. Identification of the signaling pathways that are modulated by AML1-ETO and lead to the self-renewal of immature human progenitor cells may assist in identifying compounds that can efficiently expand human stem and progenitor cells ex vivo. (Blood. 2003; 102:4369-4376)

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A Possible Role for Wnt3a in Enhancing StemRegenin1-Mediated Expansion of Hematopoietic Stem Cells

Blood ◽

10.1182/blood.v120.21.4130.4130 ◽

2012 ◽

Vol 120 (21) ◽

pp. 4130-4130

Author(s):

Lucia Duinhouwer ◽

Elwin Rombouts ◽

Nesrin Tüysüz ◽

Jan Spanholtz ◽

Derk ten Berge ◽

...

Keyword(s):

Stem Cells ◽

Hematopoietic Stem Cells ◽

Cell Biology ◽

Ex Vivo ◽

Synergistic Effects ◽

Fold Increase ◽

Differentiation Potential ◽

Self Renewal ◽

Hematopoietic Stem ◽

Cd34 Cells

Abstract Abstract 4130 Insufficient engraftment after umbilical cord blood stem cell transplantation (UCB-SCT) may be improved by the administration of ex vivo expanded UCB-derived hematopoietic stem cells (HSC). Although culturing HSC with cytokines such as SCF, Flt3L and TPO results in robust proliferation, it is accompanied with extensive differentiation and loss of self renewal capacity. Inhibition of the Aryl hydrocarbon Receptor (AhR) leads to potent expansion of hematopoietic progenitor cells (HPC) (Boitano et al. Science 2010). Reportedly, Wnt3a inhibits differentiation in different types of stem cells including hematopoietic stem cells(Reya et al. Nature 2003, Ten Berge et al. Nature Cell Biology 2011). Here, we evaluated possible additive or synergistic effects of combining the AhR antagonist StemRegenin1 (SR1) with recombinant purified Wnt3a to expand hematopoietic stem cells ex vivo. UCB-derived CD34-selected cells were cultured in serum-free Glycostem Basal Growth Medium (GBGM) supplemented with the early-acting growth factors SCF, Flt3L and TPO (SFT medium) with or without the addition of SR1 and Wnt3a. Cell number, viability and subset composition within the CD34+ cells were measured using flowcytometry. The multilineage differentiation potential and self renewal capacity of expanded CD34+HPC were evaluated in stroma-supported long-term culture-initiating cell (LTC-IC) assays. Culturing CD34+ cells in SFT medium resulted in a mean 10.2-fold increase in CD34+ cells after 1 week of culture (n=3). Addition of SR1 to the SFT-medium resulted in a 16-fold increase of CD34+ input cells within 7 days, while on the other hand, addition of Wnt3a alone to the SFT-medium resulted in a 7-fold increase in CD34+ cells. However, combining SR1 and Wnt3a in the SFT medium resulted in a 20-fold expansion of CD34+ cells compared to input. The early additive effect of Wnt3a on SFT+SR1-induced expansion of CD34+ cells, however, disappeared upon prolonged culture up to 2–3 weeks. Approximately 3–10% of UCB-derived CD34+ cells could be characterized as phenotypic HSC, as was defined by CD34+CD38lowCD45RAlowCD90+ cells. After culture, we sorted different CD34+-populations to evaluate their functional capacity. Evaluation of LTC-IC frequencies yielded a frequency of 1/23 LTC-IC in the phenotypic HSC-subset (CD34+CD38lowCD45RAlowCD90+) after 28 days of culture in SFT medium with SR1. However, no LTC-IC appeared to be present in the multipotent progenitor subset (MPP, CD34+CD38lowCD45RAlowCD90low). These results indicate that phenotypic HSC maintain their functional LTC-IC capacity after expansion culture. Collectively, our results confirm that SR1 expands HSC with preservation of self-renewal capacity and ability to differentiate into various hematopoietic lineages. In addition, we show that Wnt3a initially enhances SFT+SR1-driven expansion of CD34+ HPC, but reduces the increase in number of CD34+ cells at later stages of culture. These data may suggest that the period of expansion needed for clinical application may be shortened by combining SR1 and Wnt3a. Disclosures: Spanholtz: Glycostem Therapeutics: Employment. Groenewegen:Glycostem Therapeutics: Equity Ownership.

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Long-Term Ex Vivo Maintenance and Expansion of Transplantable Human Hematopoietic Stem Cells

Blood ◽

10.1182/blood.v94.5.1623 ◽

1999 ◽

Vol 94 (5) ◽

pp. 1623-1636 ◽

Cited By ~ 72

Author(s):

Chu-Chih Shih ◽

Mickey C.-T. Hu ◽

Jun Hu ◽

Jeffrey Medeiros ◽

Stephen J. Forman

Keyword(s):

Stem Cells ◽

Hematopoietic Stem Cells ◽

In Vitro Culture ◽

Culture System ◽

Ex Vivo ◽

Human Stem Cells ◽

Hematopoietic Stem ◽

In Vitro Culture System

Abstract We have developed a stromal-based in vitro culture system that facilitates ex vivo expansion of transplantable CD34+thy-1+ cells using long-term hematopoietic reconstitution in severe combined immunodeficient-human (SCID-hu) mice as an in vivo assay for transplantable human hematopoietic stem cells (HSCs). The addition of leukemia inhibitory factor (LIF) to purified CD34+ thy-1+ cells on AC6.21 stroma, a murine bone marrow–derived stromal cell line, caused expansion of cells with CD34+ thy-1+ phenotype. Addition of other cytokines, including interleukin-3 (IL-3), IL-6, granulocyte-macrophage colony-stimulating factor, and stem cell factor, to LIF in the cultures caused a 150-fold expansion of cells retaining the CD34+ thy-1+ phenotype. The ex vivo–expanded CD34+ thy-1+ cells gave rise to multilineage differentiation, including myeloid, T, and B cells, when transplanted into SCID-hu mice. Both murine LIF (cannot bind to human LIF receptor) and human LIF caused expansion of human CD34+ thy-1+ cells in vitro, suggesting action through the murine stroma. Furthermore, another human HSC candidate, CD34+ CD38− cells, shows a similar pattern of proliferative response. This suggests thatex vivo expansion of transplantable human stem cells under this in vitro culture system is a general phenomenon and not just specific for CD34+ thy-1+ cells.

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Interleukin-3 supports expansion of long-term multilineage repopulating activity after multiple stem cell divisions in vitro

Blood ◽

10.1182/blood.v96.5.1748 ◽

2000 ◽

Vol 96 (5) ◽

pp. 1748-1755 ◽

Cited By ~ 90

Author(s):

David Bryder ◽

Sten E. W. Jacobsen

Keyword(s):

Bone Marrow Cells ◽

Ex Vivo ◽

Calf Serum ◽

Self Renewal ◽

Hematopoietic Stem ◽

Cell Divisions ◽

Stem Cell Divisions

Abstract Although long-term repopulating hematopoietic stem cells (HSC) can self-renew and expand extensively in vivo, most efforts at expanding HSC in vitro have proved unsuccessful and have frequently resulted in compromised rather than improved HSC grafts. This has triggered the search for the optimal combination of cytokines for HSC expansion. Through such studies, c-kit ligand (KL), flt3 ligand (FL), thrombopoietin, and IL-11 have emerged as likely positive regulators of HSC self-renewal. In contrast, numerous studies have implicated a unique and potent negative regulatory role of IL-3, suggesting perhaps distinct regulation of HSC fate by different cytokines. However, the interpretations of these findings are complicated by the fact that different cytokines might target distinct subpopulations within the HSC compartment and by the lack of evidence for HSC undergoing self-renewal. Here, in the presence of KL+FL+megakaryocyte growth and development factor (MGDF), which recruits virtually all Lin−Sca-1+kit+ bone marrow cells into proliferation and promotes their self-renewal under serum-free conditions, IL-3 and IL-11 revealed an indistinguishable ability to further enhance proliferation. Surprisingly, and similar to IL-11, IL-3 supported KL+FL+MGDF-induced expansion of multilineage, long-term reconstituting activity in primary and secondary recipients. Furthermore, high-resolution cell division tracking demonstrated that all HSC underwent a minimum of 5 cell divisions, suggesting that long-term repopulating HSC are not compromised by IL-3 stimulation after multiple cell divisions. In striking contrast, the ex vivo expansion of murine HSC in fetal calf serum-containing medium resulted in extensive loss of reconstituting activity, an effect further facilitated by the presence of IL-3.

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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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Environmental Cues Can Promote Symmetrical Division of Functional Human Hematopoietic Stem Cells.

Blood ◽

10.1182/blood.v108.11.1397.1397 ◽

2006 ◽

Vol 108 (11) ◽

pp. 1397-1397

Author(s):

Nadim Mahmud ◽

Kazumi Yoshinaga ◽

Craig Beam ◽

Hiroto Araki

Keyword(s):

Stem Cells ◽

Cell Division ◽

Progenitor Cells ◽

Ex Vivo ◽

Absolute Number ◽

Self Renewal ◽

Hematopoietic Stem ◽

Differentiated Cells ◽

Cd34 Cells ◽

Cells Cultured

Abstract Widespread clinical use of ex-vivo expanded human umbilical cord blood (CB) grafts has been limited by lack of proper understanding of factors regulating self-renewal type of symmetric cell divisions. The expansion of the number of functional hematopoietic stem cells (HSC) ex-vivo requires the creation of an environment which favors symmetrical division. In our current studies, addition of late acting cytokines, (GM-CSF, IL-6, Epo) with early acting cytokines (thrombopoietin, SCF, Flt-3 ligand) resulted in loss of expansion of stem/progenitor cells. These data indicate that modification of HSC fate is not fully independent of external humoral influences. We have previously demonstrated that following treatment of CD34+ cells with 5-aza-2-deoxycytidine (5azaD) and trichostatin A (TSA) there is a 10- fold increase in the number of SCID mouse repopulating cells (SRC). This increase of SRC, however, occurred concomitantly with an increase in absolute number of CD34+CD90+ cells as well as primitive progenitors which gives rise to colony forming unit Mix lineage (CFU-Mix). We hypothesized that if the primary CD34+ cells generates CFU-Mix/CFU-GM in a ratio of ‘X’, then to observe a higher rate of symmetric cell division we would expect to see the ratio increased (>X) in the 5azaD/TSA treated cells in comparison to cells cultured in the absence of 5azaD/TSA (< X). Interestingly, analyses of our data suggest that when 5azaD/TSA treated CD34+ cells are cultured for 5 days and assayed for colonies we observed a significant increase in the ratio of CFU-Mix/CFU-GM in contrast to cells cultured in cytokines alone, 0.373 ± 0.06 and 0.066 ± 0.032 respectively. The ratio of CFU-Mix/CFU-GM of CB CD34+ cells (day 0) was 0.262 ± 0.045. These findings indicate that 5azaD/TSA treatment promotes the ratio of CFU-Mix/CFU-GM possibly by enhancing symmetric division of CFU-Mix while in the absence of 5azaD/TSA treatment the culture condition likely induces differentiation. In addition, we have also investigated the ratio of progenitor cells/differentiated cells by assessing the ratio of human CD34+ cells/CD33+ cells in the bone marrow of immunodeficient mice following transplantation (8 weeks) of equal numbers of CD34+ cells. The ratio of CD34+ cells/CD33+ cells following transplantation of 5azaD/TSA treated cells was 0.52 ± 0.14 (n = 11) while in the absence of 5azaD/TSA the ratio dropped to 0.31± 0.16 (n = 4). The ratio following transplantation of primary CD34+ (day 0) cells was 0.62 ± 0.14 (n = 6). These data suggest that 5azaD/TSA treated cells maintain the balance of generation of CD34+ cells/CD33+ cells at a comparable rate to that of primary CD34+ cells, while the CD34+ cells generated in the absence of 5azaD/TSA promotes generation of more differentiated cells. Alternatively, it is also possible that 5azaD/TSA treatment of CD34+ cells in the culture results in inhibition of myeloid differentiation at the cost of proliferation. However, the latter possibility is unlikely, since treatment of CB cells with 5azaD/TSA results in an increase in the absolute number of progenitors including SRC possessing both myeloid and lymphoid differentiation potential. Taken together, these data support our hypothesis that chromatin modifying agents in the culture is capable of promoting self-renewal type of symmetric cell division possessing in vivo multilineage marrow repopulating potential.

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Inability to Express HOXA Cluster and BCL11A Genes Compromises Self-Renewal and Multipotency of hESC-Derived Hematopoietic Cells

Blood ◽

10.1182/blood.v120.21.1190.1190 ◽

2012 ◽

Vol 120 (21) ◽

pp. 1190-1190 ◽

Cited By ~ 1

Author(s):

Diana R Dou ◽

Arazin Minasian ◽

Maria I Sierra ◽

Pamela Saarikoski ◽

Jian Xu ◽

...

Keyword(s):

Stem Cells ◽

B Cells ◽

Proliferative Potential ◽

Pluripotent Cells ◽

Hoxa Cluster ◽

Self Renewal ◽

Hematopoietic Stem ◽

Cd34 Cells ◽

Hoxa Genes

Abstract Abstract 1190 The inability to derive functional hematopoietic stem cells (HSCs) in vitro from pluripotent cells prevents widespread utilization of HSCs in the clinic; however, the molecular defects compromising the in vitro generated hematopoietic stem/progenitor cells (HSPCs) are unknown. Using a two-step differentiation method in which human embryonic stem cells (hESCs) were first differentiated into embryo bodies (EBs) and then CD34+ cells from hEBs were co-cultured on OP9M2 bone marrow mesenchymal stem cell (MSC) stroma (hEB-OP9), we were able to derive HSPCs expressing the HSC immunophenotype (CD34+CD38−CD90+CD45+) (hereafter termed CD90+HSPCs). Colony forming and stroma co-culture assays demonstrated that the hEB-OP9 CD90+HSPCs were able to differentiate into myelo-erythroid lineages and T-cells. However, when comparing CD90+HSPCs from hEB-OP9 to those from fetal liver (FL)—an in vivo source of HSCs—the former remained severely functionally limited in their proliferative potential and ability to differentiate into B-cells. To identify the basis of the proliferative and differentiation defects, we performed microarray analysis to define gene expression differences between CD90+HSPCs derived from hEB-OP9, FL, early 3–5 week placenta (PL) and an earlier stage of hESC differentiation (hEB). This analysis revealed establishment of the general hematopoietic transcription factor network (e.g. SCL, RUNX1, CMYB, ETV6, HOXB4, MYB), demonstrating the successful differentiation and identification of hematopoietic cells using our two-step culturing techniques and immunophenotype criteria. Moreover, evaluation of Spearman coefficients confirmed CD90+HSPCs isolated from hEB-OP9 culture were brought into closer resemblance of the hFL CD90+HSPCs as compared to to the developmentally immature hEB and hPL CD90+HSPCs. Encouragingly, hEB-OP9 CD90+HSPCs displayed downregulation of expression of genes related to hemogenic endothelium development associated with hEB and hPL while genes critical in HSPC function, including DNA repair and chromatin modification, were upregulated to levels comparable to hFL-HSPCs. However, a subgroup of FL HSPC genes could not be induced in hEB-OP9 HSPCs, including the HOXA cluster genes and BCL11A—implicated in HSC self-renewal and B-cell formation, respectively. Interestingly, absence of HOXA genes and BCL11A and poor proliferative potential were also observed in HSPCs from early placenta, suggesting these defects are not in vitro artifacts but instead reflect an inability of hEB-OP9 HSPCs to complete developmental maturation. To validate the necessity of HOXA genes and BCL11A in proliferation potential and multipotency, we next utilized shRNAs to target MLL—the upstream regulator of the HOXA cluster—, individual HOXA genes, or BCL11A in FL-HSPCs to test whether knockdown was sufficient to recapitulate the defects observed in hESC-derived HSPCs. Knockdown of HOXA7 resulted in the loss of CD34+ cells while HOXA9 shRNA-treated cells displayed a loss of more differentiated CD38hi cells. MLL knockdown depleted both CD38+ and CD34+ populations. BCL11A silencing resulted in the loss of B-cells. These studies identify HOXA genes and BCL11A as developmentally regulated genes essential for generating self-renewing, multipotent HSCs from pluripotent cells. Disclosures: No relevant conflicts of interest to declare.

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