Small Molecule Screens for Stem Cell Expansion

Abstract SCI-42 The identification of safe and effective methods to expand human hematopoietic stem cells (HSC) would have a major impact on the use of HSC in clinical medicine. Several features of human HSC, including the lack of a suitable cell line model and cumbersome methods for quantification, have made the identification of conditions for human HSC expansion challenging. Current culture methods using cytokine cocktails in serum-free media support the robust proliferation of CD34 positive (CD34+) cells but this is accompanied by rapid differentiation such that after 1 week of culture fewer than 20% of cells continue to express CD34. To overcome these limitations we developed a high throughput screen that uses primary human CD34+ cells and multiparameter flow cytometry to identify compounds capable of expanding human CD34 positive cells. By screening >100,000 LMW compounds we identified a molecule (SR1) that enhanced CD34 expression during ex vivo culture. Culture of CD34+ cells with cytokines and SR1 for 3 weeks leads to a >600-fold increase in the number of CD34+ cells, and a >2000-fold increase in the number of CFU compared to starting cell numbers. Importantly, cells expanded in the presence of SR1contain a 17-fold increase in the number of NOD-SCID repopulating cells compared to starting cell numbers. Mechanistic studies reveal that SR1 binds to and antagonizes the aryl hydrocarbon receptor (AHR). Knockdown of the AHR in CD34+ cells using lentiviral transduction also maintains CD34 expression. These findings suggest that AHR normally promotes HSC differentiation during ex vivo culture and that AHR antagonists can be used to promote CD34 cell expansion. To determine the clinical utility of these findings, we have begun to explore the use of SR1 to expand CD34+ cells isolated from umbilical cord blood for clinical transplantation. To this end, we have developed a GMP compatible process to manufacture CD34 positive cells expanded with SR1 for use in cord blood transplantation. In addition, we have also explored the use of SR1 to prevent HSC differentiation during HSC transduction and enable manufacturing of differentiated blood cells. These data reveal AHR antagonism and SR1 treatment as a promising method to promote HSC expansion for clinical use. Disclosures: Cooke: Novartis: Employment. Boitano:Novartis: Employment.

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SALL4 Is a Key Factor in HDAC Inhibitor Mediated Ex Vivo Expansion of Human Peripheral Blood Mobilized Stem/Progenitor CD34+CD90+ Cells

Blood ◽

10.1182/blood.v124.21.1566.1566 ◽

2014 ◽

Vol 124 (21) ◽

pp. 1566-1566 ◽

Cited By ~ 3

Author(s):

Hiro Tatetsu ◽

Fei Wang ◽

Chong Gao ◽

Shikiko Ueno ◽

Xi Tian ◽

...

Keyword(s):

Cord Blood ◽

Peripheral Blood ◽

Cell Expansion ◽

Ex Vivo ◽

Ex Vivo Expansion ◽

Self Renewal ◽

Hematopoietic Stem ◽

Cell Numbers ◽

Cd34 Cells

Abstract Hematopoietic stem cells (HSCs) possess the unique capacity to self-renew and give rise to all types of mature cells within the blood and immune systems. Despite our progress in understanding the molecular factors that support the self-renewal and differentiation of the hematopoietic system in vivo, less is known on how to modulate the factors that govern the self-renewal of hematopoietic stem/progenitor cells (HSPCs) ex vivo. Unlike in the case of embryonic stem (ES) cells, expansion of CD34+ HSPC in culture in general is at the expense of loss of “stemness”. HSPCs can be collected from cord blood (CB), mobilized peripheral blood (PBSC), and rarely bone marrow (BM) at the present practice. Due to the limited CD34+ cell number in one single cord blood unit, much of the current efforts on developing technology of ex vivo expansion of HSPC uses cord blood as a source and is clinically applied to cord blood HSPC transplants. However, there are growing needs for expanding PBSCs for transplant-related practices such as HSPC expansion from poor autologous mobilizations, gene therapy or genome-editing via TALENs or CRISPR/Cas9. Developing a technology that would allow HSPC ex vivo expansion from both CB and PBSC sources is a key step towards this goal. Several groups have reported that ex vivo culture of CB CD34+ cells with HDAC inhibitors (HDACi) can lead to expansion of a CD34+CD90+ population, which is responsible for enhanced marrow-repopulating potential. In this study, we ask whether HDACi can have a similar effect on PBSC CD34+ cells. Furthermore, we have explored the mechanism(s) mediated by HDACi in CD34+CD90+ cell expansion. First we assessed a panel of HDACi to identify the most potent molecule for CD34+CD90+ cell expansion and selected trichostatin A (TSA) for future study. Next, TSA was added to the cytokines (SCF, Flt3 ligand, IL-3 and IL-6) to further characterize its potential in PBSC CD34+CD90+ cell expansion. We observed TSA treated CD34+ cultures with cytokines yielded 4.8 times greater numbers of CD34+CD90+ cells as compared to the cultures containing cytokines with DMSO solvent control. We next examined SCID repopulating ability (SRA) to evaluate the cultured CD34+CD90+ cells in vivo. We observed that mice transplanted with 3 million CD34+ cells treated with TSA had higher degree of human cell chimerism than those treated with DMSO at 8 weeks bone marrow and peripheral blood (32% vs 18%; p < 0.05), which was further confirmed by secondary transplantation. Furthermore, these cells were capable of differentiating into cells belonging to multiple hematopoietic lineages. To investigate the molecular mechanisms responsible for the expansion of functional HSCs and progenitors that were observed following TSA treatment, we analyzed the expression levels of several HSPC related genes, which were compared between CD34+ cells treated with TSA and DMSO. Significantly higher transcript levels were detected for GATA 2 (p < 0.05), HOXB4 (p < 0.05), HOXA9 (p < 0.05), and SALL4 (p < 0.05) by real time quantitative RT-PCR in TSA expanded cells as compared with controls. To evaluate whether these transcription factors can contribute to the expansion of CD34+CD90+ cells, GATA2, HOXB4 or SALL4 shRNAs were transfected into PBSC CD34+ cells, followed by culture with TSA. Among these transcription factors, knocking down SALL4 expression led to the most significant reduction of CD34+CD90+ cell numbers (33% of reduction). In addition, overexpression of SALL4 in PBSC CD34+ cells led to an increase of CD34+CD90+ cell numbers when compared to controls (p < 0.05). Overall, our study demonstrated a novel HDACi mediated ex vivo PBSC culture technology that leads to the expansion of CD34+CD90+ cells and an increase of the marrow repopulating potential of these cells. Both gain-of-function and loss-of-function studies support that SALL4 is a key transcription factor responsible for the process. Future study on the use of HDACi or other methods to increase SALL4 expression/function will be highly beneficial to ex vivo HSPC (CB and PBSC) expansion technology. Disclosures No relevant conflicts of interest to declare.

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Engraftment and Retroviral Marking of CD34+ and CD34+CD38− Human Hematopoietic Progenitors Assessed in Immune-Deficient Mice

Blood ◽

10.1182/blood.v91.4.1243 ◽

1998 ◽

Vol 91 (4) ◽

pp. 1243-1255 ◽

Cited By ~ 69

Author(s):

Mo A. Dao ◽

Ami J. Shah ◽

Gay M. Crooks ◽

Jan A. Nolta

Keyword(s):

Stem Cells ◽

Ex Vivo ◽

Human Stem Cells ◽

Hematopoietic Stem ◽

Human Cd34 ◽

Cd34 Cells ◽

Low Levels ◽

Ex Vivo Culture ◽

Daunting Challenge

Abstract Retroviral-mediated transduction of human hematopoietic stem cells to provide a lifelong supply of corrected progeny remains the most daunting challenge to the success of human gene therapy. The paucity of assays to examine transduction of pluripotent human stem cells hampers progress toward this goal. By using the beige/nude/xid (bnx)/hu immune-deficient mouse xenograft system, we compared the transduction and engraftment of human CD34+progenitors with that of a more primitive and quiescent subpopulation, the CD34+CD38− cells. Comparable extents of human engraftment and lineage development were obtained from 5 × 105 CD34+ cells and 2,000 CD34+CD38− cells. Retroviral marking of long-lived progenitors from the CD34+ populations was readily accomplished, but CD34+CD38− cells capable of reconstituting bnx mice were resistant to transduction. Extending the duration of transduction from 3 to 7 days resulted in low levels of transduction of CD34+CD38− cells. Flt3 ligand was required during the 7-day ex vivo culture to maintain the ability of the cells to sustain long-term engraftment and hematopoiesis in the mice.

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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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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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Self-Renewal of Hematopoietic Stem Cells by a Small Molecule

Blood ◽

10.1182/blood.v112.11.210.210 ◽

2008 ◽

Vol 112 (11) ◽

pp. 210-210

Author(s):

Anthony E Boitano ◽

Peter G Schultz ◽

Michael Cooke

Keyword(s):

Cord Blood ◽

Small Molecule ◽

High Throughput ◽

Peripheral Blood ◽

Ex Vivo ◽

Fold Increase ◽

Ex Vivo Expansion ◽

Self Renewal ◽

Hematopoietic Stem ◽

Cd34 Cells

Abstract Hematopoietic stem cell (HSC) transplantation has been effectively used to manage hematopoietic malignancies and immunodeficiency. Despite the successful use several challenges remain. For autologous transplants, HSCs are routinely isolated from the peripheral blood following mobilization with G-CSF, however many patients that have been treated with chemotherapy are refractory to mobilization. In the allogeneic transplant setting, treatment related toxicity including graft vs. host disease, delayed or failed engraftment, and lack of suitable HLA-matched donors represent major challenges. Umbilical cord blood (CB) cells have great potential as an alternative source of HSCs for individuals who lack a HLA-matched donor, but at present have limited utility because of low HSC numbers per graft leading to delayed recovery. Ex-vivo expansion of HSCs is an attractive strategy to optimize autologous and allogeneic transplantation as engraftment speed (absolute neutrophil count >500/μl) and success correlates positively with HSC dose. For this reason ex-vivo HSC expansion has been a subject of intense research for the past 20 years; however, identification of culture conditions that allow HSC expansion and long-term hematopoietic reconstitution have remained elusive. Recently, several groups have reported that signals other than hematopoietic growth factors, including ligands for G protein-coupled receptors and signaling molecules sensing neighboring cells such as notch may be required for optimal HSC expansion. Manipulation of signaling pathways using low molecular weight (LMW) compounds represents an alternative approach that can be exploited to regulate ex-vivo HSC expansion. To identify such compounds, we developed and performed an unbiased high-throughput screen for small molecules that regulate HSC self-renewal. The assay took advantage of advances in screening technology developed at GNF that permit low volume (10uL) screens to be conducted in massively parallel fashion using advanced automation and imaging technologies. These advances allow screens to be conducted on purified human CD34+ HSCs isolated from normal donors and circumvent a major limitation of the field- a lack of a suitable cell line model for human HSCs. From this screen we identified a small molecule (SR1) that regulates HSC self-renewal. Mobilized peripheral blood (mPB) CD34+ HSCs cultured with SR1 for 14 days had a ten-fold increase in the number of CD34+ cells compared to cultures without compound. The expansion of mPB CD34+ cells with SR1 for 1 week was associated with increased numbers of mixed (GM and GEMM) colony forming cells (CFC) and a 9-fold increase in the number of 4 week cobblestone area forming cells (CAFC). In the NOD-SCID repopulation assay, mPB CD34+ cells expanded with SR1 for 4 days displayed >2-fold higher levels of engraftment compared to control cultures and uncultured cells. These data suggest that SR1 promotes the net expansion of NOD-SCID repopulating cells. To explore the utility of SR1 for expansion of CB HSC, CD34+ cells were isolated from CB and cultured in the presence or absence of SR1 for up to five weeks. Remarkably, SR1 supported the sustained growth of CB HSCs with >100-fold increased numbers of CD34+ cells and CD34+CD133+CD38− cells compared to control cultures (Figure 1). In vitro assays of cord blood CD34+ cells expanded for 5 weeks with SR1 showed a 65-fold increase in total CFC and >1000-fold increase in the number of GEMM CFC compared to control cultures. NOD-SCID repopulating experiments of expanded cord blood HSC are in progress. These results demonstrate that high throughput screening of LMW compound libraries is a viable approach to find novel regulators of HSC self-renewal and identify a compound class that greatly facilitates ex-vivo expansion of HSCs. Fig. 1 SR promotes sustained expansion of CB CD34 133+ CD38− cells Fig. 1. SR promotes sustained expansion of CB CD34 133+ CD38− cells

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Ex Vivo expansion Of Umbilical Cord Blood CD34+ Cells Under Hypoxic Conditions Using Novel Compound#999 With Cytokines

Blood ◽

10.1182/blood.v122.21.4508.4508 ◽

2013 ◽

Vol 122 (21) ◽

pp. 4508-4508

Author(s):

Saadiya Khan ◽

Alison L. Stewart ◽

Siddhartha Mukherjee ◽

Stuart L. Scheiber ◽

Benjamin L. Ebert ◽

...

Keyword(s):

Cord Blood ◽

Umbilical Cord ◽

Umbilical Cord Blood ◽

Ex Vivo ◽

Fold Increase ◽

Additive Effect ◽

Hematopoietic Stem ◽

Hypoxic Conditions ◽

Cd34 Cells ◽

Cells Cultured

Introduction Umbilical cord blood is an increasingly utilized source for hematopoietic stem transplantation. However the limitation is inadequate hematopoietic stem and progenitor cell (HSPC) dose leading to poor engraftment and prolonged neutropenia. Umbilical cord blood transplants (UCBT) were initially restricted to small sized children and adults. The advent of double umbilical cord blood transplants (DUCBT) led to both children and adults transplants with sufficient numbers of HSPCs. However there continue to be issues with insufficient engraftment, extended duration of cytopenia, risk of infections and prolonged duration of hospital stay. There is ongoing research to investigate optimal ex vivo umbilical cord blood (UCB) HSPC expansion with the intention to ensure sustained engraftment, reduce the prolonged periods of neutropenia and curtail the high risk of infectious complications in the immediate post-transplant period. HSPC expansion with cytokines alone produces about 7-fold increase of HSPC over 12-14days. However most IRB approved protocols require that a significant percentage of these cord blood cells be transplanted without manipulation and then the expanded cells be transplanted later. To detect a significant advantage to this expanded fraction we have calculated that HSPCs need to be expanded 8-10 fold. To achieve this we have combined an optimal cytokine combination with hypoxia and the additive of Aryl hydrocarbon Receptor (AhR) antagonist Stem Reginin1 (SR1); previously reported to facilitate HSPC expansion (Boitano et al 2010 Science). Objectives Here we evaluated if there was any potential synergistic effect of combining AhR antagonist SR1 with hypoxia for ex vivo HSPC expansion. Additionally we looked at the effect of adding #999; a small molecule identified using high-throughput screening that selectively expands murine hematopoietic stem cells. Methods UCB derived phenotypic CD34+ cells were cultured in the presence of stem cell factor (SCF), Flt3 ligand (Flt3L) and thrombopoietin (TPO) on a feeder layer of OP9 cells transduced with lentiviral vector expressing red fluorescent protein in both normoxia and hypoxia (3% oxygen). Total cell numbers (TNC) were counted, CD34+ cells were measured through flowcytometry and the self-renewal and multi-lineage differentiation was measured through week-5 cobblestone area forming (CAFC) and colony forming (CFC) assays respectively. Results CD34+ cells cultured in the presence of SCF, Flt3L and TPO (50ng/ml each) resulted in a 100fold expansion of CD34+ cells compared to input cells at 2 weeks. SR1 when added to the above cytokine cocktail led to a 200-fold expansion while #999 used with cytokines resulted in 118-fold expansion at 2weeks. Using both small molecules together in the presence of cytokines did not show an additive effect (207fold increase). Repeating the above experiments in hypoxia (3% oxygen) showed 196-fold increase with cytokines alone, 289-fold increase with SR1, 211-fold increase with #999 and again no additive effect of SR1 and #999 together. CD34+ cells cultured with SR1 or #999 with cytokines produced approximately 1.9 and 1.2 times more CFC than those with cytokines alone respectively. SR1 treated cells on week-5 CAFC showed 3-fold and #999 treated cells 1.3-fold more cobblestones compared to cytokines alone. In hypoxia CD34+ cells cultured with #999 gave rise to more colonies as compared to both SR1 (2-fold more) and cytokines (3-fold more). CAFC data for these are pending. The degree of HSPC expansion with SR1 in addition to cytokines can be increased in hypoxic conditions. # 999 when used with cytokines in hypoxia can also lead to the same degree of HSPC expansion as SR1 in normoxia. The combination of SR1 and #999 showed no additive effect in either normoxia or hypoxia. Conclusion Compound 999 when used in hypoxia leads to a significant expansion of HSPCs compared to cytokines alone or SR1 plus cytokines in normoxia. In vivo xenograft murine studies are been conducted so as to compare and evaluate the engraftment potential of these ex vivo expanded CD34+ cells in irradiated NSG mice. Disclosures: Mukherjee: Onconova Therapeutics: Research Funding. Ebert:Genoptix: Consultancy; Celgene: Consultancy.

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Wharton's Jelly- MSC As Effective Tool for Expansion of Purified Human Cord Blood CD34+ Hematopoietic Stem Cells

Blood ◽

10.1182/blood.v126.23.4770.4770 ◽

2015 ◽

Vol 126 (23) ◽

pp. 4770-4770

Author(s):

Melania Lo Iacono ◽

Elena Baiamonte ◽

Rosalia Di Stefano ◽

Barbara Spina ◽

Massimiliano Sacco ◽

...

Keyword(s):

Cord Blood ◽

Umbilical Cord ◽

Direct Contact ◽

Ex Vivo ◽

Fold Increase ◽

Contact System ◽

Hematopoietic Stem ◽

Cd34 Cells ◽

Feeder Layers ◽

Transwell System

Abstract Umbilical cord is an extra-embryonic-annex rich of both hematopoietic stem and progenitor cells (HSPC) and mesenchymal stem cells (MSC) and it is easily accessible. The HSPC derived from umbilical cord blood (UCB) are promising as graft for allogeneic bone marrow (BM) transplantation and as source of target cells for autologous HSPC gene correction. UCB-HSPC have several advantages compared to adult ones: a less risk of graft-versus-host disease, a higher frequency of progenitors with a greater clonogenic potential and more susceptibility to be transduced by lentiviral vectors. Nonetheless, the HSPC yield from single cord blood unit is not sufficient for these clinical approaches in adults. Therefore, ex-vivo expansion of HSPC in media supplemented by cytokines and/or in vitro culture systems with feeder layers, is a valid approach to exceed this limit. MSC are a component of BM-microenvironment that play a key role in supporting of hematopoiesis by ability to secrete soluble factors and probably by the direct cell-cell interaction too. In this work, we investigated the ability of umbilical cord extracellular matrix-MSC (Wharton's Jelly-MSC) to support the ex-vivo expansion of UBC- purified CD34+ cells. In particular, we evaluated the fold increase, and the frequency of CD34+ cell and CD34+subtypes during expansion at the following culture conditions: by direct contact with WJ-MSC layer, by exposure to the soluble factors secreted by WJ-MSC layer in transwell system. The fold expansion was compared with the CD34+ cells expanded in a customized serum-free medium. CD34+ cells were isolated by immuneselection from 8 fresh UCB. The WJ-MSC were isolated from UC cut-pieces by non-enzymatic procedure but thanks to their capacity to migrate to plastic substrate. At the confluence of 60-70% the WJ-MSC were treated with mytomicin-C to arrest the cell cycle. After 48h, the immune-selected CD34+ cells were seeded in WJ-MSC at the density of 5-10 x104 in 12 well plates by direct or indirect contact (by transwell system). CD34+cells were grown in absence of feeder layers at the same conditions. Early hematopoietic cytokines (Flt-3, TPO, SCF) were supplemented in all three conditions and freshly replaced every two days of culture. Numbers and frequency of CD34+cells were evaluated according to ISHAGE method and CD34+ subtyping was performed by four color method to investigated the co-expression of the primitive surface antigens (CD38, CD133, CD90). The frequency of CD34+ cells at day 5 of culture decreased only 10% and was about 50% after 8 days of culture in conditions. The expansion of CD34 + cells at direct contact with WJ-MSC was superior (5.5 fold increase) compared to that of the other two conditions (3 fold on average). At day 8of culture, the CD34+ cells expanded 12 fold at direct contact with feeder layer, about 7 fold in a transwell system and 6 fold in basic medium. No substantial differences in the grade of expansion was revealed in heterologous vs homologous co-cultures of HSPC/WJ-MSC. Noteworthy is that in the contact system in addition to the fluctuating CD34+ cells harvested from the medium (floating CD34+ cells), we found approximately 50% of the total CD34+ cells be adherent to WJ-MSC layer, these cells were released only after enzymatic proteolytic treatment. Subtyping the CD34+cell population growing in contact to the WJ-MSC or in the conditioned medium we found that the CD34+/CD133+cell population was maintained high (72% ±12 over the total CD34+ cells) as in unmanipulated CB-HSC. The CD34+CD38- cells decreased by 2,5 fold in both systems, as early as day 5 of culture. However, in the contact system this population was 3 times more represented in the attached CD34+ cell fraction. The CD34+/CD90+ subtype was also expanded (more than 8 fold) particularly in the attached fraction, as early as 5 days of culture and was maintained to the end WJ-MSC supported ex-vivo HSPC expansion with superior effect in a cell contact system. Two phenotypically different populations of HSPC developed in this system with an increased frequency of CD34+ cells that co-expressed markers typical of more early progenitors in the attached CD34+ cell fraction. We are assessing the significance of these differences by performing molecular and functional studies of WJ-MSC-supported HSPC. This work was funded by the F and P Cutino Foundation - Project RiMedRi CUP G73F12000150004 Disclosures No relevant conflicts of interest to declare.

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