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.
Disclosures: Boitano:Novartis: Employment. Schultz:Novartis: Employment. Cooke:Novartis: Employment.