Pyrimido-Indole Derivatives Are Novel Agonists of Human Cord Blood Hematopoietic Stem Cell Self-Renewal

The widespread use of cord blood (CB) unit in transplantation is limited with low number of long-term hematopoietic stem cells (LT-HSCs) and progenitors. Several approaches have been developed to expand HSC ex vivo such as automated and continuous medium delivery (fed-batch), notch delta ligand and SR1 (antagonist of aryl hydrocarbon receptor (AhR)). Concurrent with these studies, we hypothesized that small molecule with potent LT-HSC stimulating activity might be identified and potentiated in fed-batch culture system. Accordingly, we tested a library of more than 5000 small molecules for their in vitro expansion of CD34+CD45RA- cells. Most of the identified hits, except one (UM729) synthesized in our institute, suppress AhR pathway. Structure activity relationship was performed on UM729 to generate a more potent analog named UM171. This optimized molecule was 10-20 times more potent with an effective concentration of 15-20 nM when tested for its ability to expand CD34+CD45RA- cells. When compared to SR1, UM171 delivered in a fed-batch system for 12 and 16 days showed a better expansion of HSC phenotypes and lower apoptotic cell number compared to SR1 or DMSO controls. Also, UM171-expaned cultures showed higher number in multipotent progenitors (CFU-GEMM) and long term initiating cells (LTC-IC) compared to DMSO controls. Further studies showed the UM171 did not affect division rate, and its effect in expanding HSC phenotype was reversible. When combined with SR1, UM171 showed a better suppression of differentiation and led to a higher CFU-GEMM expansion compared to the single treatment of the compounds or DMOS controls. These observations suggest that UM171+SR1 cooperate to enhance ex vivo expansion of progenitor cells and suppress differentiation. To determine the in vivo activity of the expanded CD34+ CB cells, we transplanted fresh (un-manipulated) and 12-day cultured cells in NSG mice and monitored the human hematopoietic reconstitution after 20 and 30 weeks post-transplantation. Frequencies of day0 equivalent LT-HSCs were 13-fold higher in UM171 expanded cultures compared to fresh or fed-batch cultures supplemented with DMSO or SR1. Secondary experiments indicated that UM171 ex vivo treatment did not appear to affect the capability of LT-HSC to expand in primary recipients and hence similarly reconstituted secondary animals for at least 18 more weeks. This suggests that UM171 expands LT-HSC ex vivo without losing their engraftment potential. To further investigate UM171 mechanism of action, RNA- Seq expression profiling was performed. Unlike SR1 or DMSO controls, UM171 treatment was accompanied by a marked suppression of transcripts associated with erythroid and megakaryocytic differentiation and up-regulation of membrane protein transcripts such as EPCR and TEMEM 183a. In summery, UM171 is the first molecule identified so far that enables a robust ex vivo expansion of human CD34+ CB cells that sustain their in vivo activity independent of AhR suppression. Conversely, AhR suppression was limited to expand cells with less durable self-renewal potential. This study could enhance the use of small yet well HLA-matched CB units to become a prioritized source for stem cells transplantation.