Scaling up blood cell production from hPSCs is critical to advancing hPSC technologies for blood transfusion, immunotherapy, and transplantation. Here we explored the potential of the HSC agonist pyrimido-indole derivative UM171, to expand hematopoietic progenitors (HPs) derived from hPSCs in chemically defined conditions. We revealed that culture of hPSC-HPs in HSC expansion conditions (SFEM with added TPO, SCF, FLT3L, IL3 and IL6) in the presence of UM171 predominantly expanded HPs with a unique CD34 + CD41a lo CD45 + phenotype that were enriched in granulocytic progenitors (G-CFCs). In contrast, in lymphoid cultures on OP9-DLL4, in the presence of SCF, FLT3L, and IL7, UM171 selectively expanded CD34 + CD45 + CD7 + lymphoid progenitors with NK cell potential, and increased NK cell output up to 10-fold. These studies should improve our understanding of the effect of UM171 on de novo generated HPs, and facilitate development of protocols for robust granulocyte and lymphoid cell production from hPSCs, for adoptive immunotherapies.

Congenital vertebral malformations (CVMs) comprise a group of spinal abnormalities that include alterations in vertebral shape or number. Evidence suggests CVMs have a genetic link, possibly resulting from mutations in multiple genes. One candidate gene is T. T protein, a transcription factor found in a variety of animals including humans, is essential for correct embryonic development and guides the development of bone and cartilage from embryonic mesodermal tissue. T protein accumulates in the nuclei of notochord cells, interacts with DNA at specific genes, and acts as a genetic switch to activate the genes. T protein binds to the major and minor grooves of DNA as a dimer. Mutations in T (turning “off” the T protein switch) are hypothesized to result in defects in spinal development. The Cedarburg SMART (Students Modeling A Research Topic) Team has designed a partial model of T protein using 3D printing technology to investigate its structure‐function relationship, focusing primarily on the residues important for dimerization of T (Pro125, Asp126, and Pro128) and for binding DNA (Arg67). A 3D model could indicate how the location of the mutations may impact the function of T. T could consequently be a potential target for the development of treatment or prevention options. Program supported by a grant from NIH‐CTSA.

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Human pluripotent stem cell (hPSCs) have created alternative platforms for producing blood cells for transfusion, immunotherapies, and transplantation. Advancing blood cell manufacturing from hPSCs and translating hPSC-based technologies to the clinic requires improving the scalabilty of blood cell production through enhancing hematopoietic differentiation from hPSCs, and increasing expansion of lineage committed hematopoietic progenitors. The pyrimido-indole derivative UM171 has been described as one the most potent small molecules agonists for HSC expansion in vitro. However, the effect and mechanism of UM171 action on hPSC-derived hematopoietic progenitors (HPs) has not been explored. It also remains unclear whether UM171 selectively expands the most primitive multipotential HPs with lin-CD34+CD43+HSC phenotype, or affects progenitors already committed to a particular hematopoietic cell lineage. In our studies, we evaluated the effect of UM171 on the expansion and differentiation of CD34+CD43+HPs that were generated in chemically defined, serum- and feeder-free conditions from hPSCs. We revealed that culture of hPSC-derived HPs in HSC expansion conditions (SFEM with added TPO, SCF, FLT3, IL3 and IL6) in the presence of UM171 selectively expanded HPs with a unique CD34+CD41loCD45+phenotype. Isolation of this population by FACS revealed that it mostly possesses myeloid potential and is highly enriched in granulocytic progenitors (G-CFCs). In contrast, in lymphoid cultures on OP9-DLL4 in presence of SCF, Flt-3 and IL7, UM171 predominately expands CD34+CD7+CD41a-lymphoid progenitors with NK cell potential and increases up to 10-fold NK cell output in NK differentiation cultures. NK cells generated with UM171 possess strong cytotoxicity, express perforin and upregulate IFNg production, following stimulation with K562 or PMA. As determined by annexin V immunostaining, UM171 treatment decreased the number of apoptotic HPs in expansion cultures. In addition, UM171 expansion of HPs was associated with increased proliferation, as determined by BrdU assay and Ki67 staining. Extending these observations, cell cycle analysis revealed that UM171 predominantly increases the proportion of HPs in the early S phase of the cell cycle. These studies should improve our understanding of the effect of UM171 on de novo generated HPs and facilitate development of protocols for robust granulocyte and lymphoid cell production from hPSCs for adoptive immunotherapies. Disclosures Slukvin: Cynata Therapeutics Limited: Consultancy, Equity Ownership.