RUNX1c Expression Identifies an Enriched Subpopulation of Hematopoietic Progenitor Cells Derived From Human Pluripotent Stem Cells.
Authors: Ferrell Patrick I , Adlakha Mitali , Kaufman Dan S. 
Published: October 2019

Abstract
Abstract 2294
The ability of human embryonic stem cells (hESCs) to differentiate into any cell lineage makes them an important resource for studies of developmental biology. hESC-derived hematopoietic stem cells (HSCs) developed through in vitro culture systems could provide an unlimited supply of material for replacement of defective blood lineages in vivo. However, to date, there has been only limited ability to isolate functional HSCs from hESCs. Runx1 is a key hematopoietic transcription factor vital to the development of the early embryo. Previous studies have demonstrated the onset of Runx1c to correlate directly with the emergence of definitive HSCs in the murine embryo. Similarly, expression of RUNX1c increases during differentiation of hESCs to hematopoietic cells. This particular feature makes RUNX1c an especially attractive tool for tracking the development of potential HSCs from hESCs. Therefore, we have created a novel reporter system in hESCs wherein both 1 kb of the basal promoter as well as the +24 intronic enhancer of RUNX1c drive expression of the tdTomato fluorochrome. Transgenic hESC lines stably expressing tdTomato from RUNX1c regulatory elements were generated using the Sleeping Beauty transposon system. Successfully engineered clones were selected based on constitutive expression of a GFPzeocin fusion protein which was also included in the transgene. To induce differentiation, the spin-embryoid body (EB) method was used with fully defined media. Differentiating cells were analyzed for tdTomato expression. Sorted tdTomato+ cells were evaluated for RUNX1c expression and hematopoietic stem/progenitor cell potential (CFC assay). Undifferentiated RUNX1c reporter hESC clones were uniformly tdTomato-negative. These clones are shown to express the tdTomato transgene concurrent with the expression of endogenous RUNX1c, as analyzed by flow cytometry and qRT-PCR. Expression was first seen at day 12 with a peak between days 14–16 of differentiation. Cells sorted for expression of tdTomato showed a significant increase of endogenous RUNX1c expression compared to tdTomato-negative cells. Using fluorescence microscopy, we were able to detect tdTomato+ cells with hematopoietic morphology budding from the EBs after 12–15 days. Together, these results validate the accuracy of this reporter system. tdTomato+ cells were found to be enriched for hematopoietic progenitor potential, as demonstrated by increased ability to form hematopoietic colonies in methylcelluose (430 +/− 143 per 105 cells) as compared to both tdTomato negative (131 +/− 15 per 105 cells) and unsorted cells (163 +/− 28 per 105 cells). Interestingly and unexpectedly, flow cytometric analysis demonstrated that only 5–10% of CD34+CD45+ cells expressed tdTomato. The first CD45+ population was found to lack tdTomato expression. Likewise, the first emerging tdTomato+ cells did not express CD45. Of the extracellular markers examined in this study, CD31 and CD43 were found to be expressed on all tdTomato+ cells, while CD31+ and CD43+ populations, though initially tdTomato−, were found to gradually increase in tdTomato expression to 80% and 90% by day 20, respectively. Emerging CD34+ cells lack tdTomato expression with an increase to around 10% of cells, plateauing at day 15. While CD45+ cells initially lack tdTomato expression, they gain expression over time, until days 19–22, when 50–90% of CD45+ cells co-express tdTomato (RUNX1c). Therefore, this hESC RUNX1c reporter system appears to accurately define timing of RUNX1c expression. The finding that emerging RUNX1c+ cells have increased hematopoietic progenitor cell potential mimics what has been found in mice. This, combined with our finding that only a fraction of CD34+CD45+ cells express Runx1c, suggests that one reason hESC-derived hematopoietic cells fail to engraft in immunodeficient mice is that only a small percent of differentiated cells that express hematopoietic surface antigens actually have hematopoietic stem/progenitor cell activity. Current studies focus on more in-depth analysis of tdTomato expressing cells using both single-cell qRT-PCR as well as LTC-IC and engraftment assays to test for functionality. Furthermore, we are currently testing an iPSC line stably expressing the same RUNX1c:tdTomato transgene to test how its RUNX1c expression in iPSCs compares to hESCs during hematopoietic differentiation.

Disclosures:
No relevant conflicts of interest to declare.