RUNX1C-Mediated Reprogramming Of Human Bone Marrow Stromal Cells into Early Blood Progenitors

Somatic cell reprogramming to the hematopoietic lineage, either through a pluripotent state or directly, opens the possibility of production of a ready source of autologous hematopoietic stem cells (HSC) that can be used to treat/cure a wide variety of blood disorders. While it has previously been shown that dermal fibroblasts (HFF) can be directly reprogrammed to the hematopoietic lineage, the efficiency was relatively low and the resultant hematopoietic cells lacked multilineage differentiative potential. Stro1(+) isolated stromal progenitors (SIPs) can easily be isolated from the bone marrow (BM) and expanded ex-vivo to obtain clinically significant numbers of cells. In similarity to HSC, SIPs are derived from the mesoderm, and are intimately linked with HSC specification during ontogeny. As such, they are likely to be epigenetically closer to HSC than HFF, and therefore good candidates for reprogramming into hematopoietic cells. To verify the uniqueness of SIPs for reprogramming, we transduced SIPs and HFF with OCT4 and/or RUNX1C, a master transcription factor (TF) that triggers the developmental onset of definitive hematopoiesis, in the following combinations: 1) OCT4 alone; 2) RUNX1C alone; or 3) OCT4+RUNX1C. We then performed a timeline of gene/cell surface marker expression (using microarray, qRT-PCR, and flow cytometry) from day 3-16 post-transduction. Visual inspection of the cultures showed that, while reprogrammed colonies began to appear in SIPs cultures at day 9, no colonies were seen during this time period in HFF cultures. Flow cytometry and molecular analyses of colonies obtained from OCT4+RUNX1C combination demonstrated that expression of CD41, the earliest marker of commitment to the hematopoietic lineage, commenced within only 3-4 days and peaked at day 5-6, by which time ∼20% of SIPs expressed this marker. This peak in CD41 expression coincided with commencement of expression of CD34 and CD45, and maximal induction of several hematopoiesis-specific TFs and phenotypic markers such as PU.1, HOXB4, GATA2, MIXL, WNT3, KDR, CDX4, which occurred at 1-3 logs higher levels in SIPs than HFF. Further studies demonstrated that the chromatin remodeling function of OCT4 could be replaced with the histone methyltransferase inhibitor Bix-01294, with the combination of RUNX1C and Bix-01294 inducing levels of CD34 and CD41 expression by day 5 that were similar to those achieved with RUNX1C plus OCT4. The present studies thus take several important steps towards making the promise of producing autologous hematopoietic cells for transplantation via direct reprogramming a reality.