Direct conversion of human fibroblasts to multilineage blood progenitors

Similar to embryo-derived stem cells, application of human induced pluripotent stem cells (iPSCs) is limited by our understanding of lineage specification. Here, we demonstrate the ability to generate progenitors and mature cells of the hematopoietic fate directly from human dermal fibroblasts without establishing pluripotency. POU domain activation of hematopoietic transcription factors by ectopic expression of Oct-4, together with specific cytokine treatment, allowed generation of cells expressing the pan-leukocyte marker CD45. These unique fibroblast-derived cells gave rise to granulocytic, monocytic, megakaryocytic, and erythroid lineages, and demonstrated in vivo engraftment capacity. Distinct from PSC-derived hematopoietic cells, adult and not embryonic hematopoietic programs are activated, consistent with bypassing pluripotent state to generate blood fate. These findings suggest an alternative approach to cellular reprogramming for autologous cell-replacement therapies that avoids complications associated with the use of human PSCs. Molecular analysis was done on purified untransduced Fibs, Oct-4 transduced Fibs at day 4, CD45+ve Fibs at day 21 and hematopoietic cytokine treated CD45+ve Fibs at day 37. The day 4 Oct-4 transduced Fibs were isolated by puromycin selection overnight (Oct-4 vector contains puromycin resistance cassette), purity of sample was validated by staining for Oct-4 followed by Oct-4 expression analysis using flow cytometry; samples used for molecular analysis exhibited 99% Oct-4 levels. The day 21 and day 37 CD45+veFibsOct-4 were isolated based on their CD45 expression. D21 and D37 cells were stained with CD45-APC antibody (BD Biosciences) and sorted using FACSAria II (Becton- Dickinson); samples used for molecular analysis exhibited 99% CD45 levels. Analysis was performed on two biological replicates per sample.