Cooperative Effect of Erythropoietin and TGF‐β Inhibition on Erythroid Development in Human Pluripotent Stem Cells

Abstract

Patient-specific human induced-pluripotent stem cells (hiPSCs) represent important cell sources to treat patients with acquired blood disorders. To realize the therapeutic potential of hiPSCs, it is crucial to understand signals that direct hiPSC differentiation to a hematopoietic lineage fate. Our previous study demonstrated that CD34(+)CD31(+) cells derived from human pluripotent stem cells (hPSCs) contain hemato-endothelial progenitors (HEPs) that give rise to hematopoietic cells and endothelial cells. Here, we established a serum-free and feeder-free system to induce the differentiation of hPSC-derived CD34(+)CD31(+) progenitor cells to erythroid cells. We show that extracellular matrix (ECM) proteins promote the differentiation of CD34(+)CD31(+) progenitor cells into CD235a(+) erythroid cells through CD41(+)CD235a(+) megakaryocyte-erythroid progenitors (MEP). Erythropoietin (EPO) is a predominant factor for CD34(+)CD31(+) progenitor differentiation to erythroid cells, whereas transforming growth factor beta (TGF-β) inhibits the development of CD34(+)CD31(+) progenitor cells. Apoptosis of progenitor cells is induced by TGF-β in early erythroid differentiation. Suppression of TGF-β signaling by SB431542 at early stage of CD34(+)CD31(+) progenitor differentiation induces the erythroid cell generation. Together, these findings suggest that TGF-β suppression and EPO stimulation promote erythropoiesis of CD34(+)CD31(+) progenitor cells derived from hPSCs.

Definitive Hematopoietic Multipotent Progenitor Cells Are Transiently Generated From Hemogenic Endothelial Cells in Human Pluripotent Stem Cells

“…1A). The hematopoietic differentiation of hPSCs was initiated through embryoid body (EB) formation in a serum-free medium with an optimized stage-specific combination of BMP4, FGF2, and VEGF-A, as we previously described (Bai et al, 2013; Xie et al, 2015). We have previously shown that CD34 + CD31 + CD144 + cells express SCL and RUNX1 , the two key transcription factor genes required for definitive hematopoietic cell development, and contain HEPs that give rise to hematopoietic cells and endothelial cells.…”

“…For hematopoietic differentiation from hemogenic ECs, the isolated CD34 + CD31 + CD144 + cells were cultured on collagen-I coated plates (5×10 4 cells per well in 12-well plate) in serum-free endothelial cell growth medium (ECGM) (Bai et al, 2013; Xie et al, 2015). After 24 h, unattached cells were removed and adherent cells were washed with PBS and continually cultured for three additional days in ECGM or in hematopoietic cell growth medium (HCGM) containing 50 ng/ml stem cell factor (SCF), 50 ng/ml FLT3-ligand (F3L), and 50 ng/ml thrombopoietin (TPO).…”

“…For erythroid differentiation, CD34 + CD31 + CD144 + cells were subjected for erythroid differentiation culture in HCGM with 3 units/ml erythropoietin (EPO) for 6–18 days, as previously described (Xie et al, 2015). …”

“…We and others have identified hematopoietic and endothelial progenitors in differentiated hPSCs, based on markers expressed in endothelial and hematopoietic progenitor cells, including CD34, KDR (VEGFR2 or FLK1), CD31 (PECAM1), and CD144 (Kennedy et al, 2007; Choi et al, 2012; Kennedy et al, 2012; Wang et al, 2012; Bai et al, 2013; Rafii et al, 2013; Xie et al, 2015). We previously demonstrated that CD34 + CD31 + CD144 + population from hPSCs contains hemato-endothelial progenitors (HEPs) that give rise to hematopoietic cells and endothelial cells (Bai et al, 2010; Bai et al, 2013; Xie et al, 2015).…”

“…We previously demonstrated that CD34 + CD31 + CD144 + population from hPSCs contains hemato-endothelial progenitors (HEPs) that give rise to hematopoietic cells and endothelial cells (Bai et al, 2010; Bai et al, 2013; Xie et al, 2015). The key transcription factors required for definitive hematopoietic cell generation from hemogenic ECs, including SCL and RUNX1 (Lacaud et al, 2002; Patterson et al, 2005; Nottingham et al, 2007; Dzierzak and Speck, 2008; Chen et al, 2009; Lam et al, 2010; Ren and Gomez, 2010; Van et al, 2012; Ran et al, 2013; Zhen et al, 2013), are expressed in CD34 + CD31 + CD144 + cells (Bai et al, 2013).…”