Environmental Cell-Matrix Adhesion Modulates Pluripotent Stem Cell Fate Toward Definitive Hemogenic Endothelium and Hematopoietic Progenitor Cells

[Background] Human pluripotent stem cell (hPSC) technology enables regenerative transplantation therapy and establishment of in vitro disease models. hPSCs can be maintained to proliferate limitlessly and also differentiated to specific lineage cells, but hematopoietic cell differentiation efficiency varies among each hPSC lines. It has been reported the intrinsic expression level of transcription factors in hPSCs determine the commitment capacity toward hematopoietic cells (i.e. c-MYC [J Exp Med. 2010; 207: 2817-2830], IGF2 [Cell Stem Cell. 2016; 19: 341-354]). However, whether extracellular scaffold factors that contribute to maintain hPSCs influence hematopoietic differentiation potential remain elusive.

[Aim] In this study, we hypothesized that the extracellular environment such as cell-matrix adhesion signal regulates the commitment capacity toward hematopoietic cells in hPSCs. Moreover, we sought to improve the conventional hematopoietic differentiation PSC-sac method, which is simple but inefficient.

[Results] The recombinant fragment of a laminin isoform, LM511-E8, [Neurosci Res. 2018; 96: 863-874, Nature. 2016; 531: 376-380, Nat commun. 2012; 3: 1236] is used in culture to maintain pluripotency of cGMP grade hPSCs instead of Matrigel in research grade hPSCs. However, cGMP grade hPSCs maintained on LM511-E8 showed decreased differentiation towards CD34+CD43+ Hematopoietic progenitor cells (HPCs) than hPSCs maintained on Matrigel or mouse embryonic fibroblasts as feeder cells. Meanwhile, hPSCs maintained on human recombinant laminin fragments LM121-E8 or LM421-E8 yielded significantly more HPCs than hPSCs maintained on LM511-E8.

Microarray analysis revealed that hPSCs maintained on those "hematopoietic laminins (HLs)" showed upregulated activity of canonical Wnt/β-catenin signaling pathway. Activation of canonical Wnt signaling pathway by GSK3β inhibitor significantly improved the yield of HPC from hPSCs on LM511-E8. We then found using flow cytometry that hPSCs on HLs had higher cell surface expression of integrin β1 subunit (ITGB1), which is a part of integrin a6b1 complex, the laminin receptor, and also found the higher expression of ILK, pGSK3β (Ser9) and β-catenin by Western blot analysis. These results accordingly suggest that the ITGB1-ILK-pGSK3β-β-catenin axis modulate the commitment of hPSCs towards hematopoietic lineage. We also found lower concentrations of LM511-E8 increased ITGB1 surface expression and HPC differentiation on day14, without influencing the activity of pluripotency of hPSCs. These results indicate the modulation of hematopoietic capacity by extrinsic laminin at pluripotent state is determined by the affinity of laminin-integrin, not by the specificity of the laminin subunits, and this extrinsic modulation might be regulated through the expression level of ITGB1.

Next, we revised hPSC-sac method, a conventional hematopoietic differentiation method which we originally proposed [Cell Stem Cell. 2017; 20(3) : 329-344.e7. Stem Cells. 2016; 34: 1541-1552. Blood. 2011; 111(11): 5298-5306.] By adding basic fibroblast growth factor (bFGF), TGFβ inhibitor SB431542 and heparin during differentiation period, the number of HPC increased dramatically on differentiation day14. The number of definitive hemogenic endothelium (CD34+ CD31+ CD73- CD144+ CD117+ KDR+ CD41- CD43- CD45- [Cell Reports, 2012; 2: 553-567][Blood, 2013; 121(5): 770-780]) on differentiation day 9 was also increased. Simultaneously, transcription factors specific for hematopoietic lineage, ERG, SPI1, Runx1c, GFI1b, TAL1, and TEK were upregulated in hPSCs derived cells on day 9.

[Conclusion] This study revealed a mechanism in which the signals from cell-matrix adhesion by cell surface integrins and extracellular laminins modulate hematopoietic differentiation potential in hPSCs. Moreover, the revised hPSC-Sac differentiation method enables a simple, efficient and economical differentiation of hematopoietic cells. These findings thus should contribute to development of the fundamental research on hematopoietic differentiation from hPSCs towards further application.