Improved Hematopoietic Differentiation of Primate Embryonic Stem Cells

Various kinds of functional cells differentiated from embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) have recently been developed and expected for use in human regenerative medicine. However, the safety and efficacy of ESC/iPSC-based therapies must be carefully evaluated prior to clinical application, by using reliable animal models. The common marmoset (CM, Callithrix jacchus) is known to be a suitable preclinical model for clinical translation studies, and CM ESCs have already been established by us.

Hematopoietic stem/progenitor cells (HSCs/HPCs) are one of very useful cells for transplantation therapy to treat various diseases including leukemia. However the shortage of their donors becomes a huge social problem and the expansion of HSCs/HPCs in vitro is known to be very difficult. We have previously demonstrated that CM ESCs showing indefinite self-renewal can be differentiated into hematopoietic lineages by the forced expression of hematopoietic transcription factor (TAL1/SCL). However the efficiency of their hematopoietic differentiation was quite low (less than 5%). Therefore the development of new method to promote hematopoietic differentiation of CM ESCs more efficiently is needed.

To promote hematopoietic differentiation of CM ESCs, we focused on self-renewal pathway of CM ESCs and oxygen levels during EB formation. We have reported that self-renewal of CM ESCs is regulated by phosphoinositide 3-kinases (PI3Ks)-protein kinase B (AKT) pathway that is known to regulate cell cycle and cell proliferation as well as cell survival (Nii et al., 2014). On the other hand, the differentiation of mouse ESCs to hematopoietic precursors such as hemangioblasts, bipotential progenitors of endothelial and hematopoietic cells, can be enhanced by hypoxic condition (Ramírez-Bergeron et al., 2004). In addition, expansion of HSCs/HPCs can be increased by hypoxic condition in vitro (Danet et al., 2003). Thus, we hypothesized that the suppression of ESC self-renewal by the inhibition of PI3K-AKT pathway under hypoxic condition would improve hematopoietic differentiation of CM ESCs.

To test our hypothesis that the inhibition of self-renewal pathway of CM ESCs could promote their hematopoietic differentiation, we treated CM ESCs with PI3K inhibitor (LY: LY294002) for the first 4 days of EB formation and examined the proportion of CD34⁺ cells by flow cytometric analysis, and found that the populations of CD34⁺ cells were significantly increased in the presence of LY. Moreover, the day8-EBs treated with LY gave rise to significantly more hematopoietic colonies than controls in colony forming unit (CFU) assay. These results indicated that hematopoietic differentiation was significantly enhanced by the inhibition of PI3K-AKT pathway in the process of EB formation. To further promote hematopoietic differentiation of CM ESCs, we conducted EB formation assay of CM ESCs and induced their differentiation into HPCs under hypoxic condition. We found that the hypoxic condition (5% O2) significantly increased the proportion of both CD34⁺ and CD34⁺/CD117⁺ cells in day8-EBs especially when PI3K-AKT pathway was inhibited by the LY treatment. These results were also obtained from human ESCs.

In the present study, we demonstrated that transient treatment of PI3K inhibitor during EB formation under hypoxia condition promoted hematopoietic differentiation of human and CM ESCs, which might contribute to the development of the valuable experimental system using CM ESCs in order to test new strategies of human regenerative medicine.