Human endometrium derived induced pluripotent stem cells are amenable to directed erythroid differentiation

Abstract:Background: With the consistent increase in life-expectancy, excavating novel sources of regenerative medicine is an important objective to enhance disease free life expectancy. A comprehensive protocol for using human endometrium derived induced pluripotent stem cells to derive hematopoietic and erythroid lineages will be elaborated, through a two-phase culture system. Method: Discarded endometrial tissues were obtained from women receiving hysterectomy in their 4th to 5th decade due to benign uterine conditions. The endometrial cells isolated were expanded to passage 3-4 to allow stromal cells to dominate in the culture environment. pCE-Sox2, Oct4, Klf4, L-Myc and Lin28 episomal vectors were used to electrotransfection the endometrial stromal cells. The erythroblast differentiation of these established induced pluripotent stem cells (iPSCs) were conducted in two phases. The first 8 days involves commitment to hematopoietic stem cells through embryoid body with robust expansion on murine bone marrow stromal cells. The second phase involves feeder free conditions with hydrocortisone, stem cell factor, interleukin-3, and recombinant EPO. After 22 days of feeder free culture, the expression profiles of CD235a⁺, CD34⁺, CD43+ and CD 71⁺ were analyzed by flow cytometry and Wright-Giemsa staining for differential counting. The oxygen carrying capacity of cultured RBCs was measured using a hemoxanalyser. Results: As a result of inducing these cells via co-culture with murine stromal fibroblasts, all endometrium derived iPSCs were differentiated into erythroblasts with stably yielding over 80% of polychromatic and orthochromatic normoblast. The protocol for complete induction of erythroid lineage cells starting from human endometrial tissue via iPS cells has been optimized. Conclusion: Successful induction of hematopoietic cell fate followed by erythroid differentiation up to erythroblast were achieved in an effort to develop transfusion source. And a complete process of actually deriving iPS cells with discarded surgical hysterectomy specimens has significance in the possibility of expanding the scope of use of theses iPSC cell lines in the future.