Erythroblast differentiation of human endometrium derived induced pluripotent stem cells as a source of autologous transfusion

Abstract

Purpose and background: With the consistent increase in life-expectancy, the importance of regenerative medicine cannot be over-emphasized. The objective of this study is to pretreat patient derived endometrial cells with exogenous β estradiol to enhance reprogramming efficiency of endometrial stromal cell derived induced pluripotent stem (iPS) cells. Subsequently, the protocol for committing these cells into hematopoietic and erythroid lineages will be optimized through a two-phase culture system, involving feeder and non-feeder environments. 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. pMIG-human sox2, oct4, klf4 and c-Myc viral vectors, namely the Yamanaka factors were used to transduce the endometrial stromal cells. The impact of exogenous β estradiol on the reprogramming efficiencies of different endometrial donor cells were compared at concentrations of 0, 0.1 and 1μM/ml. Directed differentiation of these established iPS cells were conducted in two phases. The first 9 days involved commitment of the iPS cells to hematopoietic fate with robust expansion on murine bone marrow stromal feeder cells (OP-9). The second phase of differentiation involved feeder free conditions composed of hydrocortisone, stem cell factor, interleukin-3, recombinant erythropoietin (EPO) and poloxamer 188. After 17 days of differentiation in feeder free environment, the expression profiles of KDR (VEGF-R2), CD235a +, CD34 +, CD43 + and CD 71+ were analyzed by flow cytometry and Wright-Giemsa staining for differential counting based on morphology. Results: The treatment of donor endometrial stromal cells with β estradiol at a concentration of 0.1μM/ml resulted in iPS cell reprogramming efficiency of approximately 166 ± 5% compared with the non-treated control cells set as 100%. As a result of inducing these cells to hematopoietic fate via a 9 day co-culture with murine stromal fibroblasts, yields ranging from 8-13% was observed depending on the donor. Because the potential of these co-cultured cells to further differentiate into erythroblastic fate may not be confined to defined cell surface markers, all of the OP-9 co-cultured cells were transferred to a feeder-free system composed of hydrocortisone, stem cell factor, interleukin-3, recombinant EPO and poloxamer 188 for 17 days, stably yielding over 80% of polychromatic and orthochromatic normoblasts. Therefore, the protocol for a comprehensive induction of erythroid lineage cells starting from human endometrial tissue via iPS cell reprogramming has been fully demonstrated. Conclusion: The treatment of exogenous estradiol, unique to endometrial cells yielded higher reprogramming efficiencies of endometrial derived iPS cells compared naïve endometrial cells. From these iPS cell colonies driven from discarded endometrial cells successful induction of hematopoietic cell fate followed by erythroid differentiation up to orthochromatic normoblasts were achieved in an effort to develop autologous transfusion source.