Erythroblast differentiation of human endometrium derived induced pluripotent stem cells as a source of autologous transfusion
Other Titles
환자 자궁내막 세포로부터 제조한 유도만능줄기세포주로부터 적혈구모세포로의 분화유도
Authors
박주현
College
College of Medicine (의과대학)
Department
Dept. of Obstetrics and Gynecology (산부인과학교실)
Degree
박사
Issue Date
2017
Description
의학과/박사
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.