Transcriptome Analysis of Endometrial Stroma-Like Organoids Differentiated From Human Induced Pluripotent Stem Cells.
Presenter: Kaoru Miyazaki
OBJECTIVE: It is now appreciated that induced pluripotent stem cells (iPSCs) derived from skin biopsies or bone marrow aspirates can provide patient-specific pluripotent cells, which could be differentiated into various cells types. In fact, several bioengineered organs including kidney have recently generated through differentiation of iPSCs. view more
OBJECTIVE: It is now appreciated that induced pluripotent stem cells (iPSCs) derived from skin biopsies or bone marrow aspirates can provide patient-specific pluripotent cells, which could be differentiated into various cells types. In fact, several bioengineered organs including kidney have recently generated through differentiation of iPSCs. We view uterine tissue engineering by using endometrial cells (ECs) differentiated from iPSCs can be considered an option for treatments in patients with uterine factor infertility or endometriosis; however, no model presently exists for generating ECs from iPSCs. The purpose of this study was to develop a chemically defined protocol for differentiating human iPSCs (hiPSCs) into ECs.
DESIGN: Prospective experimental study
MATERIALS AND METHODS: hiPSCs were purchased from ATCC. Embryoid bodies (EBs) were generated from hiPSCs following 1-day culture on EB formation plate and cultured with defined chemical cocktails for 13 days, and EBs were examined for stage-specific markers every other day. Day 14 (D14) EBs were then treated with 8-bromoadenosine 39,59-cyclic monophosphate (cAMP) and medroxyprogesterone acetate (MPA) for 8 days to determine the capacity of decidualization, which characterizes ESCs. RNAseq was performed on D4, D6, D8, D14, D22 EBs, hiPSCs, and normal endometrial stromal cells to determine transcriptome of each stage.
RESULTS: Intermediate mesoderm-specific genes LHX1 and PAX2 were significantly upregulated in D4 EBs compared to hiPSCs as revealed by qPCR (N=9, p<0.05). mRNA expression of Mullerian duct marker ISL1 was significantly higher in D8 EBs than in hiPSCs (N=9, p<0.05). The expression of ESC-specific transcription factors HOXA10 and HOXA11 were upregulated in day 6 EBs and maintained thereafter. PGR, the steroid receptor predominantly expressed in ESCs, was upregulated progressively during the differentiation process until D14. After 8 days of treatment with cAMP and MPA, D22 EBs expressed significantly higher mRNA levels of decidualization markers such as FOXO1, IGFBP1, and PRL (N=9, p<0.05) compared with cells treated with vehicle control (VC). ELISA (N=9, p<0.05) confirmed the increased protein expression of FOXO1. Principal component analysis of RNAseq depicted a seamless transition of the transcriptional profiles during differentiation. Transcriptome of D14 EBs was closer to normal endometrial stromal cells compared with D8 EBs. D22 EBs shared 1404 differentially expressed genes with normal endometrial stromal cells when compared with VC.
CONCLUSIONS: We have found that EBs differentiated from hiPSCs using our chemically defined protocol have specific characteristics resembling ESCs. These results suggest that hiPSCs can be an unlimited source of ESCs. We propose these results as the first step in advancing stem cell-based therapies for uterine factor infertility or endometriosis.