O-237 In-vitro generation of primordial follicle-like structure from human germ cell-like cells derived from theca stem cell combined with ovarian somatic cells
Authors: Mirbahari S.N , Dalman A , Hasani F , Totonchi M , Rezazadeh velojerdi M 
Published: June 2022

Abstract

Study question
Does human ovarian somatic cells, improve the development of human germ cell-like cells (hGCLCs) derived from the human theca stem cell (hTSCs) after co-culture?

Summary answer
1. Yes, morphological similarities, size, and expression of germ and oocytes-specific proteins in developed hGCLCs in primordial follicle-like structure indicate the increase of in-vitro development.

What is known already
1. Somatic stem cells of the ovary can differentiate into primordial germ cell-like cells in appropriate in vitro conditions. The ovarian somatic cells secrete growth factors that can lead to further development. In ovarian dysfunction, follicle growth may be abnormal due to defects in the function of the oocyte or somatic cells. Replacement of defective oocytes or somatic cells, using the follicle-like structure technique, may “save” follicle growth. However, finding an optimal way to create a kind of co-culture that is economically viable and beneficial is a challenge.

Study design, size, duration
hTSCs which were differentiated into human germ cell-like cells hGCLCs after 12 days, were combined with ovarian somatic cells to assess the effects of ovarian somatic cells on the developmental potential of hGCLC after being placed in the structure named “primordial follicle-like structure”.

Participants/materials, setting, methods
1. The hTSCs were isolated from antral follicles of transgender individuals. Isolated hTSCs were seeded in the differentiation medium (DMEM-F12 containing 5% human follicular fluid and 5% FBS) for 40 days. The differentiated hGCLCs aggregated with somatic cells (cumulus cells and hTSCs) in a ratio of 1 to 10 and cultured in a growth medium (αMEM containing 3mg/ml  BSA, 1% ITS, 0.005 IU/ml FSH  and 0.11 mg/ml ascorbic acid) in suspension culture dish for 11 days.

Main results and the role of chance
1. After 12 days, hTSCs began to differentiate into hGCLCs and their morphology changed from spindle-shaped to round. The size of hGCLCs increased during the differentiation period (from 25-25 μm to 50 μm). The survival of hGCLC cells after differentiation and development in vitro in primordial follicle-like structures was 54%. Unlike hTSCs, hGCLCs and hGCLCs developed with ovarian somatic cells expressed the DAZL protein well (P-value&lt;0.05), hGCLCs poorly expressed the GDF9 protein. The culture of hGCLCs in primordial follicle-like structures significantly increased GDF9 expression (P-value&lt;0.05).

Limitations, reasons for caution
1. So far, there have been few studies of three-dimensional culture in the primordial follicle-like structure, all of which were performed on the oocyte. The lack of previous studies on the development of hGCLCs was a limitation that led us to set up the study from the beginning.

Wider implications of the findings
1. The hTSCs can differentiate to hGCLCs. In the presence of ovarian somatic cells, hGCLCS expressed the oocyte-specific markers which could not be expressed before. It seems that the combination of ovarian somatic cells can simulate primordial follicle structure and lead to more development of hGCLCs.

Trial registration number
25