In vitro differentiation of theca cells from ovarian cells isolated from postmenopausal women

Abstract

STUDY QUESTION Can human theca cells (TCs) be differentiated in vitro? SUMMARY ANSWER It is possible to differentiate human TCs in vitro using a medium supplemented with growth factors and hormones. WHAT IS KNOWN ALREADY There are very few studies on the origin of TCs in mammalian ovaries. Precursor TCs have been described in neonatal mice ovaries, which can differentiate into TCs under the influence of factors from oocytes and granulosa cells (GCs). On the other hand, studies in large animal models have reported that stromal cells (SCs) isolated from the cortical ovarian layer can also differentiate into TCs. STUDY DESIGN, SIZE, DURATION After obtaining informed consent, ovarian biopsies were taken from eight menopausal women (53–74 years of age) undergoing laparoscopic surgery for gynecologic disease not related to the ovaries. SCs were isolated from the ovarian cortex and in vitro cultured for 8 days in basic medium (BM) (G1), enriched with growth factors, FSH and LH in plastic (G2) or collagen substrate without (G3) or with (G4) a GC line. PARTICIPANTS/MATERIALS, SETTING, METHODS To confirm TC differentiation, relative mRNA levels for LH receptor (Lhr), steroidogenic acute regulatory protein (Star), cholesterol side-chain cleavage enzyme (Cyp11a1), cytochrome P450 17A1 (Cyp17a1), hydroxy-delta-5-steroid dehydrogenase, 3 beta- and steroid delta-isomerase 1 (Hsd3b1) and hydroxy-delta-5-steroid dehydrogenase, 3 beta- and steroid delta-isomerase 2 (Hsd3b2) were assessed. Immunohistochemistry was also performed for their protein detection and a specific marker was identified for TCs (aminopeptidase-N, CD13), as were markers for theca and small luteal cells (dipeptidyl peptidase IV (CD26) and Notch homolog 1, translocation-associated (NOTCH1)). Finally, we analyzed cell ultrastructure before (Day 0) and after in vitro culture (Day 8), and dehydroepiandrosterone (DHEA) and progesterone levels in the medium using transmission electron microscopy (TEM) and ELISA, respectively. MAIN RESULTS AND THE ROLE OF CHANCE Results obtained from qPCR showed a significant increase (P < 0.05) in mRNA levels of Lhr in F2 (floating cells in G2) and G4, Cyp17a1 in G1 and F1 (floating cells in G1) and Hsd3b2 in G1, G2, G3 and G4. Immunohistochemistry confirmed expression of each enzyme involved in the steroidogenic pathway at the protein stage. However, apart from G1, all other groups exhibited a significant (P < 0.05) rise in the number of CD13-positive cells. There was also a significant increase (P < 0.05) in NOTCH1-positive cells in G3 and G4. Ultrastructure analyses by TEM showed a distinct difference between groups and also versus Day 0. A linear trend with time revealed a significant gain (q < 0.001) in DHEA concentrations in the medium during the culture period in G1, G2, G3 and G4. It also demonstrated a statistical increase (q < 0.001) in G2, G3 and G4 groups, but G1 remained the same throughout culture in terms of progesterone levels. LARGE SCALE DATA N/A LIMITATIONS, REASONS FOR CAUTION Shorter periods of in vitro culture (e.g. 2, 4 and 6 days) could have led to increased concentrations of differentiated TCs in G2, G3 and G4. In addition, a group of cells cultured in BM and accompanied by COV434 cells would be necessary to understand their role in the differentiation process. Finally, while our results demonstrate that TCs can be differentiated in vitro from cells isolated from the cortical layer of postmenopausal ovaries, we do not know if these cells are differentiated from a subpopulation of precursor TCs present in ovarian cortex or ovarian SCs in general. It is therefore necessary to identify specific markers for precursor TCs in human ovaries to understand the origin of these cells. WIDER IMPLICATIONS OF THE FINDINGS This is a promising step toward understanding TC ontogenesis in the human ovary. Moreover, in vitro-generated human TCs can be used for studies on drug screening, as well as to understand TC-associated pathologies, such as androgen-secreting tumors and polycystic ovary syndrome. STUDY FUNDING/COMPETING INTEREST(S) This study was supported by grants from the Fonds National de la Recherche Scientifique de Belgique (FNRS) (C.A.A. is an FRS-FNRS Research Associate; grant MIS #F4535 16 awarded to C.A.A.; grant 5/4/150/5 awarded to M.M.D.; grant ASP-RE314 awarded to P.A.) and Foundation Against Cancer (grant 2018-042 awarded to A.C.). The authors declare no competing interests.

Human theca arises from ovarian stroma and is comprised of three discrete subtypes

“…Theca cells have been distinguished, mostly by morphological criteria, into two broad categories: theca externa, which provides structural support; and theca interna, which mediates the steroidogenic function of the theca, generating androgens that serve as a substrate for aromatase activity in GCs. Although developmental origins of theca have been described in mice 19 and previous studies have isolated in vitro explants of bovine 33 and human 3 , 4 theca layers, the cellular origins of TCs within the ovary, as well as their differentiation hierarchy, remain poorly understood. Here, we have used a combination of primary ovarian tissue and ovarian cortical xenografts, along with scRNA, snRNA, and scATAC sequencing, to plot the differentiation trajectory and growth kinetics of theca/stroma cells during human folliculogenesis.…”

“…Unlike GCs, which are readily accessible in an IVF setting, isolation, and culture of TCs requires direct biopsy of pre-ovulatory follicles. In addition, most studies have relied on mechanical dissociation of the theca layer from isolated antral follicles or generation of TCs from isolated ovarian stroma cells 3 , 4 , thereby yielding a heterogeneous mixture of stromal, theca, vascular and immune populations. Advances in single cell RNA sequencing (scRNASeq) can circumvent these limitations to provide high resolution snapshots of the transcriptional heterogeneity that exists within different compartments of ovarian follicles.…”

Ovarian Cell Encapsulation in an Enzymatically Crosslinked Silk-Based Hydrogel with Tunable Mechanical Properties

“…The ovaries were frozen, based on the Gallardo et al protocol [ 53 ]. After tissue thawing, stromal cells were isolated, as previously described by Asiabi et al [ 54 ]. Briefly, the tissue was minced using a tissue chopper (Campden Instruments LTD, London, UK), then transferred in DPBS with Ca 2+ and Mg 2+ (14040-091; Gibco, Paisely, UK) containing 0.28 Wünsch units/mL Liberase DH (05401089001; Sigma-Aldrich, Mannheim, Germany) and 8 Kunitz units/mL DNase (89836; Thermo Scientific, Vilnius, Lithuania).…”

Medium supplementation can influence the human ovarian cells in vitro

“…The ovarian cortical fragments were frozen using our standard method after the medullar section was removed [14]. After thawing of the ovarian samples, stromal cells were isolated following our published protocols [12,[15][16][17]. Briefly, a tissue chopper (Mickel Laboratory, the UK) set to 0.5 mm was utilized for the mechanical mincing of tissues, and then the enzymatic digestion of chopped tissues was performed by Liberase DH (05401054001; Sigma-Aldrich, Germany) and DNAse I (10,104,159,001; Sigma-Aldrich) at 37 °C for 75 minutes.…”

“…In tissue engineering, cells play a critical role in the constructed tissue. For instance, endothelial cells could accelerate vascularization, stem cells could improve regeneration, or theca cells are necessary for follicle development [1,[11][12][13]. Therefore, it is essential to identify various cell types in a tissue and their fate after culturing in different media to optimize the design of an engineered tissue.…”