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Updated: Aug 6, 2026
Extraction, Labeling, and Purification of Lineage-Specific Cells from Human Antral Follicles
Published on: November 30, 2022
Isolation of theca cells from human antral follicle membranes using ANPEP-based magnetic cell sorting
Lea Bejstrup Jensen1, Malene Louise Johannsen2,3, Jane Alrø Bøtkjær4
1Laboratory of Reproductive Biology, University Hospital of Copenhagen, Rigshospitalet, Copenhagen 2100, Denmark. lea.bejstrup.jensen@regionh.dk.
Research Question:
Can ANPEP-based magnetic activated cell sorting (MACS) be used to isolate and functionally enrich human steroidogenic theca interna cells from small antral follicle membranes?
Methods:
This experimental study used ovarian tissue from eight women (mean age 33.7 years) undergoing fertility preservation. Membranes from small antral follicles (2-9 mm) were enzymatically digested and cultured for 48 h. A mixed cell sample was obtained before MACS separation into ANPEP-positive and ANPEP-negative cell fractions, followed by an additional 48-h culture. Cell purity and viability were assessed by flow cytometry (FACS) using ANPEP and FSHR antibodies. Gene expression (ANPEP, CYP17A1, LHCGR, FSHR, CYP19A1) was analyzed by RT-qPCR. Steroid secretion (pregnenolone, progesterone, 17-OH-pregnenolone, 17-OH-progesterone, DHEA, androstenedione) was quantified using LC-MS/MS.
Results:
Before cell sorting, 29.6-82.9% of viable cells expressed ANPEP. After MACS, ~ 68% of cells in the ANPEP-positive fraction expressed ANPEP, compared with ~ 3% in the negative fraction (P < 0.05). CYP17A1 expression was higher in pre-MACS and ANPEP-positive populations than in the negative fraction. ANPEP-positive cells secreted androgen precursors (17-OH-pregnenolone, 17-OH-progesterone, DHEA, androstenedione), whereas the negative fraction produced relatively more progesterone, consistent with theca cell enrichment.
Conclusions:
ANPEP-based MACS enables enrichment of viable, steroidogenic human theca interna cells. The ANPEP-positive fraction exhibits molecular and functional characteristics consistent with theca lineage and provides a useful model for studying ovarian steroidogenesis.
