Ferran Barrachina, Bruna Paulsen, Alexander D Noblett, Christian C Kramme
OBJECTIVE: Despite the substantial impact that menopause has on women’s health, there are
few available methods of treatment, with the most popular being those that treat only specific
symptoms and hormone replacement therapy (HRT), which has limited efficacy and safety. In
this study, we detail the development of a steroidogenic and hormone responsive subdermal
ovarian implant that aims to treat menopause by integrating into native signalling pathways and
restoring hormonal balance.
MATERIALS AND METHODS: First, a set of allogeneic ovarian-like cell lines were
generated using transcription factor (TF)-directed differentiation of human induced pluripotent
stem cells (hiPSCs). Candidate clones were screened for hormone responsiveness following
exposure to follicle stimulating hormone (FSH) and androstenedione (A4) by measuring
estradiol (E2) and progesterone (P4) secretion via ELISAs. After identification of a lead clone,
parallel differentiation experiments were performed with the addition of different factors, such as
IGF1, Wnt3a, BMP4, and bFGF, to optimize hormone secretion. Next, cells were encapsulated in
an alginate matrix using a microencapsulation device and assessed for long-term viability using
live/dead staining and hormone production using qPCR analysis of CYP17A1 and CYP19A1 and
E2 and P4 ELISAs. To establish an androgen source for this implant, pure A4 crystals were
generated, embedded in a matrix, and A4 release was evaluated via ELISA. Finally, encapsulated
cells and A4 crystals were loaded into a 3D printed porous resin implant, E2 and P4 release from
the implant was assessed using ELISAs, and the immunogenicity of the implant biomaterials was
evaluated by assessing immune cell activation and measuring cytokine release.
RESULTS: The ovarian-like cell lead clone GTO-101 was selected based on the highest level of
E2 and P4 secretion in response to FSH and A4, and the differentiation conditions optimized
based on hormone secretion were selected for downstream development. Live/dead staining of
encapsulated cells, qPCR analysis, and E2 and P4 ELISAs showed that cells retained both
viability and functionality following encapsulation in the alginate matrix. Next, pure A4 crystals
were generated and embedded in a matrix, and analysis of A4 release by ELISA demonstrated
slow controlled release. After integrating the encapsulated cells and A4 crystals into the
prototype implant, analysis of hormone secretion demonstrated that cells within the implant
retained functionality, based on the production E2 and P4 in response to FSH challenge;
meanwhile, in vitro immune response assays demonstrated the biocompatibility of implant
biomaterials.
CONCLUSIONS: Collectively, these data demonstrate the successful generation of a
steroidogenic ovarian implant consisting of alginate encapsulated allogeneic ovarian-like cells.
As the in vitro results show efficacy and non-immunogenicity, the next stages in development
will include pilot in vivo studies to evaluate the safety and longevity of the implant.
IMPACT STATEMENT: This study details the generation of a steroidogenic and hormone
responsive ovarian implant for the treatment of menopause.