Moderated Poster

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MP41-19: In vitro differentiation of human spermatogonial stem cells in a three dimensional (3D) testicular organoid system

Saturday, May 13
3:30 PM - 5:30 PM
Location: BCEC: Room 156

Presentation Authors: Kara E. McAbee*, Nima Pourhabibi Zarandi, Anthony Atala, Hooman Sadri-Ardekani, Colin Bishop, Winston Salem, NC

Introduction: Young boys who have undergone chemotherapy treatment are often plagued with infertility later in life. One of the expected options to generate fertility in these cancer survivors is in vitro differentiation of the patient&[prime]s own stored testicular cells, which contain spermatogonial stem cells (SSCs). Human spermatogenesis, however, is an intricate process that has not yet been replicated in vitro. Our laboratory has recently developed novel in vitro 3D human testicular organoid systems from both mature and immature testicular cells. These systems have the potential for in vitro androgen production and spermatogenesis. Our objective was to optimize the 3D testicular organoid system created from immature human testicular cells for in vitro spermatogenesis.

Methods: Human testis tissue from a 10-year-old brain dead patient was received via the National Disease Research Interchange. Immaturity of the testicular tissue was confirmed by performing Hematoxylin and Eosin (H&E) staining, Reverse Transcriptase-PCR (RT-PCR) and immunohistochemistry for undifferentiated and differentiated germ cell markers. Mechanical and enzymatic digestion was performed on tissue in order to isolate the four major testicular cell types, spermatogonia, sertoli, leydig, and peritubular cells. The cells were seeded in 2D culture in enriched StemPro medium. Cells propagated in 2D culture were then integrated into 3D organoids. Organoids were maintained initially in a medium containing bone morphogenetic proteins and retinoic acid for the first six days and then switched to a medium containing testosterone and follicle stimulating hormone for the rest of the culture period. The morphology of the organoids was examined by H&E staining. Live/dead staining and ATP assay evaluated their viability and metabolic activity. Spermatogenic differentiation within the organoids was tested by quantitative RT-PCR for protamine 1(PRM1) as a post meiotic marker.

Results: 2D testicular cell culture was established successfully and after 4 passages the cells were integrated into a 3D system. Cell viability and ATP levels in the organoids initially dropped, then both increased over the two weeks of culture. PRM1 expression increased 30 and 53-fold in the first and second weeks of 3D culture, respectively.

Conclusions: This novel 3D testicular organoid system from human immature testicular cells has the ability to differentiate SSCs to cells expressing the post meiotic marker PRM1. The next step will be to optimize the system and focus on the analysis of these potential post meiotic germ cells.

Source Of Funding: We acknowledge the use of tissues acquired from the National Disease Research Interchange and funding sources including AFIRM II, Award No. W81XWH-13-2-0052, NIH grant 5U42RR006042 and WFIRM internal funding.

Kara E. McAbee, MD

Wake Forest Baptist Health

Kara E. McAbee, MD is a current resident at Wake Forest Baptist Health. She earned her medical degree, Cum Laude, from The Ohio State University College of Medicine in 2016. She attended Miami University (OH) for undergraduate education, earning a Bachelor of Science, Magna Cum Laude with University Honors, in Kinesiology and Health/Exercise Science.

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