In Vitro Production of Haploid Cells after Coculture of CD49f+ with Sertoli Cells from testicular sperm extraction in Nonobstructive Azoospermic Patients

Capsule:
In vitro co-culture of CD49f+ with Sertoli cells from TESE, obtained in azoospermic patients, promotes meiosis progression as demonstrated by SCP3 and CREST staining and generation of haploid cells.

Authors:
Marcia Riboldi, B.Sc., Carmen Rubio, Ph.D., Antonio Pellicer, M.D., Ph.D., Manuel Gil-Salom, Ph.D., Carlos Simón, M.D., Ph.D.

Volume 98, Issue 3, Pages 580-590.e4, September 2012

Abstract:

Objective:
To isolation of CD49f+ cells from TESE performed in azoospermic patients and induction of meiosis.

Design:
Prospective analysis

Setting:
Research center

Patients:
TESE was performed in OA and NOA patients

Interventions:
TESE was used to obtain a cells and cultured with GDNF.CD49f+ cells were sorted and co-cultured with RFP Sertoli cells in KSR media with FSH, Testosterone, GDNF and Retinoic Acid for 15 days in culture to induce meiotic progression.Cells were collected and analysed by immunofluorescence for meiosis progression (markers SCP3 and CREST) and were confirmed by FISH.

Main Outcome Measurements:
CD49f+ cells were co-culture with Sertoli cells for meiosis progression in vitro. SSCs and meiotic markers were assessed by RT-PCR, immunohistochemistry and FISH.

Results:
CD49f+ isolated from the TESE varied from 5.45% in OA to 2.36% in NOA.Sertoli cells were obtained and characterised as positive for SCF, rGDNF, WT1, GATA-4, Vimentin, presence of tight junctions and lipid droplets. After, CD49f+ cells were co-cultured with RFP Sertoli cells.Positive immunostaining for meiosis markers SCP3 and CREST on days 3 to 5 was noted in the samples obtained from a NOA patient. FISH analysis for chromosomes 13,18,21,X,Y confirmed haploid cells on day 5.

Conclusions:
In vitro co-culture of SSCs from the TESE done in NOA patients with Sertoli cells promoted meiosis induction and resulted in haploid cells. These results improve existing protocols to generate spermatogenesis in vitro and open new avenues for clinical translation in azoospermic patients.

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