In vitro production of haploid cells after coculture of CD49f+ with Sertoli cells from testicular sperm extraction in nonobstructive azoospermic patients.
OBJECTIVE
To isolate CD49f+ cells from testicular sperm extraction (TESE) samples of azoospermic patients and induce meiosis by coculturing these cells with Sertoli cells.


METHODS
Prospective analysis.


METHODS
Research center.


METHODS
Obstructive azoospermic (OA) and nonobstructive azoospermic (NOA) patients.


METHODS
TESE, with enzymatic dissociation of samples to obtain a cell suspension, which was cultured for 4 days with 4 ng/mL GDNF. The CD49f+ cells were sorted using fluorescence-activated cell sorting (FACS) as a marker to identify spermatogonial stem cells (SSCs), which were cocultured with Sertoli cells expressing red fluorescent protein (RFP) in knockout serum replacement (KSR) media with addition of 1,000 IU/mL of follicle-stimulating hormone (FSH), 1 μM testosterone, 40 ng/mL of GDNF, and 2 μM retinoic acid (RA) for 15 days in culture at 37°C and 5% CO(2) to induce meiotic progression. Cells were collected and analyzed by immunofluorescence for meiosis progression with specific markers SCP3 and CREST, and they were confirmed by fluorescence in situ hybridization (FISH).


METHODS
Isolation of CD49f+ cells and coculture with Sertoli cells, meiosis progression in vitro, assessment of SSCs and meiotic markers real-time polymerase chain reaction (RT-PCR), immunohistochemical analysis, and FISH.


RESULTS
The CD49f+ isolated from the of total cell count in the TESE samples of azoospermic patients varied from 5.45% in OA to 2.36% in NOA. Sertoli cells were obtained from the same TESE samples, and established protocols were used to characterize them as positive for SCF, rGDNF, WT1, GATA-4, and vimentin, with the presence of tight junctions and lipid droplets shown by oil red staining. After isolation, the CD49f+ cells were cocultured with RFP Sertoli cells in a 15-day time-course experiment. Positive immunostaining for meiosis markers SCP3 and CREST on days 3 to 5 was noted in the samples obtained from one NOA patient. A FISH analysis for chromosomes 13, 18, 21, X, and Y confirmed the presence of haploid cells on day 5 of the coculture.


CONCLUSIONS
In vitro coculture of SSCs from TESE samples of NOA patients along with Sertoli cells promoted meiosis induction and resulted in haploid cell generation. These results improve the existing protocols to generate spermatogenesis in vitro and open new avenues for clinical translation in azoospermic patients.
Valencia Node of the Spanish Stem Cell Bank, Prince Felipe Research Centre (CIPF), Valencia, Spain. marciariboldi@gmail.com
Marcia
M
Riboldi
Carmen
C
Rubio
Antonio
A
Pellicer
Manuel
M
Gil-Salom
Carlos
C
Simón
D053713:Azoospermia / Q000503:physiopathology / Q000628:therapy*; D002469:Cell Separation; D018920:Coculture Techniques; D006238:Haploidy; D006801:Humans; D017404:In Situ Hybridization, Fluorescence; D039503:Integrin alpha6 / Q000032:analysis*; D008297:Male; D008540:Meiosis; D011446:Prospective Studies; D012708:Sertoli Cells / Q000502:physiology*; D053625:Sperm Retrieval; D013091:Spermatogenesis; D013234:Stem Cells / Q000502:physiology*
D039503:Integrin alpha6