Document Detail


In vitro production of haploid cells after coculture of CD49f+ with Sertoli cells from testicular sperm extraction in nonobstructive azoospermic patients.
MedLine Citation:
PMID:  22732736     Owner:  NLM     Status:  Publisher    
Abstract/OtherAbstract:
OBJECTIVE: To isolate CD49f+ cells from testicular sperm extraction (TESE) samples of azoospermic patients and induce meiosis by coculturing these cells with Sertoli cells. DESIGN: Prospective analysis. SETTING: Research center. PATIENT(S): Obstructive azoospermic (OA) and nonobstructive azoospermic (NOA) patients. INTERVENTION(S): 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). MAIN OUTCOME MEASURE(S): 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. RESULT(S): 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. CONCLUSION(S): 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.
Authors:
Marcia Riboldi; Carmen Rubio; Antonio Pellicer; Manuel Gil-Salom; Carlos Simón
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Publication Detail:
Type:  JOURNAL ARTICLE     Date:  2012-6-23
Journal Detail:
Title:  Fertility and sterility     Volume:  -     ISSN:  1556-5653     ISO Abbreviation:  -     Publication Date:  2012 Jun 
Date Detail:
Created Date:  2012-6-26     Completed Date:  -     Revised Date:  -    
Medline Journal Info:
Nlm Unique ID:  0372772     Medline TA:  Fertil Steril     Country:  -    
Other Details:
Languages:  ENG     Pagination:  -     Citation Subset:  -    
Copyright Information:
Copyright © 2012 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.
Affiliation:
Valencia Node of the Spanish Stem Cell Bank, Prince Felipe Research Centre (CIPF), Valencia, Spain; Fundación IVI-Instituto Universitario IVI, Valencia, Spain.
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