PMID- 22732736
OWN - NLM
STAT- MEDLINE
DCOM- 20121107
LR  - 20220321
IS  - 1556-5653 (Electronic)
IS  - 0015-0282 (Linking)
VI  - 98
IP  - 3
DP  - 2012 Sep
TI  - In vitro production of haploid cells after coculture of CD49f+ with Sertoli cells 
      from testicular sperm extraction in nonobstructive azoospermic patients.
PG  - 580-590.e4
LID - 10.1016/j.fertnstert.2012.05.039 [doi]
AB  - 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 
      muM testosterone, 40 ng/mL of GDNF, and 2 muM retinoic acid (RA) for 15 days in 
      culture at 37 degrees 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.
CI  - Copyright (c) 2012 American Society for Reproductive Medicine. Published by 
      Elsevier Inc. All rights reserved.
FAU - Riboldi, Marcia
AU  - Riboldi M
AD  - Valencia Node of the Spanish Stem Cell Bank, Prince Felipe Research Centre 
      (CIPF), Valencia, Spain. marciariboldi@gmail.com
FAU - Rubio, Carmen
AU  - Rubio C
FAU - Pellicer, Antonio
AU  - Pellicer A
FAU - Gil-Salom, Manuel
AU  - Gil-Salom M
FAU - Simon, Carlos
AU  - Simon C
LA  - eng
PT  - Journal Article
DEP - 20120623
PL  - United States
TA  - Fertil Steril
JT  - Fertility and sterility
JID - 0372772
RN  - 0 (Integrin alpha6)
SB  - IM
MH  - Azoospermia/physiopathology/*therapy
MH  - Cell Separation
MH  - Coculture Techniques
MH  - *Haploidy
MH  - Humans
MH  - In Situ Hybridization, Fluorescence
MH  - Integrin alpha6/*analysis
MH  - Male
MH  - Meiosis
MH  - Prospective Studies
MH  - Sertoli Cells/*physiology
MH  - Sperm Retrieval
MH  - *Spermatogenesis
MH  - Stem Cells/*physiology
EDAT- 2012/06/27 06:00
MHDA- 2012/11/08 06:00
CRDT- 2012/06/27 06:00
PHST- 2011/12/08 00:00 [received]
PHST- 2012/04/15 00:00 [revised]
PHST- 2012/05/25 00:00 [accepted]
PHST- 2012/06/27 06:00 [entrez]
PHST- 2012/06/27 06:00 [pubmed]
PHST- 2012/11/08 06:00 [medline]
AID - S0015-0282(12)00593-6 [pii]
AID - 10.1016/j.fertnstert.2012.05.039 [doi]
PST - ppublish
SO  - Fertil Steril. 2012 Sep;98(3):580-590.e4. doi: 10.1016/j.fertnstert.2012.05.039. 
      Epub 2012 Jun 23.