- Cells were collected from obstructive azoospermic men with normal morphology that indicates normal in-progress spermatogenesis in these men.
- Spermatogonia appear to be round and high nuclei/cytoplasm ratio.
Flow cytometry:
- 13.3 % of the cells express SSEA-4 on their surface.
Other spermatogonial markers used in this study were CD49f (alpha-6-integrin ITGA6), CD90 (Thy-1), CD117 (c-Kit) and in combination CD49f+ CD90+ and CD1172 (Triple Stain).
=> In the adult human testes, 25 % of cells express CD49f and 13 % were CD90 positive; and <1 cells="" font="" found="" human="" in="" nbsp="" of="" population="" stained="" the="" triple="" was="">testes.
Immunohistochemistry:
- Colocalized Thy-1 and VASA in germ cells are only at the luminal part of the tubules, peritubules and interstitial cells, but not in the seminiferous tubules
- 88.3% of SSEA4 positive cells co-localized with ITGA6 and 100% SSEA4-positive cells were co-localized with VASA, but only 50% ITAG6-positive cells were co-localized with VASA. This indicated that ITAG6 is also expressed at the surface of the testicular somatic cells. Almost all ITGA6 cells do not co-localized with Thy-1.
- Only 25% of SSEA4-positive cells were co-localized with c-KIT => 2 SSEA4-positive populations in adult testis.
- ITAG6-positiive and c-KIT-positive cells were at the basement membrane of seminiferous tubules.
- LH-r is expressed only in the somatic cells and not germ cells in adult testes.
- After FACS sorting for SSEA4-positive cells, they did immunohistochemical staining for GFRa1 and PLZF. There was a 6.5x enrichment of SSCs positive for GFRa1 and 5.0x enrichment of SSCs positive for PLZF in the SSEA4-positive fraction compared to unsorted cells.
Real-time PCRs:
- c-Kit, GFRa-1, PLZF, c-RET and GPR-125 were expressed at least 3-fold and up to 7-fold greater in the SSEA4 positive population. Moreover, higher expression level of h-TERT in SSEA-4 sorted cells indicates their high level of telomerase activity and their proliferation capability. Unsorted cells showed only 10% telomerase activity while sorted cells in SSEA4-postive fraction showed 54% telomerase activity compared to hESCs (100%).
SSC transplantation:
SSEA4 MACS showed an enrichment of 40-50 folds for human SSC population, because 6.8HNP+ cells were found per tubule in mice transplanted with SSEA4 MACS sorted cells while it was only 0.15 HNP+ cells in mice transplanted with unsorted cells.
Using HNP with SSEA4, ITGA1, and c-KIT, they found that in the population of cells positive for HNP, 14% SSEA4+, 28% ITGA1+, 50% dimly c-KIT+. In addition, only a small population of cells positive for SSEA4 were c-KIT+, while 96% of SSEA4+ cells were ITAG1+.
43% of HNP+ cells co-localized with GPR-125, indicating that GPR-125 is expressed at the surface of a population of repopulating human SSCs.
20-30% of HNP cells were co-localized with Nanog, showing that 30% HNP cells repopulating mouse testes might have pluripotent characteristics
Co-localization of SSEA-4 with Nanog showed that only some of the SSEA-4 positive cells in the mouse testes express Nanog.
50% of the SSEA-4+ cells repopulating in the mouse testes co-localized GPR-125 indicating that GPR-125+ SSEA-4+ cells and GRP-125+ SSEA-4- cells are both repopulating spermatogonia.
Percentage of co-localization with HNP after human testis cell transplantation into mouse:
VASA 100
CD117 (c-kit) 49.8
GPR-125 42.8
LH-R 0
CD49f 28.1
CD29 0
SSEA-4 14.2
Nanog 28.3
Oct-4 21
TRA1-60 0
Repopulating SSCs in the adult human testes have phenotypic characteristics of SSEA4+,
ITAG6+, CD90+, GPR-125+ and c-Kit neg/low.
Rodent SSCs are only definitively identified by their ability to produce spermatogenesis when transplanted into the testes of infertile recipient mice
Rodents:
Although no SSC specific marker in rodents has been identified some markers that are expressed
by stem and/or progenitor cells have been described (e.g. GFRα 1, POU3F1, POU5F1
(OCT4), ZBTB16 (PLZF), NGN3, NANOS2, NANOS3, SOHLH1, SOHLH2, FOXO1,
ITGA6 (α 6-integrin, CD49f), LIN28, ID4, UTF1, CDH1, GPR125, ITGB1 (β 1-integrin,
CD29), EPCAM (CD326), CD9 and THY1 (CD90).
Mouse spermatogonia:
ITGA6+, ITGB1+ , THY1+, CD9+ , GFRα1+, KIT (cKIT, CD117)−, mitochondrial membrane potential high, Rhodamine 123 (Rho123) low , ITGAV (α5-Integrin, CD51)−, MHC-I− ALDH (aldehyde dehydrogenase) activity−, and CD45−.
Human:
Spermatogonia on the basement membrane of human seminiferous tubules have the phenotype of SALL4+ , ZBTB16+, UTF1+, UCHL1+, and ENO2+. The majority of cells that express SALL4, ZBTB16, UTF1, UCHL1 and ENO2, do not express the differentiation marker KIT.
Germ cell marker: VASA basement membrane
Stem cell marker: SSEA4 surface marker, basement membrane
NANOG, POU5F1, c-Myc, SOX2, KLF4, LIN28
Spermatogonia: ITAG6 (both differentiated and undifferentiated spermatogonia), basement membrane and some of the cells outside of the seminiferous tubules
Differentiated spermatogonia: c-KIT, some in basement membrane and some in luminal compartment of seminiferous tubules. Many contradictory sources studying c-KIT localization.
SSC marker: GPR-125, PLZF, UCH-L1, GFRa1 (receptor for GDNF)
Human cells: HNP
