title: Expansion and long-term culture of human spermatogonial stem cells via the activation of SMAD3 and AKT pathways.
abstract: Spermatogonial stem cells (SSCs) can differentiate into spermatids, reflecting that they could be used in reproductive medicine for treating male infertility. SSCs are able to become embryonic stem-like cells with the potentials of differentiating into numerous cell types of the three germ layers and they can transdifferentiate to mature and functional cells of other lineages, highlighting significant applications of human SSCs for treating human diseases. However, human SSCs are very rare and a long-term culture system of human SSCs has not yet established. This aim of study was to isolate, identify and culture human SSCs for a long period. We isolated GPR125-positive spermatogonia with high purity and viability from adult human testicular tissues utilizing the two-step enzymatic digestion and magnetic-activated cell sorting with antibody against GPR125. These freshly isolated cells expressed a number of markers for SSCs, including GPR125, PLZF, GFRA1, RET, THY1, UCHL1 and MAGEA4, but not the hallmarks for spermatocytes and spermatozoa, e.g. SYCP1, SYCP3, PRM1, and TNP1. The isolated human SSCs could be cultured for two months with a significant increase of cell number with the defined medium containing growth factors and hydrogel. Notably, the expression of numerous SSC markers was maintained during the cultivation of human SSCs. Furthermore, SMAD3 and AKT phosphorylation was enhanced during the culture of human SSCs. Collectively, these results suggest that human SSCs can be cultivated for a long period and expanded whilst retaining an undifferentiated status via the activation of SMAD3 and AKT pathways. This study could provide sufficient cells of SSCs for their basic research and clinic applications in reproductive and regenerative medicine.
authors.affiliation: State Key Laboratory of Oncogenes and Related Genes, Renji-Med X Clinical Stem Cell Research Center, Ren Ji Hospital, School of Medicine, Shanghai Jiao Tong University, 160 Pujian Road, Shanghai 200127, China.
authors.forename: Ying
authors.initials: Y
authors.lastname: Guo
authors.affiliation: State Key Laboratory of Oncogenes and Related Genes, Renji-Med X Clinical Stem Cell Research Center, Ren Ji Hospital, School of Medicine, Shanghai Jiao Tong University, 160 Pujian Road, Shanghai 200127, China.
authors.forename: Linhong
authors.initials: L
authors.lastname: Liu
authors.affiliation: State Key Laboratory of Oncogenes and Related Genes, Renji-Med X Clinical Stem Cell Research Center, Ren Ji Hospital, School of Medicine, Shanghai Jiao Tong University, 160 Pujian Road, Shanghai 200127, China.
authors.forename: Min
authors.initials: M
authors.lastname: Sun
authors.affiliation: State Key Laboratory of Oncogenes and Related Genes, Renji-Med X Clinical Stem Cell Research Center, Ren Ji Hospital, School of Medicine, Shanghai Jiao Tong University, 160 Pujian Road, Shanghai 200127, China.
authors.forename: Yanan
authors.initials: Y
authors.lastname: Hai
authors.affiliation: Department of Urology, Shanghai Institute of Andrology, Ren Ji Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai Human Sperm Bank, 145 Shangdong Road, Shanghai 200001, China.
authors.forename: Zheng
authors.initials: Z
authors.lastname: Li
authors.affiliation: State Key Laboratory of Oncogenes and Related Genes, Renji-Med X Clinical Stem Cell Research Center, Ren Ji Hospital, School of Medicine, Shanghai Jiao Tong University, 160 Pujian Road, Shanghai 200127, China Department of Urology, Shanghai Institute of Andrology, Ren Ji Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai Human Sperm Bank, 145 Shangdong Road, Shanghai 200001, China Shanghai Key Laboratory of Assisted Reproduction and Reproductive Genetics, Shanghai 200127, China Shanghai Key Laboratory of Reproductive Medicine, Shanghai 200025, China zupinghe@sjtu.edu.cn.
authors.forename: Zuping
authors.initials: Z
authors.lastname: He
mesh_terms: D000328:Adult; D000943:Antigens, Differentiation / Q000096:biosynthesis; D018929:Cell Culture Techniques; D005786:Gene Expression Regulation / Q000502:physiology*; D006801:Humans; D008297:Male; D051057:Proto-Oncogene Proteins c-akt / Q000378:metabolism*; D015398:Signal Transduction / Q000502:physiology*; D051900:Smad3 Protein / Q000378:metabolism*; D013093:Spermatogonia / Q000166:cytology / Q000378:metabolism
chemical_list: D000943:Antigens, Differentiation; C497790:SMAD3 protein, human; D051900:Smad3 Protein; D051057:Proto-Oncogene Proteins c-akt
keywords: Human spermatogonial stem cells; SMAD3 and AKT pathways; expansion; isolation and characterization; long-term culture