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Mei X.-X.,Shanghai JiaoTong University | Wang J.,Shanghai JiaoTong University | Wu J.,Shanghai JiaoTong University | Wu J.,Ningxia Medical University | Wu J.,Shanghai Key Laboratory of Reproductive Medicine
Asian Journal of Andrology | Year: 2015

Spermatogonial stem cells (SSCs), the stem cells responsible for male fertility, are one of a small number of cells with the abilities of both self-renewal and generation of large numbers of haploid cells. Technology improvements, most importantly, transplantation assays and in vitro culture systems have greatly expanded our understanding of SSC self-renewal and differentiation. Many important molecules crucial for the balance between self-renewal and differentiation have been recently identified although the exact mechanism(s) remain largely undefined. In this review, we give a brief introduction to SSCs, and then focus on extrinsic and intrinsic factors controlling SSCs self-renewal and differentiation. © 2015 AJA, SIMM & SJTU. All rights reserved 1008-682X. Source


Zhang C.,Shanghai JiaoTong University | Wu J.,Shanghai JiaoTong University | Wu J.,Ningxia Medical University | Wu J.,Shanghai Key Laboratory of Reproductive Medicine
Molecular Human Reproduction | Year: 2016

Study hypothesis:We investigated whether DEAD-box polypeptide 4 (DDX4) positive cells from post-natal ovaries of germline lineage reporter mice can be isolated based on endogenously expressed fluorescent proteins and used to establish a cell line for producing offspring. study finding: DDX4-positive cells frompost-natal ovaries of germline lineage reporter mice can be isolated and used to establish a cell line for producing offspring. what is known already: In recent years, female germline stem cells (FGSCs) have been isolated from the ovaries of post-natal mice by magnetic-activated cell sorting or fluorescence-activated cell sorting (FACS) relying on an antibody against DDX4. However, whether DDX4- positive cells frompost-natal ovaries of germline lineage reporter mice can be established without using an antibody, as well as a cell line established for producing offspring, remains unknown. study design, samples/materials, methods: To obtain the expected offspring (Ddx4-Cre;mT/mGmice), Ddx4-Cre micewere crossed withmT/mGmice. In the ovaries of Ddx4-Cre;mT/mGmice, germ cellswere destined to express enhanced green fluorescent protein (EGFP) while somatic cells still express tandem dimer Tomato (tdTomato). Therefore, the germ cells could be clearly distinguished from somatic cells by fluorescent proteins. Then, we investigated the pattern of fluorescent cells in the ovaries of 21-day-old Ddx4-Cre;mT/mG mice under a fluorescent microscope.Germ cells were sorted by FACS without using antibody and used to establish a FGSC line. The FGSC line was analyzed by DDX4 immunostaining, Edu (5-ethynyl-2'-deoxyuridine) labeling, and RT-PCR for germ cell markers. Finally, the physiological function of the FGSC line was examined by transplanting FGSCs into the ovaries of sterilized recipients and subsequent mating. main results and the role of chance: Firstly, we have successfully isolated FGSCs from the ovaries of 21-day-old Ddx4- Cre;mT/mG mice based on endogenously expressed fluorescent proteins. FACS was used to separate the cells and 2.3% of all viable cells was EGFP-positive germ cells. Subsequently, a FGSC line was established that was doubly positive for DDX4 immunostaining and Edu labeling. The mRNA expression of several germ cell markers in this cell line, such as Ddx4, Deleted in azoospermia-like (Dazl), B lymphocyte-induced maturation protein-1 (Blimp1), Stella and Fragilis,was detected. Lastly, the FGSClinewas proven to be functional under physiological conditions, as offspringwere produced after transplanting FGSCs into ovaries of sterilized recipients and a subsequent mating. limitations, reasons for caution: The molecular mechanisms of proliferation and differentiation of FGSCs in vivo and in vitro still need to be elucidated. wider implications of the findings: Our results confirm that DDX4-positive cells can be separated from post-natal mouse ovaries and used to establish cell lines that are functional in producing off spring, and provide further evidence for the existence of post-natal FGSCs in mammals. The Ddx4-Cre;mT/mG mouse strain is an ideal model for the isolation, characterization and propagation of FGSCs and is a useful tool for fully elucidating the molecular mechanisms of proliferation and differentiation of FGSCs in vivo and in vitro. large scale data: None. study funding and competing interest(s): This work was supported by National Basic Research Program of China (2013CB967401) and the National Nature Science Foundation of China (81370675, 81200472 and 81421061). The authors declare no competing interests. © The Author 2016. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved. Source


Hai Y.,Shanghai JiaoTong University | Hou J.,Shanghai JiaoTong University | Liu Y.,Shanghai JiaoTong University | Yang H.,Shanghai JiaoTong University | And 4 more authors.
Seminars in Cell and Developmental Biology | Year: 2014

Spermatogenesis is a complex process by which spermatogonial stem cells (SSCs) self-renew and differentiate into spermatozoa under the elaborate coordination of testicular microenvironment, namely, niche. Sertoli cells, which locate around male germ cells, are the most critical component of the niche. Significant progress has recently been made by peers and us on uncovering the effects of Sertoli cells on regulating fate determinations of SSCs. Here we addressed the roles and regulation of Sertoli cells in normal and abnormal spermatogenesis. Specifically, we summarized the biological characteristics of Sertoli cells, and we emphasized the roles of Sertoli cells in mediating the self-renewal, differentiation, apoptosis, de-differentiation, and trans-differentiation of SSCs. The association between abnormal function of Sertoli cells and impaired spermatogenesis was discussed. Finally, we highlighted several issues to be addressed for further investigation on the effects and mechanisms of Sertoli cells in spermatogenesis. Since Sertoli cells are the key supportive cells for SSCs and they are very receptive to modification, a better understanding of the roles and regulation of Sertoli cells in SSC biology and spermatogenesis would make it feasible to identify novel targets for gene therapy of male infertility as well as seek more efficient and safer strategies for male contraception. © 2014 Elsevier Ltd. Source


Guo Y.,Shanghai JiaoTong University | Hai Y.,Shanghai JiaoTong University | Gong Y.,Shanghai JiaoTong University | Li Z.,Shanghai JiaoTong University | And 3 more authors.
Journal of Cellular Physiology | Year: 2014

Spermatogenesis is a special process by which spermatogonial stem cells (SSCs) divide and differentiate to male gametes called mature spermatozoa. SSCs are the unique cells because they are adult stem cells that transmit genetic information to subsequent generations. Accumulating evidence has demonstrated that SSCs can be reprogrammed to acquire pluripotency to become embryonic stem-like cells that differentiate into all cell lineages of the three germ layers, highlighting potential important applications of SSCs for regenerative medicine. Recent studies from peers and us have made great achievements on the characterization, isolation, and culture of mouse and human SSCs, which could lead to better understanding the biology of SSCs and the applications of SSCs in both reproductive and regenerative medicine. In this review, we first compared the cell identity and biochemical phenotypes between mouse SSCs and human SSCs. Notably, the cell types of mouse and human SSCs are distinct, and human SSCs share some but not all phenotypes with mouse SSCs. The approaches for isolating SSCs as well as short- and long-term culture of mouse SSCs and short-period culture of human SSCs were also discussed. We further addressed the new advances on the self-renewal of SSCs with an aim to establish the long-term culture of human SSCs which has not yet been achieved. © 2013 Wiley Periodicals, Inc. Source


Zhang Z.,Shanghai JiaoTong University | Gong Y.,Shanghai JiaoTong University | Guo Y.,Shanghai JiaoTong University | Hai Y.,Shanghai JiaoTong University | And 9 more authors.
Cell Communication and Signaling | Year: 2013

Background: Severe shortage of liver donors and hepatocytes highlights urgent requirement of extra-liver and stem cell source of hepatocytes for treating liver-related diseases. Here we hypothesized that spermatogonial stem cells (SSCs) can directly transdifferentiate to hepatic stem-like cells capable of differentiating into mature hepatocyte-like cells in vitro without an intervening pluripotent state. Results: SSCs first changed into hepatic stem-like cells since they resembled hepatic oval cells in morphology and expressed Ck8, Ck18, Ck7, Ck19, OV6, and albumin. Importantly, they co-expressed CK8 and CK19 but not ES cell markers. Hepatic stem-like cells derived from SSCs could differentiate into small hepatocytes based upon their morphological features and expression of numerous hepatic cell markers but lacking of bile epithelial cell hallmarks. Small hepatocytes were further coaxed to differentiate into mature hepatocyte-like cells, as identified by their morphological traits and strong expression of Ck8, Ck18, Cyp7a1, Hnf3b, Alb, Tat, Ttr, albumin, and CYP1A2 but not Ck7 or CK19. Notably, these differentiated cells acquired functional attributes of hepatocyte-like cells because they secreted albumin, synthesized urea, and uptake and released indocyanine green. Moreover, phosphorylation of ERK1/2 and Smad2/3 rather than Akt was activated in hepatic stem cells and mature hepatocytes. Additionally, cyclin A, cyclin B and cyclin E transcripts and proteins but not cyclin D1 or CDK1 and CDK2 transcripts or proteins were reduced in mature hepatocyte-like cells or hepatic stem-like cells derived from SSCs compared to SSCs. Conclusions: SSCs can transdifferentiate to hepatic stem-like cells capable of differentiating into cells with morphological, phenotypic and functional characteristics of mature hepatocytes via the activation of ERK1/2 and Smad2/3 signaling pathways and the inactivation of cyclin A, cyclin B and cyclin E. This study thus provides an invaluable source of mature hepatocytes for treating liver-related diseases and drug toxicity screening and offers novel insights into mechanisms of liver development and cell reprogramming. © 2013 Zhang et al.; licensee BioMed Central Ltd. Source

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