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      Generation of Haploid Spermatids with Fertilization and Development Capacity from Human Spermatogonial Stem Cells of Cryptorchid Patients

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          Summary

          Generation of functional spermatids from azoospermia patients is of unusual significance in the treatment of male infertility. Here, we report an efficient approach to obtain human functional spermatids from cryptorchid patients. Spermatogonia remained whereas meiotic germ cells were rare in cryptorchid patients. Expression of numerous markers for meiotic and postmeiotic male germ cells was enhanced in human spermatogonial stem cells (SSCs) of cryptorchidism patients by retinoic acid (RA) and stem cell factor (SCF) treatment. Meiotic spreads and DNA content assays revealed that RA and SCF induced a remarkable increase of SCP3-, MLH1-, and CREST-positive cells and haploid cells. Single-cell RNA sequencing analysis reflected distinct global gene profiles in embryos derived from round spermatids and nuclei of somatic cells. Significantly, haploid spermatids generated from human SSCs of cryptorchid patients possessed fertilization and development capacity. This study thus provides an invaluable source of autologous male gametes for treating male infertility in azoospermia patients.

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          Highlights

          • Spermatogonia remain whereas meiotic male germ cells are rare in cryptorchid patients

          • Human SSCs of cryptorchid patients differentiate into phenotypic haploid spermatids

          • Round spermatids derived from human SSCs have fertilization and development capacity

          • Distinct gene profiles exist in embryos from round spermatid and somatic cell nuclei

          Abstract

          He, Li, and colleagues showed that spermatogonia remained whereas meiotic germ cells were rare or lost in cryptorchid patients. SSCs of cryptorchid patients were induced to differentiate into cells with phenotypic, DNA content, and fertilization and development potentials of haploid spermatids. These data demonstrate the generation of functional and autologous male gametes for treating male infertility in azoospermia patients.

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          Reconstitution of the mouse germ cell specification pathway in culture by pluripotent stem cells.

          The generation of properly functioning gametes in vitro requires reconstitution of the multistepped pathway of germ cell development. We demonstrate here the generation of primordial germ cell-like cells (PGCLCs) in mice with robust capacity for spermatogenesis. PGCLCs were generated from embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) through epiblast-like cells (EpiLCs), a cellular state highly similar to pregastrulating epiblasts but distinct from epiblast stem cells (EpiSCs). Reflecting epiblast development, EpiLC induction from ESCs/iPSCs is a progressive process, and EpiLCs highly competent for the PGC fate are a transient entity. The global transcription profiles, epigenetic reprogramming, and cellular dynamics during PGCLC induction from EpiLCs meticulously capture those associated with PGC specification from the epiblasts. Furthermore, we identify Integrin-β3 and SSEA1 as markers that allow the isolation of PGCLCs with spermatogenic capacity from tumorigenic undifferentiated cells. Our findings provide a paradigm for the first step of in vitro gametogenesis. Copyright © 2011 Elsevier Inc. All rights reserved.
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            Derivation of oocytes from mouse embryonic stem cells.

            Continuation of mammalian species requires the formation and development of the sexually dimorphic germ cells. Cultured embryonic stem cells are generally considered pluripotent rather than totipotent because of the failure to detect germline cells under differentiating conditions. Here we show that mouse embryonic stem cells in culture can develop into oogonia that enter meiosis, recruit adjacent cells to form follicle-like structures, and later develop into blastocysts. Oogenesis in culture should contribute to various areas, including nuclear transfer and manipulation of the germ line, and advance studies on fertility treatment and germ and somatic cell interaction and differentiation.
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              Human DAZL, DAZ and BOULE genes modulate primordial germ cell and haploid gamete formation

              The leading cause of infertility in men and women is quantitative and qualitative defects in human germ cell (oocyte and sperm) development. Yet, it has not been possible to examine the unique developmental genetics of human germ cell formation and differentiation due to inaccessibility of germ cells during fetal development. Although several studies have shown that germ cells can be differentiated from mouse and human embryonic stem cells, human germ cells differentiated in these studies generally did not develop beyond the earliest stages1-8. Here we used a germ cell reporter to quantitate and isolate primordial germ cells derived from both male and female hESCs. Then, by silencing and overexpressing genes that encode germ cell-specific cytoplasmic RNA-binding proteins (not transcription factors), we modulated human germ cell formation and developmental progression. We observed that human DAZL (Deleted in AZoospermia-Like) functions in primordial germ cell formation, whereas closely-related genes, DAZ and BOULE, promote later stages of meiosis and development of haploid gametes. These results are significant to the generation of gametes for future basic science and potential clinical applications.
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                Author and article information

                Contributors
                Journal
                Stem Cell Reports
                Stem Cell Reports
                Stem Cell Reports
                Elsevier
                2213-6711
                18 September 2014
                18 September 2014
                14 October 2014
                : 3
                : 4
                : 663-675
                Affiliations
                [1 ]Department of Urology, Shanghai Human Sperm Bank, Shanghai Institute of Andrology, Ren Ji Hospital, School of Medicine, Shanghai Jiao Tong University, 845 Linshan Road, Shanghai 200135, China
                [2 ]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, 1630 Dongfang Road, Shanghai 200127, China
                [3 ]Shanghai Key Laboratory of Reproductive Medicine, Shanghai 200025, China
                [4 ]Shanghai Key Laboratory of Assisted Reproduction and Reproductive Genetics, Shanghai 200001, China
                Author notes
                []Corresponding author lizhengboshi@ 123456163.com
                [∗∗ ]Corresponding author zupinghe@ 123456sjtu.edu.cn
                Article
                S2213-6711(14)00258-6
                10.1016/j.stemcr.2014.08.004
                4223697
                25358793
                92bdcd18-f42c-4542-bde5-f2d81ad7747a
                © 2014 The Authors

                This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/3.0/).

                History
                : 27 June 2014
                : 4 August 2014
                : 5 August 2014
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