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Shi L, Khan MZ, Ullah A, Liang H, Geng M, Akhtar MF, Na J, Han Y, Wang C. Advancements in Stem Cell Applications for Livestock Research: A Review. Vet Sci 2025; 12:397. [PMID: 40431490 PMCID: PMC12115878 DOI: 10.3390/vetsci12050397] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/13/2025] [Revised: 04/16/2025] [Accepted: 04/20/2025] [Indexed: 05/29/2025] Open
Abstract
Stem cells (SCs), distinguished by their capacity for self-renewal and multipotent differentiation, represent a cornerstone of regenerative medicine. These cells, which can be categorized according to their differentiation potential and developmental origin, have emerged as pivotal elements in both biomedical research and veterinary science. In herbivorous species, stem cell applications have yielded particularly promising advances across multiple domains, including reproductive biotechnology, tissue engineering and regeneration, therapeutic interventions, and immunomodulation. This review synthesizes contemporary research on stem cell biology in five economically significant herbivorous species: bovine, ovine, deer, equine, and camelid. Special emphasis is placed on stem cell isolation methodologies, culture optimization techniques, and the molecular mechanisms governing key signaling pathways. The discussion encompasses both the technical impediments facing stem cell research and the ethical framework necessary for responsible scientific advancement, with particular attention to animal welfare considerations in the development and implementation of stem cell-based technologies.
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Affiliation(s)
- Limeng Shi
- Liaocheng Research Institute of Donkey High-Efficiency Breeding and Ecological Feeding, College of Agriculture and Biology, Liaocheng University, Liaocheng 252000, China (M.Z.K.)
| | - Muhammad Zahoor Khan
- Liaocheng Research Institute of Donkey High-Efficiency Breeding and Ecological Feeding, College of Agriculture and Biology, Liaocheng University, Liaocheng 252000, China (M.Z.K.)
| | - Abd Ullah
- Liaocheng Research Institute of Donkey High-Efficiency Breeding and Ecological Feeding, College of Agriculture and Biology, Liaocheng University, Liaocheng 252000, China (M.Z.K.)
| | - Huili Liang
- Liaocheng Research Institute of Donkey High-Efficiency Breeding and Ecological Feeding, College of Agriculture and Biology, Liaocheng University, Liaocheng 252000, China (M.Z.K.)
| | - Mingyang Geng
- Yili Kazak Autonomous Prefecture Livestock General Station, Xinjiang Autonomous Region, Yili 835000, China
| | - Muhammad Faheem Akhtar
- Liaocheng Research Institute of Donkey High-Efficiency Breeding and Ecological Feeding, College of Agriculture and Biology, Liaocheng University, Liaocheng 252000, China (M.Z.K.)
| | - Jincheng Na
- Yili Kazak Autonomous Prefecture Livestock General Station, Xinjiang Autonomous Region, Yili 835000, China
| | - Ying Han
- Liaocheng Research Institute of Donkey High-Efficiency Breeding and Ecological Feeding, College of Agriculture and Biology, Liaocheng University, Liaocheng 252000, China (M.Z.K.)
| | - Changfa Wang
- Liaocheng Research Institute of Donkey High-Efficiency Breeding and Ecological Feeding, College of Agriculture and Biology, Liaocheng University, Liaocheng 252000, China (M.Z.K.)
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Tarulli GA, Tatt PRS, Howlett R, Ord S, Frankenberg SR, Pask AJ. Enrichment of spermatogonial stem cells and staging of the testis cycle in a dasyurid marsupial, the fat-tailed dunnart. Stem Cells 2025; 43:sxaf007. [PMID: 39943734 PMCID: PMC11976394 DOI: 10.1093/stmcls/sxaf007] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/11/2023] [Accepted: 12/23/2024] [Indexed: 04/09/2025]
Abstract
There is increasing interest in the use of marsupial models in research, for use in next-generation conservation by improving fitness through genetic modification, and in de-extinction efforts. Specifically, this includes dasyurid marsupials such as the Thylacine, Tasmanian devil, quolls, and the small rodent-like dunnarts. Technologies for generating genetically modified Australian marsupials remain to be established. Given the need to advance research in this space, the fat-tailed dunnart (Sminthopsis crassicaudata) is being established as a model for marsupial spermatogonial stem cell isolation, modification, and testicular transplantation. This species is small (60-90 mm body size), polyovulatory (8-12 pups per birth), and can breed in standard rodent facilities when housed in a 12:12 light cycle. To develop the fat-tailed dunnart as a model for next-generation marsupial conservation, this study aimed to enrich dunnart spermatogonial stem cells from whole testis digestions using a fluorescent dye technology and fluorescence-activated cell sorting. This approach is not dependent on antibodies or genetic reporter animals that are limiting factors when performing cell sorting on species separated from humans and mice by large evolutionary timescales. This study also assessed the development of spermatogonia and spermatogenesis in the fat-tailed dunnart, by making the first definition of the cycle of the seminiferous epithelium in any dasyurid. Overall, this is the first detailed study to assess the cycle of dasyurid spermatogenesis and provides a valuable method to enrich marsupial spermatogonial stem cells for cellular, functional, and molecular analysis.
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Affiliation(s)
- Gerard A Tarulli
- TIGRR Lab, School of BioSciences, The University of Melbourne, Parkville, Vic., Australia
| | - Patrick R S Tatt
- TIGRR Lab, School of BioSciences, The University of Melbourne, Parkville, Vic., Australia
| | - Rhys Howlett
- TIGRR Lab, School of BioSciences, The University of Melbourne, Parkville, Vic., Australia
| | - Sara Ord
- Colossal Biosciences, Dallas, TX, United States
| | - Stephen R Frankenberg
- TIGRR Lab, School of BioSciences, The University of Melbourne, Parkville, Vic., Australia
| | - Andrew J Pask
- TIGRR Lab, School of BioSciences, The University of Melbourne, Parkville, Vic., Australia
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Lee Y, Lee SW, Jeong D, Lee HJ, Choi NY, Bang JS, Ham S, Ko K. Inhibition of Class I Histone Deacetylase Enhances Self-Reprogramming of Spermatogonial Stem Cells into Pluripotent Stem Cells. Int J Stem Cells 2022; 16:27-35. [PMID: 36581367 PMCID: PMC9978831 DOI: 10.15283/ijsc22110] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/05/2022] [Revised: 08/08/2022] [Accepted: 09/21/2022] [Indexed: 12/31/2022] Open
Abstract
Background and Objectives Spermatogonial stem cells (SSCs) are the most primitive cells in spermatogenesis and are the only adult stem cells capable of passing on the genome of a given species to the next generation. SSCs are the only adult stem cells known to exhibit high Oct4 expression and can be induced to self-reprogram into pluripotent cells depending on culture conditions. Epigenetic modulation is well known to be involved in the induction of pluripotency of somatic cells. However, epigenetic modulation in self-reprogramming of SSCs into pluripotent cells has not been studied. Methods and Results In this study, we examined the involvement of epigenetic modulation by assessing whether self-reprogramming of SSCs is enhanced by treatment with epigenetic modulators. We found that second-generation selective class I HDAC inhibitors increased SSC reprogramming efficiency, whereas non-selective HDAC inhibitors had no effect. Conclusions We showed that pluripotent stem cells derived from adult SSCs by treatment with small molecules with epigenetic modulator functions exhibit pluripotency in vitro and in vivo. Our results suggest that the mechanism of SSC reprogramming by epigenetic modulator can be used for important applications in epigenetic reprogramming research.
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Affiliation(s)
- Yukyeong Lee
- Department of Stem Cell Biology, Konkuk University School of Medicine, Seoul, Korea,Center for Stem Cell Research, Institute of Advanced Biomedical Science, Konkuk University, Seoul, Korea
| | - Seung-Won Lee
- Department of Stem Cell Biology, Konkuk University School of Medicine, Seoul, Korea,Center for Stem Cell Research, Institute of Advanced Biomedical Science, Konkuk University, Seoul, Korea
| | - Dahee Jeong
- Department of Stem Cell Biology, Konkuk University School of Medicine, Seoul, Korea,Center for Stem Cell Research, Institute of Advanced Biomedical Science, Konkuk University, Seoul, Korea
| | - Hye Jeong Lee
- Department of Stem Cell Biology, Konkuk University School of Medicine, Seoul, Korea,Center for Stem Cell Research, Institute of Advanced Biomedical Science, Konkuk University, Seoul, Korea
| | - Na Young Choi
- Department of Stem Cell Biology, Konkuk University School of Medicine, Seoul, Korea,Center for Stem Cell Research, Institute of Advanced Biomedical Science, Konkuk University, Seoul, Korea
| | - Jin Seok Bang
- Department of Stem Cell Biology, Konkuk University School of Medicine, Seoul, Korea,Center for Stem Cell Research, Institute of Advanced Biomedical Science, Konkuk University, Seoul, Korea
| | - Seokbeom Ham
- Department of Stem Cell Biology, Konkuk University School of Medicine, Seoul, Korea,Center for Stem Cell Research, Institute of Advanced Biomedical Science, Konkuk University, Seoul, Korea
| | - Kinarm Ko
- Department of Stem Cell Biology, Konkuk University School of Medicine, Seoul, Korea,Center for Stem Cell Research, Institute of Advanced Biomedical Science, Konkuk University, Seoul, Korea,Research Institute of Medical Science, Konkuk University, Seoul, Korea,Correspondence to Kinarm Ko, Departement of Stem Cell Biology, Konkuk University School of Medicine, 120 Neungdong-ro, Gwanjin-gu, Seoul 05029, Korea, Tel: +82-2-2030-7888, Fax: +82-2-446-9001, E-mail:
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Ascorbic acid regulates mouse spermatogonial stem cell proliferation in a Wnt/β-catenin/ROS signaling dependent manner. Theriogenology 2022; 184:61-72. [DOI: 10.1016/j.theriogenology.2022.02.028] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/07/2020] [Revised: 02/28/2022] [Accepted: 02/28/2022] [Indexed: 11/17/2022]
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Sundaravadivelu PK, Raina K, Thool M, Ray A, Joshi JM, Kaveeshwar V, Sudhagar S, Lenka N, Thummer RP. Tissue-Restricted Stem Cells as Starting Cell Source for Efficient Generation of Pluripotent Stem Cells: An Overview. ADVANCES IN EXPERIMENTAL MEDICINE AND BIOLOGY 2021; 1376:151-180. [PMID: 34611861 DOI: 10.1007/5584_2021_660] [Citation(s) in RCA: 5] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Key Words] [Subscribe] [Scholar Register] [Indexed: 11/28/2022]
Abstract
Induced pluripotent stem cells (iPSCs) have vast biomedical potential concerning disease modeling, drug screening and discovery, cell therapy, tissue engineering, and understanding organismal development. In the year 2006, a groundbreaking study reported the generation of iPSCs from mouse embryonic fibroblasts by viral transduction of four transcription factors, namely, Oct4, Sox2, Klf4, and c-Myc. Subsequently, human iPSCs were generated by reprogramming fibroblasts as a starting cell source using two reprogramming factor cocktails [(i) OCT4, SOX2, KLF4, and c-MYC, and (ii) OCT4, SOX2, NANOG, and LIN28]. The wide range of applications of these human iPSCs in research, therapeutics, and personalized medicine has driven the scientific community to optimize and understand this reprogramming process to achieve quality iPSCs with higher efficiency and faster kinetics. One of the essential criteria to address this is by identifying an ideal cell source in which pluripotency can be induced efficiently to give rise to high-quality iPSCs. Therefore, various cell types have been studied for their ability to generate iPSCs efficiently. Cell sources that can be easily reverted to a pluripotent state are tissue-restricted stem cells present in the fetus and adult tissues. Tissue-restricted stem cells can be isolated from fetal, cord blood, bone marrow, and other adult tissues or can be obtained by differentiation of embryonic stem cells or trans-differentiation of other tissue-restricted stem cells. Since these cells are undifferentiated cells with self-renewal potential, they are much easier to reprogram due to the inherent characteristic of having an endogenous expression of few pluripotency-inducing factors. This review presents an overview of promising tissue-restricted stem cells that can be isolated from different sources, namely, neural stem cells, hematopoietic stem cells, mesenchymal stem cells, limbal epithelial stem cells, and spermatogonial stem cells, and their reprogramming efficacy. This insight will pave the way for developing safe and efficient reprogramming strategies and generating patient-specific iPSCs from tissue-restricted stem cells derived from various fetal and adult tissues.
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Affiliation(s)
- Pradeep Kumar Sundaravadivelu
- Laboratory for Stem Cell Engineering and Regenerative Medicine, Department of Biosciences and Bioengineering, Indian Institute of Technology Guwahati, Guwahati, Assam, India
| | - Khyati Raina
- Laboratory for Stem Cell Engineering and Regenerative Medicine, Department of Biosciences and Bioengineering, Indian Institute of Technology Guwahati, Guwahati, Assam, India
| | - Madhuri Thool
- Laboratory for Stem Cell Engineering and Regenerative Medicine, Department of Biosciences and Bioengineering, Indian Institute of Technology Guwahati, Guwahati, Assam, India.,Department of Biotechnology, National Institute of Pharmaceutical Education and Research Guwahati, Changsari, Guwahati, Assam, India
| | - Arnab Ray
- Laboratory for Stem Cell Engineering and Regenerative Medicine, Department of Biosciences and Bioengineering, Indian Institute of Technology Guwahati, Guwahati, Assam, India
| | - Jahnavy Madhukar Joshi
- Central Research Laboratory, SDM College of Medical Sciences and Hospital, Shri Dharmasthala Manjunatheshwara University, Dharwad, Karnataka, India
| | - Vishwas Kaveeshwar
- Central Research Laboratory, SDM College of Medical Sciences and Hospital, Shri Dharmasthala Manjunatheshwara University, Dharwad, Karnataka, India
| | - S Sudhagar
- Department of Biotechnology, National Institute of Pharmaceutical Education and Research Guwahati, Changsari, Guwahati, Assam, India
| | - Nibedita Lenka
- National Centre for Cell Science, S. P. Pune University Campus, Ganeshkhind, Pune, Maharashtra, India.
| | - Rajkumar P Thummer
- Laboratory for Stem Cell Engineering and Regenerative Medicine, Department of Biosciences and Bioengineering, Indian Institute of Technology Guwahati, Guwahati, Assam, India.
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Yao L, Peng H, Xu Z, Shi L, Li Y, Dai Y. The effect of regulating the Wnt signaling pathway on the proliferation and differentiation of spermatogonial stem cells. ANNALS OF TRANSLATIONAL MEDICINE 2020; 8:1003. [PMID: 32953803 PMCID: PMC7475508 DOI: 10.21037/atm-20-5321] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Indexed: 12/24/2022]
Abstract
Background Spermatogonial stem cells and organ engineering research has raised new hope in infertility treatment. Spermatogenesis is a complex physiological process. To observe the proliferation ability and differentiation tendency of mice spermatogonial stem cells (SSCs), to study the effect of regulating the Wnt signaling pathway on the proliferation and differentiation of SSCs, and to provide a valuable basis for the clinical application of SSCs. Methods SSCs were isolated and cultured by immunomagnetic separation. Cell surface markers were identified by flow cytometry. Axin1 was chosen as the target gene to inhibit fibrosis of SSCs by inhibiting the activity of Wnt signaling pathway. Axin-siRNA interference vector was constructed and transfected into spermatogonial stem cells. Cultured SSCs were randomly divided into six groups: control group, SSCs + TGF-β group, SSCs + DKK1 group, SSCs + Axin-RNAi group, SSCs + TGF-β + DKK1 group, SSCs + TGF-β + Axin-RNAi group. Proliferation of SSCs in each group was detected by MTT assay. Immunofluorescence, western blot and real time polymerase chain reaction analysis were used to detect protein expression in the Wnt/β catenin signaling pathways and the molecular markers of fibroblasts in SSCs. Results Flow cytometry analysis confirmed that the cultured SSCs were of high purity. MTT assay showed there was no significant difference between Axin-siRNA transfected and non-transfected cells. The proliferation ability was significantly increased in the SSCs + TGF-β group, however, it was retarded in SSCs + Axin-RNAi group. The results of immunofluorescence and western blot analysis showed that the expression levels of the Wnt signaling pathway proteins were relatively inhibited after Axin-siRNA was applied. Real-time polymerase chain reaction showed that the expression levels of the molecular markers of fibroblasts were close to the normal control group. Conclusions The Axin-siRNA constructed in this study specifically inhibited Wnt/β-catenin signal pathway activation, then inhibited the differentiation of SSCs into fibroblasts, which provides a valuable basis for the clinical application of SSCs.
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Affiliation(s)
- Leshen Yao
- Nanjing Drum Tower Hospital Clinical College of Nanjing Medical University, Nanjing, China
| | - Haiyan Peng
- Department of Chronic Communicable Disease, Jiangsu Provincial Center for Disease Control and Prevention, Nanjing, China
| | - Zhipeng Xu
- Nanjing Drum Tower Hospital Clinical College of Nanjing Medical University, Nanjing, China
| | - Liang Shi
- Nanjing Drum Tower Hospital Clinical College of Nanjing Medical University, Nanjing, China
| | - Yan Li
- Department of Chronic Communicable Disease, Jiangsu Provincial Center for Disease Control and Prevention, Nanjing, China
| | - Yutian Dai
- Nanjing Drum Tower Hospital Clinical College of Nanjing Medical University, Nanjing, China
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Xu GP, Zhang XF, Sun L, Chen EM. Current and future uses of skeletal stem cells for bone regeneration. World J Stem Cells 2020; 12:339-350. [PMID: 32547682 PMCID: PMC7280866 DOI: 10.4252/wjsc.v12.i5.339] [Citation(s) in RCA: 7] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 01/01/2020] [Revised: 04/07/2020] [Accepted: 04/18/2020] [Indexed: 02/06/2023] Open
Abstract
The postnatal skeleton undergoes growth, modeling, and remodeling. The human skeleton is a composite of diverse tissue types, including bone, cartilage, fat, fibroblasts, nerves, blood vessels, and hematopoietic cells. Fracture nonunion and bone defects are among the most challenging clinical problems in orthopedic trauma. The incidence of nonunion or bone defects following fractures is increasing. Stem and progenitor cells mediate homeostasis and regeneration in postnatal tissue, including bone tissue. As multipotent stem cells, skeletal stem cells (SSCs) have a strong effect on the growth, differentiation, and repair of bone regeneration. In recent years, a number of important studies have characterized the hierarchy, differential potential, and bone formation of SSCs. Here, we describe studies on and applications of SSCs and/or mesenchymal stem cells for bone regeneration.
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Affiliation(s)
- Guo-Ping Xu
- Department of Orthopedics, The Second Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou 310000, Zhejiang Province, China
| | - Xiang-Feng Zhang
- Department of Orthopedics, The Second Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou 310000, Zhejiang Province, China
| | - Lu Sun
- Department of Oral Medicine, Infection and Immunity, Harvard School of Dental Medicine, Harvard University, Boston, MA 02115, United States
| | - Er-Man Chen
- Department of Orthopedics, The Second Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou 310000, Zhejiang Province, China
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Yang M, Deng B, Geng L, Li L, Wu X. Pluripotency factor NANOG promotes germ cell maintenance in vitro without triggering dedifferentiation of spermatogonial stem cells. Theriogenology 2020; 148:68-75. [DOI: 10.1016/j.theriogenology.2020.02.028] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/21/2019] [Revised: 02/18/2020] [Accepted: 02/18/2020] [Indexed: 12/25/2022]
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Wang J, Xue X, Fan K, Liu Q, Zhang S, Peng M, Zhou J, Cao Z. Moderate hypoxia modulates ABCG2 to promote the proliferation of mouse spermatogonial stem cells by maintaining mild ROS levels. Theriogenology 2019; 145:149-157. [PMID: 31733931 DOI: 10.1016/j.theriogenology.2019.10.007] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/23/2019] [Revised: 09/25/2019] [Accepted: 10/09/2019] [Indexed: 12/25/2022]
Abstract
The aim of this study was to investigate the effects of different oxygen (O2) concentrations on the growth of mouse spermatogonial stem cells (SSCs) and the possible mechanisms of cell proliferation in vitro. The SSCs from testicular cells were cultured in various O2 concentrations (1%, 2.5%, 5%, and 20% O2) for 7 days. Colonies of SSCs were identified morphologically and by immunofluorescence. The number of mouse SSC colonies and the area covered by them were measured. Cell cycle progression of the SSCs was analyzed to identify the state of cell proliferation. The effects of O2 concentrations on the levels of intracellular reactive oxygen species (ROS) and expression of ATP binding cassette subfamily G member 2 (ABCG2) were also analyzed in the SSCs. Following culturing for 7 days, the SSCs were treated with Ko143 (a specific inhibitor of ABCG2) for 1 h, and the ROS level and expression of bcl-2, bax, and p53 were analyzed. The results showed that mouse SSCs formed compact colonies and had unclear borders in different O2 concentrations for 7 days, and there were no major morphologic differences between the O2 treatment groups. The expression of the SSC marker, GFR α1 was studied in each O2 treatment group. The number and area of SSC colonies, and the number of GFR α1 positive cells were the highest in the 2.5% O2 treatment group. Compared with other O2 concentrations, the number of cells in G0 cycle was significantly higher, while the level of intracellular ROS was lower at 1% O2. Moreover, the intracellular ROS levels gradually increased with increasing O2 concentration from 1% to 20%. The expression of ABCG2 in the SSCs cultured at 2.5% O2 was higher than in the other O2 groups. Inhibition of ABCG2 increased intracellular ROS generation, and the expression of the pro-apoptotic genes bax and p53, and decreased the expression of the anti-apoptotic gene bcl-2. In conclusion, moderate to low O2 tension increases ABCG2 expression to maintain mild ROS levels, triggers the expression of the anti-apoptotic genes, suppresses the proapoptotic gene pathway, and further promotes the proliferation of mouse SSCs in vitro.
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Affiliation(s)
- Juhua Wang
- College of Animal Science and Technology, Anhui Agricultural University, Hefei, China; Laboratory of Local Livestock and Poultry, Genetical Resource Conservation and Breeding in Anhui Provincial, Hefei, China; Key Laboratory of Veterinary Pathobiology and Disease Control in Anhui Provincial, Hefei, China.
| | - Xiuheng Xue
- College of Tea & Food Science and Technology, Anhui Agricultural University, Hefei, China.
| | - Kai Fan
- College of Animal Science and Technology, Anhui Agricultural University, Hefei, China
| | - Qi Liu
- College of Animal Science and Technology, Anhui Agricultural University, Hefei, China
| | - Suzi Zhang
- College of Animal Science and Technology, Anhui Agricultural University, Hefei, China
| | - Mengling Peng
- College of Animal Science and Technology, Anhui Agricultural University, Hefei, China; Key Laboratory of Veterinary Pathobiology and Disease Control in Anhui Provincial, Hefei, China
| | - Jie Zhou
- College of Animal Science and Technology, Anhui Agricultural University, Hefei, China; Key Laboratory of Veterinary Pathobiology and Disease Control in Anhui Provincial, Hefei, China
| | - Zubing Cao
- College of Animal Science and Technology, Anhui Agricultural University, Hefei, China; Laboratory of Local Livestock and Poultry, Genetical Resource Conservation and Breeding in Anhui Provincial, Hefei, China
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Lee SW, Wu G, Choi NY, Lee HJ, Bang JS, Lee Y, Lee M, Ko K, Schöler HR, Ko K. Self-Reprogramming of Spermatogonial Stem Cells into Pluripotent Stem Cells without Microenvironment of Feeder Cells. Mol Cells 2018; 41:631-638. [PMID: 29991673 PMCID: PMC6078851 DOI: 10.14348/molcells.2018.2294] [Citation(s) in RCA: 8] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/09/2017] [Revised: 05/10/2018] [Accepted: 06/25/2018] [Indexed: 01/12/2023] Open
Abstract
Spermatogonial stem cells (SSCs) derived from mouse testis are unipotent in regard of spermatogenesis. Our previous study demonstrated that SSCs can be fully reprogrammed into pluripotent stem cells, so called germline-derived pluripotent stem cells (gPS cells), on feeder cells (mouse embryonic fibroblasts), which supports SSC proliferation and induction of pluripotency. Because of an uncontrollable microenvironment caused by interactions with feeder cells, feeder-based SSC reprogramming is not suitable for elucidation of the self-reprogramming mechanism by which SSCs are converted into pluripotent stem cells. Recently, we have established a Matrigel-based SSC expansion culture system that allows long-term SSC proliferation without mouse embryonic fibroblast support. In this study, we developed a new feeder-free SSC self-reprogramming protocol based on the Matrigel-based culture system. The gPS cells generated using a feeder-free reprogramming system showed pluripotency at the molecular and cellular levels. The differentiation potential of gPS cells was confirmed in vitro and in vivo. Our study shows for the first time that the induction of SSC pluripotency can be achieved without feeder cells. The newly developed feeder-free self-reprogramming system could be a useful tool to reveal the mechanism by which unipotent cells are self-reprogrammed into pluripotent stem cells.
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Affiliation(s)
- Seung-Won Lee
- Department of Stem Cell Biology, Konkuk University School of Medicine, Seoul 05029,
Korea
- Center for Stem Cell Research, Institute of Advanced Biomedical Science, Konkuk University, Seoul 05029,
Korea
| | - Guangming Wu
- Department of Cell and Developmental Biology, Max Planck Institute for Molecular Biomedicine, Münster,
Germany
| | - Na Young Choi
- Department of Stem Cell Biology, Konkuk University School of Medicine, Seoul 05029,
Korea
- Center for Stem Cell Research, Institute of Advanced Biomedical Science, Konkuk University, Seoul 05029,
Korea
| | - Hye Jeong Lee
- Department of Stem Cell Biology, Konkuk University School of Medicine, Seoul 05029,
Korea
- Center for Stem Cell Research, Institute of Advanced Biomedical Science, Konkuk University, Seoul 05029,
Korea
| | - Jin Seok Bang
- Department of Stem Cell Biology, Konkuk University School of Medicine, Seoul 05029,
Korea
- Center for Stem Cell Research, Institute of Advanced Biomedical Science, Konkuk University, Seoul 05029,
Korea
| | - Yukyeong Lee
- Department of Stem Cell Biology, Konkuk University School of Medicine, Seoul 05029,
Korea
- Center for Stem Cell Research, Institute of Advanced Biomedical Science, Konkuk University, Seoul 05029,
Korea
| | - Minseong Lee
- Department of Stem Cell Biology, Konkuk University School of Medicine, Seoul 05029,
Korea
- Center for Stem Cell Research, Institute of Advanced Biomedical Science, Konkuk University, Seoul 05029,
Korea
| | - Kisung Ko
- Department of Medicine, College of Medicine, Chung-Ang University, Seoul 06974,
Korea
| | - Hans R. Schöler
- Department of Cell and Developmental Biology, Max Planck Institute for Molecular Biomedicine, Münster,
Germany
- Medical Faculty, University of Münster, Münster,
Germany
| | - Kinarm Ko
- Department of Stem Cell Biology, Konkuk University School of Medicine, Seoul 05029,
Korea
- Center for Stem Cell Research, Institute of Advanced Biomedical Science, Konkuk University, Seoul 05029,
Korea
- The University Open-Innovation Center, Konkuk University, Seoul 05029,
Korea
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Ryu JS, Ko K, Ko K, Kim JS, Kim SU, Chang KT, Choo YK. Roles of gangliosides in the differentiation of mouse pluripotent stem cells to neural stem cells and neural cells. Mol Med Rep 2017; 16:987-993. [DOI: 10.3892/mmr.2017.6719] [Citation(s) in RCA: 10] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/14/2016] [Accepted: 04/12/2017] [Indexed: 11/06/2022] Open
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Jeon HL, Yi JS, Kim TS, Oh Y, Lee HJ, Lee M, Bang JS, Ko K, Ahn IY, Ko K, Kim J, Park HK, Lee JK, Sohn SJ. Development of a Test Method for the Evaluation of DNA Damage in Mouse Spermatogonial Stem Cells. Toxicol Res 2017; 33:107-118. [PMID: 28443181 PMCID: PMC5402864 DOI: 10.5487/tr.2017.33.2.107] [Citation(s) in RCA: 9] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/31/2017] [Revised: 03/06/2017] [Accepted: 03/07/2017] [Indexed: 01/15/2023] Open
Abstract
Although alternative test methods based on the 3Rs (Replacement, Reduction, Refinement) are being developed to replace animal testing in reproductive and developmental toxicology, they are still in an early stage. Consequently, we aimed to develop alternative test methods in male animals using mouse spermatogonial stem cells (mSSCs). Here, we modified the OECD TG 489 and optimized the in vitro comet assay in our previous study. This study aimed to verify the validity of in vitro tests involving mSSCs by comparing their results with those of in vivo tests using C57BL/6 mice by gavage. We selected hydroxyurea (HU), which is known to chemically induce male reproductive toxicity. The 50% inhibitory concentration (IC50) value of HU was 0.9 mM, as determined by the MTT assay. In the in vitro comet assay, % tail DNA and Olive tail moment (OTM) after HU administration increased significantly, compared to the control. Annexin V, PI staining and TUNEL assays showed that HU caused apoptosis in mSSCs. In order to compare in vitro tests with in vivo tests, the same substances were administered to male C57BL/6 mice. Reproductive toxicity was observed at 25, 50, 100, and 200 mg/kg/day as measured by clinical measures of reduction in sperm motility and testicular weight. The comet assay, DCFH-DA assay, H&E staining, and TUNEL assay were also performed. The results of the test with C57BL/6 mice were similar to those with mSSCs for HU treatment. Finally, linear regression analysis showed a strong positive correlation between results of in vitro tests and those of in vivo. In conclusion, the present study is the first to demonstrate the effect of HU-induced DNA damage, ROS formation, and apoptosis in mSSCs. Further, the results of the current study suggest that mSSCs could be a useful model to predict male reproductive toxicity.
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Affiliation(s)
- Hye Lyun Jeon
- Toxicological Screening and Testing Division, National Institute of Food and Drug Safety Evaluation, Ministry of Food and Drug Safety, Osong Health Technology Administration Complex, Cheongju, Korea
| | - Jung-Sun Yi
- Toxicological Screening and Testing Division, National Institute of Food and Drug Safety Evaluation, Ministry of Food and Drug Safety, Osong Health Technology Administration Complex, Cheongju, Korea
| | - Tae Sung Kim
- Toxicological Screening and Testing Division, National Institute of Food and Drug Safety Evaluation, Ministry of Food and Drug Safety, Osong Health Technology Administration Complex, Cheongju, Korea
| | - Youkyung Oh
- Toxicological Screening and Testing Division, National Institute of Food and Drug Safety Evaluation, Ministry of Food and Drug Safety, Osong Health Technology Administration Complex, Cheongju, Korea
| | - Hye Jeong Lee
- Department of Stem Cell Biology, Konkuk University School of Medicine, Seoul, Korea
| | - Minseong Lee
- Department of Stem Cell Biology, Konkuk University School of Medicine, Seoul, Korea
| | - Jin Seok Bang
- Department of Stem Cell Biology, Konkuk University School of Medicine, Seoul, Korea
| | - Kinarm Ko
- Department of Stem Cell Biology, Konkuk University School of Medicine, Seoul, Korea
| | - Il Young Ahn
- Toxicological Screening and Testing Division, National Institute of Food and Drug Safety Evaluation, Ministry of Food and Drug Safety, Osong Health Technology Administration Complex, Cheongju, Korea
| | - Kyungyuk Ko
- Toxicological Screening and Testing Division, National Institute of Food and Drug Safety Evaluation, Ministry of Food and Drug Safety, Osong Health Technology Administration Complex, Cheongju, Korea
| | - Joohwan Kim
- Toxicological Screening and Testing Division, National Institute of Food and Drug Safety Evaluation, Ministry of Food and Drug Safety, Osong Health Technology Administration Complex, Cheongju, Korea
| | - Hye-Kyung Park
- Toxicological Evaluation and Research Department, National Institute of Food and Drug Safety Evaluation, Ministry of Food and Drug Safety, Osong Health Technology Administration Complex, Cheongju, Korea
| | - Jong Kwon Lee
- Toxicological Screening and Testing Division, National Institute of Food and Drug Safety Evaluation, Ministry of Food and Drug Safety, Osong Health Technology Administration Complex, Cheongju, Korea
| | - Soo Jung Sohn
- Toxicological Research Division, National Institute of Food and Drug Safety Evaluation, Ministry of Food and Drug Safety, Osong Health Technology Administration Complex, Cheongju, Korea
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13
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Azizi H, Skutella T, Shahverdi A. Generation of Mouse Spermatogonial Stem-Cell-Colonies in A Non-Adherent Culture. CELL JOURNAL 2017; 19:238-249. [PMID: 28670516 PMCID: PMC5412782 DOI: 10.22074/cellj.2016.4184] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Key Words] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Received: 04/24/2016] [Accepted: 08/10/2016] [Indexed: 01/15/2023]
Abstract
OBJECTIVE The properties of self-renewal and division in spermatogonial stem cells (SSCs) support spermatogenesis. There is a number of reported methods for in vitro SSC culture systems. The development of a culture system that effectively supports isolation and selfrenewal of germline stem cells (GSCs) is of tremendous benefit for clinical trials, experimental research, and as potential treatment for male infertility. The current study aims to consider the cultivation and behavior of GSCs in a non-adherent culture system. MATERIALS AND METHODS In this experimental study, we cultured testicular cells from neonatal mice in agarose coated plates in the presence of Dulbecco's modified Eagle's medium (DMEM) medium (CTRL group), 10% fetal bovine serum (FBS)+DMEM (10% group), and growth factor (G group) that contained 2% FBS, glial cell-derived neurotrophic factor (GDNF), epidermal growth factor (EGF), and fibroblast growth factor (FGF). Mouse spermatogonial stem-like colonies were isolated approximately 3 weeks after digestion of the testis tissue. After passages 2-3, the identity of the mouse spermatogonial stem-like cells was confirmed by immunocytochemistry, reverse transcription-polymerase chain reaction (RT-PCR), and flow cytometry against the germ cell markers α6, β1, c-Kit, Thy-1, c-Ret, Plzf, and Oct4. The statistical significance between mean values in different groups was determined by one-way analysis of variance (ANOVA). RESULTS We observed spermatogonial stem-like colonies in the G and 10% groups, but not the CTRL group. Immunocytochemistry, flow cytometry, and RT-PCR confirmed expressions of germ cell markers in these cells. In the spermatogonial stem-like cells, we observed a significant expression (P<0.05) of germ cell markers in the G and 10% groups versus the testis cells (T). Their proliferative and apoptotic activities were examined by Ki67 and PI/annexin V-FITC. Alkaline phosphatase assay showed that mouse spermato- gonial stem-like colonies were partially positive. CONCLUSION A non-adherent culture system could provide a favorable method for in vitro short-term culture of spermatogonial stem-like cell colonies.
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Affiliation(s)
- Hossein Azizi
- Faculty of Biotechnology, Amol University of Special Modern Technologies, Amol, Iran
| | - Thomas Skutella
- Institute for Anatomy and Cell Biology, Medical Faculty, University of Heidelberg, Heidelberg, Germany
| | - Abdolhossein Shahverdi
- Department of Stem Cells and Developmental Biology, Cell Science Research Center, Royan Institute for Stem Cell Biology and Technology, ACECR, Tehran, Iran
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An J, Zheng Y, Dann CT. Mesenchymal to Epithelial Transition Mediated by CDH1 Promotes Spontaneous Reprogramming of Male Germline Stem Cells to Pluripotency. Stem Cell Reports 2017; 8:446-459. [PMID: 28065642 PMCID: PMC5311464 DOI: 10.1016/j.stemcr.2016.12.006] [Citation(s) in RCA: 26] [Impact Index Per Article: 3.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/11/2016] [Revised: 12/05/2016] [Accepted: 12/06/2016] [Indexed: 01/15/2023] Open
Abstract
Cultured spermatogonial stem cells (GSCs) can spontaneously form pluripotent cells in certain culture conditions. However, GSC reprogramming is a rare event that is largely unexplained. We show GSCs have high expression of mesenchymal to epithelial transition (MET) suppressors resulting in a developmental barrier inhibiting GSC reprogramming. Either increasing OCT4 or repressing transforming growth factor β (TGF-β) signaling promotes GSC reprogramming by upregulating CDH1 and boosting MET. Reducing ZEB1 also enhances GSC reprogramming through its direct effect on CDH1. RNA sequencing shows that rare GSCs, identified as CDH1+ after trypsin digestion, are epithelial-like cells. CDH1+ GSCs exhibit enhanced reprogramming and become more prevalent during the course of reprogramming. Our results provide a mechanistic explanation for the spontaneous emergence of pluripotent cells from GSC cultures; namely, rare GSCs upregulate CDH1 and initiate MET, processes normally kept in check by ZEB1 and TGF-β signaling, thereby ensuring germ cells are protected from aberrant acquisition of pluripotency.
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Affiliation(s)
- Junhui An
- Department of Chemistry, Indiana University, Chemistry A025, 800 E. Kirkwood Avenue, Bloomington, IN 47405-7102, USA
| | - Yu Zheng
- Department of Chemistry, Indiana University, Chemistry A025, 800 E. Kirkwood Avenue, Bloomington, IN 47405-7102, USA
| | - Christina Tenenhaus Dann
- Department of Chemistry, Indiana University, Chemistry A025, 800 E. Kirkwood Avenue, Bloomington, IN 47405-7102, USA.
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15
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Lee HJ, Choi NY, Lee SW, Ko K, Hwang TS, Han DW, Lim J, Schöler HR, Ko K. Epigenetic alteration of imprinted genes during neural differentiation of germline-derived pluripotent stem cells. Epigenetics 2016; 11:177-83. [PMID: 26962997 DOI: 10.1080/15592294.2016.1146852] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 10/22/2022] Open
Abstract
Spermatogonial stem cells (SSCs), which are unipotent stem cells in the testes that give rise to sperm, can be converted into germline-derived pluripotent stem (gPS) by self-induction. The androgenetic imprinting pattern of SSCs is maintained even after their reprogramming into gPS cells. In this study, we used an in vitro neural differentiation model to investigate whether the imprinting patterns are maintained or altered during differentiation. The androgenetic patterns of H19, Snrpn, and Mest were maintained even after differentiation of gPS cells into NSCs (gPS-NSCs), whereas the fully unmethylated status of Ndn in SSCs was altered to somatic patterns in gPS cells and gPS-NSCs. Thus, our study demonstrates epigenetic alteration of genomic imprinting during the induction of pluripotency in SSCs and neural differentiation, suggesting that gPS-NSCs can be a useful model to study the roles of imprinted genes in brain development and human neurodevelopmental disorders.
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Affiliation(s)
- Hye Jeong Lee
- a Department of Stem Cell Biology , Konkuk University School of Medicine , Seoul , Korea.,b Center for Stem Cell Research, Institute of Advanced Biomedical Science, Konkuk University , Seoul , Korea
| | - Na Young Choi
- a Department of Stem Cell Biology , Konkuk University School of Medicine , Seoul , Korea.,b Center for Stem Cell Research, Institute of Advanced Biomedical Science, Konkuk University , Seoul , Korea
| | - Seung-Won Lee
- a Department of Stem Cell Biology , Konkuk University School of Medicine , Seoul , Korea.,b Center for Stem Cell Research, Institute of Advanced Biomedical Science, Konkuk University , Seoul , Korea
| | - Kisung Ko
- c Department of Medicine , College of Medicine, Chung-Ang University , Seoul , Korea
| | - Tae Sook Hwang
- d Department of Pathology , Konkuk University Medical Center, Konkuk University School of Medicine , Seoul , Korea
| | - Dong Wook Han
- a Department of Stem Cell Biology , Konkuk University School of Medicine , Seoul , Korea.,b Center for Stem Cell Research, Institute of Advanced Biomedical Science, Konkuk University , Seoul , Korea
| | - Jisun Lim
- e Department of Biomedical Science , Hallym University, Chuncheon , Gangwon-do , Korea
| | - Hans R Schöler
- f Department of Cell and Developmental Biology , Max Planck Institute for Molecular Biomedicine , Münster , Germany.,g Medical Faculty, University of Münster , Münster , Germany
| | - Kinarm Ko
- a Department of Stem Cell Biology , Konkuk University School of Medicine , Seoul , Korea.,b Center for Stem Cell Research, Institute of Advanced Biomedical Science, Konkuk University , Seoul , Korea.,h Research Institute of Medical Science, Konkuk University , Seoul , Korea
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16
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Tiemann U, Wu G, Marthaler AG, Schöler HR, Tapia N. Epigenetic Aberrations Are Not Specific to Transcription Factor-Mediated Reprogramming. Stem Cell Reports 2015; 6:35-43. [PMID: 26711876 PMCID: PMC4720011 DOI: 10.1016/j.stemcr.2015.11.007] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/14/2015] [Revised: 11/12/2015] [Accepted: 11/18/2015] [Indexed: 01/27/2023] Open
Abstract
Somatic cells can be reprogrammed to pluripotency using different methods. In comparison with pluripotent cells obtained through somatic nuclear transfer, induced pluripotent stem cells (iPSCs) exhibit a higher number of epigenetic errors. Furthermore, most of these abnormalities have been described to be intrinsic to the iPSC technology. Here, we investigate whether the aberrant epigenetic patterns detected in iPSCs are specific to transcription factor-mediated reprogramming. We used germline stem cells (GSCs), which are the only adult cell type that can be converted into pluripotent cells (gPSCs) under defined culture conditions, and compared GSC-derived iPSCs and gPSCs at the transcriptional and epigenetic level. Our results show that both reprogramming methods generate indistinguishable states of pluripotency. GSC-derived iPSCs and gPSCs retained similar levels of donor cell-type memory and exhibited comparable numbers of reprogramming errors. Therefore, our study demonstrates that the epigenetic abnormalities detected in iPSCs are not specific to transcription factor-mediated reprogramming.
GSCs can be converted into iPSCs and into gPSCs under specific culture conditions iPSCs and gPSCs retain the same level of donor cell-type epigenetic memory Comparable numbers of reprogramming errors can be detected in iPSCs and gPSCs Epigenetic aberrations are not specific to transcription factor-mediated reprogramming
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Affiliation(s)
- Ulf Tiemann
- Medical Faculty, Heinrich Heine University, Moorenstraße 5, 40225 Düsseldorf, Germany
| | - Guangming Wu
- Department of Cell and Developmental Biology, Max Planck Institute for Molecular Biomedicine, Röntgenstraße 20, 48149 Münster, Germany
| | - Adele Gabriele Marthaler
- Department of Cell and Developmental Biology, Max Planck Institute for Molecular Biomedicine, Röntgenstraße 20, 48149 Münster, Germany
| | - Hans Robert Schöler
- Department of Cell and Developmental Biology, Max Planck Institute for Molecular Biomedicine, Röntgenstraße 20, 48149 Münster, Germany; Medical Faculty, University of Münster, Domagkstraße 3, 48149 Münster, Germany.
| | - Natalia Tapia
- Medical Faculty, Heinrich Heine University, Moorenstraße 5, 40225 Düsseldorf, Germany.
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17
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The Controversy, Challenges, and Potential Benefits of Putative Female Germline Stem Cells Research in Mammals. Stem Cells Int 2015; 2016:1728278. [PMID: 26788065 PMCID: PMC4693009 DOI: 10.1155/2016/1728278] [Citation(s) in RCA: 10] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/03/2015] [Revised: 08/04/2015] [Accepted: 08/11/2015] [Indexed: 11/30/2022] Open
Abstract
The conventional view is that female mammals lose their ability to generate new germ cells after birth. However, in recent years, researchers have successfully isolated and cultured a type of germ cell from postnatal ovaries in a variety of mammalian species that have the abilities of self-proliferation and differentiation into oocytes, and this finding indicates that putative germline stem cells maybe exist in the postnatal mammalian ovaries. Herein, we review the research history and discovery of putative female germline stem cells, the concept that putative germline stem cells exist in the postnatal mammalian ovary, and the research progress, challenge, and application of putative germline stem cells in recent years.
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18
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Derivation of Pluripotent Cells from Mouse SSCs Seems to Be Age Dependent. Stem Cells Int 2015; 2016:8216312. [PMID: 26664410 PMCID: PMC4655302 DOI: 10.1155/2016/8216312] [Citation(s) in RCA: 17] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/20/2015] [Revised: 07/25/2015] [Accepted: 07/27/2015] [Indexed: 01/15/2023] Open
Abstract
Here, we aimed to answer important and fundamental questions in germ cell biology with special focus on the age of the male donor cells and the possibility to generate embryonic stem cell- (ESC-) like cells. While it is believed that spermatogonial stem cells (SSCs) and truly pluripotent ESC-like cells can be isolated from adult mice, it remained unknown if the spontaneous conversion of SSCs to ESC-like cells fails at some age. Similarly, there have been differences in the literature about the duration of cultures during which ESC-like cells may appear. We demonstrate the possibility to derive ESC-like cells from SSC cultures until they reach adolescence or up to 7 weeks of age, but we point out the impossibility to derive these cells from older, mature adult mice. The inability of real adult SSCs to shift to a pluripotent state coincides with a decline in expression of the core pluripotency genes Oct4, Nanog, and Sox2 in SSCs with age. At the same time genes of the spermatogonial differentiation pathway increase. The generated ESC-like cells were similar to ESCs and express pluripotency markers. In vitro they differentiate into all three germ lineages; they form complex teratomas after transplantation in SCID mice and produce chimeric mice.
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19
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Wang X, Chen T, Zhang Y, Li B, Xu Q, Song C. Isolation and Culture of Pig Spermatogonial Stem Cells and Their in Vitro Differentiation into Neuron-Like Cells and Adipocytes. Int J Mol Sci 2015; 16:26333-46. [PMID: 26556335 PMCID: PMC4661817 DOI: 10.3390/ijms161125958] [Citation(s) in RCA: 16] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/11/2015] [Revised: 10/21/2015] [Accepted: 10/23/2015] [Indexed: 12/14/2022] Open
Abstract
Spermatogonial stem cells (SSCs) renew themselves throughout the life of an organism and also differentiate into sperm in the adult. They are multipopent and therefore, can be induced to differentiate into many cells types in vitro. SSCs from pigs, considered an ideal animal model, are used in studies of male infertility, regenerative medicine, and preparation of transgenic animals. Here, we report on a culture system for porcine SSCs and the differentiation of these cells into neuron-like cells and adipocytes. SSCs and Sertoli cells were isolated from neonatal piglet testis by differential adhesion and SSCs were cultured on a feeder layer of Sertoli cells. Third-generation SSCs were induced to differentiate into neuron-like cells by addition of retinoic acid, β-mercaptoethanol, and 3-isobutyl-1-methylxanthine (IBMX) to the induction media and into adipocytes by the addition of hexadecadrol, insulin, and IBMX to the induction media. The differentiated cells were characterized by biochemical staining, qRT-PCR, and immunocytochemistry. The cells were positive for SSC markers, including alkaline phosphatase and SSC-specific genes, consistent with the cells being undifferentiated. The isolated SSCs survived on the Sertoli cells for 15 generations. Karyotyping confirmed that the chromosomal number of the SSCs were normal for pig (2n = 38, n = 19). Pig SSCs were successfully induced into neuron-like cells eight days after induction and into adipocytes 22 days after induction as determined by biochemical and immunocytochemical staining. qPCR results also support this conclusion. The nervous tissue markers genes, Nestin and β-tubulin, were expressed in the neuron-like cells and the adipocyte marker genes, PPARγ and C/EBPα, were expressed in the adipocytes.
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Affiliation(s)
- Xiaoyan Wang
- College of Animal Science & Technology, Yangzhou University, Yangzhou 225009, China.
| | - Tingfeng Chen
- College of Animal Science & Technology, Yangzhou University, Yangzhou 225009, China.
| | - Yani Zhang
- College of Animal Science & Technology, Yangzhou University, Yangzhou 225009, China.
| | - Bichun Li
- College of Animal Science & Technology, Yangzhou University, Yangzhou 225009, China.
| | - Qi Xu
- College of Animal Science & Technology, Yangzhou University, Yangzhou 225009, China.
| | - Chengyi Song
- College of Animal Science & Technology, Yangzhou University, Yangzhou 225009, China.
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20
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Maza I, Caspi I, Zviran A, Chomsky E, Rais Y, Viukov S, Geula S, Buenrostro JD, Weinberger L, Krupalnik V, Hanna S, Zerbib M, Dutton JR, Greenleaf WJ, Massarwa R, Novershtern N, Hanna JH. Transient acquisition of pluripotency during somatic cell transdifferentiation with iPSC reprogramming factors. Nat Biotechnol 2015; 33:769-74. [PMID: 26098448 PMCID: PMC4500825 DOI: 10.1038/nbt.3270] [Citation(s) in RCA: 111] [Impact Index Per Article: 11.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/27/2014] [Accepted: 06/01/2015] [Indexed: 01/20/2023]
Abstract
Somatic cells can be transdifferentiated to other cell types without passing through a pluripotent state by ectopic expression of appropriate transcription factors1,2. Recent reports have proposed an alternative transdifferentiation method in which fibroblasts are directly converted to various mature somatic cell types by brief expression of the induced pluripotent stem cell (iPSC) reprogramming factors Oct4, Sox2, Klf4 and c-Myc (OSKM) followed by cell expansion in media that promote lineage differentiation3–6. Here we test this method using genetic lineage tracing for expression of endogenous Nanog and Oct4 and for X chromosome reactivation, as these events mark acquisition of pluripotency. We show that the vast majority of reprogrammed cardiomyocytes or neural stem cells obtained from mouse fibroblasts by OSKM-induced transdifferentiation pass through a transient pluripotent state, and that their derivation is molecularly coupled to iPSC formation mechanisms. Our findings underscore the importance of defining trajectories during cell reprogramming by different methods.
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Affiliation(s)
- Itay Maza
- 1] The Department of Molecular Genetics, Weizmann Institute of Science, Rehovot, Israel. [2] The Department of Gastroenterology, Rambam Health Care Campus &Bruce Rappaport School of Medicine, Technion Institute of Technology, Haifa, Israel
| | - Inbal Caspi
- The Department of Molecular Genetics, Weizmann Institute of Science, Rehovot, Israel
| | - Asaf Zviran
- The Department of Molecular Genetics, Weizmann Institute of Science, Rehovot, Israel
| | - Elad Chomsky
- The Department of Molecular Genetics, Weizmann Institute of Science, Rehovot, Israel
| | - Yoach Rais
- The Department of Molecular Genetics, Weizmann Institute of Science, Rehovot, Israel
| | - Sergey Viukov
- The Department of Molecular Genetics, Weizmann Institute of Science, Rehovot, Israel
| | - Shay Geula
- The Department of Molecular Genetics, Weizmann Institute of Science, Rehovot, Israel
| | - Jason D Buenrostro
- Department of Genetics, Stanford University School of Medicine, Stanford, California, USA
| | - Leehee Weinberger
- The Department of Molecular Genetics, Weizmann Institute of Science, Rehovot, Israel
| | - Vladislav Krupalnik
- The Department of Molecular Genetics, Weizmann Institute of Science, Rehovot, Israel
| | - Suhair Hanna
- 1] The Department of Molecular Genetics, Weizmann Institute of Science, Rehovot, Israel. [2] The Department of Pediatrics and the Pediatric Immunology Unit, Rambam Health Care Campus &Bruce Rappaport School of Medicine, Technion Institute of Technology, Haifa, Israel
| | - Mirie Zerbib
- The Department of Molecular Genetics, Weizmann Institute of Science, Rehovot, Israel
| | - James R Dutton
- Stem Cell Institute, University of Minnesota, Minneapolis, Minnesota, USA
| | - William J Greenleaf
- The Department of Molecular Genetics, Weizmann Institute of Science, Rehovot, Israel
| | - Rada Massarwa
- The Department of Molecular Genetics, Weizmann Institute of Science, Rehovot, Israel
| | - Noa Novershtern
- The Department of Molecular Genetics, Weizmann Institute of Science, Rehovot, Israel
| | - Jacob H Hanna
- The Department of Molecular Genetics, Weizmann Institute of Science, Rehovot, Israel
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Wang H, Jiang M, Bi H, Chen X, He L, Li X, Wu J. Conversion of female germline stem cells from neonatal and prepubertal mice into pluripotent stem cells. J Mol Cell Biol 2015; 6:164-71. [PMID: 24755856 DOI: 10.1093/jmcb/mju004] [Citation(s) in RCA: 36] [Impact Index Per Article: 3.6] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/12/2023] Open
Abstract
Pluripotent stem cells derived from neonatal or adult testes are a useful tool to examine the mechanisms of pluripotency and a resource for cell-based therapies. However, therapies using these cells will only benefit males but not females. Recently, female germline stem cells (FGSCs) were discovered in ovaries. Whether FGSCs can be converted into pluripotent stem cells, similar to spermatogonial stem cells, is unknown. Here, we demonstrate that female embryonic stem-like cells (fESLCs) can be generated within 1 month from the stably proliferating FGSCs cultured in embryonic stem cell (ESC) medium. fESLCs exhibit properties similar to those of ESCs in terms of marker expression and differentiation potential. Thus, our findings suggest that generation of patient-specific fESLCs is feasible and provides a foundation for personalized regenerative applications.
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Affiliation(s)
- Hu Wang
- Key Laboratory for the Genetics of Developmental and Neuropsychiatric Disorders (Ministry of Education), Bio-X Institutes, Shanghai Jiao Tong University, Shanghai 200240, China
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22
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Zeng F, Huang F, Guo J, Hu X, Liu C, Wang H. Emerging methods to generate artificial germ cells from stem cells. Biol Reprod 2015; 92:89. [PMID: 25715792 DOI: 10.1095/biolreprod.114.124800] [Citation(s) in RCA: 15] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/05/2014] [Accepted: 02/16/2015] [Indexed: 12/29/2022] Open
Abstract
Germ cells are responsible for the transmission of genetic and epigenetic information across generations. At present, the number of infertile couples is increasing worldwide; these infertility problems can be traced to environmental pollutions, infectious diseases, cancer, psychological or work-related stress, and other factors, such as lifestyle and genetics. Notably, lack of germ cells and germ cell loss present real obstacles in infertility treatment. Recent research aimed at producing gametes through artificial germ cell generation from stem cells may offer great hope for affected couples to treat infertility in the future. Therefore, this rapidly emerging area of artificial germ cell generation from nongermline cells has gained considerable attention from basic and clinical research in the fields of stem cell biology, developmental biology, and reproductive biology. Here, we review the state of the art in artificial germ cell generation.
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Affiliation(s)
- Fanhui Zeng
- The Central Hospital of Enshi Tujia and Miao Autonomous Prefecture, Enshi, China
| | - Fajun Huang
- School of Medical Science, Hubei University for Nationalities, Enshi, China
| | - Jingjing Guo
- College of Life and Environmental Sciences, Shanghai Normal University, Shanghai, China
| | - Xingchang Hu
- College of Life and Environmental Sciences, Shanghai Normal University, Shanghai, China
| | - Changbai Liu
- The Institute of Molecular Biology, China Three Gorges University, Yichang, China
| | - Hu Wang
- Medical School, China Three Gorges University, Yichang, China
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Abstract
Pluripotent cells are a powerful tool for regenerative medicine and drug discovery. Several techniques have been developed to induce pluripotency, or to extract pluripotent cells from different tissues and biological fluids. However, the characterization of pluripotency requires tedious, expensive, time-consuming, and not always reliable wet-lab experiments; thus, an easy, standard quality-control protocol of pluripotency assessment remains to be established. Here to help comes the use of high-throughput techniques, and in particular, the employment of gene expression microarrays, which has become a complementary technique for cellular characterization. Research has shown that the transcriptomics comparison with an Embryonic Stem Cell (ESC) of reference is a good approach to assess the pluripotency. Under the premise that the best protocol is a computer software source code, here I propose and explain line by line a software protocol coded in R-Bioconductor for pluripotency assessment based on the comparison of transcriptomics data of pluripotent cells with an ESC of reference. I provide advice for experimental design, warning about possible pitfalls, and guides for results interpretation.
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24
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Establishment of recipient model for spermatogonial stem cells transplantation in Kunming mice. Tissue Cell 2014; 46:249-54. [DOI: 10.1016/j.tice.2014.05.005] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/17/2012] [Revised: 05/14/2014] [Accepted: 05/14/2014] [Indexed: 01/15/2023]
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miRNA signature in mouse spermatogonial stem cells revealed by high-throughput sequencing. BIOMED RESEARCH INTERNATIONAL 2014; 2014:154251. [PMID: 25136556 PMCID: PMC4124761 DOI: 10.1155/2014/154251] [Citation(s) in RCA: 17] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Received: 05/21/2014] [Accepted: 06/20/2014] [Indexed: 12/24/2022]
Abstract
Spermatogonial stem cells (SSCs) play fundamental roles in spermatogenesis. Although a handful of genes have been discovered as key regulators of SSC self-renewal and differentiation, the regulatory network responsible for SSC function remains unclear. In particular, small RNA signatures during mouse spermatogenesis are not yet systematically investigated. Here, using next generation sequencing, we compared small RNA signatures of in vitro expanded SSCs, testis-derived somatic cells (Sertoli cells), developing germ cells, mouse embryonic stem cells (ESCs), and mouse mesenchymal stem cells among mouse embryonic stem cells (ESCs) to address small RNA transition during mouse spermatogenesis. The results manifest that small RNA transition during mouse spermatogenesis displays overall declined expression profiles of miRNAs and endo-siRNAs, in parallel with elevated expression profiles of piRNAs, resulting in the normal biogenesis of sperms. Meanwhile, several novel miRNAs were preferentially expressed in mouse SSCs, and further investigation of their functional annotation will allow insights into the mechanisms involved in the regulation of SSC activities. We also demonstrated the similarity of miRNA signatures between SSCs and ESCs, thereby providing a new clue to understanding the molecular basis underlying the easy conversion of SSCs to ESCs.
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Limbourg A, Schnabel S, Lozanovski VJ, Napp LC, Ha TC, Maetzig T, Bauersachs J, Naim HY, Schambach A, Limbourg FP. Genetic reporter analysis reveals an expandable reservoir of OCT4+ cells in adult skin. ACTA ACUST UNITED AC 2014; 3:9. [PMID: 25408888 PMCID: PMC4230759 DOI: 10.1186/2045-9769-3-9] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/04/2013] [Accepted: 06/03/2014] [Indexed: 12/31/2022]
Abstract
The transcription factor Oct4 (Pou5f1) is a critical regulator of pluripotency in embryonic and induced pluripotent stem cells. Therefore, Oct4 expression might identify somatic stem cell populations with inherent multipotent potential or a propensity for facilitated reprogramming. However, analysis of Oct4 expression is confounded by Oct4 pseudogenes or non-pluripotency-related isoforms. Systematic analysis of a transgenic Oct4-EGFP reporter mouse identified testis and skin as two principle sources of Oct4+ cells in postnatal mice. While the prevalence of GFP+ cells in testis rapidly declined with age, the skin-resident GFP+ population expanded in a cyclical fashion. These cells were identified as epidermal stem cells dwelling in the stem cell niche of the hair follicle, which endogenously expressed all principle reprogramming factors at low levels. Interestingly, skin wounding or non-traumatic hair removal robustly expanded the GFP+ epidermal cell pool not only locally, but also in uninjured skin areas, demonstrating the existence of a systemic response. Thus, the epithelial stem cell niche of the hair follicle harbors an expandable pool of Oct4+ stem cells, which might be useful for therapeutic cell transfer or facilitated reprogramming.
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Affiliation(s)
- Anne Limbourg
- Research Group Regenerative Agents, Hannover, Germany ; REBIRTH Cluster of Excellence, Hannover, Germany ; Integrated Research Center Transplantation (IFB-Tx), Hannover, Germany ; Department of Plastic, Hand and Reconstructive Surgery, Hannover, Germany
| | - Sabine Schnabel
- Research Group Regenerative Agents, Hannover, Germany ; REBIRTH Cluster of Excellence, Hannover, Germany
| | - Vladimir J Lozanovski
- Research Group Regenerative Agents, Hannover, Germany ; REBIRTH Cluster of Excellence, Hannover, Germany ; Department of General and Transplant Surgery, University Hospital Heidelberg, Heidelberg, Germany
| | - L Christian Napp
- Research Group Regenerative Agents, Hannover, Germany ; Department of Cardiology and Angiology, Hannover, Germany
| | - Teng-Cheong Ha
- REBIRTH Cluster of Excellence, Hannover, Germany ; Institute of Experimental Hematology, OE6960 Hannover Medical School, Carl-Neuberg-Str. 1, D-30625 Hannover, Germany
| | - Tobias Maetzig
- REBIRTH Cluster of Excellence, Hannover, Germany ; Institute of Experimental Hematology, OE6960 Hannover Medical School, Carl-Neuberg-Str. 1, D-30625 Hannover, Germany
| | - Johann Bauersachs
- REBIRTH Cluster of Excellence, Hannover, Germany ; Department of Cardiology and Angiology, Hannover, Germany
| | - Hassan Y Naim
- Department of Physiological Chemistry, Hannover Veterinary School, Hannover, Germany
| | - Axel Schambach
- REBIRTH Cluster of Excellence, Hannover, Germany ; Integrated Research Center Transplantation (IFB-Tx), Hannover, Germany ; Institute of Experimental Hematology, OE6960 Hannover Medical School, Carl-Neuberg-Str. 1, D-30625 Hannover, Germany ; Division of Hematology/Oncology, Boston Children's Hospital, Harvard Medical School, Harvard, USA
| | - Florian P Limbourg
- Research Group Regenerative Agents, Hannover, Germany ; REBIRTH Cluster of Excellence, Hannover, Germany ; Department of Nephrology and Hypertension, Hannover Medical School, Hannover, Germany ; Vascular Medicine and Transplantation Research, Dept. of Nephrology and Hypertension, OE 6841, Hannover Medical School, Carl-Neuberg-Str. 1, D-30625 Hannover, Germany
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27
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Choi NY, Park YS, Ryu JS, Lee HJ, Araúzo-Bravo MJ, Ko K, Han DW, Schöler HR, Ko K. A novel feeder-free culture system for expansion of mouse spermatogonial stem cells. Mol Cells 2014; 37:473-9. [PMID: 24854861 PMCID: PMC4086341 DOI: 10.14348/molcells.2014.0080] [Citation(s) in RCA: 22] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/04/2014] [Revised: 04/26/2014] [Accepted: 04/28/2014] [Indexed: 12/24/2022] Open
Abstract
Spermatogonial stem cells (SSCs, also called germline stem cells) are self-renewing unipotent stem cells that produce differentiating germ cells in the testis. SSCs can be isolated from the testis and cultured in vitro for long-term periods in the presence of feeder cells (often mouse embryonic fibroblasts). However, the maintenance of SSC feeder culture systems is tedious because preparation of feeder cells is needed at each subculture. In this study, we developed a Matrigel-based feeder-free culture system for long-term propagation of SSCs. Although several in vitro SSC culture systems without feeder cells have been previously described, our Matrigel-based feeder-free culture system is time- and cost- effective, and preserves self-renewability of SSCs. In addition, the growth rate of SSCs cultured using our newly developed system is equivalent to that in feeder cultures. We confirmed that the feeder-free cultured SSCs expressed germ cell markers both at the mRNA and protein levels. Furthermore, the functionality of feeder-free cultured SSCs was confirmed by their transplantation into germ cell-depleted mice. These results suggest that our newly developed feeder-free culture system provides a simple approach to maintaining SSCs in vitro and studying the basic biology of SSCs, including determination of their fate.
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Affiliation(s)
- Na Young Choi
- Department of Stem Cell Biology, School of Medicine, Konkuk University, Seoul 143-701,
Korea
- Center for Stem Cell Research, Institute of Advanced Biomedical Science, Konkuk University, Seoul 143-701,
Korea
| | - Yo Seph Park
- Department of Stem Cell Biology, School of Medicine, Konkuk University, Seoul 143-701,
Korea
- Center for Stem Cell Research, Institute of Advanced Biomedical Science, Konkuk University, Seoul 143-701,
Korea
| | - Jae-Sung Ryu
- Center for Stem Cell Research, Institute of Advanced Biomedical Science, Konkuk University, Seoul 143-701,
Korea
| | - Hye Jeong Lee
- Department of Stem Cell Biology, School of Medicine, Konkuk University, Seoul 143-701,
Korea
- Center for Stem Cell Research, Institute of Advanced Biomedical Science, Konkuk University, Seoul 143-701,
Korea
| | - Marcos J. Araúzo-Bravo
- Group of Computational Biology and Bioinformatics, Biodonostia Health Research Institute, San Sebastián,
Spain
- IKERBASQUE, Basque Foundation for Science, Bilbao,
Spain
| | - Kisung Ko
- Department of Medicine, Medical Research Institute, College of Medicine, Chung-Ang University, Seoul 156-756,
Korea
| | - Dong Wook Han
- Department of Stem Cell Biology, School of Medicine, Konkuk University, Seoul 143-701,
Korea
- Center for Stem Cell Research, Institute of Advanced Biomedical Science, Konkuk University, Seoul 143-701,
Korea
| | - Hans R. Schöler
- Department of Cell and Developmental Biology, Max Planck Institute for Molecular Biomedicine, Münster 48149,
Germany
- University of Münster, Medical Faculty, 48149 Münster,
Germany
| | - Kinarm Ko
- Department of Stem Cell Biology, School of Medicine, Konkuk University, Seoul 143-701,
Korea
- Center for Stem Cell Research, Institute of Advanced Biomedical Science, Konkuk University, Seoul 143-701,
Korea
- Research Institute of Medical Science, Konkuk University, Seoul 143-701,
Korea
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28
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Chikhovskaya J, van Daalen S, Korver C, Repping S, van Pelt A. Mesenchymal origin of multipotent human testis-derived stem cells in human testicular cell cultures. ACTA ACUST UNITED AC 2013; 20:155-67. [DOI: 10.1093/molehr/gat076] [Citation(s) in RCA: 32] [Impact Index Per Article: 2.7] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/14/2022]
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29
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Takashima S, Hirose M, Ogonuki N, Ebisuya M, Inoue K, Kanatsu-Shinohara M, Tanaka T, Nishida E, Ogura A, Shinohara T. Regulation of pluripotency in male germline stem cells by Dmrt1. Genes Dev 2013; 27:1949-58. [PMID: 24029916 PMCID: PMC3792472 DOI: 10.1101/gad.220194.113] [Citation(s) in RCA: 53] [Impact Index Per Article: 4.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/07/2023]
Abstract
Spermatogonial stem cells (SSCs) have the potential to acquire pluripotency under specific culture conditions. Takashima et al. report that global DNA hypomethylation triggered by Dnmt1 depletion induces pluripotent cell derivation. Dnmt1 depletion down-regulates Dmrt1, a gene involved in sexual differentiation. Dmrt1 depletion up-regulates Sox2, which in turn up-regulates Oct4 and produces pluripotent cells. These results suggest that the Dmrt1–Sox2 axis plays a crucial role in repression of SSC pluripotency. Spermatogonial stem cells (SSCs) present the potential to acquire pluripotency under specific culture conditions. However, the frequency of pluripotent cell derivation is low, and the mechanism of SSC reprogramming remains unknown. In this study, we report that induction of global DNA hypomethylation in germline stem (GS) cells (cultured SSCs) induces pluripotent cell derivation. When DNA demethylation was triggered by Dnmt1 depletion, GS cells underwent apoptosis. However, GS cells were converted into embryonic stem (ES)-like cells by double knockdown of Dnmt1 and p53. This treatment down-regulated Dmrt1, a gene involved in sexual differentiation, meiosis, and pluripotency. Dmrt1 depletion caused apoptosis of GS cells, but a combination of Dmrt1 and p53 depletion also induced pluripotency. Functional screening of putative Dmrt1 target genes revealed that Dmrt1 depletion up-regulates Sox2. Sox2 transfection up-regulated Oct4 and produced pluripotent cells. This conversion was enhanced by Oct1 depletion, suggesting that the balance of Oct proteins maintains SSC identity. These results suggest that spontaneous SSC reprogramming is caused by unstable DNA methylation and that a Dmrt1–Sox2 cascade is critical for regulating pluripotency in SSCs.
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Affiliation(s)
- Seiji Takashima
- Department of Molecular Genetics, Graduate School of Medicine, Kyoto University, Kyoto 606-8501, Japan
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30
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Guo J, Wang H, Hu X. Reprogramming and transdifferentiation shift the landscape of regenerative medicine. DNA Cell Biol 2013; 32:565-72. [PMID: 23930590 DOI: 10.1089/dna.2013.2104] [Citation(s) in RCA: 16] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/11/2022] Open
Abstract
Regenerative medicine is a new interdisciplinary field in biomedical science, which aims at the repair or replacement of the defective tissue or organ by congenital defects, age, injury, or disease. Various cell-related techniques such as stem cell-based biotherapy are a hot topic in the current press, and stem cell research can help us to expand our understanding of development as well as the pathogenesis of disease. In addition, new technology such as reprogramming or dedifferentiation and transdifferentiation open a new area for regenerative medicine. Here we review new approaches of these technologies used for cell-based therapy and discuss future directions and challenges in the field of regeneration.
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Affiliation(s)
- Jingjing Guo
- 1 College of Life and Environmental Sciences, Shanghai Normal University , Shanghai, China
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31
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Li M, Liu C, Zhu H, Sun J, Yu M, Niu Z, Liu W, Peng S, Hua J. Expression pattern of Boule in dairy goat testis and its function in promoting the meiosis in male germline stem cells (mGSCs). J Cell Biochem 2013; 114:294-302. [PMID: 22930651 DOI: 10.1002/jcb.24368] [Citation(s) in RCA: 26] [Impact Index Per Article: 2.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/22/2012] [Accepted: 08/13/2012] [Indexed: 11/06/2022]
Abstract
Boule is a conserved gene in meiosis, which encodes RNA binding protein required for spermatocyte meiosis. Deletion of Boule was found to block meiosis in spermatogenesis, which contributes to infertility. Up to date, the expression and function of Boule in the goat testis are not known. The objectives of this study were to investigate the expression pattern of Boule in dairy goat testis and their function in male germline stem cells (mGSCs). The results first revealed that the expression level of Boule in adult testes was significantly higher than younger and immature goats, and azoospermia and male intersex testis. Over-expression of Boule promoted the expression of meiosis-related genes in dairy goat mGSCs. The expression of Stra8 was up-regulated by over-expression of Boule analyzed by Western blotting and Luciferase reporter assay. While, Cdc25a, the downstream regulator of Boule, was found not to affect the expression of Stra8, and our data illustrated that Cdc25a did not regulate meiosis via Stra8. The expression of Stra8 and Boule was up-regulated by RA induction. Taken together, results suggest the Boule plays an important role in dairy goat spermatogenesis and that over-expression of Boule may promote spermatogenesis and meiosis in dairy goat.
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Affiliation(s)
- Mingzhao Li
- College of Veterinary Medicine, Shaanxi Center of Stem Cells Engineering & Technology, Key Lab for Animal Biotechnology of Agriculture Ministry of China, Northwest A&F University, Yangling, Shaanxi 712100, China
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32
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Li M, Yu M, Zhu H, Song W, Hua J. The effects of Nanos2 on Boule and Stra8 in male germline stem cells (mGSCs). Mol Biol Rep 2013; 40:4383-9. [PMID: 23644984 DOI: 10.1007/s11033-013-2527-1] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/27/2012] [Accepted: 04/29/2013] [Indexed: 11/29/2022]
Abstract
The mitosis-meiosis switch is a key event in the differentiation of germ cells. Meiosis is important in development biology, however, it has not been clear what is the regulation mechanism in mammals. Our previous study showed that Boule could activate Stra8 directly and result in the meiosis initiation of dairy goat male germline stem cells (mGSCs). Nanos2, a RNA-binding protein, plays critical roles in the suppression of meiosis by preventing Stra8 expression and maintain the male germ cell development. The main purpose of this study was to explore whether Nanos2 represses Stra8 transcription through Boule or not. We found ectopic over-expression of Nanos2 in GC-1 and mGSCs down-regulated Stra8 transcription and translation, and Boule expression was not affected. It was in consistent with our expectation that RA could up-regulate Boule and Stra8 expression, but down-regulate Nanos2 expression in mGSCs. In dairy goat, the expression levels of Boule and Stra8 would rise with the increase of age, but the expression level of Nanos2 in 90 dpp and adult testis had not shown a clear change. In conclusion, Nanos2 represses Stra8 expression but not through Boule in dairy goat mGSCs.
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Affiliation(s)
- Mingzhao Li
- Key Lab for Animal Biotechnology of Agriculture Ministry, Shaanxi Centre of Stem Cells Engineering & Technology, College of Veterinary Medicine, Northwest A&F University, Yangling, 712100, Shaanxi, China
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33
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Ko K, Wu G, Araúzo-Bravo MJ, Kim J, Francine J, Greber B, Mühlisch J, Joo JY, Sabour D, Frühwald MC, Tapia N, Schöler HR. Autologous pluripotent stem cells generated from adult mouse testicular biopsy. Stem Cell Rev Rep 2012; 8:435-44. [PMID: 21858421 DOI: 10.1007/s12015-011-9307-x] [Citation(s) in RCA: 13] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/15/2023]
Affiliation(s)
- Kinarm Ko
- Department of Cell and Developmental Biology, Max Planck Institute for Molecular Biomedicine, Münster, Germany
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34
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Nasiri Z, Hosseini S, Hajian M, Abedi P, Bahadorani M, Baharvand H, Nasr-Esfahani M. Effects of different feeder layers on short-term culture of prepubertal bovine testicular germ cells In-vitro. Theriogenology 2012; 77:1519-28. [DOI: 10.1016/j.theriogenology.2011.11.019] [Citation(s) in RCA: 27] [Impact Index Per Article: 2.1] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/30/2011] [Revised: 11/20/2011] [Accepted: 11/20/2011] [Indexed: 01/06/2023]
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35
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Eildermann K, Gromoll J, Behr R. Misleading and reliable markers to differentiate between primate testis-derived multipotent stromal cells and spermatogonia in culture. Hum Reprod 2012; 27:1754-67. [PMID: 22442249 PMCID: PMC3357197 DOI: 10.1093/humrep/des091] [Citation(s) in RCA: 38] [Impact Index Per Article: 2.9] [Reference Citation Analysis] [Abstract] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/15/2022] Open
Abstract
BACKGROUND Several studies have reported the generation of spermatogonia-derived pluripotent stem cells from human testes. The initial aim of the present study was the derivation of equivalent stem cells from an established and experimentally accessible non-human primate model, the common marmoset monkey (Callithrix jacchus). However, an essential prerequisite in the absence of transgenic reporters in primates and man is the availability of validated endogenous markers for the identification of specific cell types in vitro. METHODS AND RESULTS We cultured marmoset testicular cells in a similar way to that described for human testis-derived pluripotent cells and set out to characterize these cultures under different conditions and in differentiation assays applying established marker panels. Importantly, the cells emerged as testicular multipotent stromal cells (TMSCs) instead of (pluripotent) germ cell-derived cells. TMSCs expressed many markers such as GFR-α, GPR125, THY-1 (CD90), ITGA6, SSEA4 and TRA-1-81, which were considered as spermatogonia specific and were previously used for the enrichment or characterization of spermatogonia. Proliferation of TMSCs was highly dependent on basic fibroblast growth factor, a growth factor routinely present in germ cell culture media. As reliable markers for the distinction between spermatogonia and TMSCs, we established VASA, in combination with the spermatogonia-expressed factors, MAGEA4, PLZF and SALL4. CONCLUSIONS Marmoset monkey TMSCs and spermatogonia exhibit an overlap of markers, which may cause erroneous interpretations of experiments with testis-derived stem cells in vitro. We provide a marker panel for the unequivocal identification of spermatogonia providing a better basis for future studies on primate, including human, testis-derived stem cells.
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Affiliation(s)
- K Eildermann
- German Primate Center, Leibniz Institute for Primate Research, Kellnerweg 4, Göttingen, Germany
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36
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van de Kamp J, Kramann R, Anraths J, Schöler HR, Ko K, Knüchel R, Zenke M, Neuss S, Schneider RK. Epithelial morphogenesis of germline-derived pluripotent stem cells on organotypic skin equivalents in vitro. Differentiation 2011; 83:138-47. [PMID: 22364881 DOI: 10.1016/j.diff.2011.11.014] [Citation(s) in RCA: 9] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/19/2011] [Revised: 11/15/2011] [Accepted: 11/16/2011] [Indexed: 11/18/2022]
Abstract
For tissue engineering, cultivation of pluripotent stem cells on three-dimensional scaffolds allows the generation of organ-like structures. Previously, we have established an organotypic culture system of skin to induce epidermal differentiation in adult stem cells. Multipotent stem cells are not able to differentiate across germinal boundaries. In contrast, pluripotent stem cells readily differentiate into tissues of all three germ layers. Germline-derived pluripotent stem cells (gPS cells) can be generated by induction of pluripotency in mouse unipotent germline stem cells without the introduction of exogenous transcription factors. In the current study, we analyzed the influence of organotypic culture conditions of skin on the epithelial differentiation of gPS cells in comparison to the well-established HM1 ES cell line. Quantitative RT-PCR data of the pluripotency gene Oct4 showed that gPS cells are characterized by an accelerated Oct4-downregulation compared to HM1 ES cells. When subjected to the organotypic culture conditions of skin, gPS cells formed tubulocystic structures lined by stratified (CK5/6(+), CK14(+), CK8/18(-)) epithelia. HM1 ES cells formed only small tubulocystic structures lined by simple, CK8/18(+) epithelia. BMP-4, an epidermal morphogen, significantly enhanced the expression of epithelial markers in HM1 ES cells, but did not significantly affect the formation of complex (squamous) epithelia in gPS cells. In HM1 ES cells the differentiation into squamous epithelium was only inducible in the presence of mature dermal fibroblasts. Both pluripotent stem cell types spontaneously differentiated into mesodermal, endodermal and into neuroectodermal cells at low frequency, underlining their pluripotent differentiation capacity. Concluding, the organotypic culture conditions of skin induce a multilayered, stratified epithelium in gPS cells, in HM1 ES cells only in the presence of dermal fibroblasts. Thus, our data show that differentiation protocols strongly depend on the stem cell type and have to be modified for each specific stem cell type.
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Affiliation(s)
- Julia van de Kamp
- Institute of Pathology, Medical Faculty, RWTH Aachen University, Aachen, Germany
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37
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Zhang S, Sun J, Pan S, Zhu H, Wang L, Hu Y, Wang J, Wang F, Cao H, Yan X, Hua J. Retinol (vitamin A) maintains self-renewal of pluripotent male germline stem cells (mGSCs) from adult mouse testis. J Cell Biochem 2011; 112:1009-21. [PMID: 21308744 DOI: 10.1002/jcb.23029] [Citation(s) in RCA: 23] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/06/2023]
Abstract
Studies have shown that male germline stem cells (mGSCs), which are responsible for maintaining spermatogenesis in the male, could be obtained from mouse and human testis. However, the traditional cultural methods were mostly dependent on serum and feeder, and the initial mGSCs were either obtained from neonatal mice or the detailed description of its potency and origin was not provided. Here we reported a novel (retinol (RE) serum-free and feeder-free) system for the successful culture of adult germline stem cells from adult Kunming mice (8-24 weeks) testis. The isolated mGSCs cultured in RE serum-free and feeder-free medium maintained the typical morphology of undifferentiated embryonic stem cells (ESCs), and they proliferated well in RE medium analyzed by proliferation assay, RT-PCR, microarray, and Western blotting. These cells also showed typical properties of ESCs (alkaline phosphatase (AP) positive, expressions of Oct4, Sox2, Nanog, and SSEA1, with the capacity to form teratomas and differentiate into various types of cells within three germ layers). Taken together, we conclude that RE promotes the self-renewal of mGSCs and maintains the pluripotency of mGSCs, the RE serum-free and feeder-free system may be useful for the culture of pluripotent stem cell lines from adult testis tissues, which provides a new resource for tissue engineering and therapy for infertility.
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Affiliation(s)
- Shanshan Zhang
- College of Veterinary Medicine, Shaanxi Centre of Stem Cells Engineering & Technology, Key Lab for Reproductive Physiology & Embryo Biotechnology of Agriculture Ministry of China, Northwest A&F University, Yangling, Shaanxi 712100, China
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38
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Nowak-Imialek M, Kues W, Carnwath JW, Niemann H. Pluripotent stem cells and reprogrammed cells in farm animals. MICROSCOPY AND MICROANALYSIS : THE OFFICIAL JOURNAL OF MICROSCOPY SOCIETY OF AMERICA, MICROBEAM ANALYSIS SOCIETY, MICROSCOPICAL SOCIETY OF CANADA 2011; 17:474-497. [PMID: 21682936 DOI: 10.1017/s1431927611000080] [Citation(s) in RCA: 32] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 05/30/2023]
Abstract
Pluripotent cells are unique because of their ability to differentiate into the cell lineages forming the entire organism. True pluripotent stem cells with germ line contribution have been reported for mice and rats. Human pluripotent cells share numerous features of pluripotentiality, but confirmation of their in vivo capacity for germ line contribution is impossible due to ethical and legal restrictions. Progress toward derivation of embryonic stem cells from domestic species has been made, but the derived cells were not able to produce germ line chimeras and thus are termed embryonic stem-like cells. However, domestic animals, in particular the domestic pig (Sus scrofa), are excellent large animals models, in which the clinical potential of stem cell therapies can be studied. Reprogramming technologies for somatic cells, including somatic cell nuclear transfer, cell fusion, in vitro culture in the presence of cell extracts, in vitro conversion of adult unipotent spermatogonial stem cells into germ line derived pluripotent stem cells, and transduction with reprogramming factors have been developed with the goal of obtaining pluripotent, germ line competent stem cells from domestic animals. This review summarizes the present state of the art in the derivation and maintenance of pluripotent stem cells in domestic animals.
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Affiliation(s)
- Monika Nowak-Imialek
- Institute of Farm Animal Genetics (FLI), Biotechnology, Mariensee, 31535 Neustadt, Germany
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39
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Kokkinaki M, Djourabtchi A, Golestaneh N. Long-term Culture of Human SSEA-4 Positive Spermatogonial Stem Cells (SSCs). ACTA ACUST UNITED AC 2011; 2. [PMID: 24466499 DOI: 10.4172/2157-7633.s2-003] [Citation(s) in RCA: 30] [Impact Index Per Article: 2.1] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/25/2022]
Abstract
Recently we and two other groups have shown that human spermatogonial stem cells (SSCs) have the potential to become pluripotent in vitro in defined culture conditions and to differentiate into cells of the three embryonic germ layers. This discovery could open new avenues for autologous cell-based therapy in degenerative diseases, bypassing the ethical and immunological problems related to the human embryonic stem cells. In addition, human SSCs could be used to treat infertility in cancer survival children. However, in order to reprogram SSCs into pluripotency, or to preserve them for repopulation of infertile testes, the first and limiting step is to have access to a highly purified human SSC population that could be multiplied and efficiently cultured in vitro maintaining their molecular and cellular characteristics. Although various studies have attempted to identify molecular markers of human SSCs, to date there is still limited information related to the specific markers that could be used for their isolation and optimized purification that allows long-term in vitro culture of isolated human SSCs. Here using SSEA-4 as an optimal marker for isolation of a subpopulation of SSCs, we show that SSEA-4 positive cells express the highest level of SSC genes compared to other subpopulations isolated with different markers, and can be maintained in culture for over 14 passages which we were unable to obtain with other SSCs markers including GPR125 and ITGA6. In addition, we have established a new technology for cell sorting and long-term culture of human SSC-SSEA-4 positive cells that maximizes the purity and viability of the sorted cells. Our findings are crucial and could be used for the most efficient isolation, purification and long-term culture of SSCs for clinical applications in regenerative medicine, or for preparation of human SSCs for autologous treatment of infertility in cancer survival children.
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Affiliation(s)
- Maria Kokkinaki
- Georgetown University School of Medicine, Department of Biochemistry and Molecular & Cellular Biology ; Lombardi Comprehensive Cancer Center, Georgetown University School of Medicine
| | - Ardalan Djourabtchi
- Georgetown University School of Medicine, Department of Biochemistry and Molecular & Cellular Biology
| | - Nady Golestaneh
- Georgetown University School of Medicine, Department of Biochemistry and Molecular & Cellular Biology ; Lombardi Comprehensive Cancer Center, Georgetown University School of Medicine
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