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      An efficient method for generating a germ cell depleted animal model for studies related to spermatogonial stem cell transplantation

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          Abstract

          Background

          Spermatogonial stem cell (SSC) transplantation (SSCT) has become important for conservation of endangered species, transgenesis and for rejuvenating testes which have lost germ cells (Gc) due to gonadotoxic chemotherapy or radiotherapy during the prepubertal phase of life. Creating a germ cell-depleted animal model for transplantation of normal or gene-transfected SSC is a prerequisite for such experimental studies. Traditionally used intraperitoneal injections of busulfan to achieve this are associated with painful hematopoietic toxicity and affects the wellbeing of the animals. Use of testicular busulfan has been reported recently to avoid this but with a very low success rate of SSCT. Therefore, it is necessary to establish a more efficient method to achieve higher SSCT without any suffering or mortality of the animals.

          Methods

          A solution of busulfan, ranging from 25 μg/20 μl to 100 μg/20 μl in 50 % DMSO was used for this study. Each testis received two diagonally opposite injections of 10 μl each. Only DMSO was used as control. Germ cell depletion was checked every 15 days. GFP-expressing SSC from transgenic donor mice C57BL/6-Tg (UBC-GFP) 30Scha/J were transplanted into busulfan-treated testis. Two months after SSCT, mice were analyzed for presence of colonies of donor-derived SSC and their ability to generate offspring.

          Results

          A dose of 75 μg of busulfan resulted in reduction of testis size and depletion of the majority of Gc of testis in all mice within 15 days post injection without causing mortality or a cytotoxic effect in other organs. Two months after SSCT, colonies of donor-derived Gc-expressing GFP were observed in recipient testes. When cohabitated with females, donor-derived offspring were obtained. By our method, 71 % of transplanted males sired transgenic progeny as opposed to 5.5 % by previously described procedures. About 56 % of progeny born were transgenic by our method as opposed to 1.2 % by the previously reported methods.

          Conclusions

          We have established an efficient method of generating a germ cell-depleted animal model by using a lower dose of busulfan, injected through two diagonally opposite sites in the testis, which allows efficient colonization of transplanted SSC resulting in a remarkably higher proportion of donor-derived offspring generation.

          Electronic supplementary material

          The online version of this article (doi:10.1186/s13287-016-0405-1) contains supplementary material, which is available to authorized users.

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          Most cited references23

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          Pluripotency of spermatogonial stem cells from adult mouse testis.

          Embryonic germ cells as well as germline stem cells from neonatal mouse testis are pluripotent and have differentiation potential similar to embryonic stem cells, suggesting that the germline lineage may retain the ability to generate pluripotent cells. However, until now there has been no evidence for the pluripotency and plasticity of adult spermatogonial stem cells (SSCs), which are responsible for maintaining spermatogenesis throughout life in the male. Here we show the isolation of SSCs from adult mouse testis using genetic selection, with a success rate of 27%. These isolated SSCs respond to culture conditions and acquire embryonic stem cell properties. We name these cells multipotent adult germline stem cells (maGSCs). They are able to spontaneously differentiate into derivatives of the three embryonic germ layers in vitro and generate teratomas in immunodeficient mice. When injected into an early blastocyst, SSCs contribute to the development of various organs and show germline transmission. Thus, the capacity to form multipotent cells persists in adult mouse testis. Establishment of human maGSCs from testicular biopsies may allow individual cell-based therapy without the ethical and immunological problems associated with human embryonic stem cells. Furthermore, these cells may provide new opportunities to study genetic diseases in various cell lineages.
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            Spermatogonial stem cell transplantation into rhesus testes regenerates spermatogenesis producing functional sperm.

            Spermatogonial stem cells (SSCs) maintain spermatogenesis throughout a man's life and may have application for treating some cases of male infertility, including those caused by chemotherapy before puberty. We performed autologous and allogeneic SSC transplantations into the testes of 18 adult and 5 prepubertal recipient macaques that were rendered infertile with alkylating chemotherapy. After autologous transplant, the donor genotype from lentivirus-marked SSCs was evident in the ejaculated sperm of 9/12 adult and 3/5 prepubertal recipients after they reached maturity. Allogeneic transplant led to donor-recipient chimerism in sperm from 2/6 adult recipients. Ejaculated sperm from one recipient transplanted with allogeneic donor SSCs were injected into 85 rhesus oocytes via intracytoplasmic sperm injection. Eighty-one oocytes were fertilized, producing embryos ranging from four-cell to blastocyst with donor paternal origin confirmed in 7/81 embryos. This demonstration of functional donor spermatogenesis following SSC transplantation in primates is an important milestone for informed clinical translation. Copyright © 2012 Elsevier Inc. All rights reserved.
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              Germline stem cell transplantation and transgenesis.

              The recently developed testis cell transplantation method provides a powerful approach to studying the biology of the male germline stem cell and its microenvironment, the stem cell niche. The technique also is being used to examine spermatogenic defects, correct male infertility, and generate transgenic animals.
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                Author and article information

                Contributors
                dr.nganguli@gmail.com
                neerja@nii.ac.in
                abulusmani@gmail.com
                dubeyneetu@hotmail.com
                nilanjana06@gmail.com
                rajeshksarkar@gmail.com
                somamghorai@gmail.com
                91-11-26703751 , subeer@nii.ac.in
                Journal
                Stem Cell Res Ther
                Stem Cell Res Ther
                Stem Cell Research & Therapy
                BioMed Central (London )
                1757-6512
                22 September 2016
                22 September 2016
                2016
                : 7
                : 142
                Affiliations
                [1 ]Embryo Biotechnology Laboratory, National Institute of Immunology, Aruna Asaf Ali Marg, New Delhi, 110067 India
                [2 ]Department of Zoology, University of Delhi, Delhi, 110 007 India
                [3 ]National Institute of Animal Biotechnology, Hyderabad, Telengana India
                Article
                405
                10.1186/s13287-016-0405-1
                5032248
                27659063
                18e74b3c-3068-4913-b369-55e5d1fe2f0d
                © The Author(s). 2016

                Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License ( http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver ( http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.

                History
                : 29 July 2016
                : 1 September 2016
                : 2 September 2016
                Funding
                Funded by: FundRef http://dx.doi.org/10.13039/501100001407, Department of Biotechnology , Ministry of Science and Technology;
                Award ID: BT/HRD/35/01/01/2010
                Award Recipient :
                Categories
                Method
                Custom metadata
                © The Author(s) 2016

                Molecular medicine
                busulfan,germ cell depletion,germ cell transplantation,fertility restoration,chemotherapy

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