scholarly journals A protocol for generation of transgenic mice by manipulating spermatogonial stem cells in vivo.

2012 ◽  
Author(s):  
LALIT SEHGAL ◽  
Lalit Sehgal ◽  
Rahul Thorat ◽  
Nileema Khapare ◽  
Amitabha Mukhopadhaya ◽  
...  
Keyword(s):  
Stem Cells ◽  
Transgenic Mice ◽  
Spermatogonial Stem Cells

Generation of Transgenic Mice by Exploiting Spermatogonial Stem Cells In Vivo

2014 ◽  
pp. 327-337 ◽  
Author(s):  
Lalit Sehgal ◽  
Abul Usmani ◽  
Sorab N. Dalal ◽  
Subeer S. Majumdar
Keyword(s):  
Stem Cells ◽  
Transgenic Mice ◽  
Spermatogonial Stem Cells

scholarly journals Lentiviral Mediated Transgenesis by In Vivo Manipulation of Spermatogonial Stem Cells

PLoS ONE ◽  
2011 ◽  
Vol 6 (7) ◽  
pp. e21975 ◽  
Author(s):  
Lalit Sehgal ◽  
Rahul Thorat ◽  
Nileema Khapare ◽  
Amitabha Mukhopadhaya ◽  
Mugdha Sawant ◽  
...  
Keyword(s):  
Stem Cells ◽  
Spermatogonial Stem Cells

scholarly journals VEGFA Isoforms Regulate the Biological Activity of Spermatogonial Stem Cells In Vivo.

2011 ◽  
Vol 85 (Suppl_1) ◽  
pp. 46-46
Author(s):  
Derek J. McLean ◽  
Kyle C. Caires ◽  
Jeanene de Avila ◽  
Andrea S. Cupp
Keyword(s):  
Stem Cells ◽  
Biological Activity ◽  
Spermatogonial Stem Cells

scholarly journals Transgene expression of steel factor in the basal layer of epidermis promotes survival, proliferation, differentiation and migration of melanocyte precursors

Development ◽  
1998 ◽  
Vol 125 (15) ◽  
pp. 2915-2923 ◽  
Author(s):  
T. Kunisada ◽  
H. Yoshida ◽  
H. Yamazaki ◽  
A. Miyamoto ◽  
H. Hemmi ◽  
...  
Keyword(s):  
Stem Cells ◽  
Transgenic Mice ◽  
Germ Cells ◽  
Expression Patterns ◽  
Basal Layer ◽  
Epidermal Keratinocytes ◽  
Oral Epithelium ◽  
Developmental Defects ◽  
Steel Factor

Mutations at the murine dominant white spotting (KitW) and steel (MgfSl) loci, encoding c-Kit receptor kinase and its ligand respectively, exert developmental defects on hematopoietic cells, melanocytes, germ cells and interstitial cells of Cajal. The expression patterns of steel factor (SLF) observed in the skin and gonads suggest that SLF mediates a migratory or a chemotactic signal for c-Kit-expressing stem cells (melanocyte precursors and primordial germ cells). By targeting expression of SLF to epidermal keratinocytes in mice, we observed extended distribution of melanocytes in a number of sites including oral epithelium and footpads where neither melanocytes nor their precursors are normally detected. In addition, enlarged pigmented spots of KitW and other spotting mutant mice were observed in the presence of the SLF transgene. These results provide direct evidence that SLF stimulates migration of melanocytes in vivo. We also present data suggesting that SLF does not simply support survival and proliferation of melanocytes but also promotes differentiation of these cells. Unexpectedly, melanocyte stem cells independent of the c-Kit signal were maintained in the skin of the SLF transgenic mice. After the elimination of c-Kit-dependent melanoblasts by function-blocking anti-c-Kit antibody, these stem cells continued to proliferate and differentiate into mature melanocytes. These melanoblasts are able to migrate to cover most of the epidermis after several months. The SLF transgenic mice described in this report will be useful in the study of melanocyte biology.

scholarly journals Autologous transplantation of spermatogonial stem cells restores fertility in congenitally infertile mice

2020 ◽  
Vol 117 (14) ◽  
pp. 7837-7844
Author(s):  
Mito Kanatsu-Shinohara ◽  
Narumi Ogonuki ◽  
Shogo Matoba ◽  
Atsuo Ogura ◽  
Takashi Shinohara
Keyword(s):  
Stem Cells ◽  
Sertoli Cells ◽  
Expression Patterns ◽  
Autologous Transplantation ◽  
Spermatogonial Stem Cells ◽  
Congenital Defects ◽  
Seminiferous Tubules ◽  
Special Environment ◽  
Deficient Mice

The blood–testis barrier (BTB) is thought to be indispensable for spermatogenesis because it creates a special environment for meiosis and protects haploid cells from the immune system. The BTB divides the seminiferous tubules into the adluminal and basal compartments. Spermatogonial stem cells (SSCs) have a unique ability to transmigrate from the adluminal compartment to the basal compartment through the BTB upon transplantation into the seminiferous tubule. Here, we analyzed the role ofCldn11, a major component of the BTB, in spermatogenesis using spermatogonial transplantation.Cldn11-deficient mice are infertile due to the cessation of spermatogenesis at the spermatocyte stage.Cldn11-deficient SSCs failed to colonize wild-type testes efficiently, andCldn11-deficient SSCs that underwent double depletion ofCldn3andCldn5showed minimal colonization, suggesting that claudins on SSCs are necessary for transmigration. However,Cldn11-deficient Sertoli cells increased SSC homing efficiency by >3-fold, suggesting that CLDN11 in Sertoli cells inhibits transmigration of SSCs through the BTB. In contrast to endogenous SSCs in intactCldn11-deficient testes, those from WT orCldn11-deficient testes regenerated sperm inCldn11-deficient testes. The success of this autologous transplantation appears to depend on removal of endogenous germ cells for recipient preparation, which reprogrammed claudin expression patterns in Sertoli cells. Consistent with this idea, in vivo depletion ofCldn3/5regenerated endogenous spermatogenesis inCldn11-deficient mice. Thus, coordinated claudin expression in both SSCs and Sertoli cells expression is necessary for SSC homing and regeneration of spermatogenesis, and autologous stem cell transplantation can rescue congenital defects of a self-renewing tissue.

Production of fertile sperm from in vitro propagating enriched spermatogonial stem cells of farmed catfish, Clarias batrachus

Zygote ◽  
2016 ◽  
Vol 24 (6) ◽  
pp. 814-824 ◽  
Author(s):  
Swapnarani Nayak ◽  
Shajahan Ferosekhan ◽  
Sangram Ketan Sahoo ◽  
Jitendra Kumar Sundaray ◽  
Pallipuram Jayasankar ◽  
...  
Keyword(s):  
Stem Cells ◽  
Gradient Centrifugation ◽  
Spermatogonial Stem Cells ◽  
Clarias Batrachus ◽  
Proliferating Cells ◽  
Complex Process ◽  
Ficoll Gradient ◽  
Mrna Expression Profiling

SummarySpermatogenesis is a highly co-ordinated and complex process. In vitro propagation of spermatogonial stem cells (SSCs) could provide an avenue in which to undertake in vivo studies of spermatogenesis. Very little information is known about the SSC biology of teleosts. In this study, collagenase-treated testicular cells of farmed catfish (Clarias batrachus, popularly known as magur) were purified by Ficoll gradient centrifugation followed by magnetic activated cell sorting using Thy1.2 (CD90.2) antibody to enrich for the spermatogonial cell population. The sorted spermatogonial cells were counted and gave ~3 × 106 cells from 6 × 106 pre-sorted cells. The purified cells were cultured in vitro for >2 months in L-15 medium containing fetal bovine serum (10%), carp serum (1%) and other supplements. Microscopic observations depicted typical morphological SSC features, bearing a larger nuclear compartment (with visible perinuclear bodies) within a thin rim of cytoplasm. Cells proliferated in vitro forming clumps/colonies. mRNA expression profiling by qPCR documented that proliferating cells were Plzf + and Pou2+, indicative of stem cells. From 60 days onwards of cultivation, the self-renewing population differentiated to produce spermatids (~6 × 107 on day 75). In vitro-produced sperm (2260 sperm/SSC) were free swimming in medium and hence motile (non-progressive) in nature. Of those, 2% were capable of fertilizing and generated healthy diploid fingerlings. Our documented evidence provides the basis for producing fertile magur sperm in vitro from cultured magur SSCs. Our established techniques of SSC propagation and in vitro sperm production together should trigger future in vivo experiments towards basic and applied biology research.

Establishment of a Labeling System for Hematopoietic Stem Cells by Nucleostemin Enhancer/Promoter-GFP Transgenic Mice.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 2240-2240
Author(s):  
Kazuhito Naka ◽  
Masako Ohmura ◽  
Toshio Suda ◽  
Atsushi Hirao
Keyword(s):  
Stem Cells ◽  
Stem Cell ◽  
Transgenic Mice ◽  
Hematopoietic Cells ◽  
Hematopoietic Stem ◽  
Fluorescent Intensity ◽  
Expression Levels ◽  
Ns Gene ◽  
Labeling System

Abstract A labeling system for tissue stem cells is a powerful tool for stem cell research and for development of stem cell-based regenerative medicine to human disease. Here we generated transgenic mice expressing green fluorescent protein (GFP) under control of enhancer/promoter activity to Nucleostemin (NS) gene. NS, a nucleolar GTP-binding protein, expresses at high levels in embryonic stem cells and neural stem cells, suggesting that NS expresses in various tissue stem cells. In fact, we found that NS highly expressed in mouse immature hematopoietic cells containing hematopoietic stem cells (HSCs) and progenitor cells compared with differentiated hematopoietic cells. To examine whether HSCs could be labeled by using enhancer/promoter activity to NS gene, we generated transgenic mice harboring putative genomic enhancer/promoter region of NS gene followed by GFP cDNA-polyA in the 3′ end (NS-GFP tg mice). Fluorescence activated cell sorting (FACS) analysis indicated that most bone marrow (BM) mononuclear cells (MNCs) showed green fluorescence in NS-GFP tg mice. To validate the relationship between fluorescent intensity of GFP and expression levels of endogenous NS mRNA, BM MNCs from NS-GFP tg mice were separated into four fractions depending on their fluorescent intensity (i.e., GFP−, GFP+, GFP++, and GFP+++) by FACS, and these cells were subjected to quantitative real-time PCR analysis. The fluorescent intensity of GFP certainly reflected the amount of endogenous NS mRNA, indicating that the enhancer/promoter region used in this study is sufficient for monitoring the expression levels of endogenous NS mRNA in vivo. We next characterized expression levels of cell-surface markers on the four fractions of BM MNCs. FACS analysis showed that the GFP+++ cells, but not the other cell populations, lost the expression of mature hematopoietic cell markers (myeloid, B cells, T cells and erythroid cells). The fluorescent intensity of GFP in c-Kit+ Sca-1+ Lineage− (KSL) cells, which represent a primitive hematopoietic cell fraction containing HSCs and progenitor cells, was much higher than that in the other cell populations, indicating that the immature hematopoietic cells highly express GFP. To investigate the biological properties of the GFP-high expressing cells, we further performed colony-forming assay in vitro and BM transplantation assay in vivo. When the sorted BM MNCs were cultured in methylcellulose medium supplemented with cytokines, GFP+++ cells showed the highest number of colony formation. To assess the repopulating capacity as stem cell function, the sorted BM MNCs were transplanted into irradiated recipient mice. Notably, only GFP+++ cells had long-term reconstitution capacity of hematopoiesis in the recipient mice on the competitive reconstitution assay. In addition, the GFP+++ cells that were sorted from the GFP+++ cells-transplanted mouse retained the reconstitution capacity after secondary BM transplantation. The reconstitutive capacity of the GFP+++ cells into multi-lineages was confirmed by detection of donor-derived B, T, and myeloid cells in the recipient mice. These results demonstrate that HSCs with self-renewal capacity and differentiation potential for multi-lineages are enriched in GFP+++ cell population. The NS-GFP tg mouse system may provide a useful tool for effective enrichment of HSCs in combination with other cell-surface markers for HSCs.

Overexpression of NF-E2 In Vivo Causes Thrombocytosis and Acute Leukemia: A Murine Model of Myeloproliferative Neoplasms.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 961-961
Author(s):  
Albert Gruender ◽  
Kai Kaufmann ◽  
Tobias Hadlich ◽  
Thomas Günther ◽  
Roland Schüle ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Transcription Factor ◽  
Transgenic Mice ◽  
Acute Leukemia ◽  
Myeloproliferative Neoplasms ◽  
Transgenic Animals ◽  
Spleen Weight ◽  
Hematopoietic Stem

Abstract Abstract 961 The transcription factor nuclear factor erythroid-2 (NF-E2) is expressed in hematopoietic stem cells as well as in myeloid, erythroid and megakaryocytic precursors. NF-E2 deficient mice display marked anemia at birth and die perinatally due to thrombopenia, demonstrating an essential role for NF-E2 in both in erythropoiesis and platelet formation. We have previously shown that NF-E2 is overexpressed in the vast majority of patients with Myeloproliferative Neoplasms (MPNs). However, the effect of augmented transcription factor activity has not been studied in vivo. We therefore engineered two independent transgenic mouse lines expressing human NF-E2 under the control of the vav-Promoter, which has previously been shown to direct transgene expression in hematopoietic stem cells as well as in precursor cells of all lineages. The two founder lines differed in the degree of NF-E2 overexpression displayed. While one line showed moderate overexpression (2 – 5-fold), the other line expressed human NF-E2 between 10 and 100-fold above the murine counterpart. Both lines paralleled observations in PV patients, where a wide range of NF-E2 overexpression was noted (median overexpression, 7-fold; range 2-fold to 40-fold; n = 59). The two founder lines show overlapping but distinct phenotypes. In both strains. moderately overexpressing NF-E2 transgenic mice (2 – 10-fold) invariably develop thrombocytosis with a latency of 14 months. In addition, megakaryocyte colony formation in the bone marrow is drastically increased. In contrast, thrombocytosis is not observed in the markedly overexpressing NF-E2 transgenic mice (above 20-fold). A similar inverse correlation between the degree of NF-E2 overexpression and platelet numbers was observed in MPN patients. In both strains, Epo-independent colony formation, a pathognomonic feature of polycythemia vera, is significantly increased in NF-E2 transgenic animals. Bone marrow histopathology shows findings characteristically seen in MPNs, including the presence of increased megakaryopoiesis with cytologically abnormal forms, often in clusters. Both NF-E2 transgenic strains display significantly increased mortality. Upon autopsy, between 15 and 20% of mice in both strains present with major gastrointestinal bleeding in conjunction with splenic atrophy. Spleen weight is reduced by over 50% (Transgenic mice: 49 +/-15 mg, wild type littermates 103 +/- 30 mg; p < 0.001, n = 8 each). One third of the remaining mice show moderate to marked splenomegaly (2 – 27 fold increase in spleen weight; mean: 434 mg, range: 124 – 2700 mg; p < 0.001 vs. wt littermates, n = 12). Histopathological examination of all spleens revealed mild to moderately expanded red pulp with increased numbers of iron containing histiocytes. This observation indicates increased red cell destruction and may explain the fact that neither hematocrit nor hemoglobin are elevated in NF-E2 transgenic animals. At 18 months of age, one mouse developed acute leukemia, which is currently being phenotyped. In summary, in a murine model moderate NF-E2 overexpression causes a phenotype resembling Essential Thrombocythemia. In addition, our preliminary data indicate that NF-E2 overexpression may predispose to the development of acute leukemia. Disclosures: No relevant conflicts of interest to declare.

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