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Purpose: To date, the methods available for isolating spermatogonial stem cells (SSCs) from porcine testicular cells have a low efficiency of cell separating. Therefore, we tried to develop a novel isolation technique with a high-yield cell separating ability to isolate SSCs from porcine testes., Methods: We confirmed the presence of SSCs by measuring alkaline phosphatase (AP) activity and SSC-specific gene expression in neonatal porcine testis-derived testicular cells. Subsequently, the isolation of SSCs from testicular cells was performed using different techniques as follows: differential plating (DP), double DP, Petri dish plating post-DP, magnetic-activated cell sorting (MACS), and MACS post-DP. Positive AP staining was used to assess and compare the isolation efficiency of each method., Results: Petri dish plating post-DP resulted in the highest isolation efficiency. The putative SSCs isolated using this method was then further characterized by analyzing the expression of SSC-specific genes and -related proteins, and germ cell-specific genes. OCT4, NANOG, EPCAM, THY1, and UCHL1 were expressed transcriptionally, and OCT4, NANOG, SOX2, TRA-1-60, TRA-1-81, and PLZF were expressed translationally in 86 % of the isolated SSCs. In contrast, no difference was observed in the percentage of cells expressing luteinizing hormone receptor (LHR), a Leydig cell-specific protein, or GATA4, a Sertoli cell-specific protein, between SSCs and negative control cells. In addition, transcriptional expression of VASA, a primordial germ cell-specific marker, and DAZL, a premeiotic germ cell-specific marker, wasn't and was detected, respectively., Conclusions: We successfully developed a novel high-yield technique to isolate SSCs from porcine testes to facilitate future porcine SSC-related research.

To study the genomic plasticity of somatic cells without ectopic genetic manipulation, we cultured mouse fibroblasts with ovarian cells, embryonic fibroblasts of different strains, and parthenogenetic embryonic stem cells (ESCs). Of 41 trials, cell aggregation resembling nascent ESC colony from inner cell mass was detected in 9 cases (22%), and 6 cases (67%) yielded fibroblast-derived colonies with ESC morphology. Cells used in coculture provided the critical (P=0.0061) inducing factor for the aggregation. These colony-forming fibroblasts (CFFs) showed similar characteristics to those in ESCs and induced pluripotent stem cells (iPSCs), including pluripotency gene expression, in vitro differentiation, and teratoma formation. Furthermore, CFFs produced somatic chimera, although none showed germline chimerism. CFFs had a tetraploid-like karyotype, and their imprinting patterns differed from parthenogenetic ESCs, thereby confirming their nongermline transmissibility. We observed dysregulation of cell cycle-related proteins, as well as both homologous and heterologous recombination of genomic single-nucleotide polymorphisms in CFFs. Our observations provide information on somatic cell plasticity, resulting in stemness or tumorigenesis, regardless of colony-forming cell progenitors in the fibroblast population. The plasticity of somatic genomes under environmental influences, as well as acquisition of pluripotency by cell fusion, is also implicated.

While cell sorting usually relies on cell-surface protein markers, molecular beacons (MBs) offer the potential to sort cells based on the presence of any expressed mRNA and in principle could be extremely useful to sort rare cell populations from primary isolates. We show here how stem cells can be purified from mixed cell populations by sorting based on MBs. Specifically, we designed molecular beacons targeting Sox2, a well-known stem cell marker for murine embryonic (mES) and neural stem cells (NSC). One of our designed molecular beacons displayed an increase in fluorescence compared to a nonspecific molecular beacon both in vitro and in vivo when tested in mES and NSCs. We sorted Sox2-MB(+)SSEA1(+) cells from a mixed population of 4-day retinoic acid-treated mES cells and effectively isolated live undifferentiated stem cells. Additionally, Sox2-MB(+) cells isolated from primary mouse brains were sorted and generated neurospheres with higher efficiency than Sox2-MB(-) cells. These results demonstrate the utility of MBs for stem cell sorting in an mRNA-specific manner.

The 21st century will see improvements in the quality of human life. The development of new therapeutic technologies will prevent prevalent diseases and enable recovery from currently incurable diseases. The development of cell and tissue replacement therapies using stem cells and their progenitors will accelerate the development of causative treatments. The effort expended thus far in developing cell therapies has revealed many technical limitations. Thus, we must explore conceptual changes in the feasibility of stem cell therapy. This paper introduces the current limitations to stem cell engineering and ways to overcome these limitations, which will provide new insight into their clinical application., (© 2011 New York Academy of Sciences.)

Objective: To evaluate whether serum replacement with growth factor-free synthetic substances contributed to the effective establishment of embryonic stem (ES) cells., Design: Randomized, prospective model study., Setting: Gamete and stem cell biotechnology laboratory at Seoul National University in Korea., Animal(s): F1 (C57BL6 x DBA2) mice., Intervention(s): Blastocysts of different origins were cultured in serum-replaced media., Main Outcome Measure(s): Embryonic stem cell establishment., Result(s): Eight batches of ES cells were established from colony-forming inner cell mass cells after the replacement of fetal bovine serum (FBS) with synthetic knockout serum replacement (KSR) in mkDMEM. The established cells were positive for ES cell markers and formed both embryoid bodies in vitro and teratomas in vivo, but the established cell batches and control (transformed) ES cells responded differently to the culture media. Higher levels of cell viability were detected after the replacement with the 75:25 FBS-KSR mixture than with any other mixtures, and a gradual decrease in viability was detected as the KSR volume ratio was increased. The 75:25 FBS-KSR mixture-containing medium supported ES cell establishment of outbred ICR, F1, and F2 of C57BL6/DBA2; F1 parthenogenetic and ES cell-complemented tetraploid blastocysts; and single ES-cell cultures., Conclusion(s): A serum-replaced medium could be used for effective ES-cell establishment of various origins.

A previous study found that undifferentiated porcine spermatogonial stem cells (SSCs) did not adhere to tenascin C, indicating that the integrin α9 and β1 subunits are inactive on the surface of porcine SSCs. However, that study used recombinant tenascin C without FNIII‐like repeats. Therefore, this study re‐evaluated the existence of integrin α9β1 actively functioning on the plasma membrane of porcine SSCs using full‐length native tenascin C with FNIII‐like repeats. The localization and function of the integrin heterodimer were confirmed using immunocytochemistry, attachment and antibody inhibition assays. In undifferentiated porcine SSCs with integrin α9β1 on the cell surface, adhesion to native tenascin C was significantly higher compared with cells lacking native tenascin C and functional blocking of integrin α9β1 significantly inhibited the attachment to native tenascin C compared with no functional blocking. Accordingly, we confirmed that the integrin α9 and β1 subunits function as an active heterodimer on the surface of porcine SSCs in the undifferentiated state. [ABSTRACT FROM AUTHOR]

Spermatogonial stem cells (SSC) are promising resources for genetic preservation and restoration of male germ cells in humans and animals. However, no studies have used SSC as donor nuclei in pig somatic cell nuclear transfer (SCNT). This study investigated the potential for use of porcine SSC as a nuclei donor for SCNT and developmental competence of SSC‐derived cloned embryos. In addition, demecolcine was investigated to determine whether it could prevent rupture of SSC during SCNT. When the potential of SSC to support embryonic development after SCNT was compared with that of foetal fibroblasts (FF), SSC‐derived SCNT embryos showed a higher (p < .05) developmental competence to the blastocyst stage (47.8%) than FF‐derived embryos (25.6%). However, when SSC were used as donor nuclei in the SCNT process, cell fusion rates were lower (p < .05) than when FF were used (61.9% vs. 75.8%). Treatment of SSC with demecolcine significantly (p < .05) decreased rupture of SSC during the SCNT procedure (7.5% vs. 18.8%) and increased fusion of cell‐oocyte couplets compared with no treatment (74.6% vs. 61.6%). In addition, SSC‐derived SCNT embryos showed higher blastocyst formation (48.4%) than FF‐derived embryos without (28.4%) and with demecolcine treatment (17.4%), even after demecolcine treatment. Our results demonstrate that porcine SSC are a desirable donor cell type for production of SCNT pig embryos and that demecolcine increases production efficiency of cloned embryos by inhibiting rupture of nuclei donor SSC during SCNT. [ABSTRACT FROM AUTHOR]

To date, in vitro culture systems able to sufficiently expand the small population of spermatogonial stem cells (SSCs), a tool for the development of sperm-mediated gene transfer techniques in transgenic pigs, in the porcine seminiferous tubule have not been reported. Therefore, as a step toward engineering a noncellular niche to support the in vitro maintenance of porcine SSC self-renewal, we investigated the types of integrin heterodimers that are expressed and functional on their membrane. The α and β integrin subunit protein expressions were analyzed using immunocytochemistry and fluorescence immunoassay, and the function of integrin heterodimers was confirmed by attachment and antibody inhibition assays. The integrin subunits, α3, α4, α5, α6, α8, α9, αV, and β1, were identified on the surface of them. Moreover, they showed significantly increased adhesion to fibronectin, laminin, and vitronectin, and functional blocking of integrin α4β1, α6β1, or αVβ1 significantly inhibited adhesion to these molecules. They showed significantly decreased adhesion to tenascin C and functional blocking of integrin α5β1 did not significantly inhibit adhesion to fibronectin. Accordingly, we confirmed that the integrin heterodimers α4β1, α6β1, and αVβ1 actively function on the surface of undifferentiated porcine SSCs, whereas α3, α5, α8, and α9 are present in inactive forms. [ABSTRACT FROM AUTHOR]

Generally, self-renewal of spermatogonial stem cells (SSCs) is maintained in vivo in a three-dimensional (3D) microenvironment consisting of the seminiferous tubule basement membrane, indicating the importance of the 3D microenvironment for in vitro culture of SSCs. Here, we report a 3D culture microenvironment that effectively maintains porcine SSC self-renewal during culture. Porcine SSCs were cultured in an agarose-based 3D hydrogel and in 2D culture plates either with or without feeder cells. Subsequently, the effects of 3D culture on the maintenance of undifferentiated SSCs were identified by analyzing cell colony formation and morphology, AP activity, and transcriptional and translational regulation of self-renewal-related genes and the effects on proliferation by analyzing cell viability and single cell-derived colony number. The 3D culture microenvironment constructed using a 0.2% (w/v) agarose-based 3D hydrogel showed the strongest maintenance of porcine SSC self-renewal and induced significant improvements in proliferation compared with 2D culture microenvironments. These results demonstrate that self-renewal of porcine SSCs can be maintained more effectively in a 3D than in a 2D culture microenvironment. Moreover, this will play a significant role in developing novel culture systems for SSCs derived from diverse species in the future, which will contribute to SSC-related research. [ABSTRACT FROM AUTHOR]

Adding a potent antioxidant, glutathione (GSH), to a cryoprotective solution consisting of dimethyl sulfoxide and ethylene glycol and/or postthaw culture medium significantly improved the postthaw viability of mouse embryonic stem cells. This effect, which was caused by a decrease in reactive oxygen species, was only induced by exposure of embryonic stem cells during cryopreservation. [ABSTRACT FROM AUTHOR]

Abstract Transgenic pigs are quite useful in many biomedical fields, such as xenotransplantation research and the production of biopharmaceutical materials. The genetic transformation of porcine spermatogonial stem cells (pSSCs) followed by differentiation into mature spermatozoa enables the effective production of transgenic pigs. Improving the transfection efficiency of pSSCs, however, has been much desired. Herein, we report the efficient genetic modification of pSSCs by using an electrically responsive Au nanowire injector (E-R Au NWI). This is the first study that shows an exogenous gene is directly delivered into the nucleus of a pSSC by using a 1-dimensional nanomaterial and then successfully expressed to produce a protein. The E-R Au NWI interfaced noninvasively with the nucleus of the pSSC, and the pEGFP-N1 plasmid was delivered by the application of an electrical stimulus without cell damage. Compared to the results of conventional nonviral vector-based gene delivery methods such as jetPEI, Lipofectamine, and electroporation, the E-R Au NWI-based method improved the pSSC transfection efficiency by at least 6.7-fold and even up to 46.7-fold. Furthermore, we successfully obtained transgenic pSSCs containing the human bone morphogenetic protein 2 gene by using E-R Au NWIs. This result suggests that the E-R Au NWI enables the efficient genetic modification of pSSCs and can be employed to produce diverse kinds of transgenic pigs. Graphical abstract Image 1 [ABSTRACT FROM AUTHOR]