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Telomeres are known as a specialized region in the end of chromosomes to protect DNA destruction, but their lengths are shortened by repetition of cell division. This telomere shortening can be preserved or be elongated by telomerase and TERT expression. Although a certain condition in the cells may affect to the cellular and molecular characteristics, the effect of differentiation induction to telomere length and telomerase activity in mesenchymal stem cells (MSCs) has been less studied. Therefore, the present study aimed to uncover periodical alterations of telomere length, telomerase activity and TERT expression in the dental pulp-derived MSCs (DP-MSCs) under condition of differentiation inductions into adipocytes and osteoblasts on a weekly basis up to 3 weeks. Shortening of telomere was significantly (p < 0.05) identified from early-middle stages of both differentiations in comparison with undifferentiated DP-MSCs by non-radioactive chemiluminescent assay and qRT-PCR method. Telomere length in undifferentiated DP-MSCs was 10.5 kb, but the late stage of differentiated DP-MSCs which can be regarded as the adult somatic cell exhibited 8.1-8.6 kb. Furthermore, the relative-quantitative telomerase repeat amplification protocol or western blotting presented significant (p < 0.05) decrease of telomerase activity since early stages of differentiations or TERT expression from middle stages of differentiations than undifferentiated state, respectively. Based on these results, it is supposed that shortened telomere length in differentiated DP-MSCs was remained along with prolonged differentiation durations, possibly due to weakened telomerase activity and TERT expression. We expect that the present study contributes on understanding differentiation mechanism of MSCs, and provides standardizing therapeutic strategies in clinical application of MSCs in the animal biotechnology.

Porcine mesenchymal stem cells (MSCs) are similar to human MSCs, hence considered a valuable model for assessing potential for cell therapy. Porcine adipose-derived MSCs (AD-MSCs) and endometrial stromal MSCs (EMSCs) displayed fibroblast-like morphology and were positive for MSC markers CD73, CD90, and CD105 and negative for hematopoietic markers CD34 and CD45. The EMSCs had similar or slightly higher growth rate compared to AD-MSCs, and similar percentage of cells of both EMSCs and AD-MSCs were at G0/G1 and G2/M phases; however, EMSCs had significantly ( P < .05) higher percentage of cells at S phase of cell cycle than AD-MSCs. Transdifferentiation ability to cardiomyocyte-like cells was confirmed in differentiated cells by the expression of lineage-specific marker genes such as DES, ACTA2, cTnT, and ACTC1 by real-time quantitative polymerase chain reaction (RT-qPCR). Furthermore, cardiomyocyte-specific protein markers cTnT and ACTC1 were expressed in completely differentiated cells. Endodermal differentiation capacity of EMSCs to pancreatic β cell-like cells was evident with the changes in morphology and the expression of β-cell-specific marker genes such as PDX1, GLUT2, SST, NKX6.1, PAX4, and NGN3 as analyzed by RT-qPCR. The differentiated cells secreted insulin and C-peptide upon glucose challenge and also they expressed insulin, PDX1, PAX4, NGN3, and GLUT2 at protein level as assessed by immunostaining confirming the successful differentiation to β cell-like cells. Porcine EMSCs possess all the characteristics of MSCs and are suitable model for studying molecular mechanisms of cellular differentiation.

There are few studies on the effects of dipeptidyl peptidase-4 inhibitors on steatohepatitis. We explored whether evogliptin (Evo), a dipeptidyl peptidase-4 inhibitor, protects against steatohepatitis in a high-fat diet (HFD)-fed mice and whether these effects involve modulation of mitophagy. Adult male C57BL/J mice were divided into the normal diet (ND), HFD (45% of energy from fat) with Evo (250 mg/kg) (HFD + Evo), and HFD groups at 4 weeks of age and were sacrificed at 20 weeks of age. The HFD group showed hepatic lipid accumulation, this was decreased in the Evo + HFD group. There was an increased 8-hydroxydeoxyguanosine (8-OHDG) expression in the HFD group compared to ND mice. However, 8-OHDG expression levels were significantly decreased in the HFD + Evo group. Expressions of the mitophagy markers PTEN-induced kinase 1 (PINK1), Parkin, and BNIP-3 (BCL2 Interacting Protein 3) were significantly increased in the HFD group. However, the expressions of these markers were lower in the HFD + Evo group than that in the HFD group. Phospho-Akt was upregulated and p53 was downregulated in the HFD + Evo group compared to the HFD group. Evogliptin may alleviate steatohepatitis in HFD-fed mice by ameliorating steatosis and oxidative stress and by modulating mitophagy in the liver.

Contrast-induced acute kidney injury (CI-AKI) is the third most common cause of hospital-acquired renal failure, with an incidence of 11%. However, the disease mechanism remains unclear, and no effective treatment is available. Paricalcitol has been reported to be effective in animal models of kidney injury. We hypothesized that paricalcitol could play a renoprotective role against CI-AKI. Rats were divided into control, paricalcitol, contrast, and paricalcitol-plus-contrast groups. We used a previously published protocol to produce CI-AKI. Paricalcitol (0.3 μg/kg) was administered intraperitoneally before 24 h and 30 min before indomethacin. We used HK-2 cells to evaluate the effects of paricalcitol on mitophagy and senescence. Ioversol triggered renal dysfunction, increasing blood urea nitrogen and serum creatinine. Significant tubular damage, increased 8-OHdG expression, and apoptosis were apparent. Ioversol injection induced high expression levels of the mitophagy markers Pink1, Parkin, and LC3 and the senescence markers β-galactosidase and p16INK4A. Paricalcitol pretreatment prevented renal dysfunction and reduced tissue damage by reducing both mitophagy and senescence. Cellular morphological changes were found, and expression of LC3B and HMGB1 was increased by ioversol in HK-2 cells. Paricalcitol countered these effects. This study showed that mitochondria might drive injury phenotypes in CI-AKI, and that paricalcitol protects against CI-AKI by decreasing mitochondrial damage.

The therapeutic efficacy of adipose mesenchymal stem cells (Ad-MSCs) for acute kidney injury (AKI) has been investigated extensively, and the anti-apoptotic, anti-inflammatory, and proangiogenic effects of heme oxygenase-1 (HO-1) reportedly ameliorate AKI. We hypothesized that the therapeutic efficacy of Ad-MSCs is dependent on their expression level of HO-1. The viability and migration ability of cisplatin-treated human renal proximal tubular epithelial cells were assessed. Sprague–Dawley rats were divided into control, cisplatin (10 mg/kg), and cisplatin plus Ad MSCs (with high and low HO-1 expression) groups. The HO-1 expression level in hAd-MSCs increased with increasing passage number, peaking at passage 4 and decreasing thereafter. The viability and migratory ability of hAd-MSCs with high HO-1 expression were greater than those of hAd-MSCs with low HO-1 expression. Renal tubular toxicity in cisplatin-treated rats was ameliorated by administration of hAd-MSCs with high HO-1 expression, although the levels of blood urea nitrogen and serum creatinine did not differ according to the level of HO-1 expression. The magnitude of reactive oxygen species induced DNA damage was lower in hAd-MSCs with high HO-1 expression than in those with low HO-1 expression. Administration of hAd-MSCs significantly suppressed cisplatin induced apoptosis. Also, hAd-MSCs with high HO-1 expression were more resistant to cisplatin-induced apoptosis than were those with low HO-1 expression. hAd MSCs with high HO-1 expression have therapeutic potential for cisplatin induced nephrotoxicity, based on our in vitro and in vivo results. These findings will facilitate the development of novel therapeutic strategies for cisplatin-induced AKI.

Radiation therapy is a standard treatment for patients with head and neck cancer. However, radiation exposure to the head and neck induces salivary gland (SG) dysfunction. Alpha lipoic acid (ALA) has been reported to reduce radiation-induced toxicity in normal tissues. In this study, we investigated the effect of ALA on radiation-induced SG dysfunction. Male Sprague&ndash, Dawley rats were assigned to the following treatment groups: control, ALA only (100 mg/kg, intraperitoneally), irradiation only, and ALA administration 24 h or 30 min prior to irradiation. The neck area, including SGs, was irradiated evenly at 2 Gy/min (total dose, 18 Gy) using a photon 6 MV linear accelerator. The rats were sacrificed at 2, 6, 8, and 12 weeks after irradiation. Radiation decreased SG weight, saliva secretion, AQP5 expression, parasympathetic innervation (GFR&alpha, 2 and AchE expression), regeneration potentials (Shh and Ptch expression), salivary trophic factor levels (brain-derived neurotrophic factor and neurturin), and stem cell expression (Sca-1). These features were restored by treatment with ALA. This study demonstrated that ALA can rescue radiation-induced hyposalivation by preserving parasympathetic innervation and regenerative potentials.

Polycaprolactone (PCL) is a biodegradable polyester that is bioresorbable and biocompatible, and is widely used in medical fields. This study examines in vitro and in vivo osteogenic activities of cultured human periosteum-derived osteoblasts (POs) seeded into growth factor (bone morphogenic protein 2 [BMP-2] or vascular endothelial growth factor [VEGF])-releasing scaffolds of PCL beads coated with Pluronic F127. Each growth factor immobilized in the PCL/F127 is cumulatively released from the beads for more than 40 days (up to 3.04 ± 0.08 ng mg-1 BMP-2 and 3.41 ± 0.18 ng mg-1 VEGF in 42 days). Long-term (∼2 years) experimental results obtained in a miniature pig model suggest that POs seeded into BMP-2 + VEGF-releasing PCL/P-F127 beads are the most effective for bone repair. In in vitro assays, osteogenic activities were higher in POs seeded into BMP-2-releasing PCL/Pluronic F127 beads at earlier time points and in POs seeded into BMP-2 + VEGF-releasing PCL/Pluronic F127 beads at later time points. These results suggest that the combination of BMP-2 and VEGF more sufficiently stimulates (in particular at late time points) osteoblast differentiation of POs seeded in the PCL/F127 in vitro and in vivo, and thus allows effective bone regeneration. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 105A: 363-376, 2017.

Aims The aim of this study was to find out a mesenchymal stem cells (MSCs) source from human dental tissues of the same donor (follicle, papilla and pulp), which exhibits higher neurogenic differentiation potential in vitro. Main methods MSCs were isolated from dental tissues (follicle, papilla and pulp) by digestion method. All MSCs were analyzed for pluripotent makers by western blot, cell surface markers by flow cytometry, adipo- and osteocytes markers by RT-qPCR. The neuronal differentiated MSCs were characterized for neuronal specific markers by RT-qPCR and immunofluorescence. Functional neuronal properties were analyzed by electrophysiology and synaptic markers expression. Key findings All MSCs expressed pluripotent markers (Oct4, Sox2 and Nanog) and were found positive for mesenymal markers (CD44, CD90, CD105) while negative for hematopoietic markers (CD34 and CD45). Furthermore, MSCs were successfully differentiated into adipocytes, osteocytes and trans-differentiated into neuronal cells. Among them, dental pulp derived MSCs exhibits higher neurogenic differentiation potential, in term of expression of neuronal specific markers at both gene and protein level, and having higher Na+ and K+ current with the expression of synaptic markers. Significance The three types of dental MSCs from a single donor broadly possessed similar cellular properties and can differentiate into neuronal cells; however, pulp derived MSCs showed higher neurogenic potential than the follicle and papilla, suggesting their use in future stem cells therapy for the treatment of neurodegenerative disorders.

There is evidence that pepsin can aggravate tonsil hypertrophy. Pepstatin is a potent inhibitor of pepsin activity and could protect patients against reflux tonsil hypertrophy by inhibiting pepsin. We examined the effects of pepstatin on the development of tonsil hypertrophy to investigate pepsin's role in the pathogenesis of tonsil lesions. We investigated whether pepstatin suppresses pepsin-mediated lymphocyte proliferation in tonsil hypertrophy. Forty-nine children with tonsil hypertrophy and twenty-two adults with tonsillitis were recruited to the study prior to surgery. Tonsil tissue from each patient was harvested and assessed for changes in the number of lymphocytes and macrophages in the presence of pepsin and pepstatin. We found that the proportions of CD4- and CD14-positive cells were significantly lower (p < 0.05), but that the proportions of CD19- and CD68-positive cells were significantly higher (p < 0.05), in children than in adults. There were significantly more CD4-positive cells after pepsin treatment, but these numbers were reduced by pepstatin. The levels of both interleukin-2 (IL-2) and interferon gamma (IFN-γ) increased significantly in response to pepsin, but were reduced when pepsin was inhibited by pepstatin. The level of IL-10 is reduced in pepsin-treated CD4 cells and the level is restored by pepstatin. IL-2 blocking reduced the increased CD4 cell number by pepsin. But, an additive or a synergic effect is not founded in combined with IL-2 blocking and pepstatin. Pepsin-positive cells did not co-localize with CD20 and CD45 cells, but they were found surrounding CD20- and CD45-positive hypertrophic tonsil cells. Pepsin-positive cells co-localized with CD68-positive cells. It is probable that pepsin from extraesophageal reflux aggravates tonsil hypertrophy and pepstatin exerts a protective effect by inhibiting pepsin activity.

Porcine mesenchymal stem cells (MSCs) are similar to human MSCs, hence considered a valuable model for assessing potential for cell therapy. Porcine adipose-derived MSCs (AD-MSCs) and endometrial stromal MSCs (EMSCs) displayed fibroblast-like morphology and were positive for MSC markers CD73, CD90, and CD105 and negative for hematopoietic markers CD34 and CD45. The EMSCs had similar or slightly higher growth rate compared to AD-MSCs, and similar percentage of cells of both EMSCs and AD-MSCs were at G0/G1 and G2/M phases; however, EMSCs had significantly ( P.05) higher percentage of cells at S phase of cell cycle than AD-MSCs. Transdifferentiation ability to cardiomyocyte-like cells was confirmed in differentiated cells by the expression of lineage-specific marker genes such as DES, ACTA2, cTnT, and ACTC1 by real-time quantitative polymerase chain reaction (RT-qPCR). Furthermore, cardiomyocyte-specific protein markers cTnT and ACTC1 were expressed in completely differentiated cells. Endodermal differentiation capacity of EMSCs to pancreatic β cell-like cells was evident with the changes in morphology and the expression of β-cell-specific marker genes such as PDX1, GLUT2, SST, NKX6.1, PAX4, and NGN3 as analyzed by RT-qPCR. The differentiated cells secreted insulin and C-peptide upon glucose challenge and also they expressed insulin, PDX1, PAX4, NGN3, and GLUT2 at protein level as assessed by immunostaining confirming the successful differentiation to β cell-like cells. Porcine EMSCs possess all the characteristics of MSCs and are suitable model for studying molecular mechanisms of cellular differentiation.

The biological properties of mesenchymal stem cells (MSCs) are influenced by donor age, gender and/or tissue sources. The present study investigated the cellular and molecular properties of porcine mesenchymal stromal/stem cells (MSCs) isolated from different tissues (adipose & dermal skin) and sex at different ages (1 week & 8 months after birth) with similar genetic and environmental backgrounds. MSCs were analyzed for alkaline phosphatase (AP) activity, CD90 and Oct3/4 expression, in vitro differentiation ability, senescence-associated β-galactosidase (SA-β-Gal) activity, telomeric properties, cell cycle status and expression of senescence (IL6, c-myc, TGFβ, p53 and p21)- and apoptosis (Bak and Bcl2)-related proteins. An age-dependent decline in AP activity and adipogenesis was observed in all MSCs, except for male A-MSCs. CD90 expression did not change, but SA-β-Gal activity increased with advancement in age, except in A-MSCs. Telomeric properties were similar in all MSCs, whereas expression levels of Oct3/4 protein declined with the advancement in age. p21 expression was increased with increase in donor age. Male derived cells have shown higher IL6 expression. The expression of p53 was slightly lower in MSCs of dermal tissue than in adipose tissue. Bak was expressed in all MSCs regardless of age, but up regulation of Bcl2 was observed in DS-MSCs derived at 1 week after birth. In conclusion, adipose tissue-derived MSCs from young female individuals were found to be more resistant to senescence under in vitro culture conditions.

Objectives To compare the effect of three different cryoprotectants on basic stem cell characteristics for the possibility of using well defined, dimethyl sulfoxide (DMSO) and serum free freezing solutions to cryopreserve human Wharton’s jelly-derived mesenchymal stem cells (WJMSCs) following controlled rate freezing protocol. Methods The mesenchymal stem cells isolated from human Wharton’s jelly were cryopreserved using 10% DMSO, 10% polyvinylpyrrolidone (PVP) and a cocktail solution comprising of 0.05 M glucose, 0.05 M sucrose and 1.5 M ethylene glycol following controlled rate freezing protocol. We investigated the post-thaw cell viability, morphology, proliferation capacity, basic stem cell characteristics, in vitro differentiation potential and apoptosis-related gene expression profile before and after cryopreservation. Results The cryoprotectant 10% DMSO has shown higher post-thaw cell viability of 81.2±0.58% whereas 10% PVP and cocktail solution have shown 62.87±0.35% and 72.2±0.23%, respectively at 0 h immediately thawing. The cell viability was further reduced in all the cryopreserved groups at 24 h later post-thaw culture. Further, the complete elimination of FBS in cryoprotectants has resulted in drastic reduction in cell viability. Cryopreservation did not alter the basic stem cell characteristics, plasticity and multipotency except proliferation rate. The expression of pro-apoptotic BAX and p53 genes were higher whilst p21 was lower in all the cryopreserved groups when compare to the control group of WJMSCs. Conclusion Although 10% DMSO has shown higher post-thaw cell viability compare to 10% PVP and cocktail solution, the present study indicates the feasibility of developing a well-defined DMSO free cryosolution which can improve storage and future broad range applications of WJMSCs in regenerative medicine without losing their basic stem cell characteristics.

Supplementary_figure for CD105+ Porcine Endometrial Stromal Mesenchymal Stem Cells Possess Differentiation Potential Toward Cardiomyocyte-Like Cells and Insulin-Producing β Cell-Like Cells In Vitro by Raghavendra Baregundi Subbarao, Sharath Belame Shivakumar, Yong-Ho Choe, Young-Bum Son, Hyeon-Jeong Lee, Imran Ullah, Si-Jung Jang, Sun-A Ock, Sung-Lim Lee, and Gyu-Jin Rho in Reproductive Sciences

To increase the overall survival rate and obtain a better prognosis for oral squamous cell carcinoma (OSCC) patients, the detection of more effective and reliable tumor prognostic markers is needed. This study is focused on the analysis of correlation between the clinicopathological features of OSCCs and the immunohistochemical (IHC) expression patterns of MIDKINE (MK) and NANOG. Sixty-two primary OSCC patients were selected and their pretreatment biopsy specimens were immunohistochemically analyzed for the MK and NANOG proteins. The IHC expression patterns, clinicopathological features, and overall survival rates were assessed to identify any correlations. MK and NANOG showed significantly similar IHC expression patterns: both demonstrated enhanced expression in histologically high-grade and clinically late-stage OSCCs. Weak or negative expression of MK and NANOG was correlated with negative neck node metastasis. Clinicopathologically, late tumor stage, neck node metastasis, high-grade tumor, and palliative treatment groups showed significantly lower overall survival rates. The enhanced expression of MK and NANOG was associated with lower overall survival rates. In particular, enhanced co-detection of MK and NANOG showed significant correlation with poor prognosis. In conclusion, enhanced IHC expression patterns of MK and NANOG in OSCC patients was significantly associated with lower overall survival rates and unfavorable clinicopathological features. These results demonstrate that analysis of IHC expression patterns of MK and NANOG in pretreatment biopsy specimens during the work-up period can provide a more definitive prognosis prediction for each OSCC patient that can help clinicians to develop a more precise individual treatment modality.

The main purpose of this study was to develop a cryopreservation method for human dental follicle tissue to maintain autologous stem cells as a resource. A modified cryoprotectant, consisting of 0.05 m glucose, 0.05 m sucrose and 1.5 m ethylene glycol in phosphate-buffered saline (PBS) was employed, with a slow-ramp freezing rate. We observed > 70% of cell survival rate after 3 months of tissue storage. Isolated and cultured human dental stem cells (hDSCs) from cryopreserved dental follicles expressed mesenchymal stem cell markers at a level similar to that of hDSCs from fresh tissue. They also successfully differentiated in vitro into the mesenchymal lineage, osteocytes, adipocytes and chondrocytes under specific inductions. Using immunohistochemistry, the early transcription factors OCT4, NANOG and SOX2 were moderately or weakly detected in the nucleus of both fresh and cryopreserved dental follicles. In addition, p63, CCND1, BCL2 and BAX protein expression levels were the same in both fresh and cryopreserved tissues. However, the positive-cell ratio and intensity of p53 protein was higher in cryopreserved tissues than in fresh tissues, indicating direct damage of the freeze-thawing process. Real-time PCR analysis of hDSCs at passage 2 from both fresh and cryopreserved dental follicles showed similar levels of mRNA for apoptosis- and transcription-related genes. Based on these results, a newly developed cryoprotectant, along with a slow ramp rate freezing procedure allows for long-term dental tissue preservation for later use as an autologous stem cell resource in regenerative cell therapy. Copyright © 2014 John Wiley & Sons, Ltd.

Polycaprolactone (PCL) is a biodegradable polyester that is bioresorbable and biocompatible, and is widely used in medical fields. This study examines in vitro and in vivo osteogenic activities of cultured human periosteum-derived osteoblasts (POs) seeded into growth factor (bone morphogenic protein 2 [BMP-2] or vascular endothelial growth factor [VEGF])-releasing scaffolds of PCL beads coated with Pluronic F127. Each growth factor immobilized in the PCL/F127 is cumulatively released from the beads for more than 40 days (up to 3.04 ± 0.08 ng mg

Mesenchymal stem cells (MSCs) have been established after isolation from various tissue sources, including bone marrow and synovial fluid. Recently, synovial-fluid-derived MSCs were reported to have multi-lineage differentiation potential and immunomodulatory features, which indicates that these cells can be used for tissue engineering and systemic treatments. This study presents a protocol for simple and non-invasive isolation of MSCs derived from the bone marrow and synovial fluid of minipigs to analyze cell surface markers for cell phenotyping and in vitro culturing. Using sexually mature six-month-old minipigs, bone marrow was extracted from the iliac crest bone using a bone marrow extractor, and the synovial fluid was aspirated from the femorotibial joint. Procedures for the collection of samples from both sources were non-invasive. The protocols for effective isolation of MSCs from harvested cell sources and for creating in vitro culture conditions to expand stable MSCs from minipigs and the application of systemic autologous treatments are provided. For cell phenotyping, the cell surface markers of both cells were analyzed using flow cytometry. In the results, the MSCs were isolated from the synovial fluid of the minipigs and showed that synovial-fluid-derived MSCs have a similar morphology and cell phenotype to bone-marrow-derived MSCs. Therefore, non-invasively obtained synovial fluid is a valuable source of MSCs.

It is commonly accepted that the sustained release of bone morphogenetic protein-2 (BMP-2) can enhance bone regeneration and minimize its safety issues. However, little is known regarding the appropriate duration of BMP-2 stimulation for sufficient osteogenic differentiation and new bone formation because of the short half-life of BMP-2 in the physiological environment and the lack of a well-defined delivery matrix that can regulate the release period of BMP-2. In this study, we prepared porous poly(lactic-co-glycolic acid) (PLGA) beads with different surface pore sizes that can regulate the release period of BMP-2 (i.e., 7, 17, and 30 days) while providing the BMP-2 concentration required for bone regeneration. Our findings in both in vitro cell culture and in vivo animal studies using these BMP-2-loaded beads demonstrate that release of BMP-2 within 7 days affects only the initial differentiation of human periosteum-derived cells (hPDCs) and does not significantly enhance their subsequent differentiation into mature functional cells. However, extending the duration of BMP-2 stimulation over 17 days can provide a suitable environment for osteogenic differentiation of hPDCs and new bone formation.

The facile nature of mesenchymal stem cell (MSC) acquisition in relatively large numbers has made Wharton's jelly (WJ) tissue an alternative source of MSCs for regenerative medicine. However, freezing of such tissue using dimethyl sulfoxide (DMSO) for future use impedes its clinical utility. In this study, we compared the effect of two different cryoprotectants (DMSO and cocktail solution) on post‐thaw cell behavior upon freezing of WJ tissue following two different freezing protocols (Conventional [−1°C/min] and programmed). The programmed method showed higher cell survival rate compared to conventional method of freezing. Further, cocktail solution showed better cryoprotection than DMSO. Post‐thaw growth characteristics and stem cell behavior of Wharton's jelly mesenchymal stem cells (WJMSCs) from WJ tissue cryopreserved with a cocktail solution in conjunction with programmed method (Prog‐Cock) were comparable with WJMSCs from fresh WJ tissue. They preserved their expression of surface markers, pluripotent factors, and successfully differentiated in vitro into osteocytes, adipocytes, chondrocytes, and hepatocytes. They also produced lesser annexin‐V‐positive cells compared to cells from WJ tissue stored using cocktail solution in conjunction with the conventional method (Conv‐Cock). Real‐time PCR and Western blot analysis of post‐thaw WJMSCs from Conv‐Cock group showed significantly increased expression of pro‐apoptotic factors (BAX, p53, and p21) and reduced expression of anti‐apoptotic factor (BCL2) compared to WJMSCs from the fresh and Prog‐Cock group. Therefore, we conclude that freezing of fresh WJ tissue using cocktail solution in conjunction with programmed freezing method allows for an efficient WJ tissue banking for future MSC‐based regenerative therapies. J. Cell. Biochem. 117: 2397–2412, 2016. © 2016 The Authors. Journal of Cellular Biochemistry published by Wiley Periodicals, Inc.

Background Recent findings have revealed that the female gonad may have regenerative activity with having germ line stem cells in juveniles and adults. Application of these germ line stem cells could be an alternative therapy for reproductive disorders in regenerative medicine. Methods To enhance the potency of differentiation into oocyte-like cells (OLCs) and folliculogenesis, we overexpressed Oct4 in ovarian stem/stromal cell (OvSCs) and examined the cellular properties related to stemness and self-renewal ability and finally demonstrated the ability of in vitro differentiation and folliculogenesis. Results Ovarian cortex included putative stem cells in terms of AP activity, cell cycle status, cell proliferation, expression of mesenchymal lineage surface markers and pluripotent transcriptional markers. Further, Oct4 transfected OvSCs (Oct4-OvSCs) were enhanced their AP activity and cell proliferation compared to OvSCs. The potential on in vitro differentiation into OLCs and in vivo folliculogenesis was also evaluated in OvSCs and Oct4-OvSCs, respectively. Oct4-OvSCs possessed higher oogenesis potential in vitro than OvSCs, in terms of expression of germ cell markers by RT-PCR and the number of OLCs. When OvSCs and Oct4-OvSCs were xeno-transplanted into infertile mice ovaries, the OvSCs transplantation induced new primary follicle formation and hormonal levels of estradiol and FSH remained similar to that of normal mice. However, Oct4-OvSCs possessed higher ability for folliculogenesis based on inducing developing follicles with thecal layer and granulosa cells and more similar estradiol level to normal mice. Conclusions These findings demonstrated that putative stem cells were present in ovarian cortex and exhibited differentiation ability into OLCs and folliculogenesis in vivo, and Oct4-overexpression enhanced these ability, suggesting their cellular models based on gene therapy in understanding the mechanisms of oogenesis and folliculogenesis, and finally in view of reproductive cell therapy.

Background In order to predict long-term prognosis and define individual treatment modalities for patients with oral squamous cell carcinoma (OSCC), more reliable tumor biomarkers are needed during the pretreatment workup period. The present study aimed to identify more reliable immunohistochemical tumor prognostic markers in the pretreatment biopsy specimens of patients with OSCC. Methods We selected 57 patients who were diagnosed with primary OSCC through histopathological analysis. Pretreatment biopsy specimens were immunohistochemically analyzed for the transcription factor NANOG, cancer stem cell marker CD44, and mutant tumor protein 53 (mutant p53). The immunostaining patterns were assessed for their association with the clinicopathological features of OSCC and overall survival rates. Results Late tumor stage, positive neck node metastasis, and high-grade differentiation were associated with significantly poorer survival rates. Enhanced expression of NANOG and mutant p53 positivity were significantly associated with clinically late-stage tumors, positive neck node metastasis, histologically high-grade tumors, and poor overall survival rates. OSCCs with strong co-detection of NANOG and mutant p53 were linked to significantly lower survival rates than those with both weak NANOG expression and p53 negativity. Increased expression of CD44 had a limited correlation with unfavorable clinicopathological features. Conclusion High expression of NANOG and positive expression of mutant p53 in the pretreatment biopsy specimens of patients with OSCC were associated with poor survival rates and unfavorable clinicopathological features. These results demonstrate that NANOG, mutant p53, and CD44 could be used as immunohistochemical markers in the pretreatment specimens of OSCC. In particular, analysis for co-expression of NANOG and mutant p53 should be made highly available as a tool for prognosis and selecting individual treatment modalities.

In our previous study, dental follicle tissues from extracted wisdom teeth were successfully cryopreserved for use as a source of stem cells. The goals of the present study were to investigate the immunomodulatory properties of stem cells from fresh and cryopreserved dental follicles (fDFCs and cDFCs, respectively) and to analyze in vivo osteogenesis after transplantation of these DFCs into experimental animals. Third passage fDFCs and cDFCs showed similar expression levels of interferon-γ receptor (CD119) and major histocompatibility complex class I and II (MHC I and MHC II, respectively), with high levels of CD119 and MHC I and nearly no expression of MHC II. Both fresh and cryopreserved human DFCs (hDFCs) were in vivo transplanted along with a demineralized bone matrix scaffold into mandibular defects in miniature pigs and subcutaneous tissues of mice. Radiological and histological evaluations of in vivo osteogenesis in hDFC-transplanted sites revealed significantly enhanced new bone formation activities compared with those in scaffold-only implanted control sites. Interestingly, at 8 weeks post-hDFC transplantation, the newly generated bones were overgrown compared to the original size of the mandibular defects, and strong expression of osteocalcin and vascular endothelial growth factor were detected in the hDFCs-transplanted tissues of both animals. Immunohistochemical analysis of CD3, CD4, and CD8 in the ectopic bone formation sites of mice showed significantly decreased CD4 expression in DFCs-implanted tissues compared with those in control sites. These findings indicate that hDFCs possess immunomodulatory properties that involved inhibition of the adaptive immune response mediated by CD4 and MHC II, which highlights the usefulness of hDFCs in tissue engineering. In particular, long-term preserved dental follicles could serve as an excellent autologous or allogenic stem cell source for bone tissue regeneration as well as a valuable therapeutic agent for immune diseases.

Endometrial stromal cells (EMSCs) obtained from porcine uterus (n = 6) were positive for mesenchymal stem cell markers (CD29, CD44 and CD90), and negative for epithelial marker CD9 and hematopoietic markers CD34, CD45 analyzed by flow cytometry. Further the cells were positive for expression of mesenchymal markers, CD105, CD140b, and CD144 by PCR. Pluripotent markers OCT4, SOX2, and NANOG were positively expressed in EMSCs analyzed by Western blotting and PCR. Further, differentiation into adipocytes and osteocytes was confirmed by cytochemical staining and lineage specific gene expression by quantitative realtime-PCR. Adipocyte (FABP, LPL, AP2) and osteocyte specific genes (ON, BG, RUNX2) in differentiated EMSCs showed significant (p &lt, 0.05) increase in expression compared to undifferentiated control cells. Neurogenic transdifferentiation of EMSCs exhibited distinctive dendritic morphology with axon projections and neuronal specific genes, NFM, NGF, MBP, NES, B3T and MAP2 and proteins, B3T, NFM, NGF, and TRKA were positively expressed in neuronal differentiated cells. Functional analysis of neuronal differentiated EMSCs displayed voltage-dependence and kinetics for transient outward K+ currents (Ito), at holding potential of −80 mV, Na+ currents and during current clamp, neuronal differentiated EMSCs was more negative than that of control EMSCs. Porcine EMSCs is a suitable model for studying molecular mechanism of transdifferentiation, assessment of electrophysiological properties and their efficiency during in vivo transplantation.

The present study compared the potential of porcine bone marrow mesenchymal stem cells (pBMSCs) at different passages as nuclear transfer (NT) donors and the developmental efficiency of NT embryos from donor cells transfected with/without Oct4 and Sox2. Early-passage pBMSCs showed higher proliferation and expression of Oct4 and Sox2 and differentiation potential into mesenchymal lineages than middle- and late-passage pBMSCs. Cleavage rate did not differ among pBMSCs at different passages, but NT embryos with early-passage pBMSCs and middle-passage pBMSCs transfected with Oct4 (Oct4-pBMSCs) had significantly (p

To study gene expression and to determine distinctive characteristics of embryos produced by different methods, normalisation of the gene(s) of interest against reference gene(s) has commonly been employed. Therefore, the present study aimed to assess which reference genes tend to express more stably in single porcine blastocysts produced in vivo (IVO) or by parthenogenetic activation (PA), in vitro fertilisation (IVF) and somatic cell nuclear transfer (SCNT) using different analysis programs, namely geNorm, Normfinder and Bestkeeper. Commonly used reference genes including 18S rRNA (18S), H2A histone family, member Z (H2A), hypoxanthine phosphoribosyltransferase1 (HPRT1), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), ribosomal protein 4 (RPL4), peptidylprolyl isomerase A (PPIA), beta actin (ACTB), succinate dehydrogenase complex, subunit A (SDHA) and hydroxymethylbilane synthase (HMBS2) were analysed; most of them resulted in significantly (P

The main purpose of this study was to develop a cryopreservation method for human dental follicle tissue to maintain autologous stem cells as a resource. A modified cryoprotectant, consisting of 0.05 m glucose, 0.05 m sucrose and 1.5 m ethylene glycol in phosphate-buffered saline (PBS) was employed, with a slow-ramp freezing rate. We observed 70% of cell survival rate after 3 months of tissue storage. Isolated and cultured human dental stem cells (hDSCs) from cryopreserved dental follicles expressed mesenchymal stem cell markers at a level similar to that of hDSCs from fresh tissue. They also successfully differentiated in vitro into the mesenchymal lineage, osteocytes, adipocytes and chondrocytes under specific inductions. Using immunohistochemistry, the early transcription factors OCT4, NANOG and SOX2 were moderately or weakly detected in the nucleus of both fresh and cryopreserved dental follicles. In addition, p63, CCND1, BCL2 and BAX protein expression levels were the same in both fresh and cryopreserved tissues. However, the positive-cell ratio and intensity of p53 protein was higher in cryopreserved tissues than in fresh tissues, indicating direct damage of the freeze-thawing process. Real-time PCR analysis of hDSCs at passage 2 from both fresh and cryopreserved dental follicles showed similar levels of mRNA for apoptosis- and transcription-related genes. Based on these results, a newly developed cryoprotectant, along with a slow ramp rate freezing procedure allows for long-term dental tissue preservation for later use as an autologous stem cell resource in regenerative cell therapy. Copyright © 2014 John WileySons, Ltd.

Dental tissues provide an alternative autologous source of mesenchymal stem cells (MSCs) for regenerative medicine. In this study, we isolated human dental MSCs of follicle, pulp and papilla tissue from a single donor tooth after impacted third molar extraction by excluding the individual differences. We then compared the morphology, proliferation rate, expression of MSC-specific and pluripotency markers, and in vitro differentiation ability into osteoblasts, adipocytes, chondrocytes and functional hepatocyte-like cells (HLCs). Finally, we analyzed the protein expression profiles of undifferentiated dental MSCs using 2DE coupled with MALDI-TOF-MS. Three types of dental MSCs largely shared similar morphology, proliferation potential, expression of surface markers and pluripotent transcription factors, and differentiation ability into osteoblasts, adipocytes, and chondrocytes. Upon hepatogenic induction, all MSCs were transdifferentiated into functional HLCs, and acquired hepatocyte functions by showing their ability for glycogen storage and urea production. Based on the proteome profiling results, we identified nineteen proteins either found commonly or differentially expressed among the three types of dental MSCs. In conclusion, three kinds of dental MSCs from a single donor tooth possessed largely similar cellular properties and multilineage potential. Further, these dental MSCs had similar proteomic profiles, suggesting their interchangeable applications for basic research and call therapy.