Insensitive Effects of Inflammatory Cytokines on the Reference Genes of Synovial Fluid Resident-Mesenchymal Stem Cells Derived from Rheumatoid Arthritis Patients.

Mesenchymal stem cells derived from rheumatoid arthritis patients (RA-MSCs) provide an understanding of a variety of cellular and immunological responses within the inflammatory milieu. Sustained exposure of MSCs to inflammatory cytokines is likely to exert an influence on genetic variations, including reference genes (RGs). The sensitive effect of cytokines on the reference genes of RA-SF-MSCs may be a variation factor affecting patient-derived MSCs as well as the accuracy and reliability of data. Here, we comparatively evaluated the stability levels of nine RG candidates, namely GAPDH, ACTB, B2M, EEF1A1, TBP, RPLP0, PPIA, YWHAZ, and HPRT1, to find the most stable ones. Alteration of the RG expression was evaluated in MSCs derived from the SF of healthy donors (H-SF-MSCs) and in RA-SF-MSCs using the geNorm and NormFinder software programs. The results showed that TBP, PPIA, and YWHAZ were the most stable RGs for the normalization of H-SF-MSCs and RA-SF-MSCs using RT-qPCR, whereas ACTB, the most commonly used RG, was less stable and performed poorly. Additionally, the sensitivity of RG expression upon exposure to proinflammatory cytokines (TNF-α and IL-1ß) was evaluated. RG stability was sensitive in the H-SF-MSCs exposed to TNF-α and IL-1ß but insensitive in the RA-SF-MSCs. Furthermore, the normalization of IDO expression using ACTB falsely diminished the magnitude of biological significance, which was further confirmed with a functional analysis and an IDO activity assay. In conclusion, the results suggest that TBP, PPIA, and YWHAZ can be used in SF-MSCs, regardless of their exposure to inflammatory cytokines.

Hematological patterns and histopathological assessment of Miniature Pigs in the experiments on human mesenchymal stem cell transplantation.

Background: Multipotent and immune privileged properties of mesenchymal stem cells (MSCs) were investigated for the treatment of various clinical diseases. For the years, many researches into the animal studies evaluated human stem cell therapeutic capacity related to the regenerative medicine. However, there were limited reports on immune privileged properties of human MSCs in animal studies. The present study investigated hematological and biochemical parameter and lymphocyte subset in mini-pigs following human MSCs transplantation as a means of validation of reliability that influence the animal test results. Methods: The miniature pigs were transplanted with human MSCs seeded with scaffold. After transplantation, all animals were evaluated by CBC, biochemistry and lymphocyte subset test. After 9 weeks, all pigs were sacrificed and organs were histologically analyzed. Results: CBC test showed that levels of RBC were decreased and reticulocyte, WBC and neutrophil were increased in transient state initially after transplantation, but returned to normal value. The proportion of B lymphocyte and cytotoxic T cell were also initially enhanced within the normal range temporarily. The female and male miniature pigs showed normal ranges for blood chemistry assessments. During the 9 weeks post-operative period, the animals showed a continuous increase in body weight and length. Furthermore, no abnormal findings were observed from the histological analysis of sacrificed pigs. Conclusions: Overall, miniature pigs transplanted with human MSCs seeded with scaffold were found to have physiologically similar results to normal animals. This result might be a reliable indicator of the animal experiments using miniature pigs with human MSCs.

Female Germ Cell Development, Functioning and Associated Adversities under Unfavorable Circumstances.

In the present era, infertility is one of the major issues which restricts many couples to have their own children. Infertility is the inability to achieve a clinical pregnancy after regular unprotected sexual intercourse for the period of one year or more. Various factors including defective male or female germ cell development, unhealthy and improper lifestyles, diseases like cancer and associated chemo-or-radiation therapies, congenital disorders, etc., may be responsible for infertility. Therefore, it is highly important to understand the basic concepts of germ cell development including primordial germ cell (PGC) formation, specification, migration, entry to genital ridges and their molecular mechanisms, activated pathways, paracrine and autocrine signaling, along with possible alteration which can hamper germ cell development and can cause adversities like cancer progression and infertility. Knowing all these aspects in a proper way can be very much helpful in improving our understanding about gametogenesis and finding possible ways to cure related disorders. Here in this review, various aspects of gametogenesis especially female gametes and relevant factors causing functional impairment have been thoroughly discussed.

Evaluation of odonto/osteogenic differentiation potential from different regions derived dental tissue stem cells and effect of 17ß-estradiol on efficiency.

BACKGROUND: The dentin is a tissue, which is formed by odontoblasts at the pulp interface of the teeth that supports the enamel. Odontoblasts, the cranial neural crest cells are derived from ectodermal mesenchymal stem cells (MSCs) and are long and polarized cells. They are present at the outer surface of dentin and play a prominent role about dentin formation. Recently, attention has been focused on induction of odontoblast using various type of MSCs and effects of the 17ß-estradiol supplementation. In this study, we establish an efficient odonto/osteoblast differentiation protocol using 17ß-estradiol supplementation while comparing the odonto/osteoblast ability of various dental MSCs. METHODS: Same donor derived four types of dental MSCs namely dental pulp stem cells (DPSCs), stem cells from apical papilla (SCAP), dental follicle stem cells (DFSCs), and periodontal ligament stem cells (PDLSCs) were evaluated for their stemness characteristics and potency towards odonto/osteoblast (Induced odonto/osteoblast) differentiation. Then 17ß-estradiol supplementation of 0 and 10 µM was applied to the odonto/osteoblast differentiation media for 14 days respectively. Furthermore, mRNA and protein levels of odonto/osteoblast markers were evaluated. RESULTS: All of the experimental groups displayed stemness characteristics by showing adipocyte and chondrocyte differentiation abilities, expression for cell surface markers and cell proliferation capacity without any significant differences. Moreover, all dental derived MSCs were shown to have odonto/osteoblast differentiation ability when cultured under specific conditions and also showed positive expression for odontoblast markers at both mRNA and protein level. Among all, DPSCs revealed the higher differentiation potential than other dental MSCs. Furthermore, odonto/osteoblast differentiation potential was enhanced by supplementing the differentiation media with 17ß-estradiol (E2). CONCLUSIONS: Thus, DPSCs possess higher odonto/osteogenic potential than the SCAPs, DFSCs, PDLSCs and their differentiation capacity can by further enhanced under E2 supplementation.

Small Molecule-Induced Pancreatic ß-Like Cell Development: Mechanistic Approaches and Available Strategies.

Diabetes is a metabolic disease which affects not only glucose metabolism but also lipid and protein metabolism. It encompasses two major types: type 1 and 2 diabetes. Despite the different etiologies of type 1 and 2 diabetes mellitus (T1DM and T2DM, respectively), the defining features of the two forms are insulin deficiency and resistance, respectively. Stem cell therapy is an efficient method for the treatment of diabetes, which can be achieved by differentiating pancreatic ß-like cells. The consistent generation of glucose-responsive insulin releasing cells remains challenging. In this review article, we present basic concepts of pancreatic organogenesis, which intermittently provides a basis for engineering differentiation procedures, mainly based on the use of small molecules. Small molecules are more auspicious than any other growth factors, as they have unique, valuable properties like cell-permeability, as well as a nonimmunogenic nature; furthermore, they offer immense benefits in terms of generating efficient functional beta-like cells. We also summarize advances in the generation of stem cell-derived pancreatic cell lineages, especially endocrine ß-like cells or islet organoids. The successful induction of stem cells depends on the quantity and quality of available stem cells and the efficient use of small molecules.

Subpopulations of miniature pig mesenchymal stromal cells with different differentiation potentials differ in the expression of octamer-binding transcription factor 4 and sex determining region Y-box 2.

OBJECTIVE: Human mesenchymal stromal cells (MSCs) exhibit variable differentiation potential and can be divided accordingly into distinct subpopulations whose ratios vary with donor age. However, it is unknown whether the same is true in pigs. This study investigated MSC subpopulations in miniature pig and compared their characteristics in young (2 to 3 months) and adult (27 to 35 months) pigs. METHODS: Osteogenic, chondrogenic, and adipogenic capacity of isolated MSCs was evaluated by von Kossa, Alcian blue, and oil red O staining, respectively. Cell surface antigen expression was determined by flow cytometry. Proliferative capacity was assessed with the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Expression of marker genes was detected by quantitative real-time polymerase chain reaction. RESULTS: Porcine MSCs comprised cells with trilineage and bilineage differentiation potential (tMSCs and bMSCs, respectively) and non-differentiating stromal cells (NDSCs). The tMSC and bMSC fractions were smaller in adult than in young pigs (63.0% vs 71.2% and 11.6% vs 24.0%, respectively, p<0.05); NDSCs showed the opposite trend (25.4% vs 4.8%; p<0.05). Subpopulations showed no differences in morphology, cell surface antigen expression, or proliferative capacity, but octamer-binding transcription factor 4 (OCT4) expression was higher in tMSCs than in bMSCs and NDSCs (p<0.05), whereas sex determining region Y-box 2 (SOX2) expression was higher in tMSCs and bMSCs than in NDSCs (p<0.05). Aging had no effect on these trends. CONCLUSION: Porcine MSCs comprise distinct subpopulations that differ in their differentiation potential and OCT4 and SOX2 expression. Aging does not affect the characteristics of each subpopulation but alters their ratios.

TATA box binding protein and ribosomal protein 4 are suitable reference genes for normalization during quantitative polymerase chain reaction study in bovine mesenchymal stem cells.

OBJECTIVE: Quantitative polymerase chain reaction (qPCR) has been extensively used in the field of mesenchymal stem cell (MSC) research to elucidate their characteristics and clinical potential by normalization of target genes against reference genes (RGs), which are believed to be stably expressed irrespective of various experimental conditions. However, the expression of RGs is also variable depending on the experimental conditions, which may lead to false or contradictory conclusions upon normalization. Due to the current lack of information for a clear list of stable RGs in bovine MSCs, we conducted this study to identify suitable RGs in bovine MSCs. METHODS: The cycle threshold values of ten traditionally used RGs (18S ribosomal RNA [18S], beta-2-microglobulin [B2M], H2A histone family, member Z [H2A], peptidylprolyl isomerase A [PPIA], ribosomal protein 4 [RPL4], succinate dehydrogenase complex, subunit A [SDHA], beta actin [ACTB], glyceraldehyde-3-phosphate dehydrogenase [GAPDH], TATA box binding protein [TBP], and hypoxanthine phosphoribosyltrasnfrase1 [HPRT1]) in bovine bone marrow-derived MSCs (bBMMSCs) were validated for their stabilities using three types of RG evaluation algorithms (geNorm, Normfinder, and Bestkeeper). The effect of validated RGs was then verified by normalization of lineage-specific genes (fatty acid binding protein 4 [FABP4] and osteonectin [ON]) expressions during differentiations of bBMMSCs or POU class 5 homeobox 1 (OCT4) expression between bBMMSCs and dermal skins. RESULTS: Based on the results obtained for the three most stable RGs from geNorm (TBP, RPL4, and H2A), Normfinder (TBP, RPL4, and SDHA), and Bestkeeper (TBP, RPL4, and SDHA), it was comprehensively determined that TBP and RPL4 were the most stable RGs in bBMMSCs. However, traditional RGs were suggested to be the least stable (18S) or moderately stable (GAPDH and ACTB) in bBMMSCs. Normalization of FABP4 or ON against TBP, RPL4, and 18S presented significant differences during differentiation of bBMMSCs. However, although significantly low expression of OCT4 was detected in dermal skins compared to that in bBMMSCs when TBP and RPL4 were used in normalization, normalization against 18S exhibited no significance. CONCLUSION: This study proposes that TBP and RPL4 were suitable as stable RGs for qPCR study in bovine MSCs.

Pancreatic endocrine-like cells differentiated from human umbilical cords Wharton's jelly mesenchymal stem cells using small molecules.

Following success of pancreatic islet transplantation in the treatment of Type I diabetes mellitus, there is a growing interest in using cell-based treatment approaches. However, severe shortage of donor islets-pancreas impeded the growth, and made researchers to search for an alternative treatment approaches. In this context, recently, stem cell-based therapy has gained more attention. The current study demonstrated that epigenetic modification improves the in vitro differentiation of Wharton's jelly mesenchymal stem cells (WJMSCs) into pancreatic endocrine-like cells. Here we used two histone deacetylase (HDAC) inhibitors namely trichostatin A (TSA) and TMP269. TSA inhibits both class I and II HDACs whereas TMP269 inhibits only class IIa HDACs. WJMSCs were differentiated using a multistep protocol in a serum-free condition with or without TSA pretreatment. A marginal improvement in differentiation was observed after TSA pretreatment though it was not significant. However, exposing endocrine precursor-like cells derived from WJMSCs to TMP269 alone has significantly improved the differentiation toward insulin-producing cells. Further, increase in the expression of paired box 4 (PAX4), insulin, somatostatin, glucose transporter 2 (GLUT2), MAF bZIP transcription factor A (MAFA), pancreatic duodenal homeobox 1 (PDX-1), and NKX6.1 was observed both at messenger RNA and protein levels. Nevertheless, TMP269-treated cells secreted higher insulin upon glucose challenge, and demonstrated increased dithizone staining. These findings suggest that TMP269 may improve the in vitro differentiation of WJMSCs into insulin-producing cells.

Differentiation potential of different regions-derived same donor human Wharton's jelly mesenchymal stem cells into functional smooth muscle-like cells.

The present study evaluates the transdifferentiation potential of different region-derived same donor Wharton's jelly MSCs (WJMSCs) into functional smooth muscle-like cells (SMLCs). All regions showed baseline expression for early smooth muscle cell (SMC) markers (αSMA and SM22-α) whereas mid marker CALPONIN gradually reduced during in vitro culture expansion and late marker myosin heavy chain type-11 (MHY-11) was completely absent. Furthermore, WJMSCs were induced to SMLCs using DMEM containing 10% FBS supplemented with different concentrations/combinations of TGF-ß1 and PDGF-BB under normoxia (20% O2) condition. Three treatment groups namely group A: 2.5 ng/ml TGF-ß1, group B: 5 ng/ml PDGF-BB and group C: 2.5 ng/ml TGF-ß1 + 5 ng/ml PDGF-BB were used for the induction of WJMSCs into SMLCs. Cells were evaluated for SMC-specific marker expression at different time intervals. Finally, selection of the SMC-specific highly potent region along with the most suitable treatment group was done on the basis of highest outcome in terms of SMC-specific marker expression and functional competence of transdifferentiated cells. Among all regions, baby region-derived WJMSCs (B-WJMSCs) exhibited highest SMC marker expression and functional ability. To mimic the in vivo physiological conditions, hypoxic conditions (3% O2) were used to evaluate the effect of low oxygen on the SMLC differentiation potential of selected WJMSCs using previously used same parameters. Annexin-V assay was performed to check the effect of cytokines and different oxygen concentrations, which revealed no significant differences. It was concluded that different induction conditions have different but positive effects on the functional SMLC differentiation ability of WJMSCs.

Oxygen-Releasing Microparticles for Cell Survival and Differentiation Ability under Hypoxia for Effective Bone Regeneration.

Sufficient oxygen delivery into tissue-engineered three-dimensional (3D) scaffolds to produce clinically applicable tissues/organs remains a challenge for researchers and clinicians. One potential strategy to overcome this limitation is the use of an oxygen releasing scaffold. In the present study, we prepared hollow microparticles (HPs) loaded with an emulsion of the oxygen carrier perfluorooctane (PFO; PFO-HPs) for the timely supply of oxygen to surrounding cells. These PFO-HPs prolonged the survival and preserved the osteogenic differentiation potency of human periosteal-derived cells ( hPDCs) under hypoxia. hPDCs seeded onto PFO-HPs formed new bone at a faster rate and with a higher bone density than hPDCs seeded onto phosphate buffered saline-loaded control HPs. These findings suggest that PFO-HPs provide a suitable environment for the survival and maintenance of differentiation ability of hPDCs at bony defects without vascular networks until new blood vessel ingrowth occurs, thus enhancing bone regeneration. PFO-HPs are a promising system for effective delivery of various functional cells, including stem cells and progenitor cells, to regenerate damaged tissues/organs.

Comparative analysis of human Wharton's jelly mesenchymal stem cells derived from different parts of the same umbilical cord.

Easy isolation, lack of ethical issues, high proliferation, multi-lineage differentiation potential and immunomodulatory properties of umbilical cord (UC)-derived mesenchymal stem cells (MSCs) make them a valuable tool in stem cell research. Recently, Wharton's jelly (WJ) was proven as the best MSC source among various compartments of UC. However, it is still unclear whether or not Wharton's jelly-derived MSCs (WJMSCs) from different parts of the whole cord exhibit the same characteristics. There may be varied MSCs present in different parts of WJ throughout the length of the UC. For this purpose, using an explant attachment method, WJMSCs were isolated from three different parts of the UC, mainly present towards the placenta (mother part), the center of the whole cord (central part) and the part attached to the fetus (baby part). WJMSCs from all three parts were maintained in normal growth conditions (10% ADMEM) and analyzed for mesenchymal markers, pluripotent genes, proliferation rate and tri-lineage differentiation potential. All WJMSCs were highly proliferative, positively expressed CD90, CD105, CD73 and vimentin, while not expressing CD34, CD45, CD14, CD19 or HLA-DR, differentiated into adipocytes, osteocytes and chondrocytes and expressed pluripotency markers OCT-4, SOX-2 and NANOG at gene and protein levels. Furthermore, MSCs derived from all the parts were shown to have potency towards hepatocyte-like cell differentiation. Human bone marrow-derived MSCs were used as a positive control. Finally, we conclude that WJMSCs derived from all the parts are valuable sources and can be efficiently used in various fields of regenerative medicine.

The Role of Human Umbilical Vein Endothelial Cells in Osteogenic Differentiation of Dental Follicle-Derived Stem Cells in In Vitro Co-cultures.

Angiogenesis and vascularization are essential for the growth and survival of most tissues. Engineered bone tissue requires an active blood vessel network for survival and integration with mature host tissue. Angiogenesis also has an effect on cell growth and differentiation in vitro. However, the effect of angiogenic factors on osteoprogenitor cell differentiation remains unclear. We studied the effects of human umbilical vein endothelial cells (HUVECs) on osteogenic differentiation of dental follicle-derived stem cells (DFSCs) in vitro by co-culturing DFSCs and HUVECs. Cell viability, based on metabolic activity and DNA content, was highest for co-cultures with a DFSC/HUVEC ratio of 50:50 in a 1:1 mixture of mesenchymal stem cell growth medium and endothelial cell growth medium. Osteoblastic and angiogenic phenotypes were enhanced in co-cultures with a DFSC/HUVEC ratio of 50:50 compared with DFSC monocultures. Increased expression of angiogenic phenotypes and vascular endothelial growth factor (VEGF) levels were observed over time in both 50:50 DFSC/HUVEC co-cultures and DFSC monocultures during culture period. Our results showed that increased angiogenic activity in DFSC/HUVEC co-cultures may stimulate osteoblast maturation of DFSCs. Therefore, the secretion of angiogenic factors from HUVECs may play a role in the osteogenic differentiation of DFSCs.

Cholinergic Nerve Differentiation of Mesenchymal Stem Cells Derived from Long-Term Cryopreserved Human Dental Pulp In Vitro and Analysis of Their Motor Nerve Regeneration Potential In Vivo.

The reduction of choline acetyltransferase, caused by the loss of cholinergic neurons, leads to the absence of acetylcholine (Ach), which is related to motor nerve degeneration. The aims of the present study were to evaluate the in vitro cholinergic nerve differentiation potential of mesenchymal stem cells from cryopreserved human dental pulp (hDPSCs-cryo) and to analyze the scale of in vivo motor nerve regeneration. The hDPSCs-cryo were isolated and cultured from cryopreserved dental pulp tissues, and thereafter differentiated into cholinergic neurons using tricyclodecane-9-yl-xanthogenate (D609). Differentiated cholinergic neurons (DF-chN) were transplanted into rats to address sciatic nerve defects, and the scale of in vivo motor nerve regeneration was analyzed. During in vitro differentiation, the cells showed neuron-like morphological changes including axonal fibers and neuron body development, and revealed high expression of cholinergic neuron-specific markers at both the messenger RNA (mRNA) and protein levels. Importantly, DF-chN showed significant Ach secretion ability. At eight weeks after DF-chN transplantation in rats with sciatic nerve defects, notably increased behavioral activities were detected with an open-field test, with enhanced low-affinity nerve growth factor receptor (p75NGFR) expression detected using immunohistochemistry. These results demonstrate that stem cells from cryopreserved dental pulp can successfully differentiate into cholinergic neurons in vitro and enhance motor nerve regeneration when transplanted in vivo. Additionally, this study suggests that long-term preservation of dental pulp tissue is worthwhile for use as an autologous cell resource in the field of nerve regeneration, including cholinergic nerves.

Selection of reference genes for quantitative real-time polymerase chain reaction in porcine embryos.

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<0.05) different cycle threshold (CT) values in porcine embryos except for SDHA and H2A. In evaluation of stable reference genes across in vivo and in vitro porcine blastocysts, three kinds of programs showed slightly different results; however, there were similar patterns about the rankings of more or less stability overall. In conclusion, SDHA and H2A were determined as the most appropriate reference genes for reliable normalisation in order to find the comparative gene expression in porcine blastocysts produced by different methods, whereas 18S was regarded as a less-stable reference gene. The present study has evaluated the stability of commonly used reference genes for accurate normalisation in porcine embryos to obtain reliable results.

Effects of Osteogenic-Conditioned Medium from Human Periosteum-Derived Cells on Osteoclast Differentiation.

Stem/progenitor cell-based regenerative medicine using the osteoblast differentiation of mesenchymal stem cells (MSCs) is regarded as a promising approach for the therapeutic treatment of various bone defects. The effects of the osteogenic differentiation of stem/progenitor cells on osteoclast differentiation may have important implications for use in therapy. However, there is little data regarding the expression of osteoclastogenic proteins during osteoblastic differentiation of human periosteum-derived cells (hPDCs) and whether factors expressed during this process can modulate osteoclastogenesis. In the present study, we measured expression of RANKL in hPDCs undergoing osteoblastic differentiation and found that expression of RANKL mRNA was markedly increased in these cells in a time-dependent manner. RANKL protein expression was also significantly enhanced in osteogenic-conditioned media from hPDCs undergoing osteoblastic differentiation. We then isolated and cultured CD34+ hematopoietic stem cells (HSCs) from umbilical cord blood (UCB) mononuclear cells (MNCs) and found that these cells were well differentiated into several hematopoietic lineages. Finally, we co-cultured human trabecular bone osteoblasts (hOBs) with CD34+ HSCs and used the conditioned medium, collected from hPDCs during osteoblastic differentiation, to investigate whether factors produced during osteoblast maturation can affect osteoclast differentiation. Specifically, we measured the effect of this osteogenic-conditioned media on expression of osteoclastogenic markers and osteoclast cell number. We found that osteoclastic marker gene expression was highest in co-cultures incubated with the conditioned medium collected from hPDCs with the greatest level of osteogenic maturation. Although further study will be needed to clarify the precise mechanisms that underlie osteogenic-conditioned medium-regulated osteoclastogenesis, our results suggest that the osteogenic maturation of hPDCs could promote osteoclastic potential.

Stem Cells from Cryopreserved Human Dental Pulp Tissues Sequentially Differentiate into Definitive Endoderm and Hepatocyte-Like Cells in vitro.

We previously described a novel tissue cryopreservation protocol to enable the safe preservation of various autologous stem cell sources. The present study characterized the stem cells derived from long-term cryopreserved dental pulp tissues (hDPSCs-cryo) and analyzed their differentiation into definitive endoderm (DE) and hepatocyte-like cells (HLCs) in vitro. Human dental pulp tissues from extracted wisdom teeth were cryopreserved as per a slow freezing tissue cryopreservation protocol for at least a year. Characteristics of hDPSCs-cryo were compared to those of stem cells from fresh dental pulps (hDPSCs-fresh). hDPSCs-cryo were differentiated into DE cells in vitro with Activin A as per the Wnt3a protocol for 6 days. These cells were further differentiated into HLCs in the presence of growth factors until day 30. hDPSCs-fresh and hDPSCs-cryo displayed similar cell growth morphology, cell proliferation rates, and mesenchymal stem cell character. During differentiation into DE and HLCs in vitro, the cells flattened and became polygonal in shape, and finally adopted a hepatocyte-like shape. The differentiated DE cells at day 6 and HLCs at day 30 displayed significantly increased DE- and hepatocyte-specific markers at the mRNA and protein level, respectively. In addition, the differentiated HLCs showed detoxification and glycogen storage capacities, indicating they could share multiple functions with real hepatocytes. These data conclusively show that hPDSCs-cryo derived from long-term cryopreserved dental pulp tissues can be successfully differentiated into DE and functional hepatocytes in vitro. Thus, preservation of dental tissues could provide a valuable source of autologous stem cells for tissue engineering.

The involvement of histone methylation in osteoblastic differentiation of human periosteum-derived cells cultured in vitro under hypoxic conditions.

Although oxygen concentrations affect the growth and function of mesenchymal stem cells (MSCs), the impact of hypoxia on osteoblastic differentiation is not understood. Likewise, the effect of hypoxia-induced epigenetic changes on osteoblastic differentiation of MSCs is unknown. The aim of this study was to examine the in vitro hypoxic response of human periosteum-derived cells (hPDCs). Hypoxia resulted in greater proliferation of hPDCs as compared with those cultured in normoxia. Further, hypoxic conditions yielded decreased expression of apoptosis- and senescence-associated genes by hPDCs. Osteoblast phenotypes of hPDCS were suppressed by hypoxia, as suggested by alkaline phosphatase activity, alizarin red-S-positive mineralization, and mRNA expression of osteoblast-related genes. Chromatin immunoprecipitation assays showed an increased presence of H3K27me3, trimethylation of lysine 27 on histone H3, on the promoter region of bone morphogenetic protein-2. In addition, mRNA expression of histone lysine demethylase 6B (KDM6B) by hPDCs was significantly decreased in hypoxic conditions. Our results suggest that an increased level of H3K27me3 on the promoter region of bone morphogenetic protein-2, in combination with downregulation of KDM6B activity, is involved in the suppression of osteogenic phenotypes of hPDCs cultured in hypoxic conditions. Although oxygen tension plays an important role in the viability and maintenance of MSCs in an undifferentiated state, the effect of hypoxia on osteoblastic differentiation of MSCs remains controversial. In addition, evidence regarding the importance of epigenetics in regulating MSCs has been limited. This study was to examine the role hypoxia on osteoblastic differentiation of hPDCs, and we examined whether histone methylation is involved in the observed effect of hypoxia on osteogenic differentiation of hPDCs.

Midkine and NANOG Have Similar Immunohistochemical Expression Patterns and Contribute Equally to an Adverse Prognosis of Oral Squamous Cell Carcinoma.

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.

DMSO- and Serum-Free Cryopreservation of Wharton's Jelly Tissue Isolated From Human Umbilical Cord.

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 Wiley Periodicals, Inc.

Development of Porous Beads to Provide Regulated BMP-2 Stimulation for Varying Durations: In Vitro and In Vivo Studies for Bone Regeneration.

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.