SSEA-3 Research Articles

Investigation of VASA Gene and Protein Expression in Neonate and Adult Testicular Germ Cells in Mice In Vivo and In Vitro.

Objective We aimed to examine the expression levels of the VASA gene and protein in testis sections of neonate and adult mice as well as testicular cell cultures. Materials and Methods In this experimental study, in order to investigate the expression of this germ cell marker gene in more detail, we analyzed the expression of VASA by immunocytochemistry, immunohistochemistry and fluidigm reverse transcription-polymerase chain reaction (RT-PCR). Results The immunohistochemical assays showed that the VASA protein was exclusively expressed in germ cells in the seminiferous tubules of the neonate and adult testis and not in somatic cells. VASA was not detectable in PLZF positive spermatogonial stem cells (SSCs), was weakly expressed in proliferating spermatogonia, and became abundant in spermatocytes and round spermatozoa. Counting VASA-positive cells in the seminiferous tubules of the neonate and adult testis depicted significant higher expression (P<0.05) of VASA in the adult testis in comparison to its neonate counterpart. SSC colonies were established in vitro after digestion of the testis and characterized by immunocytochemistry for CD90 and stage-specific embryonic antigens 3 (SSEA3). Immunocytochemistry confirmed that in contrast to the not detectable signal in vivo, VASA protein was strongly localized in the cytoplasm of both neonate and adult mouse SSCs under in vitro conditions. The results of Fluidigm RT-PCR revealed a significant higher expression of the germ cell gene VASA in adult SSCs in comparison to neonate SSCs in cell culture (P<0.05). ConclusionThe VASA protein is, therefore, an extremely specific marker of testicular germ cell differentiation in vivo and mostly expressed in the adult testis in spermatocytes and round spermatids. The immunohistochemical signal in spermatogonia is very low. So, PLZF positive SSCs are negative for VASA in vivo, while in contrast, once isolated from the testicular niche VASA is also strongly expressed in SSCs under in vitro conditions.

Rescue from Stx2-Producing E.coli-Associated Encephalopathy by Intravenous Injection of Muse Cells in NOD-SCID Mice.

Shiga toxin-producing Escherichia coli (STEC) causes hemorrhagic colitis, hemolytic uremic syndrome, and acute encephalopathies that may lead to sudden death or severe neurologic sequelae. Current treatments, including immunoglobulin G (IgG) immunoadsorption, plasma exchange, steroid pulse therapy, and the monoclonal antibody eculizumab, have limited effects against the severe neurologic sequelae. Multilineage-differentiating stress-enduring (Muse) cells are endogenous reparative non-tumorigenic stem cells that naturally reside in the body and are currently under clinical trials for regenerative medicine. When administered intravenously, Musecells accumulate to the damaged tissue, where they exert anti-inflammatory, anti-apoptotic, anti-fibrotic, and immunomodulatory effects, and replace damaged cells by differentiating into tissue-constituent cells. Here, severely immunocompromised non-obese diabetic/severe combined immunodeficiency (NOD-SCID) mice orally inoculated with 9 × 109 colony-forming units of STEC O111 and treated 48 h later with intravenous injection of 5 × 104 Muse cells exhibited 100% survival and no severe after-effects of infection. Suppression of granulocyte-colony-stimulating factor (G-CSF) by RNAi abolished the beneficial effects of Muse cells, leading to a 40% death and significant body weight loss, suggesting the involvement of G-CSF in the beneficial effects of Muse cells in STEC-infected mice. Thus, intravenous administration of Muse cells could be a candidate therapeutic approach for preventing fatal encephalopathy after STEC infection.

Hierarchical and Programmable One-Pot Oligosaccharide Synthesis.

This article presents a general experimental protocol for programmable one-pot oligosaccharide synthesis and demonstrates how to use Auto-CHO software for generating potential synthetic solutions. The programmable one-pot oligosaccharide synthesis approach is designed to empower fast oligosaccharide synthesis of large amounts using thioglycoside building blocks (BBLs) with the appropriate sequential order of relative reactivity values (RRVs). Auto-CHO is a cross-platform software with a graphical user interface that provides possible synthetic solutions for programmable one-pot oligosaccharide synthesis by searching a BBL library (containing about 150 validated and >50,000 virtual BBLs) with accurately predicted RRVs by support vector regression. The algorithm for hierarchical one-pot synthesis has been implemented in Auto-CHO and uses fragments generated by one-pot reactions as new BBLs. In addition, Auto-CHO allows users to give feedback for virtual BBLs to keep valuable ones for further use. One-pot synthesis of stage-specific embryonic antigen 4 (SSEA-4), which is a pluripotent human embryonic stem cell marker, is demonstrated in this work.

DNA-PKcs modulates progenitor cell proliferation and fibroblast senescence in idiopathic pulmonary fibrosis

BackgroundRecent studies have highlighted the contribution of senescent mesenchymal and epithelial cells in Idiopathic Pulmonary Fibrosis (IPF), but little is known regarding the molecular mechanisms that regulate the accumulation of senescent cells in this disease. Therefore, we addressed the hypothesis that the loss of DNA repair mechanisms mediated by DNA protein kinase catalytic subunit (DNA-PKcs) in IPF, promoted the accumulation of mesenchymal progenitors and progeny, and the expression of senescent markers by these cell types.MethodsSurgical lung biopsy samples and lung fibroblasts were obtained from patients exhibiting slowly, rapidly or unknown progressing IPF and lung samples lacking any evidence of fibrotic disease (i.e. normal; NL). The expression of DNA-Pkcs in lung tissue was assessed by quantitative immunohistochemical analysis. Chronic inhibition of DNA-PKcs kinase activity was mimicked using a highly specific small molecule inhibitor, Nu7441. Proteins involved in DNA repair (stage-specific embryonic antigen (SSEA)-4+ cells) were determined by quantitative Ingenuity Pathway Analysis of transcriptomic datasets (GSE103488). Lastly, the loss of DNA-PKc was modeled in a humanized model of pulmonary fibrosis in NSG SCID mice genetically deficient in PRKDC (the transcript for DNA-PKcs) and treated with Nu7441.ResultsDNA-PKcs expression was significantly reduced in IPF lung tissues. Chronic inhibition of DNA-PKcs by Nu7441 promoted the proliferation of SSEA4+ mesenchymal progenitor cells and a significant increase in the expression of senescence-associated markers in cultured lung fibroblasts. Importantly, mesenchymal progenitor cells and their fibroblast progeny derived from IPF patients showed a loss of transcripts encoding for DNA damage response and DNA repair components. Further, there was a significant reduction in transcripts encoding for PRKDC (the transcript for DNA-PKcs) in SSEA4+ mesenchymal progenitor cells from IPF patients compared with normal lung donors. In SCID mice lacking DNA-PKcs activity receiving IPF lung explant cells, treatment with Nu7441 promoted the expansion of progenitor cells, which was observed as a mass of SSEA4+ CgA+ expressing cells.ConclusionsTogether, our results show that the loss of DNA-PKcs promotes the expansion of SSEA4+ mesenchymal progenitors, and the senescence of their mesenchymal progeny.

Isolation and identification of urine-derived stem cells from female patients with interstitial cystitis/bladder pain syndrome

Objective To establish the method for isolation and culture of urine-derived stem cells (USCs) from female patients with interstitial cystitis/bladder pain syndrome(IC/BPS). Methods The USCs were collected from fresh midstream urine samples from 6 female IC/BPS patients admitted to our hospital from June 2018 to December 2018.The 6 patients were 33-55 years old (average 41.5 years old), and their course of illness was 2-18 years (average 8 years). The USCs were isolated from the urine through times of centrifugation and cultured in specific medium. Growth curve and cell cycle of USCs were observed.The expression of surface markers of USCs was analyzed by flow cytometry and immunofluorescence, the smooth muscle and epithelial differentiation potential of USCs were detected by immunofluorescence staining of surface markers of smooth muscle cells and epithelial cells. Results USCs were successfully extracted from 3 of 6 female patients, and the success rate reached 50% by once extraction.USCs showed a rice-grain spindle appearance and showed logarithmic growth. USCs expressed surface markers associated with mesenchymal stem cells( e. g. CD44, CD73, CD105, CD133) and embryonic stem cells [e.g. stage-specific embryonic antigen 4 (SSEA4)] and pericytes[e.g. CD146, platelet derived growth factor beta receptor (PDGFRB) and NG2], but didn’t express hematopoietic stem cell surface markers(e.g. CD31, CD34 and CD45). When induced to smooth muscle cells or epithelial cells, the cells expressed the surface markers of smooth muscle cells [e.g. desmin, myosin, alpha-smooth muscle actin(α-SMA) and vimentin ] and epithelial cells(e.g. uroplakin 1A, uroplakin 3B, AE1/AE3 and cytokeratin 13). Conclusions The method of isolation and culture of USCs from female IC/BPS patients was successfully established, and it provides a preliminary technical method for exploring the application of USCs in the clinical study of IC/BPS patients with autologous treatment. Key words: Cystitis, interstitial; Bladder pain syndrome; Urine; Stem Cells; Isolation; Identification

De novo generation of a functional human thymus from induced pluripotent stem cells

A proper functional thymus is required for generation of T cell–mediated immunity. This is dramatically illustrated by patients lacking a thymus, such as children with complete DiGeorge syndrome, which is fatal if left untreated.1-3 Therapeutic options for such patients are limited and confined to transplantation of small fragments from allogeneic neonatal thymi.

Neurotrophic Factor Secretion and Neural Differentiation Potential of Multilineage-differentiating Stress-enduring (Muse) Cells Derived from Mouse Adipose Tissue

Multilineage-differentiating stress-enduring (Muse) cells are endogenous pluripotent stem cells that can be isolated based on stage-specific embryonic antigen-3 (SSEA-3), a pluripotent stem cell-surface marker. However, their capacities for survival, neurotrophic factor secretion, and neuronal and glial differentiation are unclear in rodents. Here we analyzed mouse adipose tissue-derived Muse cells in vitro. We collected mesenchymal stem cells (MSCs) from C57BL/6 J mouse adipose tissue and separated SSEA-3+, namely Muse cells, and SSEA-3–, non-Muse cells, to assess self-renewability; pluripotency marker expression (Nanog, Oct3/4, Sox2, and SSEA-3); spontaneous differentiation into endodermal, mesodermal, and ectodermal lineages; and neural differentiation capabilities under cytokine induction. Neurally differentiated Muse and non-Muse cell functions were assessed by calcium imaging. Antioxidant ability was measured to assess survival under oxidative stress. Brain-derived neurotrophic factor (BDNF), vascular endothelial cell growth factor (VEGF), and hepatocyte growth factor (HGF) secretion were analyzed in enzyme-linked immunosorbent assays. SSEA-3+ Muse cells (6.3 ± 1.9% of mouse adipose-MSCs), but not non-Muse cells, exhibited self-renewability, spontaneous differentiation into the three germ layers, and differentiation into cells positive for Tuj-1 (27 ± 0.9%), O4 (17 ± 3.4%), or GFAP (23 ± 1.3%) under cytokine induction. Neurally differentiated Muse cells responded to KCl depolarization with greater increases in cytoplasmic Ca2+ levels than non-Muse cells. Cell survival under oxidative stress was significantly higher in Muse cells (50 ± 2.7%) versus non-Muse cells (22 ± 2.8%). Muse cells secreted significantly more BDNF, VEGF, and HGF (273 ± 12, 1479 ± 7.5, and 6591 ± 1216 pg/mL, respectively) than non-Muse cells (133 ± 4.0, 1165 ± 20, and 2383 ± 540 pg/mL, respectively). Mouse Muse cells were isolated and characterized for the first time. Muse cells showed greater pluripotency-like characteristics, survival, neurotrophic factor secretion, and neuronal and glial-differentiation capacities than non-Muse cells, indicating that they may have better neural-regeneration potential.

Stage-specific embryonic antigen-4 is a histological marker reflecting the malignant behavior of prostate cancer

Stage-specific embryonic antigen-4 (SSEA-4), a specific marker for pluripotent stem cells, plays an important role in the malignant behavior of several cancers. Here, SSEA-4 expression was evaluated by immunohistochemistry using monoclonal antibody RM1 specific to SSEA-4 in 181 and 117 prostate cancer (PC) specimens obtained by biopsy and radical prostatectomy (RP), respectively. The relationships between SSEA-4 expression in cancer cells or the presence of SSEA-4-positive tumor-infiltrating immune cells (TICs) and clinicopathological parameters were analyzed. SSEA-4 expression in cancer cells was significantly associated with Gleason score, local progression, and lymph node and distant metastasis. In RP specimens, high SSEA-4 expression in cancer cells and the presence of SSEA-4-positive TICs were significant predictors of pT3, i.e., invasion and worse biochemical recurrence (BCR) after RP, respectively, in univariate analysis. In contrast, combination of high SSEA-4 expression in cancer cells and the presence of SSEA-4-positive TICs was an independent predictor for pT3 and BCR in multivariate analysis. Biologically this combination was also independently associated with suppression of apoptosis. Thus, the co-expression of SSEA-4 in cancer cells and TICs may have crucial roles in the malignant aggressiveness and prognosis of PC. Invasive potential and suppression of apoptosis may be linked to SSEA-4 expression.

Role of c-MET Inhibitors in Overcoming Drug Resistance in Spheroid Models of Primary Human Pancreatic Cancer and Stellate Cells.

Pancreatic stellate cells (PSCs) are a key component of tumor microenvironment in pancreatic ductal adenocarcinoma (PDAC) and contribute to drug resistance. c-MET receptor tyrosine kinase activation plays an important role in tumorigenesis in different cancers including PDAC. In this study, effects of PSC conditioned medium (PCM) on c-MET phosphorylation (by immunocytochemistry enzyme-linked immunosorbent assay (ELISA)) and drug response (by sulforhodamine B assay) were investigated in five primary PDAC cells. In novel 3D-spheroid co-cultures of cyan fluorescence protein (CFP)-firefly luciferase (Fluc)-expressing primary human PDAC cells and green fluorescence protein (GFP)-expressing immortalized PSCs, PDAC cell growth and chemosensitivity were examined by luciferase assay, while spheroids’ architecture was evaluated by confocal microscopy. The highest phospho-c-MET expression was detected in PDAC5 and its subclone sorted for “stage specific embryonic antigen-4” (PDAC5 (SSEA4)). PCM of cells pre-incubated with PDAC conditioned medium, containing increased hepatocyte growth factor (HGF) levels, made PDAC cells significantly more resistant to gemcitabine, but not to c-MET inhibitors. Hetero-spheroids containing both PSCs and PDAC5 (SSEA4) cells were more resistant to gemcitabine compared to PDAC5 (SSEA4) homo-spheroids. However, c-MET inhibitors (tivantinib, PHA-665752 and crizotinib) were equally effective in both spheroid models. Experiments with primary human PSCs confirmed the main findings. In conclusion, we developed spheroid models to evaluate PSC–PDAC reciprocal interaction, unraveling c-MET inhibition as an important therapeutic option against drug resistant PDAC.

Approach for Profiling of Glycosphingolipid Glycosylation by Multiplexed Capillary Gel Electrophoresis Coupled to Laser-Induced Fluorescence Detection To Identify Cell-Surface Markers of Human Pluripotent Stem Cells and Derived Cardiomyocytes.

Application of human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) as tissue transplants in regenerative medicine depends on cell-surface marker-based characterization and/or purification. Glycosphingolipids (GSLs) are a family of highly diverse surface-exposed biomolecules that have been neglected as potential surface markers for hiPSC-CMs due to significant analytical challenges. Here, we describe the development of a novel and high-throughput-compatible workflow for the analysis of GSL-derived glycans based on ceramide glycanase digestion, 8-aminopyrene-1,3,6-trisulfonic acid (APTS) labeling, and multiplexed capillary gel electrophoresis coupled to laser-induced fluorescence detection (xCGE-LIF). GSL glycans were detected with highly reproducible migration times after repeated analysis by xCGE-LIF. We built up a migration time database comprising 38 different glycan species, and we showed exemplarily that as few as 10 pg of fucosyl lactotetra was detectable. GSL glycan profiling could be performed with 105 human induced pluripotent stem cells, and we quantitatively dissected global alterations of GSL glycosylation of human induced pluripotent stem cells (hiPSCs) and hiPSC-CMs by employing xCGE-LIF. In our study, we observed a general switch from complex GSLs with lacto- and globo-series core structures comprising the well-known human pluripotent stem cell marker stage-specific embryonic antigen 3 (SSEA3) and SSEA4 in hiPSCs toward the simple gangliosides GM3 and GD3 in hiPSC-CMs. This is the first description of GM3 and GD3 being highly abundant GSLs on the cell surface of stem cell-derived cardiomyocytes.

Quantitative Analysis of SSEA3+ Cells from Human Umbilical Cord after Magnetic Sorting

Multilineage-differentiating stress-enduring (Muse) cells are a population of pluripotent stage-specific embryonic antigen 3 (SSEA3)+ mesenchymal stem cells first described by Mari Dezawa in 2010. Although some investigators have reported SSEA3+ mesenchymal cells in umbilical cord tissues, none have quantitatively compared SSEA3+ cells isolated from Wharton’s jelly (WJ) and the cord lining (CL) of human umbilical cords (HUCs). We separated WJ and the CL from HUCs, cultured mesenchymal stromal cells (MSCs) isolated from these two tissues with collagenase, and quantified the percentage of SSEA3+ cells over three passages. The first passage had 5.0% ± 4.3% and 5.3% ± 5.1% SSEA3+ cells from WJ and the CL, respectively, but the percentage of SSEA3+ cells decreased significantly (P < 0.05) between P0 and P2 in the CL group and between P0 and P1 in the WJ group. Magnetic-activated cell sorting (MACS) markedly enriched SSEA3+ cells to 91.4% ± 3.2%. Upon culture of the sorted population, we found that the SSEA3+ percentage ranged from 62.5% to 76.0% in P2–P5 and then declined to 42.0%–54.7% between P6 and P9. At P10, the cultures contained 37.4% SSEA3+ cells. After P10, we resorted the cells and achieved 89.4% SSEA3+ cells in culture. The procedure for MACS-based enrichment of SSEA3+ cells, followed by expansion in culture and a re-enrichment step, allows the isolation of many millions of SSEA3+ cells in relatively pure culture. When cultured, the sorted SSEA3+ cells differentiated into embryoid spheres and survived 4 weeks after transplant into a contused Sprague-Dawley rat spinal cord. The transplanted SSEA3+ cells migrated into the injury area from four injection points around the contusion site and did not produce any tumors. The umbilical cord is an excellent source of fetal Muse cells, and our method allows the practical and efficient isolation and expansion of relatively pure populations of SSEA3+ Muse cells that can be matched by human leukocyte antigen for transplantation in human trials.

Increased Expression of Cell Surface SSEA-1 is Closely Associated with Naïve-Like Conversion from Human Deciduous Teeth Dental Pulp Cells-Derived iPS Cells.

Stage-specific embryonic antigen 1 (SSEA-1) is an antigenic epitope (also called CD15 antigen) defined as a Lewis X carbohydrate structure and known to be expressed in murine embryonal carcinoma cells, mouse embryonic stem cells (ESCs), and murine and human germ cells, but not human ESCs/induced pluripotent stem cells (iPSCs). It is produced by α1,3-fucosyltransferase IX gene (FUT9), and F9 ECCs having a disrupted FUT9 locus by gene targeting are reported to exhibit loss of SSEA-1 expression on their cell surface. Mouse ESCs are pluripotent cells and therefore known as “naïve stem cells (NSCs).” In contrast, human ESCs/iPSCs are thought to be epiblast stem cells (EpiSCs) that are slightly more differentiated than NSCs. Recently, it has been demonstrated that treatment of EpiSCs with several reprograming-related drugs can convert EpiSCs to cells similar to NSCs, which led us to speculate that SSEA-1 may have been expressed in these NSC-like EpiSCs. Immunocytochemical staining of these cells with anti-SSEA-1 revealed increased expression of this epitope. RT-PCR analysis also confirmed increased expression of FUT9 transcripts as well as other stemness-related transcripts such as REX-1 (ZFP42). These results suggest that SSEA-1 can be an excellent marker for human NSCs.

Identification and characterization of splenic adherent cells forming densely-packed colonies.

It is thought that the spleen contains stem cells that differentiate into somatic cells other than immune cells. We investigated the presence of these hypothetical splenic cells with stem cell characteristics and identified adherent cells forming densely-packed colonies (Splenic Adherent Colony-forming Cell; SACC) in the spleen. Splenic Adherent Colony-forming Cell was positive for alkaline phosphatase staining and stage-specific embryonic antigen (SSEA)-1 antigen. However, the self-renewal properties of SACCs were limited because they stopped cell proliferation once colonies visible to the naked eye were formed. Gene expression analyses by semi-quantitative RT-PCR revealed the significant expression of c-Myc and Klf4, whereas faint or no expression was evident for Nanog, Oct3/4, and Sox2. Global expression analyses by DNA microarray and subsequent gene ontology analyses revealed that the expression levels of genes related to the immune system were significantly lower in SACCs than in control splenic cells. In contrast, genes unrelated to the immune system, such as those involved in cell adhesion and axon guidance, were relatively highly expressed in SACCs compared with control splenic cells. Taken together, we identified a novel cell type residing in the spleen that is different from the hypothetical splenic stem cell, but which bears some, but not all, characteristics that represent an undifferentiated state.

Isolation, characterization and germline chimera preparation of primordial germ cells from the Chinese Meiling chicken.

Primordial germ cells (PGCs) are precursors of germline cells that can generate sperm and eggs in adults, making them promising tools for transgenic animal preparation and germplasm preservation, especially in avians. In this study, we purified the PGCs from circulating embryonic blood of Chinese Meiling chickens using Nycodenz density centrifugation, and characterized them by alkaline phosphatase (AKP) staining, periodic acid-Schiff (PAS) staining and stage-specific embryonic antigen-1 (SSEA-1) immunostaining and PGC-specific gene amplification. The purified PGCs were also labeled with PKH26 and transferred into donor chicken embryos at the Hamburger-Hamilton (HH) stage 14 to 16, and cells with red fluorescence were observed in the gonads of 8-d-old embryos. When using about 200 PGCs isolated from Chinese Meiling chickens, microinjection into the dorsal aortas of recipient chickens with white feathers at stage HH14 to 16 resulted in germline chimeras that hatched and attained sexual maturity. The frequency of donor-derived yellow-feathered offspring from germline chimeric chickens was 12.6 ± 2.6% after mating with the white-feathered chickens. These results demonstrate that we had successfully purified the PGCs from the Chinese Meiling chicken. These germline cells could be used to preserve Chinese Meiling chickens.

Direct differentiation of insulin-producing cells from human urine-derived stem cells.

The loss of pancreatic β-cells is a cause of diabetes. Therefore, replacement of pancreatic β-cells is a logical strategy for the treatment of diabetes, and the generation of insulin-producing cells (IPCs) from stem cells has been widely investigated as an alternative source for pancreatic β-cells. Here, we isolated stem cells from human urine and investigated their differentiation potential into IPCs. We checked the expression of surface stem cell markers and stem cell transcription factors, and found that the isolated human urine-derived stem cells (hUDSCs) expressed the stem cell markers CD44, CD90, CD105 and stage-specific embryonic antigen (SSEA)-4. In addition, these cells expressed octamer binding transcription factor (Oct)4 and vimentin. hUDSCs could differentiate into adipocytes and osteocytes, as evidenced by Oil-red O staining and Alizarin Red S-staining of differentiated cells, respectively. When we directly differentiated hUDSCs into IPCs, the differentiated cells expressed mRNA for pancreatic transcription factors such as neurogenin (Ngn)3 and pancreatic and duodenal homeobox (Pdx)1. Differentiated IPCs expressed insulin and glucagon mRNA and protein, and these IPCs also secreted insulin in response to glucose stimulation. In conclusion, we found that hUDSCs can be directly differentiated into IPCs, which secrete insulin in response to glucose.

Differentiation potential of the cells in the macula flava of the human vocal fold mucosa

The latest research suggests cells in the maculae flavae located at both ends of the lamina propria of the human vocal fold mucosa have stemness. This study investigated the differentiation potential of the cells in the maculae flavae of the human vocal fold mucosa. Four normal human adult vocal folds from surgical specimens were used. After extraction of the anterior maculae flavae located at the anterior end of the lamina propria of the human vocal fold mucosa under microscope, the maculae flavae were minced, cultured and proliferated in mesenchymal stem cell growth medium and morphological features were assessed. Cell surface markers were detected using flow cytometry. Cell differentiation into adipogenic, chondrogenic and osteogenic lineages was performed. Cell’s differentiation potential was assessed using a human pluripotent stem cell functional identification kit and immunohistochemistry. Subcultured cells formed a colony-forming unit. Subcultured cells expressed CD90, CD105 and CD73 and lacked expression of CD45, CD34, CD11b, CD19 and HLA-DR. They differentiated into adipogenic, chondrogenic and osteogenic lineages. Consequently, the cell features in the maculae flavae meet the minimal criteria defining mesenchymal stromal cells. In addition, subcultured cells differentiated into ectoderm, mesoderm and endoderm and expressed stage-specific embryonic antigen 3 (SSEA-3). The results of this study are consistent with the hypothesis that the cells in the maculae flavae in the lamina propria of the human vocal fold mucosa are putative stem cells.

In vitro differentiation of human multilineage differentiating stress-enduring (Muse) cells into insulin producing cells.

Mesenchymal stem cells (MSCs) is a heterogeneous population. Muse cells is a rare pluripotent subpopulation within MSCs. This study aims to evaluate the pulirpotency and the ability of Muse cells to generate insulin producing cells (IPCs) after in vitro differentiation protocol compared to the non-Muse cells. Muse cells were isolated by FACSAria III cell sorter from adipose-derived MSCs and were evaluated for its pluripotency. Following in vitro differentiation, IPCs derived from Muse and non-Muse cells were evaluated for insulin production. Muse cells comprised 3.2 ± 0.7% of MSCs, approximately 82% of Muse cells were positive for anti stage-specific embryonic antigen-3 (SSEA-3). Pluripotent markers were highly expressed in Muse versus non-Muse cells. The percentage of generated IPCs by flow cytometric analysis was higher in Muse cells. Under confocal microscopy, Muse cells expressed insulin and c-peptide while it was undetected in non-Muse cells. Our results introduced Muse cells as a new adult pluripotent subpopulation, which is capable to produce higher number of functional IPCs.

MiR-21 Contributes to Human Amniotic Membrane-Derived Mesenchymal Stem Cell Growth and Human Amniotic Membrane-Derived Mesenchymal Stem Cell-Induced Immunoregulation.

Human amniotic membrane-derived mesenchymal stem cells (hAM-MSCs) are considered a new and favorable source of stem cells for cell replacement-based therapy. Some microRNAs (miRNAs) have been reported to participate in the regulation of immune responses. Our aim was to investigate the effects of miR-21 on the biological characteristics, immunoregulatory properties, and potential mechanisms of hAM-MSCs. hAM-MSCs were isolated from the placental amnion membrane of a newborn. Cell proliferation, cell cycle, apoptosis, and expressions of cell surface markers were measured by CCK-8 and flow cytometric assays in hAM-MSCs. The expression of mesenchymal-specific antigens vimentin and stage-specific embryonic antigen-4 (SSEA-4) were identified by immunofluorescence staining. Tumor necrosis factor alpha (TNF-α), monocyte chemotactic protein-1 (MCP-1), and interleukin-10 (IL-10) expressions in the cocultured supernatant of hAM-MSCs and peripheral blood mononuclear cells (PBMC) were detected via enzyme-linked immunosorbent assays (ELISA). Flow cytometric analyses revealed that the positive expression rates of the cell surface markers CD29, CD44, CD73, and CD90 in hAM-MSCs were 97.3%, 96.3%, 97.8%, and 98.2%, respectively, while the rates of CD34 and CD45 expression were only 0.6% and 0.84%, respectively. The immunofluorescent staining results showed that vimentin and SSEA-4 were positive in hAM-MSCs. CCK-8 assays revealed that miR-21 overexpression significantly promoted hAM-MSC proliferation. Cell cycle analyses revealed that the number of hAM-MSCs-miR-21+ cells during the synthesis phase (S phase) was significantly increased. miR-21 overexpression also significantly inhibited apoptosis in hAM-MSCs. The ELISA analyses revealed that miR-21 overexpression enhanced the inhibitory effect of hAM-MSCs on the secretion of TNF-α and MCP-1 as well as the promotive effect on the secretion of IL-10 in PBMC cocultured with miR-21-hAM-MSCs. In addition, miR-21 downregulation reduced the inhibitory effect of hAM-MSCs on the secretion of TNF-α, MCP-1, and the promotive effect on the secretion of IL-10 in PBMC cocultured with anti-miR-21-hAM-MSCs. Our data showed that miR-21 promoted hAM-MSCs proliferation, inhibited apoptosis, and was involved in controlling the immunoregulatory capacity of hAM-MSCs.

Multilineage-differentiating stress-enduring (Muse)-like cells exist in synovial tissue.

Cartilage regeneration is a promising therapy for restoring joint function in patients with cartilage defects. The limited availability of autologous chondrocytes or chondrogenic progenitor cells is an obstacle to its clinical application. We investigated the existence and chondrogenic potential of synovial membrane-derived multilineage-differentiating stress-enduring (Muse)-like cells as an alternative cell source for cartilage regeneration. Cells positive for stage-specific embryonic antigen-3 (SSEA-3), a marker of Muse cells, were isolated from the synovial membranes of 6 of 8 patients (median age, 53.5 years; range 36-72 years) by fluorescence-activated cell sorting. SSEA-3-positive cells were cultured in methylcellulose to examine their ability to form Muse clusters that are similar to the embryoid bodies formed by human embryonic stem cells. Muse clusters were expanded and chondrogenic potential of M-cluster-derived MSCs examined using a pellet culture system. Chondrogenic differentiation was evaluated by proteoglycan, safranin O, toluidine blue and type II collagen staining. To evaluate the practicality of the procedure for isolating Muse-like cells, we compared chondrogenic potential of M-cluster derived MSCs with expanded cells derived from the clusters formed by unsorted synovial cells. Synovial membranes contained SSEA-3-positive cells that after isolation exhibited Muse-like characteristics such as forming clusters that expressed NANOG, OCT3/4, and SOX2. In the pellet culture system, cell pellets created from the M-cluster-derived MSCs exhibited an increase in wet weight, which implied an increase in extracellular matrix production, displayed metachromasia with toluidine blue and safranin O staining and were aggrecan-positive and type II collagen-positive by immunostaining. Unsorted synovial cells also formed clusters in methylcellulose culture, and the expanded cell population derived from them exhibited chondrogenic potential. The histological and immunohistochemical appearance of chondrogenic pellet created from unsorted synovial cell-derived cells were comparable with that from M-cluster-derived MSCs. Muse-like cells can be isolated from the human synovial membrane, even from older patients, and therefore may provide a source of multipotent cells for regenerative medicine. In addition, the cluster-forming cell population within synovial cells also has excellent chondrogenic potential. These cells may provide a more practical option for cartilage regeneration.

EFFECT OF NATURAL AND SYNTHETIC FOOD COLORANTS ON SPERMATOGENESIS AND THE EXPRESSION OF ITS CONTROLLING GENES

The use of food additives is controversial. However, data regarding their effects on fertility genes are still very sparse. The present study was designed to assess the effects of two coloring agents, carmoisine (synthetic) and curcumin (natural) on the expression of some genes with fertility impact. Sixty three male Sprague-Dawley albino rats were orally administered carmoisine and curcumin in three doses, acceptable daily intake (ADI), 5x- and 10x- ADI for 15, 30, and 45 days. Sperm analysis and testicular expression level of biomarkers Testin, Glial cell derived neurotrophic factor (GDNF), tyrosine kinase receptor (c-KIT), follicle stimulating hormone receptor (FSHR), A kinase anchor protein 3 (PRKA3), spermatogenesis associated 7 (Spata7), Stage-specific embryonic antigen-1 (SSEA1) genes were measured and supported with histopathological studies on rat testes tissues. The recorded results revealed significant down regulation of the tested genes in rats supplemented with carmoisine in time and dose dependent manner. However, these declines were also observed after treatment with medium and high doses of curcumin. Sperm counts were significantly decreased after carmiosine treatment in a dose dependent manner, it was 74.6±6.36, 74.00±6.63, 49.00±0.28 and 147.00±3.2 for ADI, 5xADI, 10xADI and control group respectively, without any changes after curcumin treatment. Also, histopathological studies indicated deleterious effect with medium and high doses of carmiosine. In Conclusion, carmoisine induced hazardous effects on fertility at different levels when consumed in concentrations higher than the acceptable daily-authorized level (50 mg/kg b.wt). However, curcumin as a natural food color is saver than carmoisine up to certain levels.