Mesenchymal Stem Cell Surface Markers Research Articles

Mesenchymal stem cells provide paracrine neuroprotective resources that delay degeneration of co-cultured organotypic neuroretinal cultures

Through the paracrine effects of stem cells, including the secretion of neurotrophic, immunomodulatory, and anti-apoptotic factors, cell-based therapies offer a new all-encompassing approach to treatment of neurodegenerative diseases. In this study, we used physically separated co-cultures of porcine neuroretina (NR) and human mesenchymal stem cells (MSC) to evaluate the MSC paracrine neuroprotective effects on NR degeneration. NR explants were obtained from porcine eyes and cultured alone or co-cultured with commercially available MSCs from Valladolid (MSCV; Citospin S.L.; Valladolid, Spain), currently used for several approved treatments. Cultures were maintained for 72 h. MSC surface markers were evaluated before and after co-culture with NRs. Culture supernatants were collected and the concentration of brain-derived neurotrophic factor (BDNF), ciliary neurotrophic factor (CNTF), and glial-derived neurotrophic factor (GDNF) were determined by enzyme-linked immunosorbent assays. NR sections were stained by haematoxylin/eosin or immunostained for terminal deoxynucleotidyl transferase dUTP nick end labelling (TUNEL), glial fibrillary acidic protein, β-tubulin III, and neuronal nuclei marker. NR morphology, morphometry, nuclei count, apoptosis rate, retinal ganglion cells, and glial cell activation were evaluated. Treatment effects were statistically analysed by parametric or non-parametric tests. The MSCs retained stem cell surface markers after co-culture with NR. BDNF and CNTF concentrations in NR-MSCV co-cultures were higher than other experimental conditions at 72 h (p < 0.05), but no GDNF was detected. NR general morphology, total thickness, and cell counts were broadly preserved in co-cultures, and the apoptosis rate determined by TUNEL assay was lower than for NR monocultures (all p < 0.05). Co-cultures with MSCV also protected retinal ganglion cells from degenerative changes and reduced reactive gliosis (both p < 0.05). In this in vitro model of spontaneous NR degeneration, the presence of co-cultured MSCs retarded neuroglial degeneration. This effect was associated with elevated concentrations of the neurotrophic factors BDNF and CNTF. Our data suggest that the paracrine secretion of these, and possibly other molecules, are a potential resource for the treatment of several neuroretinal diseases.

Pathological ASXL1 Mutations and Protein Variants Impair Neural Crest Development.

SummaryThe neural crest (NC) gives rise to a multitude of fetal tissues, and its misregulation is implicated in congenital malformations. Here, we investigated molecular mechanisms pertaining to NC-related symptoms in Bohring-Opitz syndrome (BOS), a developmental disorder linked to mutations in the Polycomb group factor Additional sex combs-like 1 (ASXL1). Genetically edited human pluripotent stem cell lines that were differentiated to NC progenitors and then xenotransplanted into chicken embryos demonstrated an impairment of NC delamination and emigration. Molecular analysis showed that ASXL1 mutations correlated with reduced activation of the transcription factor ZIC1 and the NC gene regulatory network. These findings were supported by differentiation experiments using BOS patient-derived induced pluripotent stem cell lines. Expression of truncated ASXL1 isoforms (amino acids 1–900) recapitulated the NC phenotypes in vitro and in ovo, raising the possibility that truncated ASXL1 variants contribute to BOS pathology. Collectively, we expand the understanding of truncated ASXL1 in BOS and in the human NC.

COMPARISON OF HUMAN PLATELET LYSATE AND FETAL BOVINE SERUM IN CULTURE MEDIA FOR HUMAN DENTAL PULP STEM CELL PROLIFERATION

Objective: Ex vivo and in vitro cell cultures require a basal medium with added supplements containing growth factors, proteins, and enzymes tosupport attachment, growth, and proliferation. Fetal bovine serum (FBS) is used to supplement cell culture media. However, human platelet lysate(hPL) represents an attractive alternative as it is nonxenogeneic.Methods: Human third molars were collected from six healthy donors (19–35 years old) with no history of regular alcohol consumption or smoking.Human dental pulp stem cells (hDPSCs) at the second passage were divided into two culture media groups, 10% FBS and 5% hPL, as well as a controlgroup after 24 h of serum starvation. A flow cytometry analysis was conducted to measure CD90, CD105, CD73, CD34, CD45, and Human LeukocyteAntigen-DR isotype (HLA-DR). Cellular proliferation was evaluated on days 1, 3, and 5.Results: The flow cytometry analysis revealed that the majority of the cells expressed positive mesenchymal stem cell surface markers, includingCD73 (98.5%), CD90 (98.3%), and CD105 (71.0%), and lacked CD34, CD45, and HLA-DR. There were significant differences among the 5% hPL, 10%FBS, and control groups on days 1, 3, and 5.Conclusion: For a nonxenogeneic culture, 5% hPL can be used as an alternative in culture media for hDPSC proliferation.

Diabetes mellitus type 2 reduces the viability, proliferation, and angiogenic marker of adipose-derived stem cells cultured in low-glucose anti-oxidant-serum supplemented medium

Introduction: Hyperglycemia in diabetic patients induces elevated pro-inflammatory cytokine production, resulting in cellular damage, which may affect the regenerative function of mesenchymal stem cells (MSCs), such as adipose-derived stem cells (ADSCs). Identifying the effect of diabetes on ADSCs and optimization of culture conditions is therefore an important starting point for the application of autologous stem cells to improve clinicial outcomes. The aim of this study was to investigate the effect of diabetes on ADSCs that cultured in low-glucose anti-oxidant-serum supplemented medium.&#x0D; Methods: In this study, freshly isolated stromal vascular fraction (SVF) and expanded ADSCs were compared between diabetic and non-diabetic donors. SVF were isolated from the abdominal fat, and total viable cells and viability were estimated. Fresh SVF were cultured in low-glucose (100 mg/dL) culture medium supplemented with an anti-oxidant and fetal bovine serum (complete culture medium) at a low density for 14 days for the colony formation unit-fibroblast (CFU-F) assay. The remaining SVF were expanded to obtain ADSCs in the complete culture medium, which were evaluated based on MSCs surface marker expression and three lineage differentiation potential. Diabetic and non-diabetic ADSCs were compared with respect to population doubling time and viability after serial passage.&#x0D; Results: Total viable counts (0.97 +/- 0.39 x 109 cells/10 mL of adipose tissue, 0.56 +/- 0.39 x 109 cells/10 mL of adipose tissue, p=0.02, independent t-test), but not viability (98.63 +/- 1.12%, 98.20 +/- 1.21%, p= 0.38, independent t-test), were significantly higher for SVF cells from adipose tissues of non-diabetic donors than diabetic donors. Fewer CFU-F were obtained from cultured diabetic SVF than from non-diabetic SVF. Diabetic and non-diabetic ADSCs had similar differentiation potency and CD73 (99.44 +/- 0.34%, 97.15 +/- 5.37%, p= 0.21, Mann-Whitney U test) and CD90 (97.30 +/- 2.86%, 95.06 +/- 6.32%, p= 0.90, Mann- Whitney U test) expression, but significantly fewer diabetic ADSCs expressed CD105 or endoglin, a marker for angiogenesis (89.91 +/- 7.14%, 57.90 +/- 21.36% for non-diabetic and diabetic groups, p&lt; 0.001, Mann-Whitney U test). Diabetic ADSCs tended to exhibit slower proliferation (4.43 +/- 2.70 days, 3.04 +/- 0.55 days, p= 0.27 in passage 2 (P2); 3.95 +/- 1.55 days, 2.96 +/- 0.91 days, p= 0.21 in P3, independent t-test) and lower viability than those of non-diabetic ADSCs (77.65 +/- 10.61%, 87.13 +/- 10.06%, p= 0.25 in P2; 82.70 +/- 8.07%, 91.15 +/- 3.77%, p= 0.04 in P3, independent t-test). Culture in low-glucose anti-oxidant-serum supplemented medium did not improve CD105 expression (65.14 +/- 5.86%, 71.06 +/- 10.27%, 64.05 +/- 10.04%, p= 0.70, for P1, P2, and P3, respectively, repeated measure ANOVA) and cell proliferation (p= 0.50 for P2 vs. P3, paired t-test) of diabetic ADSCs.&#x0D; Conclusions: Overall, diabetes reduced CD105 expression and ADSCs proliferation, suggesting that the angiogenic potency of diabetic ADSCs is reduced. The diabetic ADSCs in this study were also more prone to cell death caused by handling technique compared to non-diabetic ADSCs. Therefore, more advanced culture techniques should be applied to expand ADSCs from diabetic patients to achieve expected clinical outcomes.&#x0D;

Isolation and Characterization of an Adult Stem Cell Population from Human Epidural Fat.

Study Design Isolation and characterization of human epidural fat (HEF) stem/progenitor cells. Objective To identify a progenitor population within HEF and to determine if they meet the minimal criteria of a mesenchymal stem cell (MSC). Summary of Background Data The biological function, if any, has yet to be determined for HEF. The presence of MSCs within HEF may indicate a regenerative potential within the HEF. Methods HEF was isolated from 10 patients during elective spinal surgery. HEF cells were differentiated along osteo-, adipo-, and chondrogenic lineages, with differentiation analyzed via qPCR and histology. The cell surface receptor profile of HEF cells was examined by flow cytometry. HEF cells were also assayed through the collagen contraction assay. Prx1CreERT2GFP:R26RTdTomato MSC lineage-tracking mice were employed to identify EF MSCs in vivo. Results HEF cell lines were obtained from all 10 patients in the study. Cells from 2/10 patients demonstrated full MSC potential, while cells from 6/10 patients demonstrated progenitor potential; 2/10 patients presented with cells that retained only adipogenic potential. HEF cells demonstrated MSC surface marker expression. All patient cell lines contracted collagen gels. A Prx1-positive population in mouse epidural fat that appeared to contribute to the dura of the spinal cord was observed in vivo. Conclusions MSC and progenitor populations are present within HEF. MSCs were not identified in all patients examined in the current study. Furthermore, all patient lines demonstrated collagen contraction capacity, suggesting either a contaminating activated fibroblast population or HEF MSCs/progenitors also demonstrating a fibroblast-like phenotype. In vivo analysis suggests that these cell populations may contribute to the dura. Overall, these results suggest that cells within epidural fat may play a biological role within the local environment above providing a mechanical buffer.

Immunophenotypic characterization, multi-lineage differentiation and aging of zebrafish heart and liver tissue-derived mesenchymal stem cells as a novel approach in stem cell-based therapy

Mesenchymal stem cells (MSCs) are a good model for preclinical and clinical investigations, and alternative sources of MSCs are subject to intensive experiments. In this study, mesenchymal stem cells (MSCs) were isolated from heart and liver tissue of Zebrafish (Danio rerio). The flow-cytometry as well as RT-PCR were used to analyze the expression of a panel of cell surface markers CD44, CD90, CD31 and CD34. In the following, alizarin red, oil red-O and toluidine blue staining were carried out to evaluate the multi-lineage differentiation of zebrafish heart and liver tissue-derived MSCs. Subsequently, the gene and protein expression of Oct4, Sox2 and Nanog as pluri-potent markers were analyzed by RT-PCR and western blotting, respectively. In addition, MTT assay was used for cell proliferation potential and population doubling time (PDT) assessment. Also, the aging of cells was investigated by β-galactosidase activity assay. The results showed that, like other MSCs, zebrafish heart and liver tissue-derived MSCs were positive for mesenchymal, negative for hematopoietic markers and expressed pluripotent markers Oct4, Sox2 and Nanog. Moreover, these cells were differentiated to osteocyte, adipocyte, and chondrocyte lineages following directed differentiation. It was found that PDT of zebrafish heart and liver tissue-derived MSCs were 50.67 and 46.61 h, respectively. These cells had significantly more rapid growth on day 4. Our results show that zebrafish heart and liver tissue-derived MSCs exhibited typical MSC characteristics including fibroblast morphology, multi-lineage differentiation capacity, pluripotency potential and expression of a typical set of classic MSC surface markers.

Differentiation of adult human mesenchymal stem cells into dopaminergic neurons.

The striatal dopamine (DA) deficiency is known as the main cause of the clinical picture of Parkinson’s disease (PD). The disease is a progressive degeneration of dopaminergic neurons in the striatum. The treatment of PD is based on compensation for the brain’s supply of DA lost by drug therapy, deep brain stimulation, surgery, gene and cell therapies. Clinical studies have focused on the utility of stem cell-based therapies in PD. Embryonic and mesenchymal stem cells (MSCs) are widely used. Recently, human adipose derived stem cells (hADSCs) have been considered as a suitable source of tissue for this purpose. In this project, hADSCs differentiated into dopaminergic neurons and the specificity of the cell preparations was examined. Human adipose tissues were collected from healthy volunteers undergoing liposuction and hADSCs were isolated by collagenase-based enzymatic method. Flow cytometry was performed using the surface cluster of differentiation (CD) markers to confirm the cell typical properties. Then hADSCs were differentiated to dopaminergic neurons in neurobasal medium in the presence of differentiation factors and confirmed by immunocytochemistry via neuronal and dopaminergic markers. The isolated hADSCs were cultured and identified by the expression of MSCs surface markers including CD90, and CD44. These cells did not express hematopoietic surface markers such as CD45 and CD14. Differentiated cells express neuronal marker NeuN and dopaminergic marker tyrosine hydroxylase (TH). It is concluded that hADSCs can be easily taken from the patient’s own body and differentiated into dopaminergic cells having a lower risk of transplant rejection.

Isolation of Multipotent Mesenchymal Stem Cells from Human Extraocular Muscle Tissue.

Mesenchymal stem cells (MSCs) have attracted significant attention as potential therapeutic cells to treat various diseases ranging from tissue injuries, graft versus host disease, degenerative diseases and cancer. Since the initial discovery of MSCs in the bone marrow cells, MSCs have been successfully isolated from various adult and neo-natal tissues, albeit the procedures are often coupled with difficulties in harvesting tissue and produce low yield of cells, requiring extensive expansion in vitro. Here, we explored extra-ocular muscle tissues obtained from patients as a novel source of MSCs which express characteristic cell surface markers of MSCs and show multilineage differentiation potential with high proliferation capacity.

In vitro enhancement and functional characterization of neurite outgrowth by undifferentiated adipose-derived stem cells.

Adipose‑derived stem cells(ASCs) can easily be obtained and expanded invitro for use in autologous cell therapy. Via their production of cytokines and neurotrophic factors, transplanted ASCs provide neuroprotection, neovascularization and induction of axonal sprouting. However, the influencing mechanism of undifferentiated ASCs on nerve regeneration is currently only partially understood. In the present study, undifferentiated ASCs and cutaneous primary afferent dorsal root ganglion(DRG) neurons were co‑cultured in order to investigate their interaction. ASCs were isolated from adult rat fat tissue. The presence of characteristic stem cell markers was determined by flow cytometry in three subsequent passages. Adipogenic, osteogenic, chondrogenic and glial differentiation was performed in order to evaluate their differentiation capacity. A direct co‑culture system with DRG cells was established to determine the effect of undifferentiated pluripotent ASCs on neurite elongation. Neurite outgrowth, length and number was examined in the co‑culture and compared with single‑culture cells and cells stimulated with nerve growth factor (NGF). In ASC cultures, NGF expression was assessed by ELISA. The present results demonstrated that the specific mesenchymal stem cell surface markers CD44, CD73 and CD90 were detected in all three subsequent passages of the isolated ASCs. In accordance, ASC differentiation into adipogenic, osteogenic, chondrogenic and Schwann cell phenotype was conducted successfully. Neurite outgrowth of DRG neurons was enhanced following co‑culture with ASCs, resulting in increased neurite length after 24h of cultivation. Furthermore, neurite outgrowth of DRG neurons was directed towards the undifferentiated ASC and direct cell‑to‑cell contact was observed. In summary, the results of the present study revealed an interaction between the two cell types with guidance of neurite growth towards the undifferentiated ASC. These findings suggest that the use of undifferentiated ASC optimizing tissue‑engineered constructs may be promising for peripheral nerve repair.

Electrosprayed genipin cross-linked alginate-chitosan microcarriers for ex vivo expansion of mesenchymal stem cells.

Mesenchymal stem cells (MSCs) are potential therapeutic candidates, owing to their ability to differentiate into several cell types. However, the gap between availability and demand of MSCs requires alternative expansion methods from 2D culture flasks. Microcarriers are a promising approach for MSC expansion due to their high surface area-to-volume ratio. However, current commercial microcarriers do not provide the highest cell yield due to low cell attachment efficiencies and difficulty in cell detachment. This study developed a hydrogel-based microcarrier from genipin cross-linked alginate-chitosan beads. Alginate beads were produced by electrospraying before being coated with chitosan and cross-linked in genipin. The degree of cross-linking was determined through fluorescence reading of the genipin-chitosan conjugates. MSCs cultured on these microcarriers had a 26% higher cell attachment and twice the proliferation rate compared to the commercial microcarrier Cytodex 1. Cells easily detached from the hydrogel beads and did not require extended incubation periods or intense agitation during cell harvest. There was no significant difference in gene expression between the two microcarriers for the positive MSC surface markers CD-90, CD-105, and CD-73 as well as showing either low or no signal for negative MSC surface markers CD-45 and CD-34. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 107A: 122-133, 2019.

Comparison of the biological characteristics of human mesenchymal stem cells derived from exfoliated deciduous teeth, bone marrow, gingival tissue, and umbilical cord.

Different sources of mesenchymal stem cells (MSCs) may differ in their biological characteristics, which are important for their clinical application. In the present study, MSCs were isolated from human exfoliated deciduous teeth (SHED), bone marrow, gingival tissue and umbilical cord tissue, and their biological characteristics including surface markers, proliferation capacity, tumorigenicity and immunogenicity were analyzed by flow cytometric analysis, ELISA and co-culture with human lymphocytes, respectively. The results indicated that all four types of stem cells obtained from different sources expressed MSC surface markers, and they did not show tumorigenicity either in vivo or in vitro. Stem cells from SHED exhibited the strongest proliferation capacity. Umbilical cord-derived MSCs displayed the strongest immunomodulatory ability, while bone marrow MSCs exhibited the best antigen-presenting potential in response to interferon-γ stimulation. These results provide information on MSCs derived from different tissues, which may be helpful in their clinical application.

Reversible secretome and signaling defects in diabetic mesenchymal stem cells from peripheral arterial disease patients

ObjectiveRegenerative medicine seeks to stall or to reverse the pathologic consequences of chronic diseases. Many people with diabetes have peripheral arterial disease (PAD), which increases their already high risk of major amputation. Cellular therapies are a promising regenerative medicine approach to PAD that can be used to focally inject regenerative cells to endangered tissue beds. Mesenchymal stem cells (MSCs) are known to promote tissue regeneration through stromal support and paracrine stimulation of new blood vessels (angiogenesis). Whereas little is known about human diabetic MSCs (dMSCs), particularly those from patients with PAD, dMSCs have a limited expansion capacity but can be improved with human platelet lysate (PL) supplementation. PL is rich in many growth factors, including epidermal growth factor (EGF), which is known to be important to cell proliferation and survival signaling pathways. We hypothesize that dMSCs have a reversible defect in EGF receptor pathways. The objective of this work was to test this hypothesis using dMSCs from PAD patients. MethodsThe secretome expression of EGF and prominent angiogens was characterized from bone marrow (BM)-derived and adipose tissue-derived (ATD) dMSCs from five patients (six limbs) undergoing major amputation. Western blot was used to characterize the AKT and extracellular signal-regulated protein kinases 1 and 2 expression in dMSCs under standard culture (5% fetal bovine serum plus fibroblast growth factor 2 [FGF2]), 5% human PL, or 5% fetal bovine serum plus EGF. Healthy donor MSCs were control cells. The angiogenic activity of BM- and ATD-dMSCs was tested on human umbilical vein endothelial cells (ECs). Paired t-test, analysis of variance, and Kruskal-Wallis tests were used as appropriate. ResultsBoth BM- and ATD-dMSCs had typical MSC surface marker expression and similar expansion profiles, and they did not express EGF in their secretome. PL supplementation of dMSCs improved AKT signaling, but they were resistant to FGF2 activation of extracellular signal-regulated protein kinases 1 and 2. EGF supplementation led to similar AKT expression as with PL, but PL had greater phosphorylation of AKT at 30 and 60 minutes. The conditioned media from both BM- and ATD-dMSCs had robust levels of prominent angiogens (vascular endothelial growth factor, monocyte chemoattractant protein 1, hepatocyte growth factor), which stimulated EC proliferation and migration, and the co-culture of dMSCs with ECs led to significantly longer EC sprouts in three-dimensional gel than EC-alone pellets. ConclusionsPL and EGF supplementation improves AKT expression in dMSCs over that of FGF2, but PL improved pAKT over that of EGF. Thus, PL supplementation strategies may improve AKT signaling, which could be important to MSC survival in cellular therapies. Furthermore, BM- and ATD-dMSCs have similar secretomes and robust in vitro angiogenic activity, which supports pursuing dMSCs from both reservoirs in regenerative medicine strategies.

Regenerative potential of human nucleus pulposus resident stem/progenitor cells declines with ageing and intervertebral disc degeneration.

Numerous studies have demonstrated the presence of resident nucleus pulposus stem/progenitor cells (NPSCs) in the tissue of the intervertebral disc (IVD). However, the cellular identity of NPSCs during IVD degeneration and ageing are poorly defined at present, despite significant progress in the understanding of NPSC biology. In the present study, NPSCs were isolated from human degenerated IVD and were characterized by flow cytometry, gene expression assays and proliferation and multipotency analysis. The results of the present study demonstrated that NPSCs isolated from human degenerated IVD may be divided into two groups according to the expression of mesenchymal stem cell (MSC) surface markers: The high expression of MSC surface markers group (H‑NPSCs) was highly positive for CD29, CD44, CD73, CD90 and CD105 at rates >95%, and the low expression of MSC markers surface markers group (L‑NPSCs), with the expression of CD29 and CD105 exhibiting individual variability, however, all at rates <95%. The donors for H‑NPSCs were aged <20years, while the majority of donors for L‑NPSCs were aged >25years, with one exception aged <20years. The results highlighted that the low expression of MSC surface markers in NPSCs from aged and degenerated NP tissues were associated with a low rate of proliferation and reduced differentiation potential, as well as downregulation of the NP progenitor marker Tie2 and higher expression of NP cell‑specific markers. These findings demonstrated that the regenerative potential of human NPSCs declines with ageing and degeneration of the IVD.

Mesenchymal Stem Cell Levels of Human Spinal Tissues.

Systematic review. The aim of this study was to investigate, quantify, compare, and compile the various mesenchymal stem cell (MSC) tissue sources within human spinal tissues to act as a compendium for clinical and research application. Recent years have seen a dramatic increase in academic and clinical understanding of human MSCs. Previously limited to cells isolated from bone marrow, the past decade has illicited the characterization and isolation of human MSCs from adipose, bone marrow, synovium, muscle, periosteum, peripheral blood, umbilical cord, placenta, and numerous other tissues. As researchers explore practical applications of cells in these tissues, the absolute levels of MSCs in specific spinal tissue will be critical to guide future research. The PubMED, MEDLINE, EMBASE, and Cochrane databases were searched for articles relating to the harvest, characterization, isolation, and quantification of human MSCs from spinal tissues. Selected articles were examined for relevant data, categorized according to type of spinal tissue, and when possible, standardized to facilitate comparisons between sites. Human MSC levels varied widely between spinal tissues. Yields for intervertebral disc demonstrated roughly 5% of viable cells to be positive for MSC surface markers. Cartilage endplate cells yielded 18,500 to 61,875 cells/0.8 mm thick sample of cartilage end plate. Ligamentum flavum yielded 250,000 to 500,000 cells/g of tissue. Annulus fibrosus fluorescence activated cell sorting treatment found 29% of cells positive for MSC marker Stro-1. Nucleus pulposus yielded mean tissue samples of 40,584 to 234,137 MSCs per gram of tissue. Numerous tissues within and surrounding the spine represent a consistent and reliable source for the harvest and isolation of human MSCs. Among the tissues of the spine, the annulus fibrosus and ligamentum flavum each offer considerable levels of MSCs, and may prove comparable to that of bone marrow. 5.

116 - Hypoxia increases vegf secretion in multiple sources of mesenchymal stem cell

It is already known worldwide that mesenchymal stem cell (MSC) is rich in paracrine factors. In fact, numbers of paracrine factors have been discovered including chemokines and cytokines as well as pro-angiogenic factors contained therein. Co-culturing MSC with endothelial progenitor cell (EPC) improved EPC function without it was differentiated. This indicated that MSC secreted pro-angiogenic factors such as Vascular Endothelial Growth Factor (VEGF) which influence this phenomenon. The aim of this study is to investigate the influence of oxygen tension in VEGF secreted by MSC. Multiple MSCs; bone marrow (BM), adipose tissue (AD), and umbilical cord (UC)-derived mesenchymal stem cells were cultured up to 6th passage in Minimum Essential Medium (MEM) with 10% FBS and 1% Antibiotic-Antimycotic. After 80% confluency was reached, the culture medium was replaced with serum-free medium and incubated for 72 hours. These steps were done simultaneously in two different conditions; normoxia (21% O2) and hypoxia (3% O2). MSCs‘ secretome were collected and quantified by Enzyme-Linked Immunosorbent Assay (ELISA) method. VEGF secreted by hypoxic-conditioned MSCs increased more than 50 times compared to normoxic-conditioned ones. Our result showed that VEGF secreted by UC-MSC, BM-MSC, AD-MSC in normoxia were 2.436 pg/mL, 385.782 pg/mL, and 105.682 pg/mL respectively. On the other hand, VEGF secreted by UC-MSC, BM-MSC, AD-MSC in hypoxic condition were 2,795.982 pg/mL, 1,588.982 pg/mL, and 1,402.982 pg/mL respectively. After the culture was accomplished, the cells were also harvested to be checked further their immunophenotype and gene expressions. Surface markers of all three MSCs in both conditions showed no significant differences. Hypoxia-induced Factor 1-alpha (HIF1A) and VEGFA genes were expressed more intense in hypoxic-conditioned UC-MSCs than normoxic-conditioned ones. It can be concluded that in hypoxic condition, MSCs derived from umbilical cord, bone marrow, and adipose tissue increase VEGF secretion which possibly impacts their clinical outcome as cell therapies towards vascular diseases. Figure 2Genes expression (VEGFA and HIF1A) of umbilical cord derived mesenchymal stem cell in normoxia vs hypoxia 3%. View Large Image Figure Viewer Download Hi-res image Table IImmunophenotyping of UC-MSC, BM-MSC, AD-MSC in Normoxia (21% O2) Condition. MSC SURFACE MARKERS UC-MSC BM-MSC AT-MSC CD 105 90.75% 95.28% 96.5% CD 73 94.65% 99.34% 96.28% CD 90 92.76% 99.97% 99.85% Open table in a new tab Table IIImmunophenotyping of UC-MSC, BM-MSC, AD-MSC in Hypoxia (3% O2) Condition. MSC SURFACE MARKERS UC-MSC BM-MSC AT-MSC CD 105 95.52% 99.32% 95.37% CD 73 99.58% 99% 95.29% CD 90 99.47% 99.8% 95.27% Open table in a new tab

Down regulation of ITGA4 and ITGA5 genes after formation of 3D spherules by human Wharton's jelly stem cells (hWJSCs).

Human Wharton's jelly mesenchymal stem cells (hWJSCs) are multipotent stem cells that could be aggregated into 3D spherules. ITGA4 and ITGA5 genes encode α4 and α5 subunits of integrins, respectively. In this study, we analyzed expression levels of ITGA4 and ITGA5 gene mRNAs in undifferentiated and 3D spherules forming hWJSCs in order to determine their expression pattern for possible future treatment of cancer cells in a co-culture fashion. For the purpose of obtaining hWJSCs, umbilical cords were collected from patients with caesarian section at full term delivery. The cells were then characterized according to cell surface markers using flow cytometry. Furthermore pluripotency of the obtained cells was verified. Subsequently the cells were aggregated in 3D spherules using hanging drop cultures. Expression levels of ITGA4 and ITGA5 gene mRNAs were determined by RT-PCR and Real time PCR, both in the initial undifferentiated cells and those aggregated in the spherules. The obtained hWJSCs demonstrated pluripotency, differentiating to adipogenic and osteogenic cells. They also expressed mesenchymal stem cell surface markers. Following the aggregation of these cells and formation of 3D spherules, mRNA expression levels of both genes were significantly reduced (P < 0.05) compared with the initial undifferentiated state. The results of this study demonstrated that aggregation of hWJSCs into spherules alters their expression of ITGA4 and ITGA5. The implications of such an alteration would require further research.

Activated Yes-Associated Protein Accelerates Cell Cycle, Inhibits Apoptosis, and Delays Senescence in Human Periodontal Ligament Stem Cells.

Objectives: To provide insight into the biological effects of activated Yes-associated protein (YAP) on the proliferation, apoptosis, and senescence of human periodontal ligament stem cells (h-PDLSCs).Methods: h-PDLSCs were isolated by the limiting dilution method, and their surface markers were quantified by flow cytometry. Enhanced green fluorescence protein (EGFP)-labeled lentiviral vector was used to activate YAP in h-PDLSCs, then qRT-PCR and Western blotting were used to evaluate the expression level of YAP. Immunofluorescence was used to detect the location of YAP in h-PDLSCs. The proliferation activity was detected by cell counting kit-8 (CCK-8) and 5-ethynyl-2'-deoxyuridine (EdU), and the cell cycle was determined by flow cytometry. Apoptosis was analyzed by Annexin V-APC staining. Cell senescence was detected by β-galactosidase staining. Proteins in ERK, Bcl-2, and p53 signaling pathways were detected by Western blotting.Results: h-PDLSCs were isolated successfully and were positive for human mesenchymal stem cell surface markers. After YAP was activated by lentiviral vector, the mRNA and protein of YAP were highly expressed, and more YAP translocated into the nucleus. When YAP was overexpressed in h-PDLSCs, proliferation activity was improved; early and late apoptosis rates decreased (P<0.05); the proportion of cells in G2/M phases increased (P<0.05), while that in G0/G1 phase decreased (P<0.05); cellular senescence was delayed (P<0.01); the expression of P-MEK, P-ERK, P-P90RSK and P-Msk increased, while the expression of Bcl-2 family members (Bak, Bid and Bik) decreased.Conclusions: Activated YAP promotes proliferation, inhibits apoptosis, and delays senescence of h-PDLSCs. The Hippo-YAP signaling pathway can influence ERK and Bcl-2 signaling pathways.

Mesenchymal stem cells from periapical lesions modulate cytokine production by local immune cells

Background/Aim. Mesenchymal stem cells (MSCs) have been shown to suppress immune and inflammatory reactions. However, it is not known whether MSCs from inflammatory tissues, such as periapical lesions (PLs) have similar effects. This question was addressed in this study in which the aim was to examine the capacity of PL-MSCs for modulating cytokine production by local immune cells. Methods. PL-MSCs were isolated from asymptomatic (as) and symptomatic (sy) PLs. Their phenotype was analyzed by flow cytometry by detecting MSC surface markers. Anti-inflammatory and immunomodulatory properties of PL-MSCs were examined by measuring cytokine production in direct co-culture experiments with mononuclear cells (MNCs) isolated from asPLs and syPLs, respectively. The levels of cytokines in supernatants were determined by specific ELISA kits. Results. Both PL-MSCs lines were characterized by typical MSC phenotype, with the predominance of CD29, CD44, CD90, CD105 and CD166. However, the lines, independently of their similar phenotype had the same modulatory effect on cytokine production, but the response of asPL-MNCs and syPL-MNCs was different, in spite of similar composition of these MNCs. Both MSC lines inhibited the production of inflammatory cytokines, such as interleukin-1_ (IL-1_) and tumor necrosis factor-_ (TNF-_). However, IL-8 was only down-regulated in the co-culture of these MSC lines with syPL-MNCs. The PL-MSCs also modulated the production of immunoregulatory cytokines. Transforming growth factor-_ (TGF-_) was up-regulated by both as- and syPL-MNCs but IL-10 was up-regulated only by asPL-MNCs. Conclusion. Our results showed that PL-MSCs contribute to the restriction of local inflammatory and immune responses, but this effect is probably less efficient during the exacerbation of PL inflammation.

One-step Derivation of Functional Mesenchymal Stem Cells from Human Pluripotent Stem Cells.

Mesenchymal stem cells (MSCs) are invaluable cell sources for understanding stem cell biology and potential application in tissue engineering and regenerative medicine. The current issues of MSCs that demand to be further addressed are limited donors, tissue sources and limited capacity of ex vivo expansion. Here, we describe a simple and easy protocol for generating functional mesenchymal stem cells from human pluripotent stem cells (hPSCs) via one-step low glucose medium switch strategy in feeder-free culture system. In this protocol, human induced pluripotent stem cells (hiPSCs) and H9 human embryonic stem cells (hESCs) were successfully differentiated into MSCs, named hiPSC-MSCs and hESC-MSCs, respectively. The derived hiPSC-MSCs and hESC-MSCs exhibited common MSC characteristics as MSCs derived from human bone marrow (hBM-MSCs), including expressing MSC surface markers and possessing capability of tri-lineage differentiation in vitro (adipogenesis, osteogenesis and chondrogenesis). As compared with other available protocols, our protocol can be applied to generate a large number of MSCs from hPSCs with high efficiency, low-cost manner, moreover, not involving embryoid body, mouse feeder-cell, flow sorting, and pathway inhibitors (such as SB203580 and SB431542). We believe that this protocol could provide a robust platform to reach the future demand for producing the industrial scale of MSC from hPSCs for autologous cell-based therapy.

The Origin and Identification of Mesenchymal Stem Cells in Teeth: from Odontogenic to Non-odontogenic.

Mesenchymal stem cells (MSCs) in teeth have been exploited as vital seed cells for stem cell-based dental medicine. To date, several mesenchymal stem cell populations originated from odontogenic tissue have been isolated and characterized by their expression of MSC surface markers and capacity of multi-lineage differentiation, including dental pulp stem cells (DPSCs), stem cells from human exfoliated deciduous teeth (SHED), stem cells from apical papilla (SCAP) and so on. However, their identity in vivo remains elusive, which hinders further understanding of their application in stem cell-based tooth regeneration. Label retaining and lineage tracing analyses, which serve as gold standards for identification of stem cells in vivo, provide feasibility for identifying MSCs in teeth. In this review, we will discuss the issues of MSCs, including the origin and identification of both odontogenic and non-odontogenic MSCs, and address the role of nerve-derived Sonic hedgehog (Shh) in the regulation of MSCs in the neurovascular bundle (NVB). Based on label retaining and lineage tracing analyses, latest studies have found new populations of non-odontogenic MSCs in teeth, periarterial-derived and glial-derived, regulated by the Shh derived from nerves in the NVB, which provides a new hope for tooth regeneration.