Identification Of Mesenchymal Stem Cells Research Articles

Study on the mechanism of quercetin inducing mesenchymal stem cells to differentiate into fibroblasts through TGF-β1 and IGF-1

BackgroundStem cell differentiation has opened up new avenues for disease treatment, tissue repair, and drug development in the study of regenerative medicine, and has huge application prospects. This study aimed to explore the mechanism of quercetin on the differentiation of mesenchymal stem cells (MSCs) into fibroblasts. MethodsIn this study, cell differentiation experiments and flow cytometry were used to detect the successful isolation of bone marrow MSCs from SD rats. Quercetin at 5, 10, and 20 μM was used as low, medium, and high doses to intervene in MSCs. The cell viability changes of ligament fibroblasts at 24, 48, and 72 hours after quercetin treatment were detected using a CCK-8 cell counting kit. Cell proliferative capacity was determined by flow cytometry. RT-qPCR measured the relative expression levels of TGF-β1, IGF-1, COL-Ⅰ, COL-Ⅲ, FN (fibronectin), and TNMD (Tenomodulin) in different experimental groups. Molecular docking experiments were conducted to explore the binding effect of quercetin on TGF-β1 and IGF-1 proteins. ResultsFlow cytometry verified the successful isolation of MSCs, which had high expression of CD29 and CD73, while lower expression of CD90 and CD45. Experimental results show that low and medium doses of quercetin can enhance cell proliferation, while high doses have no significant effect on cells. Detection of cell proliferation through flow cytometry yielded similar results to CCK-8. Transwell experiments have shown that low and medium doses of quercetin can increase cell migration ability. In addition, RT-qPCR detection showed that quercetin can increase the mRNA expression of TGF-β1 and IGF-1, and promote the expression of COL-Ⅰ, COL-Ⅲ, FN, and TNMD genes in ligament fibroblasts. Molecular docking results showed that quercetin can bind firmly to TGF-β1 and IGF-1. ConclusionOverall, this study revealed the morphological characteristics and identification of MSCs, as well as the regulatory mechanism of quercetin on the behavior of ligament fibroblasts. Quercetin affects the proliferation and gene expression of ligament fibroblasts by regulating the expression of TGF-β1 and IGF-1, which may provide a new perspective for biomedical research on the skeletal system.

PSVII-18 Applying the Marbling Window Concept to Enhance Intramuscular Fat Development in Beef Cattle

Abstract The increase in marbling deposition without increasing the overall fatness of the carcass is one of the most challenge goals in meat industry. Previous studies have suggested the nutritional interventions at the neonatal stages as a strategy to enhance the commitment of muscular mesenchymal stem cells to adipogenesis, which may contribute to enhance marbling deposition at the finishing phase. As such the objective of this study was designed the evaluate the impact nutrient supplementation during the pre-weaning stage of beef calves has on intramuscular adipogenic determination. Thirty-four female calves were assigned to two experimental treatments: Control (CON, n = 17), where calves received only a mineral mixture as a supplement; Supplemented (SUP, n = 17), where calves received an energy-protein supplement (0.5 kg·100 kg of BW-1·day-1; the same mineral mixture was used in the CON supplement). Calves were supplemented from 100 to 250 days of age. Once animals reached 250 days of age, skeletal muscle tissue samples were biopsied from the 10th and 11th rib and used for identification of mesenchymal stem cells and preadipocytes through immunofluorescence, mRNA and protein abundance of adipo-fibrogenic markers, and abundance of candidate miRNA controlling expression of adipogenic commitment. Data were analyzed considering the completely randomized design of the experiment. Heifers were randomized across paddocks, and paddocks were randomized across treatments. The PROC MIXED of SAS 9.2 was used for all analyses. The average daily gain (P = 0.08) and final body weight (P = 0.07) tended to be greater in SUP than CON calves. The number of fibro-adipogenic progenitor cells (P = 0.69) did not differ between treatments, while a greater number of intramuscular pre-adipocytes were observed in SUP than CON calves (P = 0.01). The expression of miRNA-4429 (P = 0.20) did not differ between treatments, while the expression of miRNA-129-5p (P = 0.09) and miRNA-129-2-3p (P = 0.05) tended to be greater in CON than SUP calves. Our findings demonstrate that energy-protein supplementation from 100 to 250 days of age is capable to enhance intramuscular adipogenesis in beef cattle. Moreover, our results suggest that regulation of adipogenic commitment occurs by post-transcriptional regulation through miR-129 family.

Expression Profile of New Marker Genes Involved in Differentiation of Human Wharton's Jelly-Derived Mesenchymal Stem Cells into Chondrocytes, Osteoblasts, Adipocytes and Neural-like Cells.

Wharton's jelly (WJ) contains mesenchymal stem cells (MSCs) exhibiting broad immunomodulatory properties and differentiation capacity, which makes them a promising tool for cellular therapies. Although the osteogenic, chondrogenic and adipogenic differentiation is a gold standard for proper identification of MSCs, it is important to elucidate the exact molecular mechanisms governing these processes to develop safe and efficient cellular therapies. Umbilical cords were collected from healthy, full-term deliveries, for subsequent MSCs (WJ-MSCs) isolation. WJ-MSCs were cultivated in vitro for osteogenic, chondrogenic, adipogenic and neurogenic differentiation. The RNA samples were isolated and the transcript levels were evaluated using NovaSeq platform, which led to the identification of differentially expressed genes. Expression of H19 and SLPI was enhanced in adipocytes, chondrocytes and osteoblasts, and NPPB was decreased in all analyzed groups compared to the control. KISS1 was down-regulated in adipocytes, chondrocytes, and neural-like cells compared to the control. The most of identified genes were already implicated in differentiation of MSCs; however, some genes (PROK1, OCA2) have not yet been associated with initiating final cell fate. The current results indicate that both osteo- and adipo-induced WJ-MSCs share many similarities regarding the most overexpressed genes, while the neuro-induced WJ-MSCs are quite distinctive from the other three groups. Overall, this study provides an insight into the transcriptomic changes occurring during the differentiation of WJ-MSCs and enables the identification of novel markers involved in this process, which may serve as a reference for further research exploring the role of these genes in physiology of WJ-MSCs and in regenerative medicine.

Isolation and Identification of Mesenchymal Stem Cells Derived from Adipose Tissue of Sprague Dawley Rats.

Adult mesenchymal cells have revolutionized molecular and cell biology in recent decades. They can differentiate into different specialized cell types, in addition to their great capacity for self-renewal, migration, and proliferation. Adipose tissue is one of the least invasive and most accessible sources of mesenchymal cells. It has also been reported to have higher yields compared to other sources, as well as superior immunomodulatory properties. Recently, different procedures for obtaining adult mesenchymal cells from different tissue sources and animal species have been published. After evaluating the criteria of some authors, we standardized a methodology applicable to different purposes and easily reproducible. A pool of stromal vascular fraction (SVF) from perirenal and epididymal adipose tissue allowed us to develop primary cultures with optimal morphology and functionality. The cells were observed adhered to the plastic surface for 24 h, and exhibited a fibroblast-like morphology, with prolongations and a tendency to form colonies. Flow cytometry (FC) and immunofluorescence (IF) techniques were used to assess the expression of the membrane markers CD105, CD9, CD63, CD31, and CD34. The ability of adipose-derived stem cells (ASCs) to differentiate into the adipogenic lineage was also assessed using a cocktail of factors (4 µM insulin, 0.5 mM 3-methyl-iso-butyl-xanthine, and 1 µM dexamethasone). After 48 h, a gradual loss of fibroblastoid morphology was observed, and at 12 days, the presence of lipid droplets positive to oil red staining was confirmed. In summary, a procedure is proposed to obtain optimal and functional ASC cultures for application in regenerative medicine.

Identification of mesenchymal stem cells based on the passaging effect using machine learning approaches.

For diseases that lack efficient therapeutic approaches, stem cell therapy is an emerging alternative treatment, wherein cell quality assessment is an essential step before cell transplantation into patients. Cell quality is generally evaluated using approaches, such as immunohistochemistry and flow cytometry, which involve complex laboratory routines. In this milieu, several studies have demonstrated the feasibility of applying image processing and analysis to assist cell quality assessment more efficiently. We developed an image-based analytical tool using cell tracking, cytometric analyses, and gating with time-lapse microscopy to investigate the migratory behavior of human mesenchymal stem cells (hMSCs) cultured on chitosan membranes. Using this tool, systematic studies were carried out to inspect variations in cell quality by extracting two morphological and seven migratory features of cells of interest. We found that chitosan membrane-induced multi-cellular spheroids move at a higher speed and in a more consistent direction than single cells. The hMSCs showed a tendency to aggregate with strong 3-D spheroid formability and increased mobility during maturation; however, both these tendencies decreased with degeneration. Furthermore, we automated the cell quality assessment procedure by utilizing machine learning. We selected 570 hMSCs, 390 adipose-derived adult stem cells (hADSCs) and 180 human placenta-derived multipotent cells (hPDMCs), with complete movement trajectories from 25 videos, at three different passages. The differentiation accuracy between hADSCs and hPDMCs in the mature passage was 96.3%, which was higher than that in the early (82.6%) and late (80.5%) passages, whereas the accuracy of passage identification was 62.8% for hADSCs and 70.6% for hPDMCs. The limited accuracy of passage identification may be owing to the insufficient number of morphological features. This study shows the potential of applying machine learning in monitoring the quality of stem cells during in vitro culture.

MESENCHYMAL STEM CELL MARKERS IN PERIODONTAL TISSUES AND PERIAPICAL LESIONS

Objectives Mesenchymal stem cells (MSCs) are characterized by the potential to differentiate into multiple cell lineages, high proliferation rates, and self-renewal capacity, in addition to the ability to maintain their undifferentiated state. The criteria used for the identification of MSCs include the positive expression of specific surface antigens, such as CD73, CD90, CD105, CD44, CD146, STRO-1, CD166, NANOG, and OCT4. The aim of this review was to explore the literature on markers able to identify MSCs and the presence of these cells in the healthy periodontal ligament and chronic periapical lesions (CPLs), highlighting their role in regenerative medicine and implications in the evolution of these lesions. Study Design Narrative literature review searching the PubMed and Medline databases was done. Articles published in English between 1974 and 2020 were retrieved. Results The included studies confirmed the presence of MSCs in the healthy periodontal ligament and in CPLs. Several surface markers are used for the characterization of these cells, which, although not specific, are effective in cell recognition. Conclusions MSCs participate in tissue repair, exerting anti-inflammatory, immunosuppressive, and proangiogenic effects, and are therefore involved in the progression and attenuation of CPLs or in the persistence of these lesions. Mesenchymal stem cells (MSCs) are characterized by the potential to differentiate into multiple cell lineages, high proliferation rates, and self-renewal capacity, in addition to the ability to maintain their undifferentiated state. The criteria used for the identification of MSCs include the positive expression of specific surface antigens, such as CD73, CD90, CD105, CD44, CD146, STRO-1, CD166, NANOG, and OCT4. The aim of this review was to explore the literature on markers able to identify MSCs and the presence of these cells in the healthy periodontal ligament and chronic periapical lesions (CPLs), highlighting their role in regenerative medicine and implications in the evolution of these lesions. Narrative literature review searching the PubMed and Medline databases was done. Articles published in English between 1974 and 2020 were retrieved. The included studies confirmed the presence of MSCs in the healthy periodontal ligament and in CPLs. Several surface markers are used for the characterization of these cells, which, although not specific, are effective in cell recognition. MSCs participate in tissue repair, exerting anti-inflammatory, immunosuppressive, and proangiogenic effects, and are therefore involved in the progression and attenuation of CPLs or in the persistence of these lesions.

Cobalt Ferrite Magnetic Nanoparticles for Tracing Mesenchymal Stem Cells in Tissue: A Preliminary Study

Therapy with mesenchymal stem cells (MSCs) is promising in many diseases. Evaluation of their efficacy depends on adequate follow-up of MSCs after transplantation. Several studies have shown that MSCs can be labeled and subsequently visualized with magnetic nanoparticles (NPs). We investigated the homing of MSCs labeled with magnetic cobalt ferrite NPs in experimentally induced acute kidney injury in mice. To explore the homing of MSCs after systemic infusion into mice, we developed a pre-infusion strategy for optimal tracing and identification of MSCs with polyacrylic acid-coated cobalt ferrite (CoFe2O4) NPs by light and transmission electron microscopy (TEM) in various organs of mice with cisplatin-induced acute kidney injury and control mice. By correlative microscopy, we detected MSCs labeled with NPs in the lungs, spleen, kidney, and intestine of cisplatin-treated mice and in the lungs and spleen of control mice. Our results confirm that labeling MSCs with metal NPs did not affect the ultrastructure of MSCs and their ability to settle in various organs. This study demonstrates the usefulness of cobalt ferrite NPs in ex vivo visualization of MSCs and offers correlative microscopy as a useful method in routine histopathology laboratories for tracing MSCs in paraffin-embedded tissue.

Growth and mineralization of osteoblasts from mesenchymal stem cells on microporous membranes: Epithelial-like growth with transmembrane resistance and pH gradient

Osteoblasts in vivo form an epithelial-like layer with tight junctions between cells. Bone formation involves mineral transport into the matrix and acid transport to balance pH levels. To study the importance of the pH gradient in vitro, we used Transwell inserts composed of polyethylene terephthalate (PET) membranes with 0.4 μm pores at a density of (2 ± 0.4) x 106 pores per cm2. Mesenchymal stem cells (MSCs) prepared from murine bone marrow were used to investigate alternative conditions whereby osteoblast differentiation would better emulate in vivo bone development. MSCs were characterized by flow cytometry with more than 90% CD44 and 75% Sca-1 labeling. Mineralization was validated with paracellular alkaline phosphatase activity, collagen birefringence, and mineral deposition confirming MSCs identity. We demonstrate that MSCs cultured and differentiated on PET inserts form an epithelial-like layer while mineralizing. Measurement of the transepithelial resistance was ∼1400 Ω•cm2 at three weeks of differentiation. The pH value of the media above and under the cells were measured while cells were in proliferation and differentiation. In mineralizing cells, a difference of 0.145 pH unit was observed between the medium above and under the cells indicating a transepithelial gradient. A significant difference in pH units was observed between the medium above and below the cells in proliferation compared to differentiation. Data on pH below membranes were confirmed by pH-dependent SNARF1 fluorescence. Control cells in proliferative medium did not form an epithelial-like layer, displayed low transepithelial resistance, and there was no significant pH gradient. By transmission electron microscopy, membrane attached osteoblasts in vitro had abundant mitochondria consistent with active transport that occurs in vivo by surface osteoblasts. In keeping with osteoblastic differentiation, scanning electron microscopy identified deposition of extracellular collagen surrounded by hydroxyapatite. This in vitro model is a major advancement in modeling bone in vivo for understanding of osteoblast bone matrix production.

The hernia sac—A suitable source for obtaining mesenchymal stem cells

BackgroundInguinal hernia sac, extended tissue from peritoneum, gradually enlarged in size with hernia disease time and prolapsed tissue volume. We hypothesize that mesenchymal stem cells are present in the development of hernia sac. The current study aimed to test the hypothesis that hernia sac, which is often resected and discarded as medical waste, contains mesenchymal stem cells and thus might be a suitable source to harvest mesenchymal stem cells. MethodsBetween July 2019 and June 2020, 4 hernia sacs were resected during hernia surgery and then obtained for mesenchymal extraction using the Miltenyi gentleMACS Dissociator. The presence of mesenchymal stem cells was determined by the markers CD105, CD73, and CD90, with assessment of the expressions ≥95%, whereas markers CD45, CD34, CD11b, CD19, and HLA-DR were used to assess lack expression (≤2%). Moreover, von Kossa staining, Alcian blue staining, and Oil Red O staining were used to verify the cells' ability for differentiation. ResultsCells retrieved from the hernia sacs displayed a spindle-shaped morphology and exhibited adherence to plastics. The cell surface immunophenotypic profile was confirmed using surface markers APC-A (CD73), FITC-A (CD90), and PerCP-Cy5-5-A (CD105), with results showing 100%, 100%, and 99.2%, respectively, strongly indicating the presence of mesenchymal stem cells. Moreover, staining of in vitro cell cultures showed in vitro differentiation of precursor cells into osteoblasts, adipocytes, and chondroblasts, suggesting positive differentiation ability and identification of mesenchymal stem cells. ConclusionInguinal hernia sac is a novel source of mesenchymal stem cells that can be easily obtained and stored for future usage.

An improved methodology for efficient isolation of mesenchymal stem cells from Caprine bone marrow

The aim of this study was to investigate the effect of increasing volume of bone marrow (BM) aspirate of goats on yield of mesenchymal stem cells (MSCs). For this, yield of MSCs in seven groups (Gr; 1-7 Grs, including a control Gr) were compared. After isolation and enrichment of BM MSCs with density gradient centrifugation using increasing volume of Histopaque-1077, the properties of MSCs to adhere the plastic surfaces and expression of alkaline phosphatase (ALP) were used for the identification of MSCs. The results indicated that 18 ml of BM aspirate (Gr 5) provided maximum number of MSCs using 3 ml of histopaque compared to the control and other Gr. The yield in Gr 5 was about 4.4 folds higher compared to the control Gr. In conclusion, these results demonstrated that in a single procedure the modified protocol yield significantly higher number of caprine MSCs from goat BM aspirate compared to the standard protocol. Thus, it can be used for isolation of more number of MSCs from a large quantity of bone marrow with least volume of consumables required for density-gradient centrifugation.

Exosomes from Kartogenin-Pretreated Infrapatellar Fat Pad Mesenchymal Stem Cells Enhance Chondrocyte Anabolism and Articular Cartilage Regeneration.

Objective To evaluate the effect of Kartogenin-pretreated exosomes derived from infrapatellar fat pad mesenchymal stem cells on chondrocyte in vitro and articular cartilage regeneration in vivo. Methods Infrapatellar fat pad mesenchymal stem cells (IPFP-MSCs) were isolated from rabbits to harvest exosomes. After identification of mesenchymal stem cells and exosomes, rabbit chondrocytes were divided into three groups for further treatment: the EXO group (chondrocytes treated with exosomes isolated from infrapatellar fat pad mesenchymal stem cells), KGN-EXO group (chondrocytes treated with exosomes isolated from infrapatellar fat pad mesenchymal stem cells pretreated with KGN), and control group. After processing and proliferation, phenotypic changes of chondrocytes were measured. In the in vivo study, 4 groups of rabbits with articular cartilage injury were treated with KGN-EXO, EXO, IPFP-MSCs, and control. Macroscopic evaluation and histological evaluation were made to figure out the different effects of the 4 groups on cartilage regeneration in vivo. Results The proliferation rate of chondrocytes in the EXO or KGN-EXO group was significantly higher than that in the control group (P < 0.05). The qRT-PCR results showed that the expression of Sox-9, Aggrecan, and Col II was the highest in the KGN-EXO group compared with the EXO group and the control group (P < 0.05). The results of Western blot were consistent with the results of qRT-PCR. In vivo, the cartilage defects in the KGN-EXO group showed better gross appearance and improved histological score than those in IPFP-MSC groups, EXO groups, and control groups (P < 0.05). At 12 weeks, the defect site in the KGN-EXO group was almost completely repaired with a flat and smooth surface, while a large amount of hyaline cartilage-like structures and no obvious cracks were observed. Conclusion Our study demonstrates that the exosomes isolated from infrapatellar fat pad mesenchymal stem cells pretreated with KGN have potent ability to induce chondrogenic differentiation of stem cells, effectively promoting the proliferation and the expression of chondrogenic proteins and genes of chondrocytes. The KGN-EXO can also promote the repair of articular cartilage defects more effectively, which can be used as a potential therapeutic method in the future.

Comparison of senescence progression in mesenchymal cells from human umbilical cord walls measured by immunofluorescence and flow cytometry of p16 and p21.

ABSTRACTObjective To follow the expansion of mesenchymal stem cells from umbilical cords by two classic senescence markers, p16 (INK4A) and p21 (CDKN1A), using practical, fast, and less expensive methods than the gold standard Western blotting technique, to evaluate its applicability in the laboratory.Methods Mesenchymal stem cells from umbilical cords were isolated from Wharton’s jelly and, after quality control, morphological and immunophenotypic characterization by flow cytometry, were expanded in culture until coming close to cell cycle arrest (replicative senescence).Results A comparison was made between young cells, at passage 5, and pre-senescent cells, at passage 10, evaluating the protein expression of the classic cell senescence markers p16 and p21, comparing the results obtained by Western blotting with those obtained by flow cytometry and indirect immunofluorescence.Conclusion Follow-up of cell cultures, through indirect p16 immunofluorescence, allows the identification of mesenchymal stem cells from umbilical cord cultures at risk of reaching replicative senescence.

Comparison of the effects of intramyocardial and intravenous injections of human mesenchymal stem cells on cardiac regeneration after heart failure.

Objective(s):Existing studies have demonstrated that intravenous and intramyocardial-administrated mesenchymal stem cells (MSCs) lead to tissue repair after cardiac disorders. We compared the efficiency of both administration methods.Materials and Methods:A rat model of isoproterenol-induced heart failure (ISO-HF) was established to compare the effects of intravenous and intramyocardial-administrated MSCs on cardiac fibrosis and function. The animals were randomly assigned into six groups: i) control or normal, ii) ISO-HF (HF) iii) ISO-HF rats treated with intramyocardial administration of culture medium (HF+IM/CM), iv) ISO-HF rats treated with intravenous administration of culture medium ( HF+IV/CM), v) ISO-HF rats treated with intravenous administration of MSCs (HF+IV/MSCs), vi) ISO-HF rats treated with intramyocardial administration of MSCs ( HF+IM/MSCs). Cultured MSCs and culture medium were administrated at 4 weeks after final injection of ISO. Heart function, identification of MSCs, osteogenic differentiation, adipogenic differentiation, cardiac fibrosis and tissue damage were evaluated by echocardiography, flow-cytometery, von Kossa, oil red O, Masson’s trichrome and H & E staining, respectively. Results:Both intravenous and intramyocardial MSCs therapy significantly improved heart function and reduced cardiac fibrosis and tissue damage (P<0.05), whereas the cultured medium had no beneficial effects. Conclusion:In sum, our results confirm the validity of both administration methods in recovery of HF, but more future research is required.

Injectable hydrogel combined with nucleus pulposus-derived mesenchymal stem cell for the treatment of degenerative intervertebral disc in rats

Objective To investigate the effect of injectable hydrogel combined with nucleus pulposus-derived mesenchymal stem cell (NPMSC) transplantation on the degenerative intervertebral disc in rats. Methods NPMSC was isolated from the caudal spinal nucleus pulposus of Sprague-Dawley rats and identification of mesenchymal stem cells was determined. After 2 weeks of intervertebral disc degeneration model was established by annulus fibrosus puncture, the groups were divided: control group (untreated), treatment group (injection of hydrogel loaded NPMSC into the intervertebral disc), phosphate buffer solution (PBS) group (injection of the same amount of PBS), hydrogel group (injection of the same amount of hydrogel), cell group (injection of the same amount of NPMSC suspension). At 4 and 8 weeks after intervention, relative disc height index (DHI%) and magnetic resonance imaging (MRI) index were detected. At 8 weeks after intervention, histological score, the expression of collagen type Ⅱ and aggrecan was detected by immunofluorescence and reverse transcription-polymerase chain reaction. One-way ANOVA was used for comparison between groups, and LSD-t test was used for pairwise comparison. Results At 8 weeks after intervention, for the DHI% and MRI index, the experiment group [(87.80±3.40)%, 3 313.84±227.80] was higher than cell group [(79.65±2.33)%, 3 006.83±147.92] (t=3.411, 3.127, P<0.05); cell group was higher than hydrogel group [(69.49±4.79)%, 1 288.00±202.09] (t=4.252, 17.504, P<0.05); for histological score, the cell group (9.00±1.29) was higher than experiment group (7.16±0.68) (t=3.550, P<0.05), the hydrogel group (12.50±0.95) was higher than cell group (t=6, 753, P<0.05); for the protein express of collagen type Ⅱ and aggrecan, the cell group (0.30±0.02, 0.58±0.03) was less than experiment group (0.38±0.03, 0.69±0.05) (t=-4.000, -3, 267, P<0.05), the hydrogel group (0.14±0.03, 0.14±0.04) was less than cell group (t=-8.000, -13.307, P<0.05). Conclusion Injectable hydrogel combined with NPMSC transplantation can delay the level of disc degeneration in animal model and promote the repair and regeneration of degenerative disc. Key words: Hydrogel; Nucleus pulposus-derived mesenchymal stem cell; Intervertebral disc degeneration; Cell transplantation

In vivo analysis of the presence of heme oxygenase-1, transcription factor Jun-D and CD90+/CD73+/CD105+/CD45- cells in the pulp of bleached teeth.

To investigate hydrogen peroxide (H2 O2 )-induced responsiveness in pulp cells using heme oxygenase-1 (HO-1) immunolabelling, Jun-D immunolabelling to study the effects of H2 O2 on odontoblastic differentiation and CD90+/CD73+/CD105+/CD45- cell counting for in vivo identification of mesenchymal stem cells in the pulp. The maxillary molars of 50 rats were treated with a bleaching gel (35% H2 O2 , 1×30min) or placebo gel (control groups). At 2, 3, 7, 15 and 30days after the treatment (n=10), inflammation in pulp tissue was analysed by haematoxylin-eosin staining, HO-1- and Jun-D-immunolabelled cells were counted in each third of the pulp chamber, and the number of CD90+/CD73+/CD105+/CD45- cells was quantified by immunofluorescence. The results were assessed using the Paired t-test or Wilcoxon signed-rank test (P<0.05). Significant H2 O2 -induced inflammation was noted at 2 and 3days (P<0.05), with tertiary dentine formation occurring from 7days. The bleached specimens had greater HO-1 immunolabelling in the middle and cervical thirds of the coronal pulp at 2 and 3days, in all thirds at 7days, and in the occlusal third at 15days (P<0.05), and significant nuclear Jun-D immunolabelling in the cervical third at 2 and 3days and in the occlusal and middle thirds at 7days (P<0.05). Bleached and control groups had low numbers of CD90+/CD73+/CD105+/CD45- cells in the pulp at all periods (P>0.05). Pulp cells responded to oxidative stress by expressing HO-1 during the post-bleaching inflammation phase until the beginning of the repair phase. Jun-D expression occurred during the reduction of inflammation and the beginning of tertiary dentine production. The presence of oxidative stress did not influence the number of CD90+/CD73+/CD105+/CD45- cells identified in vivo in the dental pulp.

Tendon contains more stem cells than bone at the rotator cuff repair site

The rotator cuff (RC) repair failure rate is high. Tendon and bone represent sources of mesenchymal stem cells (MSCs), but the number of MSCs from each has not been compared. Bone channeling may increase bone-derived MSC numbers participating in enthesis re-formation at the "footprint" repair site. The effect of preoperative channeling on increasing bone MSC numbers has never been reported. We asked (1) whether bone contains more MSCs than tendon at the time of arthroscopic repair and (2) whether bone preoperative channeling at the RC repair site increases the number of bone-derived MSCs at the time of surgery. In 23 participants undergoing arthroscopic RC repair, bone was sampled from the footprint and tendon was sampled from the distal supraspinatus. We randomized participants to the channeling or no-channeling group5 to 7 days before surgery. We enumerated MSCs from both tissues using the colony-forming unit-fibroblast (CFU-F) assay (10 per group). We identified MSC identity using flow cytometry and MSC tri-differentiation capacity (n = 3). Tendon CFU-F per gram exceeded bone CFU-F per gram for both groups (479 ± 173 CFU-F/g vs. 162 ± 54 CFU-F/g for channeling [P = .036] and 1334 ± 393 CFU-F/g vs. 284 ± 88 CFU-F/g for no channeling [P = .009]). Ninety-nine percent of cultured cells satisfied the MSC definition criteria. The distal supraspinatus tendon contained more MSCs per gram than the humeral footprint. Tendon may represent an important and overlooked MSC source for postoperative enthesis re-formation. Further studies are needed to evaluate the repair role of tendon MSCs and to recommend bone channeling in RC repair.

Chromatin accessibility identifies diversity in mesenchymal stem cells from different tissue origins

Mesenchymal stem cells (MSCs), which can differentiate into tri-lineage (osteoblast, adipocyte, and chondrocyte) and suppress inflammation, are promising tools for regenerative medicine. MSCs are phenotypically diverse based on their tissue origins. However, the mechanisms underlying cell-type-specific gene expression patterns are not fully understood due to the lack of suitable strategy to identify the diversity. In this study, we investigated gene expression programs and chromatin accessibilities of MSCs by whole-transcriptome RNA-seq analysis and an assay for transposase-accessible chromatin using sequencing (ATAC-seq). We isolated MSCs from four tissues (femoral and vertebral bone marrow, adipose tissue, and lung) and analysed their molecular signatures. RNA-seq identified the expression of MSC markers and both RNA-seq and ATAC-seq successfully clustered the MSCs based on their tissue origins. Interestingly, clustering based on tissue origin was more accurate with chromatin accessibility signatures than with transcriptome profiles. Furthermore, we identified transcription factors potentially involved in establishing cell-type specific chromatin structures. Thus, epigenome analysis is useful to analyse MSC identity and can be utilized to characterize these cells for clinical use.

Pathogenetic Characteristics of Mesenchymal Stem Cells in Hidradenitis Suppurativa

Hidradenitis suppurativa (HS) is a disease of the terminal hair follicle in apocrine gland-enriched skin areas, where immunobiology dysregulation of mesenchymal stem cells (MSCs) may have a key role. To investigate the MSC profile in patients with HS and in healthy controls. In this prospective case-control study, patients with HS were recruited from the Dermatological Clinic at the Department of Clinical and Molecular Sciences, Università Politecnica delle Marche, Ancona, Italy. Biopsy specimens were analyzed at the Histology Section of the Department of Clinical and Molecular Sciences. Participants included 11 patients with HS and 9 healthy controls, who were recruited into the study between January 20, 2015, and September 20, 2016, and underwent punch biopsy from axillary skin. None of the participants had received any antibiotics (systemic or topical therapy) within almost 12 weeks before the study. The immunophenotypic profile of MSCs was characterized following the minimal criteria established by the International Society for Cellular Therapy for the identification of MSCs. Levels of 12 cytokines belonging to helper T-cell subtypes 1, 2, and 17 pathways were examined on the secretome of isolated cells by enzyme-linked immunoabsorbent assay. Skin MSCs were characterized in 11 patients with HS (8 women and 3 men; mean [SD] age, 35.8 [7.9] years) and 9 healthy controls (7 women and 2 men; mean [SD] age, 36.7 [6.9] years). The healthy controls were matched with patients with HS for body mass index. Mesenchymal stem cells isolated from patients with HS (HS-MSCs) and from healthy controls (C-MSCs) met the International Society for Cellular Therapy minimal criteria. Compared with C-MSCs, cytokine analyses of HS-MSCs revealed statistically significant overexpression of interleukin (IL) 6 (median [interquartile range {IQR}], 8765.00 [7659.00-9123.00] vs 2849.00 [2609.00-3001.00] pg/mL; P = .008), IL-10 (median [IQR], 29.46 [26.35-35.79] vs 21.36 [19.89-23.33] pg/mL; P = .004), IL-12 (median [IQR], 15.25 [13.27-16.25] vs 11.89 [10.73-12.33] pg/mL; P = .03), IL-17A (median [IQR], 15.24 [13.23-17.24] vs 11.24 [10.28-11.95] pg/mL; P = .008), tumor necrosis factor (median [IQR], 42.54 [42.20-43.94] vs 32.55 [31.78-33.28] pg/mL; P = .004), transforming growth factor β1 (median [IQR], 1728.00 [1535.00-1979.00] vs 500.80 [465.00-634.50] pg/mL; P = .004), and interferon γ (median [IQR], 11.49 [10.71-12.35] vs 9.45 [9.29-10.01] pg/mL; P = .005). Mesenchymal stem cells isolated from the skin of patients with HS seem to be activated toward an inflammatory status. The imbalance between proinflammatory and anti-inflammatory activities of MSCs favors the hypothesis of their pathogenic involvement in HS.

Location, Isolation, and Identification of Mesenchymal Stem Cells from Adult Human Sweat Glands.

Sweat glands (SGs) are spread over almost the entire surface of the human body and are essential for thermoregulation. Theoretically, tissue-specific stem cells (TSSCs) are excellent candidate cells for the regeneration of SGs due to their genetic stability and differentiation ability. Herein, we attempted to isolate TSSCs derived from adult human sweat glands (ahSGs). ahSGs were localized and identified by H&E staining, double immunofluorescence staining, transmission electron microscope (TEM), and immuno-TEM. We found a population of cells with stem cell characteristics (SGSCs), located in basal myoepithelial cells of the secretory portion of the solenoid bulb. The SGSCs expressed alpha-smooth muscle actin (α-SMA) and showed the typical characteristics of mesenchymal stem cells (MSCs), with a positive antigen profile for CD44, CD73, CD90, and CD105, and had the multilineage differentiation potential to osteoblasts and adipocytes. In addition, the isolated α-SMA positive cells remained stably phenotypic and proliferative cycles at passage 12. This is the first report of successful isolation of MSC-like cells from ahSGs, which may contribute to wound repair and SG regeneration.

PR358: Identification of mesenchymal stem cells with bright expression of LNGFR and THY‐1 in neural crest‐like cells derived from human iPS cells

PR358: Identification of mesenchymal stem cells with bright expression of LNGFR and THY‐1 in neural crest‐like cells derived from human iPS cells