Adipogenic Differentiation Potential Research Articles

Exploring SSEA3 as an emerging biomarker for assessing the regenerative potential of dental pulp-derived stem cells

BackgroundHuman dental pulp-derived stem cells (hDPSCs) have emerged as a promising source for adult stem cell-based regenerative medicine. Stage-specific embryonic antigen 3 (SSEA3) is a cell surface marker associated with Multilineage-differentiating stress-enduring (Muse) cells, a subpopulation of human bone marrow-derived stem cells (hBMSCs), known for their potent regenerative potential and safety profile. In this study, we investigated the influence of the prolonged culture period and the number of culture passages on the regenerative capacity of hDPSCs and explored the association between SSEA3 expression and their regenerative abilities. MethodshDPSCs were isolated and cultured for up to 20 passages. Cell proliferation, migration, and osteogenic, adipogenic and neurogenic differentiation potential were assessed at passages 5, 10, and 20. Flow cytometry and immunofluorescence were employed to analyze SSEA3 expression. RNA sequencing (RNA-seq) was performed on SSEA3-positive and SSEA3-negative hDPSCs to identify differentially expressed genes and associated pathways. ResultsOur findings demonstrated a progressive decline in hDPSCs proliferation and migration capacity with increasing passage number. Conversely, cell size exhibited a positive correlation with passage number. Early passage hDPSCs displayed superior osteogenic and adipogenic differentiation potential. Notably, SSEA3 expression exhibited a significant negative correlation with passage numbers, reflecting the observed decline in differentiation capacity. RNA-seq analysis revealed distinct transcriptional profiles between SSEA3-positive and SSEA3-negative hDPSCs. SSEA3-positive cells displayed upregulation of genes associated with ectodermal differentiation and downregulation of genes involved in cell adhesion. ConclusionsThis study elucidates the impact of passaging on hDPSC behavior and suggests SSEA3 as a valuable biomarker for evaluating stemness and regenerative potential. SSEA3-positive hDPSCs, functionally analogous to Muse cells, represent a promising cell population for developing targeted regenerative therapies with potentially improved clinical outcomes.

Characterization of cells in blood evoked from periapical tissues in immature teeth with pulp necrosis and their potential for autologous cell therapy in Regenerative Endodontics

ObjectiveThe objectives of this study were to isolate, characterize progenitor cells from blood in the root canals of necrotic immature permanent teeth evoked from periapical tissues and evaluate the applicable potential of these isolated cells in Regenerative Endodontics. DesignTen necrotic immature permanent teeth from seven patients were included. Evoked bleeding from periapical tissues was induced after chemical instrumentation of the root canals. Cells were isolated from the canal blood and evaluated for cell surface marker expression, multilineage differentiation potential, proliferation ability, and target protein expression. Cell sheets formed from these cells were transferred into human root segments, and then transplanted into nude mice. Histological examination was performed after eight weeks. Data analysis was conducted using one-way ANOVA followed by Tukey’s post-hoc comparison, considering p < 0.05 as statistically significant. ResultsThe isolated cells exhibited characteristics typical of fibroblastic cells with colony-forming efficiency, and displayed Ki67 positivity and robust proliferation. Flow cytometry data demonstrated that at passage 3, these cells were positive for CD73, CD90, CD105, CD146, and negative for CD34 and CD45. Vimentin expression indicated a mesenchymal origin. Under differentiation media specific differentiation media, the cells demonstrated osteogenic, adipogenic, and chondrogenic differentiation potential. Subcutaneous root canals with cell sheets of isolated cells in nude mice showed the formation of pulp-like tissues. ConclusionsThis study confirmed the presence of progenitor cells in root canals following evoked bleeding from periapical tissues of necrotic immature teeth. Isolated cells exhibited similar immunophenotype and regenerative potential with dental mesenchymal stromal cells in regenerative endodontic therapy.

Mesenchymal stem cells derived from CHIR99021 and TGF‑β induction remained on the colicomentum and improved cardiac function of a rat model of acute myocardium infarction.

Human induced pluripotent stem cells (hiPSCs) have been regarded as a potential stem cell source for cell therapy. However, the production of cells with mesenchymal potential from hiPSCs through spontaneous differentiation is time consuming and laborious. In the present study, the combined use of the GSK-3 inhibitor CHIR99021 and TGF-β was used to obtain mesenchymal stem cell (MSC)-like cells from hiPSCs. During the induction process, the transcription of epithelial-mesenchymal transition (EMT)-related genes N-cadherin and Vimentin in the transformed cells was upregulated, whereas the transcription of E-cadherin and pluripotency-related transcription factors SOX2, OCT4 and NANOG did not change significantly. This indicated that whilst cells were pluripotent, EMT was initiated by the upregulation of transcription of EMT promoting genes. Both SMAD-dependent and independent signalling pathways were significantly activated by the combined induction treatment compared with the single factor induction. The hiPSC-derived MSC-like cells (hiPSC-MSCs) expressed MSC-related markers and acquired osteogenic, chondrogenic and adipogenic differentiation potentials. After being injected into the peritoneal cavity of rats, the hiPSC-MSCs secreted angiogenic and immune-regulatory factors and remained on the colicomentum for 3 weeks. Within an 11-week period, four intraperitoneal hiPSC-MSC injections (1x107 cells/injection) into acute myocardial infarction (AMI) model rats significantly increased the left ventricular ejection fraction, left ventricular fractional shortening and angiogenesis and significantly reduced scar size and the extent of apoptosis in the infarcted area compared with that of the control PBS injection. Symptoms of hiPSC-MSC-induced immune reaction or tumour formation were not observed over the course of the experiment in the hiSPC-MSC treated rats. In conclusion, the CHIR99021 and TGF-β combined induction was a rapid and effective method to obtain MSC-like cells from hiPSCs and multiple high dose intraperitoneal injections of hiPSC-derived MSCs were safe and effective at restoring cardiac function in an AMI rat model.

Silencing of Jumonji domain-containing 1C inhibits the osteogenic differentiation of bone marrow mesenchymal stem cells via nuclear factor-κB signaling.

Osteoporosis is a common metabolic bone disorder induced by an imbalance between osteoclastic activity and osteogenic activity. During osteoporosis, bone mesenchymal stem cells (BMSCs) exhibit an increased ability to differentiate into adipocytes and a decreased ability to differentiate into osteoblasts, resulting in bone loss. Jumonji domain-containing 1C (JMJD1C) has been demonstrated to suppress osteoclastogenesis. To examine the effect of JMJD1C on the osteogenesis of BMSCs and the potential underlying mechanism. BMSCs were isolated from mouse bone marrow tissues. Oil Red O staining, Alizarin red staining, alkaline phosphatase staining and the expression of adipogenic and osteogenic-associated genes were assessed to determine the differentiation of BMSCs. Bone marrow-derived macrophages (BMMs) were incubated with receptor activator of nuclear factor-kappa Β ligand to induce osteoclast differentiation, and osteoclast differentiation was confirmed by tartrate-resistant acid phosphatase staining. Other related genes were measured via reverse transcription coupled to the quantitative polymerase chain reaction and western blotting. Enzyme-linked immunosorbent assays were used to measure the levels of inflammatory cytokines, including tumor necrosis factor alpha, interleukin-6 and interleukin-1 beta. The osteogenic and adipogenic differentiation potential of BMSCs isolated from mouse bone marrow samples was evaluated. JMJD1C mRNA and protein expression was upregulated in BMSCs after osteoblast induction, while p-nuclear factor-κB (NF-κB) and inflammatory cytokines were not significantly altered. Knockdown of JMJD1C repressed osteogenic differentiation and enhanced NF-κB activation and inflammatory cytokine release in BMSCs. Moreover, JMJD1C expression decreased during BMM osteoclast differentiation. The JMJD1C/NF-κB signaling pathway is potentially involved in BMSC osteogenic differentiation and may play vital roles in the pathogenesis of osteoporosis.

The Impact of Centrifugal Force on Isolation of Bone Marrow Mononuclear Cells Using Density Gradient Centrifugation.

Bone marrow mononuclear cells (BMMNCs) have great potential in bone regenerative therapy. The main method used today to obtain BMMNCs is Ficoll density gradient centrifugation. However, the centrifugal force for this isolation method is still suboptimal. To determine the optimal centrifugal force in Ficoll density gradient centrifugation of bone marrow (BM) to achieve high stem/progenitor cell content BMMNCs for regenerative therapy. BM was aspirated from nine minipigs and divided into three groups according to different centrifugal forces (200g, 300g and 400g). Immediately after BMMNCs were obtained from each group by Ficoll density gradient centrifugation, residual red blood cell (RBC) level, nucleated cell counting, viability and flow cytometric analyses of apoptosis and reactive oxygen species (ROS) generation were measured. The phenotypic CD90 and colony formation analyses of BMMNCs of each group were performed as well. Bone marrow-derived mesenchymal stem cells (BMSCs) were harvested at passage 2, then morphology, cell phenotype, proliferation, adipogenic, chondrogenic and osteogenic lineage differentiation potential of BMSCs from each group were compared. The 300g centrifugal force was able to isolate BMMNCs from BM with the same efficiency as 400g and provided significantly higher yields of CD90+ BMSCs and fibroblastic colony-forming units of BMSC (CFU-f(BMSC)), which is more crucial for the regenerative efficacy of BMMNCs. Meanwhile, 200g hosted the most RBC contamination and minimum CFU-f (BMSC) yield, which will be disadvantageous for BMMNC-based cell therapy. As for in vitro cultured BMSCs which were isolated from BMMNCs by different centrifugal forces, no significant differences were found on morphology, cell proliferation rate, phenotypic marker, adipogenic, chondrogenic and osteogenic differentiation potential. 300g may be the optimal centrifugal force when using Ficoll density gradient centrifugation to isolate BMMNCs for bone regenerative therapy. This journal requires that authors assign a level of evidence to each submission to which Evidence-Based Medicine rankings are applicable. This excludes Review Articles, Book Reviews and manuscripts that concern Basic Science, Animal Studies, Cadaver Studies and Experimental Studies. For a full description of these Evidence-Based Medicine ratings, please refer to the Table of Contents or the online Instructions to Authors www.springer.com/00266.

Comparative Analysis of Dental Stem Cells from a Single Donor: Differences in Adipogenic Differentiation and Protein Profile

Dental stem cells (DSCs) are multipotent cells with high proliferation capacity and multilineage differentiation. Few studies have compared the cellular characteristics and adipogenic differentiation potential of DSCs derived from tissues of the same individual. The objective of this work was to evaluate the differences in growth characteristics, expression of mesenchymal stem cells (MSCs)-specific markers,&#x0D; and the proteomic profile in response to adipogenic differentiation, of dental pulp and periodontal ligament cells obtained from a single donor. Dental cells were isolated from the third molar of a single donor using the outgrowth method. To obtain the proliferation curve of the cells was evaluated by trypan blue analysis. After the cells were cultured in adipogenic medium, morphological changes were monitored by oil red O staining, as well as adipogenic markers PPARγ and adiponectin by RT-qPCR. Finally, a two-dimensional electrophoresis of the proteins isolated from these cells was performed to analyze the proteomic profile. The two types of DSCs share similar cellular characteristics; however, their capacity for adipogenic differentiation is different. Based on the protein profiling results, we identified five differentially expressed proteins between both types of stem cells. Our findings showed that dental pulp and periodontal ligament stem cells from a single donor have similar cellular characteristics but a different response to adipogenesis, which would explain the differences in the expression of their proteins.

Synthesis of bone biocompatible implants using human adipose-derived mesenchymal stem cells (hADMSCs) and PCL/laminin scaffold substrate.

Bone tissue engineering is considered a new method in the treatment of bone defects and can be an effective alternative to surgery and bone grafting. The use of adipose tissue mesenchymal stem cells (ADMSCs) on synthetic polymer scaffolds is one of the new approaches in bone tissue engineering. In this study, we aimed to investigate the effect of laminin coating on biocompatibility and osteogenic differentiation of ADMSCs seeded on polycaprolactone (PCL) scaffolds. The morphology of the electrospun scaffold was evaluated using a scanning electron microscope (SEM). Cell proliferation and cytotoxicity were determined by MTT assay. The adipogenic and osteogenic differentiation potential of the cells was evaluated. The osteogenic differentiation of ADMSCs cultured on the PCL scaffold coated with laminin was assessed by evaluating the level of alkaline phosphatase (ALP) activity, intracellular calcium content, and expression of bone-specific genes. The results showed that the ADMSCs cultured on PCL/laminin showed enhanced osteogenic differentiation compared to those cultured on non-coated PCL or control medium (P<0.05). It seems that laminin enhances the physicochemical properties and biocompatibility of PCL nanofiber scaffolds; and by modifying the surface of the scaffold, improves the differentiation of ADMSCs into osteogenic cells.

Effect of High BMI on Human Bone Marrow-Derived Mesenchymal Stromal Cells.

Bone marrow-derived mesenchymal stromal cells (BMSCs) are attractive candidates in tissue engineering and regenerative medicine. Growing evidence has suggested that a high body mass index (BMI) can affect the properties of BMSCs, resulting in a reduced quality of the cells. However, the results are not consistent. Therefore, this study aimed to investigate the influences of high BMI on human BMSCs (hBMSCs). To avoid gender bias, BMSCs from females and males were studied independently. Finally, hBMSCs from 89 females and 152 males were separately divided into the normal BMI group (18.5 kg/m2 ≤ BMI < 25 kg/m2) and the high BMI group (BMI > 25 kg/m2). The cells were analyzed for the colony-forming potential; proliferation capacity; in vitro adipogenic, osteogenic, and chondrogenic differentiation potentials; and the expression of 32 common surface antigens. The results showed that high BMI did not change the number of colonies at passage 1 in females and males. In contrast, significantly reduced colony numbers at passage 4 (P4) were found in both female and male donors with high BMI. The doubling time of hBMSCs was comparable between the normal and the high BMI groups of females and males. Furthermore, the results of trilineage differentiation did not differ between the different BMI groups of males. In females, the high and the normal BMI groups also showed similar adipogenic and chondrogenic differentiation, while osteogenic differentiation was significantly enhanced in the high-BMI group. Regarding the expression of surface antigens, the expressions of CD200 and SSEA4 on hBMSCs were reduced in the high-BMI group of females and males, respectively. In conclusion, high BMI suppressed the clonogenicity of female and male hBMSCs at P4, improved the in vitro osteogenesis of female hBMSCs, and decreased the expressions of CD200 on hBMSCs in females and SSEA4 in males.

Proliferation and Differentiation Potential of Bone Marrow-Derived Mesenchymal Stem Cells From Children With Polydactyly and Adults With Basal Joint Arthritis.

This study compared the proliferation and differentiation potential of bone marrow-derived mesenchymal stem cells (BMSCs) derived from infants with polydactyly and adults with basal joint arthritis. The proliferation rate of adult and infant BMSCs was determined by the cell number changes and doubling times. The γH2AX immunofluorescence staining, age-related gene expression, senescence-associated β-galactosidase (SA-β-gal) staining were analyzed to determine the senescence state of adult and infant BMSCs. The expression levels of superoxide dismutases (SODs) and genes associated with various types of differentiation were measured using Real-Time Quantitative Polymerase Chain Reaction (RT-qPCR). Differentiation levels were evaluated through histochemical and immunohistochemical staining. The results showed that infant BMSCs had a significantly higher increase in cell numbers and faster doubling times compared with adult BMSCs. Infant BMSCs at late stages exhibited reduced γH2AX expression and SA-β-gal staining, indicating lower levels of senescence. The expression levels of senescence-related genes (p16, p21, and p53) in infant BMSCs were also lower than in adult BMSCs. In addition, infant BMSCs demonstrated higher antioxidative ability with elevated expression of SOD1, SOD2, and SOD3 compared with adult BMSCs. In terms of differentiation potential, infant BMSCs outperformed adult BMSCs in chondrogenesis, as indicated by higher expression levels of chondrogenic genes (SOX9, COL2, and COL10) and positive immunohistochemical staining. Moreover, differentiated cells derived from infant BMSCs exhibited significantly higher expression levels of osteogenic, tenogenic, hepatogenic, and neurogenic genes compared with those derived from adult BMSCs. Histochemical and immunofluorescence staining confirmed these findings. However, adult BMSCs showed lower adipogenic differentiation potential compared with infant BMSCs. Overall, infant BMSCs demonstrated superior characteristics, including higher proliferation rates, enhanced antioxidative activity, and greater differentiation potential into various lineages. They also exhibited reduced cellular senescence. These findings, within the context of cellular differentiation, suggest potential implications for the use of allogeneic BMSC transplantation, emphasizing the need for further in vivo investigation.

CD49f and CD146: A Possible Crosstalk Modulates Adipogenic Differentiation Potential of Mesenchymal Stem Cells.

The lack of appropriate mesenchymal stem cells (MSCs) selection methods has given the challenges for standardized harvesting, processing, and phenotyping procedures of MSCs. Genetic engineering coupled with high-throughput proteomic studies of MSC surface markers arises as a promising strategy to identify stem cell-specific markers. However, the technical limitations are the key factors making it less suitable to provide an appropriate starting material for the screening platform. A more accurate, easily accessible approach is required to solve the issues. This study established a high-throughput screening strategy with forward versus side scatter gating to identify the adipogenesis-associated markers of bone marrow-derived MSCs (BMSCs) and tonsil-derived MSCs (TMSCs). We classified the MSC-derived adipogenic differentiated cells into two clusters: lipid-rich cells as side scatter (SSC)-high population and lipid-poor cells as SSC-low population. By screening the expression of 242 cell surface proteins, we identified the surface markers which exclusively found in lipid-rich subpopulation as the specific markers for BMSCs and TMSCs. High-throughput screening of the expression of 242 cell surface proteins indicated that CD49f and CD146 were specific for BMSCs and TMSCs. Subsequent immunostaining confirmed the consistent specific expression of CD49f and CD146 and in BMSCs and TMSCs. Enrichment of MSCs by CD49f and CD146 surface markers demonstrated that the simultaneous expression of CD49f and CD146 is required for adipogenesis and osteogenesis of mesenchymal stem cells. Furthermore, the fate decision of MSCs from different sources is regulated by distinct responses of cells to differentiation stimulations despite sharing a common CD49f+CD146+ immunophenotype. We established an accurate, robust, transgene-free method for screening adipogenesis associated cell surface proteins. This provided a valuable tool to investigate MSC-specific markers. Additionally, we showed a possible crosstalk between CD49f and CD146 modulates the adipogenesis of MSCs.

Preparation of porous GelMA microcarriers by microfluidic technology for Stem-Cell culture

Gelatin methacrylamide porous microcarriers (GelMA PMS), one of the most promising three-dimensional cell culture scaffolds, are limited in large-scale applications due to lack of ideal preparation method, unclear regulation of pore size on cell behavior and a lack of research on the entire cell culture process. Herein, combining microfluidic synchronous photo-crosslinking and ice-templating method, a low-cost, easy-assembly and suitable for scalability method is proposed to prepare GelMA PMS for bone marrow mesenchymal stem cells (BMSCs) expansion. The pore size of GelMA PMS can be controlled by adjusting the freezing temperature (-20 ℃, −60 ℃, −196℃). As a result, PMS 60 (frozen at −60 ℃) with the optimized pore size of 25.3 ± 3.2 μm exhibited the highest cell attachment ratio (90.2%) and the largest cell spreading area (0.516 mm2/MS). More importantly, PMS 60 displayed higher cell attachment efficiency, proliferation rate, adipogenic, osteogenic, and chondrogenic differentiation potential than commercial microcarriers Cytodex-1. Furthermore, BMSCs not only achieved bead-to-bead transfer on PMS 60 but also exhibited potent proliferation rate after transfer to new blank microcarriers. Cultured BMSCs were efficiently harvested from PMS 60 without damaging cell viability. In addition, the cryopreservation and thawing process did not affect the PMS 60 to support cell proliferation, while cells loaded on Cytodex-1 could not continue to proliferate. Therefore, GelMA PMS fabricated in this work shows great potential application and provides insights for MSCs culture.

Characteristics of Human Nasal Turbinate Stem Cells under Hypoxic Conditions.

This study investigated the influence of hypoxic culture conditions on human nasal inferior turbinate-derived stem cells (hNTSCs), a subtype of mesenchymal stem cells (MSCs). It aimed to discern how hypoxia affected hNTSC characteristics, proliferation, and differentiation potential compared to hNTSCs cultured under normal oxygen levels. After obtaining hNTSCs from five patients, the samples were divided into hypoxic and normoxic groups. The investigation utilized fluorescence-activated cell sorting (FACS) for surface marker analysis, cell counting kit-8 assays for proliferation assessment, and multiplex immunoassays for cytokine secretion study. Differentiation potential-osteogenic, chondrogenic, and adipogenic-was evaluated via histological examination and gene expression analysis. Results indicated that hNTSCs under hypoxic conditions preserved their characteristic MSC phenotype, as confirmed by FACS analysis demonstrating the absence of hematopoietic markers and presence of MSC markers. Proliferation of hNTSCs remained unaffected by hypoxia. Cytokine expression showed similarity between hypoxic and normoxic groups throughout cultivation. Nevertheless, hypoxic conditions reduced the osteogenic and promoted adipogenic differentiation potential, while chondrogenic differentiation was relatively unchanged. These insights contribute to understanding hNTSC behavior in hypoxic environments, advancing the development of protocols for stem cell therapies and tissue engineering.

Rat Plantar Fascia Stem/Progenitor Cells Showed Lower Expression of Ligament Markers and Higher Pro-Inflammatory Cytokines after Intensive Mechanical Loading or Interleukin-1β Treatment In Vitro.

The pathogenesis of plantar fasciitis is unclear, which hampers the development of an effective treatment. The altered fate of plantar fascia stem/progenitor cells (PFSCs) under overuse-induced inflammation might contribute to the pathogenesis. This study aimed to isolate rat PFSCs and compared their stem cell-related properties with bone marrow stromal cells (BMSCs). The effects of inflammation and intensive mechanical loading on PFSCs' functions were also examined. We showed that plantar fascia-derived cells (PFCs) expressed common MSC surface markers and embryonic stemness markers. They expressed lower Nanog but higher Oct4 and Sox2, proliferated faster and formed more colonies compared to BMSCs. Although PFCs showed higher chondrogenic differentiation potential, they showed low osteogenic and adipogenic differentiation potential upon induction compared to BMSCs. The expression of ligament markers was higher in PFCs than in BMSCs. The isolated PFCs were hence PFSCs. Both IL-1β and intensive mechanical loading suppressed the mRNA expression of ligament markers but increased the expression of inflammatory cytokines and matrix-degrading enzymes in PFSCs. In summary, rat PFSCs were successfully isolated. They had poor multi-lineage differentiation potential compared to BMSCs. Inflammation after overuse altered the fate and inflammatory status of PFSCs, which might lead to poor ligament differentiation of PFSCs and extracellular matrix degeneration. Rat PFSCs can be used as an in vitro model for studying the effects of intensive mechanical loading-induced inflammation on matrix degeneration and erroneous stem/progenitor cell differentiation in plantar fasciitis.

Effects of Age on the Biological Properties of Cryopreserved Adipose-Derived Stem Cells and ASC-Enriched Fat Grafts.

Donor age is an important factor affecting the biological characteristics of human adipose-derived stem cells. The aim of this study was to compare the effects of age on the biological properties of cryopreserved adipose-derived stem cells and fat survival of cell-assisted lipotransfer. Human lipoaspirates were obtained from 60 healthy female patients (aged 18-65years) who underwent abdominal liposuction. Samples were divided into three groups according to donor age: group A, 18-29years; group B, 30-49years; and group C, 50-65years. Adipose-derived stem cells were obtained by in vitro culture at the second passage and cryopreserved for 4weeks. The cryopreserved ASCs were examined for biological characteristics, including cell proliferation, wound healing and adipogenic differentiation. Then, the fat survival of cryopreserved ASC-assisted fat transplantation was compared at different ages. SVF viability decreased with increasing age. Moreover, there was a decline in cell proliferation and migration of ASCs with increasing age. A significant difference was found in the adipogenic differentiation of ASCs in the three groups. There were significant differences in graft retention in different age groups. ASC-assisted fat grafting was more effective in young people than in elderly people. Honor age affects the proliferation and migration of adipose-derived stem cells but not the adipogenic differentiation potential of ASCs. Cryopreserved ASCs from younger people more effectively improved the fat survival of grafts. This journal requires that authors assign a level of evidence to each article. For a full description of these Evidence-Based Medicine ratings, please refer to the Table of Contents or the online Instructions to Authors www.springer.com/00266 .

Small Extracellular Vesicles Maintain Homeostasis of Senescent Mesenchymal Stem Cells at Least Through Excreting Harmful Lipids.

Mesenchymal stem cells (MSCs) play an essential role in multiple physiological processes in vivo and a promising cell-based therapy for various diseases. Nonetheless, MSCs suffer from senescence with expansion culture, leading to a limitation for their clinical application. Recently, it was reported that small extracellular vesicles (sEVs) are involved in regulation of senescence in tumor cells and fibroblasts. However, the biological roles of sEVs in senescent MSCs (Sen MSCs) are poorly understood. In this study, we established a replicative senescence model of MSCs by successive passages and compared the phenotypic changes between presenescent MSCs (Pre-Sen MSCs) and Sen MSCs and found that Sen MSCs exhibited a diminished adipogenic and osteogenic differentiation potential and elevated senescence-associated secretory phenotype levels. In addition, we found that sEV secretion was increased in Sen MSCs, and inhibition of sEV secretion led to apoptosis, DNA damage, and decreased cell viability, suggesting that increased sEV secretion plays an important role in maintaining Sen MSC homeostasis. To further investigate the molecular mechanisms, metabolomic profiling of Pre-Sen MSC-derived sEVs (Pre-Sen-sEVs) and Sen MSC-derived sEVs (Sen-sEVs) was performed. The results showed that lipid metabolites were significantly increased in Sen-sEVs and these significantly upregulated lipid metabolites were shown to be toxic for inducing cellular senescence and apoptosis in previous studies. Kyoto Encyclopedia of Genes and Genomes analysis revealed enrichment of differential metabolites between Pre-Sen-sEVs and Sen-sEVs mainly in 25 signaling pathways, of which 21 metabolic pathways have been shown to be closely associated with senescence. Taken together, our findings suggested that increased sEV secretion maintains Sen MSC homeostasis, at least in part, by excreting harmful lipids, thus providing new insights into the regulation of senescence by sEVs.

Galactosylation of rat natural scaffold for MSC differentiation into hepatocyte-like cells: A comparative analysis of 2D vs. 3D cell culture techniques

The liver plays a crucial role in drug detoxification, and the main source of liver transplants is brain-dead patients. However, the demand for transplants exceeds the available supply, leading to controversies in selecting suitable candidates for acute liver diseases. This research aimed to differentiate mesenchymal stem cells (MSCs) into hepatocyte-like cells using galactosylated rat natural scaffolds and comparing 2-D and 3-D cell culture methods. The study involved isolating and culturing Wharton's jelly cells from the umbilical cord, examining surface markers and adipogenic differentiation potential of MSCs, and culturing mesenchymal cells on galactosylated scaffolds. The growth and proliferation of stem cells on the scaffolds were evaluated using the MTT test, and urea synthesis was measured in different culture environments. Changes in gene expression were analyzed using real-time PCR. Flow cytometry results confirmed the presence of specific surface antigens on MSCs, indicating their identity, while the absence of a specific antigen indicated their differentiation into adipocytes. The MTT test revealed higher cell attachment to galactosylated scaffolds compared to the control groups. Urea secretion was observed in differentiated cells, with the highest levels in cells cultured on galactosylated scaffolds. Gene expression analysis showed differential expression patterns for OCT-4, HNF1, ALB, AFP, and CYP genes under different conditions. The findings indicated that hepatocyte-like cells derived from 3D cultures on galactosylated scaffolds exhibited superior characteristics compared to cells in other culture conditions. These cells demonstrated enhanced proliferation, stability, and urea secretion ability. The study also supported the differentiation potential of MSCs derived from Wharton's jelly umbilical cord into liver-like cells.

Comparison of stem cell characteristics between perichondral-derived stem cells and periosteal stem cells in postnatal rats

Bone marrow mesenchymal stem cells (BMSCs), periosteal stem cells (PSCs), and other bone stem cells originate from embryonic bone formation, but their function and stem cell characteristics such as proliferation ability and differentiation ability change at different anatomical locations. Perichondral-derived stem cells (PCSCs) are more closely related to PSCs in origin and function, usually used to be studied together with PSCs as one type of stem cell. However, this leads to the ignoration of the PCSCs' characteristics. Since the anatomical locations of these two types of stem cells diverse, PCSCs should have some differences from PSCs. In this study, the PCSCs in the perichondrium surrounding the growth plate cartilage expressed CTSK and CD200 same as PSCs. However, when compared the stem cell characteristics of PCSCs with that of PSCs, PCSCs were more elongated than PSCs in morphology and have stronger self-renewal ability, as well as stronger chondrogenic and adipogenic differentiation potentials. This study revealed the stem cell characteristics of PCSCs distinguished from PSCs, which may indicate PCSCs and PSCs should not be treated as one type of cell to research in the future.

Angelica sinensis polysaccharides alleviate the oxidative burden on hematopoietic cells by restoring 5-fluorouracil-induced oxidative damage in perivascular mesenchymal progenitor cells

Context 5-Fluorouracil (5-FU)-injured stromal cells may cause chronic bone marrow suppression; however, the underlying mechanism remains unclear. Angelica sinensis polysaccharide (ASP), the main biologically active ingredient of the Chinese herb, Angelica sinensis (Oliv.) Diels (Apiaceae), may enrich the blood and promote antioxidation. Objective This study investigated the protective antioxidative effects of ASP on perivascular mesenchymal progenitors (PMPs) and their interactions with hematopoietic cells. Materials and methods PMPs were dissociated from C57BL/6 mouse femur and tibia and were subsequently divided into the control, ASP (0.1 g/L), 5-FU (0.025 g/L), and 5-FU + ASP (pre-treatment with 0.1 g/L ASP for 6 h, together with 0.025 g/L 5-FU) then cultured for 48 h. Hematopoietic cells were co-cultured on these feeder layers for 24 h. Cell proliferation, senescence, apoptosis, and oxidative indices were detected, along with stromal osteogenic and adipogenic differentiation potentials. Intercellular and intracellular signaling was analyzed by real-time quantitative reverse transcription polymerase chain reaction and Western blotting. Results ASP ameliorated the reactive oxygen species production/scavenge balance in PMPs; improved osteogenic differentiation; increased SCF, CXCL12, VLA-4/VCAM-1, ICAM-1/LFA1, and TPO/MPL, Ang-1/Tie-2 gene expression. Further, the ASP-treated feeder layer alleviated hematopoietic cells senescence (from 21.9 ± 1.47 to 12.1 ± 1.13); decreased P53, P21, p-GSK-3β, β-catenin and cyclin-D1 protein expression, and increased glycogen synthase kinase (GSK)-3β protein expression in co-cultured hematopoietic cells. Discussion and conclusions ASP delayed oxidative stress-induced premature senescence of 5-FU-treated feeder co-cultured hematopoietic cells via down-regulation of overactivated Wnt/β-catenin signaling. These findings provide a new strategy for alleviating myelosuppressive stress.

Isolation methods, proliferation, and adipogenic differentiation of adipose-derived stem cells from different fat depots in bovines.

The adipose-derived stem cells (ASCs) are a valuable resource for regenerative medicine and essential materials for research in fat deposition. However, the isolation procedure of ASCs has not been standardized and needs to be harmonized; differences in proliferation and adipogenic differentiation of ASCs obtained from different fat depots have not been well characterized. In the present study, we compared the efficiency of ASCs isolation by enzymatic treatment and explant culture methods and the proliferation ability and adipogenic differentiation potential of ASCs isolated from subcutaneous and visceral fat depots. The explant culture method was simple and with no need for expensive enzymes while the enzymatic treatment method was complex, time consuming and costly. By the explant culture method, a larger number of ASCs were isolated from subcutaneous and visceral fat depots. By contrast, fewer ASCs were obtained by the enzymatic treatment method, especially from visceral adipose. ASCs isolated by the explant culture method performed well in cell proliferation and adipogenic differentiation, though they were slightly lower than those by the enzymatic treatment method. ASCs isolated from visceral depot demonstrated higher proliferation ability and adipogenic differentiation potential. In total, the explant culture method is simpler, more efficient, and lower cost than the enzymatic treatment method for ASCs isolation; compared with visceral adipose, subcutaneous adipose is easier to isolate ASCs; however, the visceral ASCs are superior to subcutaneous ASCs in proliferation and adipogenic differentiation.

Multilineage Differentiation Potential of Equine Adipose-Derived Stromal/Stem Cells from Different Sources.

The investigation of multipotent stem/stromal cells (MSCs) in vitro represents an important basis for translational studies in large animal models. The study's aim was to examine and compare clinically relevant in vitro properties of equine MSCs, which were isolated from abdominal (abd), retrobulbar (rb) and subcutaneous (sc) adipose tissue by collagenase digestion (ASCs-SVF) and an explant technique (ASCs-EXP). Firstly, we examined proliferation and trilineage differentiation and, secondly, the cardiomyogenic differentiation potential using activin A, bone morphogenetic protein-4 and Dickkopf-1. Fibroblast-like, plastic-adherent ASCs-SVF and ASCs-EXP were obtained from all sources. The proliferation and chondrogenic differentiation potential did not differ significantly between the isolation methods and localizations. However, abd-ASCs-EXP showed the highest adipogenic differentiation potential compared to rb- and sc-ASCs-EXP on day 7 and abd-ASCs-SVF a higher adipogenic potential compared to abd-ASCs-EXP on day 14. Osteogenic differentiation potential was comparable at day 14, but by day 21, abd-ASCs-EXP demonstrated a higher osteogenic potential compared to abd-ASCs-SVF and rb-ASCs-EXP. Cardiomyogenic differentiation could not be achieved. This study provides insight into the proliferation and multilineage differentiation potential of equine ASCs and is expected to provide a basis for future preclinical and clinical studies in horses.