Differentiation-inducing Medium Research Articles

Geniposide modulates GSK3β to inhibit Th17 differentiation and mitigate endothelial damage in intracranial aneurysm.

Intracranial aneurysm (IA) is a common cerebrovascular disease. Immune system disorders and endothelial dysfunction are essential mechanisms of its pathogenesis. This study aims to explore the therapeutic effect and mechanism of Geniposide (Gen)on IA, which has a protective impact on endothelial cells and cardiovascular and cerebrovascular diseases. IA mouse models were administered intraperitoneal injections of geniposide for 2 weeks following elastase injection into the right basal ganglia of the brain for intervention. The efficacy of Gen in treating IA was evaluated through pathological testing and transcriptome sequencing analysis of Willis ring vascular tissue. The primary mechanism of action was linked to the expression of GSK3β in Th17 cells. The percentage of splenic Th17 cell differentiation in IA mice was significantly inhibited by Gen. GSK3β/STAT3, and other pathway protein expression levels were also significantly inhibited by Gen. Additionally, TNF-α and IL-23 cytokine contents were significantly downregulated after Gen treatment. These results indicated that Gen significantly inhibited the percentage of Th17 cell differentiation, an effect that was reversed upon overexpression of the GSK3B gene. Furthermore, Gen-treated, Th17 differentiation-inducing cell-conditioned medium significantly up-regulated the expression of tight junction proteins ZO-1, Occludin, and Claudin-5 in murine aortic endothelial cells. Administering the GSK3β inhibitor Tideglusib to IA mice alleviated the severity of IA disease pathology and up-regulated aortic tight junction protein expression. In conclusion, Gen inhibits Th17 cell differentiation through GSK3β, which reduces endothelial cell injury and up-regulates tight junction protein expression.

Integrating Mechanics and Bioactivity: A Detailed Assessment of Elasticity and Viscoelasticity at Different Scales in 2D Biofunctionalized PEGDA Hydrogels for Targeted Bone Regeneration.

Methods for promoting and controlling the differentiation of human mesenchymal stem cells (hMSCs) in vitro before in vivo transplantation are crucial for the advancement of tissue engineering and regenerative medicine. In this study, we developed poly(ethylene glycol) diacrylate (PEGDA) hydrogels with tunable mechanical properties, including elasticity and viscoelasticity, coupled with bioactivity achieved through the immobilization of a mixture of RGD and a mimetic peptide of the BMP-2 protein. Despite the key relevance of hydrogel mechanical properties for cell culture, a standard for its characterization has not been proposed, and comparisons between studies are challenging due to the different techniques employed. Here, a comprehensive approach was employed to characterize the elasticity and viscoelasticity of these hydrogels, integrating compression testing, rheology, and atomic force microscopy (AFM) microindentation. Distinct mechanical behaviors were observed across different PEGDA compositions, and some consistent trends across multiple techniques were identified. Using a photoactivated cross-linker, we controlled the functionalization density independently of the mechanical properties. X-ray photoelectrin spectroscopy and fluorescence microscopy were employed to evaluate the functionalization density of the materials before the culturing of hMSCs on them. The cells cultured on all functionalized hydrogels expressed an early osteoblast marker (Runx2) after 2 weeks, even in the absence of a differentiation-inducing medium compared to our controls. Additionally, after only 1 week of culture with osteogenic differentiation medium, cells showed accelerated differentiation, with clear morphological differences observed among cells in the different conditions. Notably, cells on stiff but stress-relaxing hydrogels exhibited an overexpression of the osteocyte marker E11. This suggests that the combination of the functionalization procedure with the mechanical properties of the hydrogel provides a potent approach to promoting the osteogenic differentiation of hMSCs.

Effects of Semiconductor Laser Irradiation on Differentiation of Human Dental Pulp Stem Cells in Co-Culture with Dentin.

This study aimed to determine the effect of photobiomodulation therapy induced by semiconductor laser irradiation on human dental pulp stem cell (hDPSC) proliferation and their differentiation into odontoblast-like cells (OLCs). The effects of various semiconductor laser irradiation conditions on hDPSCs were examined. Three groups were evaluated: a single laser irradiation at 6 h post-seeding, multiple laser irradiations up to four times every 4 days after the first dose, and a control with no laser irradiation. The cells were irradiated at 10, 30, and 150 mW using a semiconductor laser. The effect of laser irradiation on hDPSC differentiation into OLCs was also determined. Four groups were evaluated, including co-culture using basic medium and dentin discs, simple culture using OLC differentiation-inducing medium, co-culture using OLC differentiation-inducing medium and dentin discs, and control culture with basic medium. The expression of the nestin, ALP, DSPP, and DMP-1 genes was measured using real-time PCR. The multiple irradiation group irradiated at 30 mW exhibited significantly more cell proliferation than the control. The expression of nestin associated with differentiation into OLCs during each culture period tended to be lower, whereas DSPP and ALP expression was higher compared with that of the control. Multiple laser irradiations at a low power of 30 mW induced significant hDPSC proliferation and might induce differentiation into OLCs.

Cooling‐promoted myogenic differentiation of murine bone marrow mesenchymal stem cells through TRPM8 activation in vitro

AbstractTRPM8 agonist has been reported to promote osteogenic differentiation of mesenchymal stem cells (MSCs), therefore we evaluated whether cooling‐induced activation of TRPM8 promotes myogenic differentiation of MSCs. We used 5‐azacytidine as a myogenic differentiation inducer in murine bone marrow‐derived MSCs. Addition of menthol, a TRPM8 agonist, to the differentiation induction medium significantly, increased the percentage of MyoD‐positive cells, a specific marker of myogenic differentiation. We performed intracellular Ca2+ imaging experiments using fura‐2 to confirm TRPM8 activation by cooling stimulation. The results confirmed that intracellular Ca2+ concentration ([Ca2+]i) increases due to TRPM8 activation, and TRPM8 antagonist inhibits increase in [Ca2+]i at medium temperatures below 19°C. We also examined the effect of cooling exposure time on myogenic differentiation of MSCs using an external cooling stimulus set at 17°C. The results showed that 60 min of cooling had an acceleratory effect on differentiation (2.18 ± 0.27 times). We observed that the TRPM8 antagonist counteracted the differentiation‐promoting effect of the cooling. These results suggest that TRPM8 might modulate the multiple differentiation pathways of MSCs, and that cooling is an effective way of activating TRPM8, which regulates MSCs differentiation in vitro.

Notoginsenoside R1 promotes osteogenic differentiation of human bone marrow mesenchymal stem cells via ERα/GSK-3β/β-catenin signalling pathway.

Human bone marrow mesenchymal stem cells (hBMSCs) are attractive therapeutic agents for bone tissue regeneration owing to their osteogenic differentiation potential. Notoginsenoside R1 (NGR1) is a novel phytoestrogen with diverse pharmacological activities. Here, we probed whether NGR1 has an effect on the osteogenic differentiation of hBMSCs. EdU, CCK-8 and Transwell assays were used to measure proliferation and migration of hBMSCs after treatment with different doses of NGR1. hBMSCs were treated with osteogenic differentiation induction medium for osteogenesis. Alizarin red S (ARS) and alkaline phosphatase (ALP) staining were used to measure mineralized nodule formation and ALP activity in hBMSCs, respectively. ICI 182780, an antagonist of oestrogen receptor alpha (ERα) was used to inhibit ERα expression. The results showed that NGR1 enhanced hBMSC proliferation and migration. NGR1 increased ALP activity and mineralized nodule formation as well as promoting ALP, RUNX2 and OCN expression in hBMSCs. NGR1 enhanced ERα expression and promoted GSK-3β/β-catenin signal transduction in hBMSCs. ICI 182780 reversed NGR1-mediated activation of the GSK-3β/β-catenin signalling and promoted an effect on hBMSC behaviour. Thus NGR1 promotes proliferation, migration and osteogenic differentiation of hBMSCs via the ERα/GSK-3β/β-catenin signalling pathway.

Ruxolitinib altered IFN-β induced necroptosis of human dental pulp stem cells during osteoblast differentiation

ObjectiveThis study aimed to evaluate the role of ruxolitinib in the interferon beta (IFN-β) mediated osteoblast differentiation using human dental pulp stem cells (hDPSCs). DesignhDPSCs from five deciduous teeth of healthy patients were stimulated by adding human recombinant IFN-β protein (1 or 2 ng/ml) to the osteogenic differentiation induction medium. Substrate formation was determined using Alizarin Red staining, calcium concentration, and osteoblast marker expression levels. Ruxolitinib was used to inhibit the Janus kinase/signal transducers and activators of transcription (JAK-STAT) pathway. Apoptosis was detected using terminal deoxynucleotidyl nick-end labeling (TUNEL) staining, and necroptosis was detected using propidium iodide staining and phosphorylated mixed lineage kinase domain-like protein (pMLKL) expression. ResultsIn the IFN-β-treated group, substrate formation was inhibited by a reduction in alkaline phosphatase (ALP) expression in a concentration-dependent manner. Although the proliferation potency was unchanged between the IFN-β-treated and control groups, the cell number was significantly reduced in the experimental group. TUNEL-positive cell number was not significantly different; however, the protein level of necroptosis markers, interleukin-6 (IL-6) and pMLKL were significantly increased in the substrate formation. Cell number and ALP expression level were improved in the group administered ruxolitinib, a JAK-STAT inhibitor. Additionally, ruxolitinib significantly suppressed IL-6 and pMLKL levels. ConclusionRuxolitinib interfered with the IFN-β-mediated necroptosis and osteogenic differentiation via the JAK-STAT pathway.

Docosahexaenoic acid contributes to increased CaMKII protein expression and a tendency to increase nNOS protein expression in differentiated NG108-15 cells.

Docosahexaenoic acid (DHA; 22:6n-3), an n-3 polyunsaturated fatty acid, has various important roles in brain functions. Nitric oxide (NO) produced by neuronal NO synthase (nNOS) and Ca2+/calmodulindependent protein kinase II (CaMKII) is also involved in brain functions. We investigated the influence of DHA on nNOS and CaMKII protein expression in differentiated NG108-15 cells. NG108-15 cells were seeded in 12-well plates, and after 24 h, the medium was replaced with Dulbecco's modified Eagle's medium containing 1% fetal bovine serum, 0.2 mM dibutyryl cyclic adenosine monophosphate and 100 nM dexamethasone as differentiation-inducing medium. When cells were cultured in differentiation-inducing medium, neurite-like outgrowths were observed on days 5 and 6. However, no significant difference in morphology was observed in cells with or without DHA treatment. With or without DHA addition, nNOS protein expression was increased on days 5 and 6 compared with day 0. This increase tended to be enhanced by DHA. CaMKII protein expression did not change after differentiation without DHA, but was significantly increased on day 6 compared with day 0 with DHA addition. These data indicate that DHA is involved in brain functions by regulating CaMKII and nNOS protein expression.

In vitro myogenesis activation of specific muscle-derived stem cells from patients with Duchenne muscular dystrophy

BackgroundMuscle-derived stem cells (MDSCs) contribute to the repair of injured muscles. However, the myogenicity of MDSCs generated from patients with Duchenne muscular dystrophy (DMD) relative to healthy individuals remains unclear. MethodsA human DMD model was established using the stem cells prepared from muscle derived from patients with DMD (DMD-hMDSCs). The expression of myogenic lineage-specific markers in MDSCs was examined with immunofluorescence, real-time polymerase chain reaction, and western blotting. ResultsIt was demonstrated that, compared with cells from healthy subjects, DMD-hMDSCs are primed to self-differentiate in growth-inducing medium (GM) and robustly differentiate into myotubes in differentiation-inducing medium(DM). This feature was termed “myogenesis activation,” and it was speculated that it contributes to the depletion of myogenic progenitors. Furthermore, MDSCs consistently express pax7, but the time-course of this expression does not correlate with the expression of the myogenic lineage-specific markers. ConclusionsThe myogenesis activation in DMD-hMDSCs demonstrated in this study may provide novel mechanistic insights into DMD pathogenesis and potential therapies.

DNMT1/PKR double knockdowned HepG2 (HepG2-DP) cells have high hepatic function and differentiation ability

HepG2 cells are widely used as a human hepatocytes model, but their functions, including drug metabolism, are inferior to primary hepatocytes. We previously reported that the hepatic gene expressions in HepG2 cells were upregulated by treatment with zebularine, which is an inhibitor of DNA methylation, through the inhibition of both DNA methyltransferase 1 (DNMT1) and double-stranded RNA-dependent protein kinase (PKR). In this study, we established a new HepG2 cell subline, HepG2-DP cells, by stable double knockdown of DNMT1 and PKR and evaluated its function. Albumin production, expression of CYP1A2 genes, and accumulation of lipid droplets were increased in HepG2-DP cells compared with the original HepG2 cells. Comprehensive gene expression analysis of transcription factors revealed that the expression of important genes for hepatic function, such as HNF1β, HNF4α, ONECUT1, FOXA1, FOXA2, FOXA3, and various nuclear receptors, was upregulated in HepG2-DP cells. These results indicate that the newly established HepG2-DP cells are a highly functional hepatocyte cell line. In addition, we investigated whether HepG2-DP cells are able to mature by differentiation induction, since HepG2 cells are derived from hepatoblastoma. The gene expression of major CYPs and Phase II, III drug-metabolizing enzyme genes was significantly increased in HepG2-DP cells cultured in differentiation induction medium. These results suggest that HepG2-DP cells can be further matured by the induction of differentiation and could therefore be applied to studies of drug metabolism and pharmacokinetics.

Wnt Signaling Inhibition Induces Lateral Lumens in Enteroids

BackgroundTo overcome the constant shedding of the intestinal epithelium, the stem cells at the base of the crypt rapidly proliferate and differentiate into epithelial cells necessary for proper intestinal function. A Wnt:Notch signaling gradient exists along the crypt‐villus axis, where distinct levels of Wnt relative to Notch signaling is necessary for the differentiation of epithelial cell populations. In our recent publication, mice lacking intestinal epithelial myosin 5B (MYO5B) have intracellular microvillus inclusions and cell differentiation deficits. The intestinal epithelium of these MYO5B knockout mice have significantly less transcript levels for genes associated with Wnt signaling relative to those associated with Notch signaling, possibly explaining these differentiation deficits. Here, we hypothesized that mice lacking germline MYO5B would have a similar lack of Wnt‐associated gene expression, and that Wnt inhibition in wildtype mouse crypt‐derived enteroids would give rise to microvillus inclusions, as observed n MYO5B knockouts.MethodsSegments of duodenum and jejunum were isolated from postnatal day (PND) 4 germline MYO5B knockout and littermate control mice. RNA was isolated from each tissue and transcript expression of Wnt ligands were analyzed by qPCR. A segment of jejunum was collected from adult wildtype C57Bl/6 mice, and the crypt was isolated to generate enteroids. After passaging in expansion media, the enteroids were cultured in differentiation‐inducing media containing Wnt inhibitors for three days. Enteroids were fixed and whole mount stained to visualize overall structure, tight junctions, and basolateral markers using confocal microscopy.ResultsLoss of germline MYO5B resulted in a decrease in transcript levels of most Wnt ligands in the duodenum and jejunum, however only the loss of Wnt4 expression was significant. Wnt secretion inhibitor (C59) enhanced microvillus inclusion formation in MYO5B deficient enteroids. Unexpectedly, C59 treatment in both MYO5B deficient and wildtype mouse enteroids induced the formation of large, intercellular lateral lumens, rather than small, intracellular microvillus inclusions (Fig. 1). Enteroids generated from adult wildtype mice similarly contained lateral lumens induced by C59, but in less numbers than that seen in neonatal mouse‐derived enteroids. These lateral lumens were not present in vehicle‐treated enteroids. Immunofluorescence studies illustrated that most of these lateral lumens expressed tight junction (ZO‐1) and basolateral (E‐cadherin) markers, again suggesting these structures are lateral lumens rather than microvillus inclusions. Similarly, treatment of wildtype enteroids with canonical Wnt signaling inhibitors, salinomycin and pyrvinium, gave rise to a similar lateral lumen phenotype.ConclusionsGermline MYO5B expression is critical for the intestinal expression of some (i.e., Wnt 4), but not all Wnt ligands. Wnt inhibition in enteroids induced the formation of lateral lumens, rather than microvillus inclusions, in wild type differentiated enteroids. This unexpected phenotype suggests that Wnt signaling promotes the establishment of proper polarity in intestinal epithelial cells.

DNA/Magnetic Nanoparticles Composite to Attenuate Glass Surface Nanotopography for Enhanced Mesenchymal Stem Cell Differentiation.

Mesenchymal stem cells (MSCs) have extensive pluripotent potential to differentiate into various cell types, and thus they are an important tool for regenerative medicine and biomedical research. In this work, the differentiation of hTERT-transduced adipose-derived MSCs (hMSCs) into chondrocytes, adipocytes and osteoblasts on substrates with nanotopography generated by magnetic iron oxide nanoparticles (MNPs) and DNA was investigated. Citrate-stabilized MNPs were synthesized by the chemical co-precipitation method and sized around 10 nm according to microscopy studies. It was shown that MNPs@DNA coatings induced chondrogenesis and osteogenesis in hTERT-transduced MSCs. The cells had normal morphology and distribution of actin filaments. An increase in the concentration of magnetic nanoparticles resulted in a higher surface roughness and reduced the adhesion of cells to the substrate. A glass substrate modified with magnetic nanoparticles and DNA induced active chondrogenesis of hTERT-transduced MSC in a twice-diluted differentiation-inducing growth medium, suggesting the possible use of nanostructured MNPs@DNA coatings to obtain differentiated cells at a reduced level of growth factors.

RETRACTED ARTICLE: Saxagliptin enhances osteogenic differentiation in MC3T3-E1 cells, dependent on the activation of AMP-activated protein kinase α (AMPKα)/runt-related transcription factor-2 (Runx-2)

Statement of Retraction Qiang Wang, Xiaoxing Xie, Dehua Zhang, Feng Mao, Shaobo Wang, Yi Liao- 2022, Saxagliptin enhances osteogenic differentiation in MC3T3-E1 cells, dependent on the activation of AMP-activated protein kinase α (AMPKα)/runt-related transcription factor-2 (Runx-2), Bioengineered 13(1), doi: 10.1080/21655979.2021.2008667 Since publication, significant concerns have been raised about the integrity of the data and reported results in the article. When approached for an explanation, the authors did not provide their original data or any necessary supporting information. As verifying the validity of published work is core to the integrity of the scholarly record, we are therefore retracting the article. All authors listed in this publication have been informed. We have been informed in our decision-making by our editorial policies and the COPE guidelines. The retracted article will remain online to maintain the scholarly record, but it will be digitally watermarked on each page as ‘Retracted’.

Differentiated PDGFRα-Positive Cells: A Novel In-Vitro Model for Functional Studies of Neuronal Nitric Oxide Synthase

It is evident that depletion of interstitial cells and dysfunction of nitric oxide (NO) pathways are key players in development of several gastrointestinal (GI) motility disorders such as diabetic gastroparesis (DGP). One of the main limitations of DGP research is the lack of isolation methods that are specific to interstitial cells, and therefore conducting functional studies is not feasible. The present study aims (i) to differentiate telomerase transformed mesenchymal stromal cells (iMSCs) into platelet-derived growth factor receptor-α-positive cells (PDGFRα-positive cells) using connective tissue growth factor (CTGF) and L-ascorbic acids; (ii) to investigate the effects of NO donor and inhibitor on the survival rate of differentiated PDGFRα-positive cells; and (iii) to evaluate the impact of increased glucose concentrations, mimicking diabetic hyperglycemia, on the gene expression of neuronal nitric oxide synthase (nNOS). A fibroblastic differentiation-induction medium supplemented with connective tissue growth factor was used to differentiate iMSCs into PDGFRα-positive cells. The medium was changed every day for 21 days to maintain the biological activity of the growth factors. Gene and protein expression, scanning electron and confocal microscopy, and flow cytometry analysis of several markers were conducted to confirm the differentiation process. Methyl tetrazolium cell viability, nitrite measurement assays, and immunostaining were used to investigate the effects of NO on PDGFRα-positive cells. The present study, for the first time, demonstrated the differentiation of iMSCs into PDGFRα-positive cells. The outcomes of the functional studies showed that SNAP (NO donor) increased the survival rate of differentiated PDGFRα-positive cells whereas LNNA (NO inhibitor) attenuated these effects. Further experimentations revealed that hyperglycemia produced a significant increase in expression of nNOS in PDGFRα-positive cells. Differentiation of iMSCs into PDGFRα-positive cells is a novel model to conduct functional studies and to investigate the involvement of NO pathways. This will help in identifying new therapeutic targets for treatment of DGP.

Pulsed-electromagnetic-field induced osteoblast differentiation requires activation of genes downstream of adenosine receptors A2A and A3.

Pulsed-electromagnetic-field (PEMF) treatment was found to enhance cellular differentiation of the mouse preosteoblast, MC3T3-E1, to a more osteoblastic phenotype. Differentiation genes such as Alp, BSPI, cFos, Ibsp, Osteocalcin, Pthr1 and Runx2 showed increased expression in response to PEMF stimulation. Detailed molecular mechanisms linking PEMF to the activation of these genes are limited. Two adenosine receptors known to be modulated in response to PEMF, Adora2A and Adora3, were functionally impaired by CRISPR-Cas9-mediated gene disruption, and the consequences of which were studied in the context of PEMF-mediated osteoblastic differentiation. Disruption of Adora2A resulted in a delay of Alp mRNA expression, but not alkaline phosphatase protein expression, which was similar to that found in wild type cells. However, Adora3 disruption resulted in significantly reduced responses at both the alkaline phosphatase mRNA and protein levels throughout the PEMF stimulation period. Defects observed in response to PEMF were mirrored using a chemically defined growth and differentiation-inducing media (DM). Moreover, in cells with Adora2A disruption, gene expression profiles showed a blunted response in cFos and Pthr1 to PEMF treatment; whereas cells with Adora3 disruption had mostly blunted responses in AlpI, BSPI, Ibsp, Osteocalcin and Sp7 gene activation. To demonstrate specificity for Adora3 function, the Adora3 open reading frame was inserted into the ROSA26 locus in Adora3 disrupted cells culminating in rescued PEMF responsiveness and thereby eliminating the possibility of off-target effects. These results lead us to propose that there are complementary and parallel positive roles for adenosine receptor A2A and A3 in PEMF-mediated osteoblast differentiation.

Wnt antagonist secreted frizzled-related protein I (sFRP1) may be involved in the osteogenic differentiation of periodontal ligament cells in chronic apical periodontitis.

To explore the mechanism of secreted frizzled-related protein I (sFRP1) involvement in the osteogenic differentiation of human periodontal ligament cells (hPDLCs) under inflammatory conditions. hPDLCs were cultured in an osteogenic differentiation-inducing medium (odi) and subjected to the stimulation ofPorphyromonas gingivalis lipopolysaccharide (P.gingivalisLPS) with or without the inhibition of sFRP1. Quantitative real-time polymerase chain reaction, Western blot and enzyme-linked immunosorbent assay were carried out to evaluate the expression of osteogenic markers as well as the classic Wnt signalling pathway. Periapical periodontitis was induced in Wistar rats to further confirm the effect of sFRP1 inhibitor on bone loss in vivo. After the Shapiro-Wilk normality test, data were analysed by Student's paired t-test or one-way Anova test with a P value less than 0.05 as the level of statistical significance. Significantly decreased mRNA and protein expression of osteogenic markers were detected in hPDLCs treated withP.gingivalisLPS during osteogenic induction (P<0.001). Increased expression of sFRP1 was observed (P<0.01), whilst Wnt/β-catenin signalling pathway was inhibited by the addition of P.gingivalisLPS (P<0.01). After the addition of the sFRP1 inhibitor, the decrease of osteogenic markers (P<0.05) and the inhibition of Wnt/β-catenin signalling pathway (P<0.05) were reversed significantly. The animal experiment further confirmed thatthe sFRP1 inhibitor significantly reduced bone loss of periapical lesions in vivo (P<0.0001). Wnt antagonist sFRP1 was involved in the osteogenic differentiation of hPDLCs under inflammation. Modulation of the Wnt/β-catenin signalling pathway through the inhibition of sFRP1 may offer a new perspective on the treatment of chronic apical periodontitis.

Regeneration in Experimental Alveolar Bone Defect Using Human Umbilical Cord Mesenchymal Stem Cells

Cleft lip and palate is a congenital disorder including cleft lip, and/or cleft palate, and/or alveolar cleft, with high incidence.The alveolar cleft causes morphological and functional abnormalities. To obtain bone bridge formation and continuous structure between alveolar clefts, surgical interventions are performed from infancy to childhood. However, desirable bone bridge formation is not obtained in many cases. Regenerative medicine using mesenchymal stem cells (MSCs) is expected to be a useful strategy to obtain sufficient bone bridge formation between alveolar clefts. In this study, we examined the effect of human umbilical cord-derived MSCs by transplantation into a rat experimental alveolar cleft model. Human umbilical cords were digested enzymatically and the isolated cells were collected (UC-EZ cells). Next, CD146-positive cells were enriched from UC-EZ cells by magnetic-activated cell sorting (UC-MACS cells). UC-EZ and UC-MACS cells showed MSC gene/protein expression, in vitro. Both cells had multipotency and could differentiate to osteogenic, chondrogenic, and adipogenic lineages under the differentiation-inducing media. However, UC-EZ cells lacked Sox2 expression and showed the lower ratio of MSCs than UC-MACS cells. Thus, UC-MACS cells were transplanted with hydroxyapatite and collagen (HA + Col) into alveolar cleft model to evaluate bone formation in vivo. The results of micro computed tomography and histological staining showed that UC-MACS cells with HA + Col induced more abundant bone formation between the experimental alveolar clefts than HA + Col implantation only. Cells immunopositive for osteopontin were accumulated along the bone surface and some of them were embedded in the bone. Cells immunopositive for human-specific mitochondria were aligned along the newly formed bone surface and in the new bone, suggesting that UC-MACS cells contributed to the bone bridge formation between alveolar clefts. These findings indicate that human umbilical cords are reliable bioresource and UC-MACS cells are useful for the alveolar cleft regeneration.

The Inhibitory Effects of Juglanin on Adipogenesis in 3T3-L1 Adipocytes.

IntroductionDeregulation of adipogenesis plays an important role in obesity and other metabolism disorders. PPAR, C/EBP and SREBP1c are key transcriptional factors involved in adipogenesis and lipogenesis. Juglanin is a natural compound belonging to flavonoids, and it has been reported that juglanin has a potent inhibitory effect on inflammation and certain type of cancers. However, the effects of juglanin in adipogenesis have not been reported before.Materials and Methods3T3-L1 preadipocytes were incubated with differentiation induction medium in the presence or absence of 0.5, 2.5, or 5 µM juglanin for an 8-day differentiation period. The lipid droplets accumulated in the cytoplasm were monitored by Oil Red O staining on days 0, 2, 5, and 8. The regulatory effects of juglanin on adipogenesis-related genes and proteins were investigated by real-time polymerase chain reaction and Western blot analysis.ResultsJuglanin significantly decreased lipid accumulation in differentiated adipocytes. Our findings show that juglanin reduced the expression of C/EBPα, C/EBPβ, and SREBP-1c without affecting PPARα or PPARγ expression. Additionally, juglanin increased the activation of the SIRT1/AMPK signaling pathway through the phosphorylation of AMPKα. Finally, we performed an AMPK inhibitor experiment, which revealed that the inhibitory effects of juglanin on adipogenesis are mediated through AMPK.DiscussionJuglanin can prevent adipogenesis by suppressing lipid accumulation and the differentiation of preadipocytes. The mechanism of juglanin regulating adipogenesis requires further investigation. Future clinical study in vivo could shed more light on its implication in modulating obesity and metabolic disorders.

Effects of platelet-rich plasma on mesenchymal stem cells isolated from rat uterus.

BackgroundPlatelet-rich plasma (PRP), which represents a valuable source of growth factors, is increasingly being applied in regenerative medicine. Recent findings suggest the feasibility of using PRP in the treatment of infertility secondary to refractory thin endometrium. Mesenchymal stem/stromal cells (MSCs) of the endometrium are an essential cellular component responsible for extracellular matrix remodeling, angiogenesis, cell-to-cell communication, and postmenstrual tissue repair. Using a rat model, we examine the effects of autologous PRP on MSCs isolated from the uterus and compare them with the effects of autologous ordinary plasma (OP) and complete growth medium.MethodsMSCs were isolated from uterine tissues via enzymatic disaggregation. Flow cytometry immunophenotyping of the primary cell cultures was complemented by immunocytochemistry for Ki-67 and vimentin. The ability of MSCs to differentiate in osteo-, chondro-, and adipogenic directions was assessed using differentiation-inducing media. The levels of autophagy and apoptosis markers, as well as the levels of matrix metalloproteinase 9 (MMP9) and estrogen receptor α, were assessed by western blotting.ResultsAfter 24 h incubation, the proliferation index of the PRP-treated MSC cultures was significantly higher than that of the MSC cultures treated with complete growth medium. PRP treatment elevated production of LC3B protein, an autophagy marker, while OP treatment upregulated the expression of stress-induced protein p53 and extracellular enzyme MMP9. The results indicate practical relevance and validity for PRP use in the treatment of infertility.

Endosialin defines human stem Leydig cells with regenerative potential.

STUDY QUESTIONIs endosialin a specific marker of human stem Leydig cells (SLCs) with the ability to differentiate into testosterone-producing Leydig cells (LCs) in vitro and in vivo?SUMMARY ANSWEREndosialin is a specific marker of human SLCs which differentiate into testosterone-producing LCs in vitro and in vivo.WHAT IS KNOWN ALREADYHuman SLCs have been identified and isolated using the marker platelet-derived growth factor receptor α (PDGFRα) or nerve growth factor receptor (NGFR). However, the specificity was not high; thus, LCs and germ cells could be mistakenly sorted as SLCs if PDGFRα or NGFR was used as a marker for human SLCs isolation.STUDY DESIGN, SIZE, DURATIONFirstly, we re-evaluated the specificity of PDGFRα and NGFR for SLCs in adult human testes. Then we analysed the previously published single-cell sequencing data and found that endosialin may identify human SLCs. Subsequently, we sorted endosialin+ cells from four human donors and characterized their self-renewal and multipotent properties. To assess whether endosialin+ cells have the potential to differentiate into functional LCs in vitro, these cells were stimulated by differentiation-inducing medium. We next assessed the in vivo regenerative potential of human endosialin+ cells after xenotransplantation into the testes of immunodeficient mice.PARTICIPANTS/MATERIALS, SETTING, METHODSSingle-cell sequencing analysis, immunofluorescence and flow cytometry were used to characterize human testis tissues. In vitro colony formation, multipotent differentiation (adipogenic, osteogenic and chondrogenic) and Leydig cell-lineage induction were used to assess stem cell activity. Xenotransplantation into 3-week-old immunodeficient mice was used to determine in vivo regenerative potential. Endpoint measures included testosterone measurements, cell proliferation, immunofluorescence, flow cytometry and quantitative RT–PCR.MAIN RESULTS AND THE ROLE OF CHANCEThe results indicate that endosialin is a specific marker of SLCs compared with PDGFRα and NGFR. Additionally, endosialin+ cells isolated from human testes show extensive proliferation and differentiation potential in vitro: their self-renewal ability was inferred by the formation of spherical clones derived from a single cell. Moreover, these cells could differentiate into functional LCs that secreted testosterone in response to LH in a concentration-dependent manner in vitro. These self-renewal and differentiation properties reinforce the proposal that human testicular endosialin+ cells are SLCs. Furthermore, transplanted human endosialin+ cells appear to colonize the murine host testes, localize to peritubular and perivascular regions, proliferate measurably and differentiate partially into testosterone-producing LCs in vivo.LARGE SCALE DATANA.LIMITATIONS, REASONS FOR CAUTIONOwing to the difficulty in collecting human testis tissue, the sample size was limited. The functions of endosialin on SLCs need to be elucidated in future studies.WIDER IMPLICATIONS OF THE FINDINGSA discriminatory marker, endosialin, for human SLCs purification is a prerequisite to advance research in SLCs and logically promote further clinical translation of SLCs-based therapies for male hypogonadism.STUDY FUNDING/COMPETING INTEREST(S)A.P.X. was supported by the National Key Research and Development Program of China (2017YFA0103802 and 2018YFA0107200). C.D. was supported by the National Natural Science Foundation of China (81971314) and the Natural Science Foundation of Guangdong Province, China (2018B030311039). The authors declare no conflict of interest.

Stiffness-mediated mesenchymal stem cell fate decision in 3D-bioprinted hydrogels.

BackgroundHydrogels with tuneable mechanical properties are an attractive material platform for 3D bioprinting. Thus far, numerous studies have confirmed that the biophysical cues of hydrogels, such as stiffness, are known to have a profound impact on mesenchymal stem cell (MSC) differentiation; however, their differentiation potential within 3D-bioprinted hydrogels is not completely understood. Here, we propose a protocol for the exploration of how the stiffness of alginate–gelatin (Alg-Gel) composite hydrogels (the widely used bioink) affects the differentiation of MSCs in the presence or absence of differentiation inducing factors.MethodsTwo types of Alg-Gel composite hydrogels (Young’s modulus: 50 kPa vs. 225 kPa) were bioprinted independently of porosity. Then, stiffness-induced biases towards adipogenic and osteogenic differentiation of the embedded MSCs were analysed by co-staining with alkaline phosphatase (ALP) and oil red O. The expression of specific markers at the gene level was detected after a 3-day culture.ResultsConfocal microscopy indicated that all tested hydrogels supported MSC growth and viability during the culture period. Higher expression of adipogenic and osteogenic markers (ALP and lipoprotein lipase (LPL)) in stiffer 3D-bioprinted matrices demonstrated a more significant response of MSCs to stiffer hydrogels with respect to differentiation, which was more robust in differentiation-inducing medium. However, the LPL expression in stiffer 3D-bioprinted constructs was reduced at day 3 regardless of the presence of differentiation-inducing factors. Although MSCs embedded in softer hydrogels to some extent proceeded toward adipogenic and osteogenic lineages within a few days, their differentiation seemed to be slower and more limited. Interestingly, the hydrogel itself (without differentiation-inducing factors) exhibited a slight effect on whether MSCs differentiated towards an adipogenic or an osteogenic fate. Considering that the mechano-regulated protein Yes-associated protein (YAP) is involved in MSC fate decisions, we further found that inhibition of YAP significantly downregulated the expression of ALP and LPL in MSCs in stiffer constructs regardless of the induced growth factors present.ConclusionsThese results demonstrate that the differentiation of MSCs in 3D-bioprinted matrices is dependent on hydrogel stiffness, which emphasizes the importance of biophysical cues as a determinant of cellular behaviour.