Differentiation Of hADSCs Research Articles

Promoting effect of nano hydroxyapatite and vitamin D3 on the osteogenic differentiation of human adipose-derived stem cells in polycaprolactone/gelatin scaffold for bone tissue engineering

Tissue engineering knowledge is a step towards the treatment of irreversible damages to human beings. In the present study, PCL/Gel, PCL/Gel/nHA, PCL/Gel/Vit D3 and PCL/Gel/nHA/Vit D3 (Polycaprolactone/Gelatin/Nanohydroxyapatite/Vitamin D3) composite scaffolds were successfully constructed using electrospinning method. The proliferation and differentiation of hADSCs into the bone phenotype were determined using MTT method, ALP activity, Von Kossa and Alizarin red staining, and qRT-PCR test. The simultaneous presence of nHA and vitamin D3 led to the increased activity of ALP in the early stages (on the 14th day) and increased mineralization in the late stages (on the 21st day) in differentiated hADSCs. Further, it was found that the use of nHA and vitamin D3 resulted in increased expression of BGLAP and COLL I and reduced expression of ALP and RUNX2 in hADSCs for 21 days. The results indicated that nHA and vitamin D3 have a synergistic effect on the osteogenic differentiation of hADSCs.

Inhibitory effect of celastrol on adipogenic differentiation of human adipose-derived stem cells

Control of adipogenesis in mesenchymal stem cells (MSCs) offers enormous potential for management of obesity- and aging-related diseases. Celastrol, the traditional Chinese medicine extracted from Tripterygium wilfordi, exhibits anti-obesity effects in in vitro and in vivo murine models. This study describes how celastrol affects multilineage differentiation potential of human adipose-derived stem cells (hADSCs). We performed in vitro adipogenic differentiation of hADSCs and investigated how celastrol-induced lipid accumulation and expression of adipocyte differentiation markers varied with dose, duration, and donor age. In addition, we assessed the effect of celastrol on osteogenic and chondrogenic differentiation of hADSCs. During adipogenic induction of hADSCs, the inhibitory effect of celastrol on lipid accumulation and adipogenesis depended on dose, duration, time of administration, and individual donor. Inhibition was mediated by proliferator-activated receptor-γ (PPARG) and CCAAT/enhancer-binding protein alpha (CEBPA). Celastrol also suppressed differentiation of hADSCs into the osteogenic and chondrogenic lineages. Celastrol plays a regulatory role in multilineage differentiation of human MSCs. Our findings provide important insights regarding management of obesity and stem cell therapy.

MiR-1307-3p suppresses the chondrogenic differentiation of human adipose-derived stem cells by targeting BMPR2.

MicroRNAs (miRs) are involved in several physiological processes, including chondrogenic differentiation, however, their expression and roles in the chondrogenic differentiation of human adipose-derived stem cells (hADSCs) remain to be fully elucidated to date. Our previous study showed that miR-1307-3p was significantly downregulated during chondrogenic differentiation by microarray and northern blot analysis. The present study aimed to investigate the effects of miR-1307-3p on chondrogenic differentiation and the underlying mechanisms. First, the decreased expression of miR-1307-3p was confirmed by reverse transcription-quantitative polymerase chain reaction analysis. Subsequently, gain- and loss-of-function of miR-1307-3p experiments showed that the overexpression of miR-1307-3p suppressed the deposition of cartilage matrix proteoglycans and decreased the expression of cartilage-related markers, including sex determining region Y-box 9, collagen type II α1 chain and aggrecan, whereas the knockdown of miR-1307-3p had the opposite effect. In addition, bone morphogenetic protein receptor type 2 (BMPR2) was identified as a target of miR-1307-3p. Further mechanistic investigations showed that miR-1307-3p attenuated the chondrogenic differentiation of hADSCs at least partly by inhibiting BMPR2-mothers against decapentaplegic signaling pathways. In conclusion, the findings revealed that miR-1307-3p inhibited the chondrogenic differentiation of hADSCs by targeting BMPR2 and its down-stream signaling pathway, which may provide novel therapeutic clues for the treatment of cartilage injury.

Suppressive Role of Boron on Adipogenic Differentiation and Fat Deposition in Human Mesenchymal Stem Cells

Over the past years, adipose tissue has become an invaluable source of mesenchymal stem cells (MSCs) due to development of improved isolation methodologies. In a recent work, our group established a primary culture of human adipose-derived stem cells (hADSCs), which were characterized for their stem cell characteristics in detail and studied their myogenic differentiation potential in presence of boron. In the current study, we focused on the effects of a boron-containing compound, sodium pentaborate pentahydrate (NaB), on the adipogenic differentiation of hADSCs. Incorporation of boron in various chemical derivates has been a novel interest in drug-discovery attempts due to increasing number of reports on their anticancer, antibacterial, antiviral, and antifungal activities. In this report, a striking suppressive activity of boron on adipogenic differentiation of hADSCs is observed in a dose-dependent manner. Higher concentrations of NaB (20, 50, and 100μg/mL (68, 170 and 340μM)) resulted in a progressive decrease of lipid deposition, suppressed master regulators of adipogenesis transcriptional programming at the mRNA and protein levels, while having no evident cytotoxicity on the cells. The findings of this study are encouraging to undertake further investigations on potential beneficial effects boron in terms of its impact on normal and dysfunctional adipose biology. In that respect, these results pave the path to evaluate boron-based compounds in prevention and treatment of obesity which is a modern age pandemic that is predominant worldwide and found in strong association with comorbidities, including type 2 diabetes, hypertension, cardiovascular disease, cancers, and others."

ZNF521 Has an Inhibitory Effect on the Adipogenic Differentiation of Human Adipose-Derived Mesenchymal Stem Cells.

Mesenchymal stem cells (MSCs) are multipotent progenitors present in the bone marrow stroma and in subcutaneous abdominal fat, an abundant and easily accessible source of MSCs with the ability to differentiate along multiple lineage pathways. The stem cell-associated transcription co-factor Zinc Finger Protein 521 (ZNF521/zfp521) has been implicated in the control of the homeostasis of hematopoietic, neural and osteo-adipogenic progenitors. Here we document through the analysis of a panel of human adipose-derived stem cells (hADSCs), that ZNF521 strongly inhibits the generation of mature adipocytes. Enforced overexpression of ZNF521 in these cells resulted in a significant delay and reduction in adipocyte differentiation upon exposure to inducers of adipogenesis. Of particular relevance, ZNF521 was able to inhibit the expression of ZNF423, recently identified as an essential commitment factor necessary for the generation of pre-adipocytes. Conversely, silencing of ZNF521 was found to significantly enhance the adipogenic differentiation of hADSCs. Inhibition of adipogenesis by ZNF521 was at least in part due to inhibition of EBF1. Taken together, these results confirm a role for ZNF521 as a key negative regulator of adipocyte differentiation of hADSCs.

Differentiation of adipose derived stem cells to keratinocyte-like cells on an advanced collagen wound matrix

Despite advances in tissue engineering and regenerative medicine, skin regeneration and cutaneous wound healing remains a significant medical challenge. A bioengineered skin that stimulates the body's natural regeneration capability is needed to address the current lack of treatment options. To this end, a biocompatible collagen wound matrix was developed using an electrochemical deposition fabrication process. The advanced collagen wound matrix has relatively high tensile strength compared to normal collagen matrix made by the heat gelation process and open porosity, and serves as an excellent platform for cellular growth and differentiation. Human adipose derived stem cells (hADSCs) were cultured on this collagen matrix and a co-culture system with primary keratinocytes and keratinocyte conditioned media was developed for differentiation of the hADSCs to keratinocyte-like cells. After fifteen days, hADSCs in co-culture began to exhibit a "cobblestone-like" morphology, indicating preliminary signs of differentiation to a keratinocyte-like cell. Based on morphological analysis at day 30, the co-culture with keratinocyte conditioned media system shows promising preliminary evidence of hADSC differentiation to a keratinocyte-like cell on an electrochemically aligned collagen wound matrix.

Cardiac extracellular matrix hydrogel together with or without inducer cocktail improves human adipose tissue-derived stem cells differentiation into cardiomyocyte–like cells

Studies have demonstrated that differentiation of stem cells into cardiomyocytes is a complex phenomenon that requires sufficient inducing factors at various time points. Cardiac extracellular matrix (cECM) could provide tissue specific microenvironment and act as an inductive template for efficient cell differentiation. The aim of this study was to investigate the effect of cECM on differentiation of human adipose tissue-derived stem cells (hADSCs) into cardiomyocytes using cECM hydrogel in combination with a cardiac inductive cocktail. hADSCs were cultured on ECM-coated plates with and without inductive cocktail for 3weeks. qRT-PCR and western blot analysis were used to evaluate the expression pattern of special cardiac genes and proteins. When hADSCs were cultured in the presence of cECM cardiac genes including GATA4, HAND1, HAND2, NKX2.5, Troponin I, βMHC, Connexin43 were highly expressed in differentiated cells. Also Connexin43, cTnI and βMHC proteins were expressed as well. We could show that cECM by itself could affect viability, proliferation and differentiation of hADSCs. However, combination of cECM with a cardiac inducing cocktail could improve the results.

Role of LncRNA ROR in Ad36-induced browning of human adipose-derived stem cells

Objective To investigate the role of LncRNA ROR in Ad36-induced browning of human adipose-derived stem cells(hADSC). Methods After hADSC was induced by cocktail and Ad36 for 2, 4, 6, and 8 days, Oil red O staining was performed for observing the adipogenic status. The mRNA expressions of LncRNA ROR, uncoupling protein 1(UCP1), and PRDM16 were detected by real- time PCR and the protein expressions of UCP1 and PRDM16 were detected by Western-blot. After LncRNA ROR was knocked down by siRNA, UCP1 and PRDM16 mRNA and protein expression levels in the process of Ad36-induced adipocyte differentiation were detected by real- time PCR and Western-blot. Results Oil red O staining showed that fat droplets in the cocktail-induced group were larger than those in the Ad36-induced group. Compared with the cocktail group, the mRNA expressions of LncRNA ROR, UCP1 and PRDM16, and the protein expressions of UCP1 and PRDM16 in Ad36 group were significantly increased(P<0.05). The mRNA and protein expressions of UCP1 and PRDM16 in LncRNA ROR knockdown group were significantly lower than those in control group(P<0.05). Conclusion In the process of Ad36-induced hADSC differentiation, the up-regulation of LncRNA ROR may stimulate UCP1 and PRDM16 expression, and thus promote the browning of hADSC. (Chin J Endocrinol Metab, 2018, 34: 318-324) Key words: Human adipose-derived stem cells; Browning; Cocktail; Ad36; Long noncoding RNA

MiR-450b Promotes Osteogenic Differentiation In Vitro and Enhances Bone Formation In Vivo by Targeting BMP3.

Osteoporosis is characterized by deterioration of bone microarchitecture and low bone mass. One of the primary causes of osteoporosis is the decrease in the osteogenic differentiation of mesenchymal stem cells (MSCs). Tissue engineering therapy with genetically modified MSCs has attracted much attention in the study of bone regeneration. In this study, we found that the expression level of miR-450b was upregulated during osteogenic differentiation of human adipose-derived mesenchymal stem cells (hADSCs). To explore the effect of miR-450b on the osteogenesis of hADSCs, we performed a series of gain- and loss-of-function analyses and demonstrated that miR-450b not only promoted the process of hADSC differentiation to osteoblasts in vitro but also enhanced ectopic bone formation in vivo. Bone morphogenetic protein 3 (BMP3), the most abundant BMP member in bone, was identified as a direct target of miR-450b. Downregulation of the endogenous expression of BMP3 could mimic the effect of miR-450b upregulation on the osteogenic differentiation of hADSCs. Overall, our study first demonstrated that a novel microRNA miR-450b was essential for hADSC differentiation, which could promote osteogenic differentiation in vitro and enhance bone formation in vivo by directly suppressing BMP3.

The Anti‐adipogenic Effects of Neohesperidin Dihydrochalcone Derivatives on Human Adipose‐Derived Stem Cells

Obesity, described by an excess accumulation of adipocyte, has emerged as one of the major public health problems. Excess adipocyte increases the risk for multiple pathological conditions and metabolic disorders such as dyslipidemia, hypertension and cancer. Oxygen free radicals are highly reactive and generated by incomplete reduction of oxygen molecule during oxidative phosphorylation which is expected to cause damage to various cellular functions. Mitochondrial electron transport chain is thought to be as a major intracellular generator of reactive oxygen species (ROS). Adipose‐derived stem cells (ADSCs) are generally extracted from adipose tissue and have the ability to differentiate into multiple mesodermal cell types, such as adipocytes, osteoblasts, chondrocytes and neuronal cells. ROS positively stimulate adipogenic differentiation, it is significant to degrade the level of ROS for the preventing above diseases. Nuclear factor erythroid 2‐related factor‐2 (Nrf2), a transcription factor defined as one of the key antioxidant regulators. In the cytoplasm, under normal conditions, Nrf2 is constructively bound to Kelch‐like ECH associating protein 1 (Keap 1) protein. But exposuring to ROS, Nrf2 is dissociated from the Keap‐Nrf2 complex and translocated from the cytoplasm to the nucleus. Subsequently, Nrf2 promotes expression of antioxidant enzyme genes such as heme oxygenase 1 (HO‐1) and NAD(P)H:Quinone Oxidoreductase 1 (NQO‐1) by binding to the antioxidant response element (ARE). Neohesperidin is a flavonoid compounds originated from peel of citrus fruits and its hydrogenated form, Neohesperidin dihydrochalcone (NHDC) has antioxidant activity. Thus, we observed the anti‐adipogenic effects of NHDC derivatives on human adipose‐derived stem cells (hADSC) through Nrf2 activation. Cell viability and compounds' proper concentrations were set by MTS assay. The effect of these compounds on suppressing oxidative stress, lipid accumulation, and adipogenic differentiation from hADSCs was investigated using Oil Red O staining, reverse‐transcriptase polymerase chain reaction (RT‐PCR), quantitative real‐time PCR (qPCR), and western blotting. And also intracellular ROS production was measured using 2′, 7′‐dichlorofluorescin diacetate (DCF‐DA). It was first found that NHDC derivatives treatment during adipogenic differentiation of hADSCs reduced lipid droplets accumulation and increased expression level of anti‐oxidant marker (Nrf2, HO‐1, NQO‐1). But expression of adipogenic markers, peroxisome proliferator‐activated receptor gamma (PPAR γ) and CCAAT/enhancer‐binding protein α (C/EBP α) were downregulated. The results demonstrate that NHDC derivatives play an efficacious role in inhibiting adipogenic differentiation of hADSCs through reducing cellular oxidative stress. Therefore, using these compounds as therapeutic applications including development of functional foods and pharmaceutical agents is worthy of note for health promoting.Support or Funding InformationThis research was supported by Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by Ministry of Science, ICT &amp; Future Planning (NRF‐2015R1C1A1A01053726)This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Polylactic Acid Nanopillar Array-Driven Osteogenic Differentiation of Human Adipose-Derived Stem Cells Determined by Pillar Diameter.

Numerous studies have determined that physical cues, especially the nanotopography of materials, play key roles in directing stem cell differentiation. However, most research on nanoarrays for stem cell fate regulation is based on nonbiodegradable materials, such as silicon wafers, TiO2, and poly(methyl methacrylate), which are rarely used as tissue engineering biomaterials. In this study, we prepared biodegradable polylactic acid (PLA) nanopillar arrays with different diameters but the same center-to-center distance using a series of anodic aluminum oxide nanowell arrays as templates. Human adipose-derived stem cells (hADSCs) were selected to investigate the effect of the diameter of PLA nanopillar arrays on stem cell differentiation. By culturing hADSCs without the assistance of any growth factors or osteogenic-induced media, the differentiation tendencies of hADSCs on the nanopillar arrays were assessed at the gene and protein levels. The assessment results suggested that the osteogenic differentiation of hADSCs can be driven by nanopillar arrays, especially by nanopillar arrays with a diameter of 200 nm. Moreover, an in vivo animal model of the samples demonstrated that PLA film with the 200 nm pillar array exhibits an improved ectopic osteogenic ability compared with the planar PLA film after 4 weeks of ectopic implantation. This study has provided a new variable to investigate in the interaction between stem cells and nanoarray structures, which will guide the bone regeneration clinical research field. This work paves the way for the utility of degradable biopolymer nanoarrays with specific geometrical and mechanical signals in biomedical applications, such as patches and strips for spine fusion, bone crack repair, and restoration of tooth enamel.

Fibrin scaffold could promote survival of the human adipose-derived stem cells during differentiation into cardiomyocyte-like cells.

Human adipose-derived stem cells (hADSCs) are capable of differentiation into many cells including cardiac cells. Different types of scaffolds are used for cell differentiation but the best is yet to be determined. In this study, fibrin scaffold (3D) was fabricated using human plasma fibrinogen and compared with culture plates (2D) for the growth and differentiation of hADSCs into cardiomyocyte-like cells. For this purpose, after obtaining the properties of the isolated hADSCs and fibrin scaffold, four biochemical tests were employed to determine the relative growth rate of hADSCs in 2D and 3D cultures. To examine the effects of two different culture systems on cardiomyogenic differentiation, hADSCs were treated with 10 or 50μM 5-azacytidine (5-Aza) for 24h and followed until 10weeks. The results indicated that the growth of hADSCs in 3D significantly increased after the seventh day (P < 0.05). Western blot, qRT-PCR and immunochemistry assays were used to evaluate the rate of cardiac differentiation, which showed significantly higher expression of special cardiac genes such as NKX2.5, Cx43, MLC2v, βMHC, HAND1, HAND2 and cTnI (P < 0.05) in the treated hADSCs with 50μM 5-Aza in the 3D group. However, the expression level of the specific cardiac proteins in 3D was not significant using western blot and immunofluorescence staining. In conclusion, this study suggests that the fibrin scaffold with a compressive stress of 107.74kPa can keep the cells alive for 10weeks and also allows a higher and sooner differentiation of hADSCs into cardiomyocyte-like cells treated with 50μM 5-Aza.

Injectable and detachable heparin-based hydrogel micropatches for hepatic differentiation of hADSCs and their liver targeted delivery

A micropatterned heparin-based hydrogel system that can provide sustained release of multiple growth factors upon one time loading was prepared via photopolymerization and lithography and it was employed as a culture matrix for differentiating hADSCs into hepatic lineage. Mature differentiation of hADSCs into hepatic lineage in terms of gene expression and immunofluorostaining of hepatic markers, and functional characteristics such as glycogen storage ability and production of albumin and urea was observed on the soft hydrogel (∼400 Pa) when the gel elasticity was modulated. This optimal heparin-based hydrogel was used to prepare micropatches containing hepatic-differentiated cells by 1) micropatterning of the gel on a polyelectrolyte multilayer (PEM), 2) seeding of hADSCs and inducing hepatic differentiation, and 3) electrochemical retrieval of cell-attached micropatches. Upon i.v. injection, the retrieved cell micropatches showed a prolonged retention in the liver and promoted function compared to single cell injection in a rat model. In conclusion, this injectable and detachable miropatterned heparin-based hydrogel system could serve as a total platform for the stem cell differentiation under well-controlled microenvironment in vitro and for targeted delivery of the differentiated cells in vivo.

Exendin-4 modifies adipogenesis of human adipose-derived stromal cells isolated from omentum through multiple mechanisms.

Abdominal obesity is considered a major factor in the development of metabolic disorders. Glucagon-like peptide-1 (GLP-1) has been reported to have positive effects on improving body metabolism and to reducing insulin resistance. However, it remains less clear whether GLP-1 plays a role in the adipogenesis process of visceral fat. Here, we analyzed the in vitro actions and probable mechanisms of Exendin-4, a GLP-1 receptor agonist, on human adipose-derived stromal cells (hADSCs) isolated from omentum. Our results demonstrated that Exendin-4 improved cell viability via promoting proliferation and inhibiting apoptosis in hADSCs isolated from omentum. Mechanistically, the activation of MAPK/ ERK1/2, Akt/GSK-3β, and PKA/CREB pathways and downstream consequences induced are involved in the proliferative and anti-apoptotic roles of Exendin-4. More intriguingly, Exendin-4 could promote the differentiation of omental hADSCs. Underlying mechanisms of the differentiation of hADSCs are associated with the upregulation of the expression of pro-adipogenic genes and downregulation of the expression of anti-adipogenic genes. Our data demonstrate that Exendin-4 modifies adipogenesis of hADSCs isolated from omentum through multiple mechanisms, these effects could contribute to the protective actions of GLP-1 receptor agonist body metabolism and insulin sensitivity.

Histone deacetylase inhibition-mediated neuronal differentiation via the Wnt signaling pathway in human adipose tissue-derived mesenchymal stem cells

Histone deacetylase (HDAC) inhibitors, which have an effect on cell homeostasis, cell cycle progression, and terminal differentiation, can act to promote self-renewal and enhance directed differentiation of several lineages of stem cells. However, the roles of HDAC inhibitors on neurogenic differentiation and the mechanisms of Wnt signaling following treatment with HDAC inhibitors remain unclear in stem cells. We hypothesized that HDAC inhibitors regulate downstream Wnt signaling and neurogenic differentiation of mesenchymal stem cells. Following neural induction with supplementary factors, human adipose tissue-derived mesenchymal stem cells (hADSCs) were differentiated into neurogenic cells in vitro. We examined the neurogenic differentiation induced by the HDAC inhibitors, MS-275, sodium butyrate (NaB), trichostatin A (TSA), and valproic acid (VPA), by RT-PCR and western blot analysis. Based on RT-PCR analysis, the expressions of NEUROG2 and NEFL were highly increased following HDAC inhibitor treatment compared with control medium. Most of the neuronal marker genes were expressed when neural-induced hADSCs (NI-hADSCs) were treated with the HDAC inhibitors individually. Interestingly, expression of most of the Wnt-related genes were highly increased following treatment with the HDAC inhibitors, especially with MS-275 treatment. Further, the protein level of Wnt5 was upregulated after neurogenic induction with MS-275 and VPA treatment, based on western blot analysis. Furthermore, we found that c-Jun expression was increased after treatment with the HDAC inhibitors, except with NaB. The protein levels of phosphor-JNK and phosphor-GSK-3β were upregulated considerably. In conclusion, the HDAC inhibitors could induce neurogenic differentiation of hADSCs by activating canonical Wnt or non-canonical Wnt signaling pathways.

MiRNA-10b Reciprocally Stimulates Osteogenesis and Inhibits Adipogenesis Partly through the TGF-β/SMAD2 Signaling Pathway.

As the population ages, the medical and socioeconomic impact of age-related bone disorders will further increase. An imbalance between osteogenesis and adipogenesis of mesenchymal stem cells (MSCs) can lead to various bone and metabolic diseases such as osteoporosis. Thus, understanding the molecular mechanisms underlying MSC osteogenic and adipogenic differentiation is important for the discovery of novel therapeutic paradigms for these diseases. miR-10b has been widely reported in tumorigenesis, cancer invasion and metastasis. However, the effects and potential mechanisms of miR-10b in the regulation of MSC adipogenic and osteogenic differentiation have not been explored. In this study, we found that the expression of miR-10b was positively correlated with bone formation marker genes ALP, RUNX2 and OPN, and negatively correlated with adipogenic markers CEBPα, PPARγ and AP2 in clinical osteoporosis samples. Overexpression of miR-10b enhanced osteogenic differentiation and inhibited adipogenic differentiation of human adipose-derived mesenchymal stem cells (hADSCs) in vitro, whereas downregulation of miR-10b reversed these effects. Furthermore, miR-10b promoted ectopic bone formation in vivo. Target prediction and dual luciferase reporter assays identified SMAD2 as a potential target of miR-10b. Silencing endogenous SMAD2 expression in hADSCs enhanced osteogenesis but repressed adipogenesis. Pathway analysis indicated that miR-10b promotes osteogenic differentiation and bone formation via the TGF-β signaling pathway, while suppressing adipogenic differentiation may be primarily mediated by other pathways. Taken together, our findings imply that miR-10b acts as a critical regulator for balancing osteogenic and adipogenic differentiation of hADSCs by repressing SMAD2 and partly through the TGF-β pathway. Our study suggests that miR-10b is a novel target for controlling bone and metabolic diseases.

Differentiation of human adipose-derived stem cells into cardiomyocyte-like cells in fibrin scaffold by a histone deacetylase inhibitor

BackgroundHuman adipose-derived stem cells (hADSCs) are capable of differentiating into many cells such as cardiac cells. Different types of inducers are used for cardiac cell differentiation, but this question still remains to be investigated, which one is the best. The aim of this paper was to investigate the effect of combination of fibrin scaffold and trichostatin A (TSA), for differentiation of hADSCs into cardiomyocyte-like cells.MethodsAfter approval of characteristics of hADSCs and fibrin scaffold, hADSCs were cultured in fibrin scaffold with 10 µM TSA for 72 h and kept in standard conditions for 4 weeks. QRT-PCR and immunostaining assay were performed for evaluating the expression pattern of special cardiac genes and proteins.ResultsIn particular, our study showed that fibrin scaffold alongside TSA enhanced expression of the selected genes and proteins.ConclusionsWe concluded that the TSA alone or with fibrin scaffold can lead to the generation of cardiac like cells in a short period of time.

Zinc sulfate contributes to promote telomere length extension via increasing telomerase gene expression, telomerase activity and change in the TERT gene promoter CpG island methylation status of human adipose-derived mesenchymal stem cells.

The use of mesenchymal stem cells (MSCs) for cell therapy and regenerative medicine has received widespread attention over the past few years, but their application can be complicated by factors such as reduction in proliferation potential, the senescent tendency of the MSCs upon expansion and their age-dependent decline in number and function. It was shown that all the mentioned features were accompanied by a reduction in telomerase activity and telomere shortening. Furthermore, the role of epigenetic changes in aging, especially changes in promoter methylation, was reported. In this study, MSCs were isolated from the adipose tissue with enzymatic digestion. In addition, immunocytochemistry staining and flow cytometric analysis were performed to investigate the cell-surface markers. In addition, alizarin red-S, sudan III, toluidine blue, and cresyl violet staining were performed to evaluate the multi-lineage differentiation of hADSCs. In order to improve the effective application of MSCs, these cells were treated with 1.5 × 10−8 and 2.99 × 10−10 M of ZnSO4 for 48 hours. The length of the absolute telomere, human telomerase reverse transcriptase (hTERT) gene expression, telomerase activity, the investigation of methylation status of the hTERT gene promoter and the percentage of senescent cells were analyzed with quantitative real-time PCR, PCR-ELISA TRAP assay, methylation specific PCR (MSP), and beta-galactosidase (SA-β-gal) staining, respectively. The results showed that the telomere length, the hTERT gene expression, and the telomerase activity had significantly increased. In addition, the percentage of senescent cells had significantly decreased and changes in the methylation status of the CpG islands in the hTERT promoter region under treatment with ZnSO4 were seen. In conclusion, it seems that ZnSO4 as a proper antioxidant could improve the aging-related features due to lengthening of the telomeres, increasing the telomerase gene expression, telomerase activity, decreasing aging, and changing the methylation status of hTERT promoter; it could potentially beneficial for enhancing the application of aged-MSCs.

One-Dimensional Hydroxyapatite Nanostructures with Tunable Length for Efficient Stem Cell Differentiation Regulation.

It is well-accepted that most osteogenic differentiation processes do need growth factors assistance to improve efficiency. As a material cue, hydroxyapatite (HAp) can promote osteogenic differentiation of stem cells only in a way. Up to now, rare work related to the relationship between HAp nanostructures and stem cells in osteogenic differentiation process without the assistance of growth factors has been reported. In this study, one-dimensional (1D) HAp nanostructures with tunable length were synthesized by an oleic acid assisted solvothermal method by adjusting the alcohol/water ratio (η). The morphology of 1D HAp nanostructures can be changed from long nanowires into nanorods with the η value change. Different substrates constructed by 1D HAp nanostructures were prepared to investigate the effect of morphology of nanostructured HAp on stem cell fate without any growth factors or differentiation induce media. Human adipose-derived stem cells (hADSCs), a kind of promising stem cell for autologous stem cell tissue engineering, were used as the stem cell model. The experiments prove that HAp morphology can determine the performance of hADSCs cultured on different substrates. Substrate constructed by HAp nanorods (100 nm) is of little benefit to osteogenic differentiations. Substrate constructed on HAp long nanowires (50 μm) causes growth and spread inhibition of hADSCs, which even causes most cells death after 7 days of culture. However, substrate constructed by HAp short nanowires (5 μm) can destine the hADSCs differentiation to osteoblasts efficiently in normal medium (after 3 weeks) without any growth factors. It is surprise that hADSCs have changed to polyhedral morphology and exhibited the tendency to osteogenic differentiation after only 24 h culture. Hydroxyapatite nanostructures mediated stem cell osteogenic differentiation excluding growth factors provides a powerful cue to design biomaterials with special nanostructures, and helps to elucidate the interaction of stem cell and biomaterials nanostructures. The results from this study are promising for application in bone tissue engineering.

The combination of miR-122 overexpression and Let-7f silencing induces hepatic differentiation of adipose tissue-derived stem cells.

Human adipose tissue-derived stem cells (hADSCs) have been considered as a promising source for cell therapy of liver diseases due to their accessibility, abundance, and expression of hepatocyte markers. Currently, the important role of certain microRNAs (miRNAs) has been reported during hepatic differentiation of stem cells. However, the combination effect of miRNAs on hepatic differentiation of these cells needs to be more investigated. The present study seeks to determine whether the combination of miRNAs can enhance hepatic differentiation of hADSCs in the absence of any other stimulation. First, lentiviral transduction was used to overexpress miR-122 and silence d let-7f in hADSCs for up to 21 days. Then, hepatic functionality was evaluated by analyzing specific hepatocyte genes and biochemical markers at different time points of differentiation induction. Stable miR-122 overexpression and let-7f silencing together in hADSCs resulted in increased expression of hepatocyte markers including ALB, AFP, CK18, CK19, and HNF4a. In addition, urea and albumin production, immunocytochemistry, and glycogen staining confirmed that the treated cells differentiated toward hepatocyte-like cells. Therefore, our findings demonstrate the possibility of using microRNAs to induce hADSCs into functional hepatocyte-like cells.