Expression of liver‐specific markers in naïve adipose‐derived mesenchymal stem cells

Background: Increasing evidence suggests that adipose tissue contains mesenchymal stem cells (MSC) that possess the ability to transdifferentiate into other cell types including hepatocytes, similar to bone marrow‐derived stem cells. The existence of precommitted cells in the MSC population may explain transdifferentiation. Aims: Our aim was to identify a population of putative hepatocyte‐like precursor cells in human adipose tissue. Methods: We analysed the ‘basal’ hepatic potential of undifferentiated, naïve human adipose‐derived mesenchymal stem cells (hADMSC). hADMSC were isolated from human adipose tissue and characterized for cell surface markers and for liver‐specific gene expression. Results: The isolated undifferentiated naïve hADMSCs expressed MSC surface markers. They also expressed α‐fetoprotein, CK18, CK19 and HNF4, which are known as early liver expressing genes. Interestingly, the undifferentiated naïve hADMSC were also positive for albumin, G‐6‐P and α‐1‐antitrypsin (AAT), which are all known to be predominantly expressed in adult liver cells. These cells acquired a hepatocyte‐specific phenotype and function upon treatment with a differentiation medium, resulting in the upregulation of albumin, G‐6‐P and AAT. Moreover, urea production, glycogen storage ability and cellular uptake of indocyanine green, which were absent in the basal state, were evident in the treated cells. Conclusions: Our findings suggest the presence of cells with hepatocyte‐like properties that are isolated from human adipose tissue and that can readily acquire hepatocyte‐like functions. Adipose tissue could thus be an exciting alternative means for repopulating the liver after various injuries, and might serve as a source for the transplantation of liver cells.     

Insight into the development of obesity: functional alterations of adipose‐derived mesenchymal stem cells

Obesity is associated with a variety of disorders including cardiovascular diseases, diabetes mellitus and cancer. Obesity changes the composition and structure of adipose tissue, linked to pro‐inflammatory environment, endocrine/metabolic dysfunction, insulin resistance and oxidative stress. Adipose‐derived mesenchymal stem cells (ASCs) have multiple functions like cell renewal, spontaneous repair and homeostasis in adipose tissue. In this review article, we have summarized the recent data highlighting that ASCs in obesity are defective in various functionalities and properties including differentiation, angiogenesis, motility, multipotent state, metabolism and immunomodulation. Inflammatory milieu, hypoxia and abnormal metabolites in obese tissue are crucial for impairing the functions of ASCs. Further work is required to explore the precise molecular mechanisms underlying its alterations and impairments. Based on these data, we suggest that deregulated ASCs, possibly also other mesenchymal stem cells, are important in promoting the development of obesity. Restoration of ASCs/mesenchymal stem cells might be an additional strategy to combat obesity and its associated diseases.     

Antiinflammatory and chondroprotective effects of intraarticular injection of adipose‐derived stem cells in experimental osteoarthritis

Objective

In experimental collagenase‐induced osteoarthritis (OA) in the mouse, synovial lining macrophages are crucial in mediating joint destruction. It was recently shown that adipose‐derived stem cells (ASCs) express immunosuppressive characteristics. This study was undertaken to explore the effect of intraarticular injection of ASCs on synovial lining thickness and its relation to joint pathology in experimental mouse OA.

Methods

ASCs were isolated from fat surrounding the inguinal lymph nodes and cultured for 2 weeks. Experimental OA was induced by injection of collagenase into the knee joints of C57BL/6 mice. OA phenotypes were measured within 8 weeks after induction. Histologic analysis was performed, and synovial thickening, enthesophyte formation, and cartilage destruction were measured in the knee joint.

Results

ASCs were injected into the knee joints of mice 7 days after the induction of collagenase‐induced OA. On day 1, green fluorescent protein–labeled ASCs were attached to the lining layer in close contact with macrophages. Thickening of the synovial lining, formation of enthesophytes associated with medial collateral ligaments, and formation of enthesophytes associated with cruciate ligaments were significantly inhibited on day 42 after ASC treatment, by 31%, 89%, and 44%, respectively. Destruction of cartilage was inhibited on day 14 (65%) and day 42 (35%). In contrast to early treatment, injection of ASCs on day 14 after OA induction showed no significant effect on synovial activation or joint pathology on day 42.

Conclusion

These findings indicate that a single injection of ASCs into the knee joints of mice with early‐stage collagenase‐induced OA inhibits synovial thickening, formation of enthesophytes associated with ligaments, and cartilage destruction.

     

Protective effect of melatonin‐supported adipose‐derived mesenchymal stem cells against small bowel ischemia‐reperfusion injury in rat

We tested the hypothesis that combined melatonin and autologous adipose‐derived mesenchymal stem cells (ADMSC) was superior to either alone against small bowel ischemia‐reperfusion (SBIR) injury induced by superior mesenteric artery clamping for 30 min followed by reperfusion for 72 hr. Male adult Sprague Dawley rats (n = 50) were equally categorized into sham‐operated controls SC, SBIR, SBIR‐ADMSC (1.0 × 10⁶ intravenous and 1.0 × 10⁶ intrajejunal injection), SBIR‐melatonin (intraperitoneal 20 mg/kg at 30 min after SI ischemia and 50 mg/kg at 6 and 18 hr after SI reperfusion), and SBIR‐ADMSC‐melatonin groups. The results demonstrated that the circulating levels of TNF‐α, MPO, LyG6+ cells, CD68+ cells, WBC count, and gut permeability were highest in SBIR and lowest in SC, significantly higher in SBIR‐ADMSC group and further increased in SBIR‐melatonin group than in the combined therapy group (all P < 0.001). The ischemic mucosal damage score, the protein expressions of inflammation (TNF‐α, NF‐κB, MMP‐9, MPO, and iNOS), oxidative stress (NOX‐1, NOX‐2, and oxidized protein), apoptosis (APAF‐1, mitochondrial Bax, cleaved caspase‐3 and PARP), mitochondrial damage (cytosolic cytochrome C) and DNA damage (γ‐H2AX) markers, as well as cellular expressions of proliferation (PCNA), apoptosis (caspase‐3, TUNEL assay), and DNA damage (γ‐H2AX) showed an identical pattern, whereas mitochondrial cytochrome C exhibited an opposite pattern compared to that of inflammation among all groups (all P < 0.001). Besides, antioxidant expressions at protein (NQO‐1, GR, and GPx) and cellular (HO‐1) levels progressively increased from SC to the combined treatment group (all P < 0.001). In conclusion, combined melatonin‐ADMSC treatment offered additive beneficial effect against SBIR injury.     

TIPE2-modified human amnion-derived mesenchymal stem cells promote the efficacy of allogeneic heart transplantation through inducing immune tolerance

BackgroundImmune rejection of heart transplantation has been regarded as the biggest challenge encountered by a patient suffering from end-stage heart disease. The transplantation of human amnion-derived mesenchymal stem cells (hAD-MSCs) has exhibited promising application prospects in organ transplantation. However, its persistent unsatisfactory tolerance has limited the widespread application of this technology. We aim to investigate the role of tumor necrosis factor-α-induced protein-8 like-2 (TIPE2)-mediated hAD-MSCs in immune tolerance in heart transplantation and its molecular regulatory mechanisms.MethodsThis project detected the effect of TIPE2 on immune tolerance by constructing an allogeneic heart transplantation mouse model through which TIPE2-overexpressed hAD-MSCs were injected into recipients. The fluorescence distribution of TIPE2-hAD-MSCs in mice was observed by a small animal in vivo imaging system. Pathological changes of the transplanted heart were detected by hematoxylin and eosin (HE) staining. Flow cytometry was performed to detect the content of cardiac lymphocytes. The expression of immune-induced related factors was measured by quantitative real-time PCR (qRT-PCR) and western blot assays.ResultsTIPE2-hAD-MSCs protected myocardial tissue structures, reduced the spleen and thymus indexes in recipient mice, minimized the content of cardiac lymphocytes, reduced expressions of ERK, p38, and IFN-γ, and elevated expressions of both IL-10 and TGF-β, markedly improving the survival time and survival rates of recipient mice.ConclusionsTIPE2-hAD-MSCs induce immune tolerance and improve the survival rates of allogeneic heart transplantation in mice. This study is expected to offer an ideal source and target of cells for organ transplantation.     

Potent induction of chondrocytic differentiation of human adipose‐derived adult stem cells by bone morphogenetic protein 6

Objective

Recent studies have identified an abundant source of multipotent progenitor cells in subcutaneous human adipose tissue, termed human adipose‐derived adult stem cells (ADAS cells). In response to specific media formulations, including transforming growth factor β1 (TGFβ1), these cells exhibit significant ability to differentiate into a chondrocyte‐like phenotype, expressing cartilage‐specific genes and proteins such as aggrecan and type II collagen. However, the influence of other growth factors on the chondrogenic differentiation of ADAS cells is not fully understood. This study was undertaken to investigate the effects of TGFβ1, TGFβ3, insulin‐like growth factor 1, bone morphogenetic protein 6 (BMP‐6), and dexamethasone, in various combinations, on the chondrogenic potential of ADAS cells in alginate beads.

Methods

The chondrogenic response of alginate‐encapsulated ADAS cells was measured by quantitative polymerase chain reaction, ³H‐proline and ³⁵S‐sulfate incorporation, and immunolabeling for specific extracellular matrix components.

Results

Significant differences in chondrogenesis were observed under the different culture conditions for all outcomes measured. Most notably, BMP‐6 up‐regulated AGC1 and COL2A1 expression by an average of 205‐fold and 38‐fold, respectively, over day‐0 controls, while down‐regulating COL10A1 expression by ∼2‐fold.

Conclusion

These findings suggest that BMP‐6 is a potent inducer of chondrogenesis in ADAS cells, in contrast to mesenchymal stem cells, which exhibit increased expression of type X collagen and a hypertrophic phenotype in response to BMP‐6. Combinations of growth factors containing BMP‐6 may provide a novel means of regulating the differentiation of ADAS cells for applications in the tissue‐engineered repair or regeneration of articular cartilage.

     

Melatonin facilitates adipose‐derived mesenchymal stem cells to repair the murine infarcted heart via the SIRT1 signaling pathway

Mesenchymal stem cells (MSCs)‐based therapy provides a promising therapy for the ischemic heart disease (IHD). However, engrafted MSCs are subjected to acute cell death in the ischemic microenvironment, characterized by excessive inflammation and oxidative stress in the host's infarcted myocardium. Melatonin, an indole, which is produced by many organs including pineal gland, has been shown to protect bone marrow MSCs against apoptosis although the mechanism of action remains elusive. Using a murine model of myocardial infarction (MI), this study was designed to evaluate the impact of melatonin on adipose‐derived mesenchymal stem cells (AD‐MSCs)‐based therapy for MI and the underlying mechanism involved with a focus on silent information regulator 1(SIRT1) signaling. Our results demonstrated that melatonin promoted functional survival of AD‐MSCs in infarcted heart and provoked a synergetic effect with AD‐MSCs to restore heart function. This in vivo effect of melatonin was associated with alleviated inflammation, apoptosis, and oxidative stress in infarcted heart. In vitro studies revealed that melatonin exert cytoprotective effects on AD‐MSCs against hypoxia/serum deprivation (H/SD) injury via attenuating inflammation, apoptosis, and oxidative stress. Mechanistically, melatonin enhanced SIRT1 signaling, which was accompanied with the increased expression of anti‐apoptotic protein Bcl2, and decreased the expression of Ac‐FoxO1, Ac‐p53, Ac‐NF‐ΚB, and Bax. Taken together, our findings indicated that melatonin facilitated AD‐MSCs‐based therapy in MI, possibly through promoting survival of AD‐MSCs via SIRT1 signaling. Our data support the promise of melatonin as a novel strategy to improve MSC‐based therapy for IHD, possibly through SIRT1 signaling evocation.     

Additional benefit of combined therapy with melatonin and apoptotic adipose‐derived mesenchymal stem cell against sepsis‐induced kidney injury

This study tested whether combined therapy with melatonin and apoptotic adipose‐derived mesenchymal stem cells (A‐ADMSCs) offered additional benefit in ameliorating sepsis‐induced acute kidney injury. Adult male Sprague–Dawley rats (n = 65) were randomized equally into five groups: Sham controls (SC), sepsis induced by cecal‐ligation and puncture (CLP), CLP‐melatonin, CLP‐A‐ADMSC, and CLP‐melatonin‐A‐ADMSC. Circulating TNF‐α level at post‐CLP 6 hr was highest in CLP and lowest in SC groups, higher in CLP‐melatonin than in CLP‐A‐ADMSC and CLP‐melatonin‐A‐ADMSC groups (all P < 0.001). Immune reactivity as reflected in the number of splenic helper‐, cytoxic‐, and regulatory‐T cells at post‐CLP 72 hr exhibited the same pattern as that of circulating TNF‐α among all groups (P < 0.001). The histological scoring of kidney injury and the number of F4/80+ and CD14+ cells in kidney were highest in CLP and lowest in SC groups, higher in CLP‐melatonin than in CLP‐A‐ADMSC and CLP‐melatonin‐A‐ADMSC groups, and higher in CLP‐A‐ADMSC than in CLP‐melatonin‐A‐ADMSC groups (all P < 0.001). Changes in protein expressions of inflammatory (RANTES, TNF‐1α, NF‐κB, MMP‐9, MIP‐1, IL‐1β), apoptotic (cleaved caspase 3 and PARP, mitochondrial Bax), fibrotic (Smad3, TGF‐β) markers, reactive‐oxygen‐species (NOX‐1, NOX‐2), and oxidative stress displayed a pattern identical to that of kidney injury score among the five groups (all P < 0.001). Expressions of antioxidants (GR+, GPx+, HO‐1, NQO‐1+) were lowest in SC group and highest in CLP‐melatonin‐A‐ADMSC group, lower in CLP than in CLP‐melatonin and CLP‐A‐ADMSC groups, and lower in CLP‐melatonin‐ than in CLP‐A‐ADMSC‐tretaed animals (all P < 0.001). In conclusion, combined treatment with melatonin and A‐ADMSC was superior to A‐ADMSC alone in protecting the kidneys from sepsis‐induced injury.     

Melatonin treatment improves adipose‐derived mesenchymal stem cell therapy for acute lung ischemia–reperfusion injury

This study investigated whether melatonin‐treated adipose‐derived mesenchymal stem cells (ADMSC) offered superior protection against acute lung ischemia–reperfusion (IR) injury. Adult male Sprague‐Dawley rats (n = 30) were randomized equally into five groups: sham controls, lung IR–saline, lung IR–melatonin, lung IR–melatonin–normal ADMSC, and lung IR–melatonin–apoptotic ADMSC. Arterial oxygen saturation was lowest in lung IR–saline; lower in lung IR–melatonin than sham controls, lung IR–melatonin–normal ADMSC, and lung IR–melatonin–apoptotic ADMSC; lower in lung IR–melatonin–normal ADMSC than sham controls and lung IR–melatonin–apoptotic ADMSC; lower in lung IR–melatonin–apoptotic ADMSC than sham controls (P < 0.0001 in each case). Right ventricular systolic blood pressure (RVSBP) showed a reversed pattern among all groups (all P < 0.0001). Changes in histological scoring of lung parenchymal damage and CD68+ cells showed a similar pattern compared with RVSBP in all groups (all P < 0.001). Changes in inflammatory protein expressions such as VCAM‐1, ICAM‐1, oxidative stress, TNF‐α, NF‐κB, PDGF, and angiotensin II receptor, and changes in apoptotic protein expressions of cleaved caspase 3 and PARP, and mitochondrial Bax, displayed identical patterns compared with RVSBP in all groups (all P < 0.001). Numbers of antioxidant (GR+, GPx+, NQO‐1+) and endothelial cell biomarkers (CD31+ and vWF+) were lower in sham controls, lung IR–saline, and lung IR–melatonin than lung IR–melatonin–normal ADMSC and lung IR–melatonin–apoptotic ADMSC, and lower in lung IR–melatonin–normal ADMSC than lung IR–melatonin–apoptotic ADMSC (P < 0.001 in each case). In conclusion, when the animals were treated with melatonin, the apoptotic ADMSC were superior to normal ADMSC for protection of lung from acute IR injury.     

Melatonin treatment further improves adipose‐derived mesenchymal stem cell therapy for acute interstitial cystitis in rat

This study tests the hypothesis that combined melatonin and adipose‐derived mesenchymal stem cell (ADMSC, 1.2 × 10⁶ given intravenously) treatment offer superior protection against cyclophosphamide (CYP 150 mg/kg)‐induced acute interstitial cystitis (AIC) in rats. Male adult Sprague‐Dawley rats were treated as follows: sham controls, AIC alone, AIC + melatonin, AIC + ADMSC, and AIC + melatonin +ADMSC. When melatonin was used, it was given as follows: 20 mg/kg at 30 min after CYP and 50 mg/kg at 6 and 18 hr after CYP. Twenty‐four‐hour urine volume, urine albumin level, and severity of hematuria were highest in AIC rats and lowest in the controls; likewise urine volume was higher in AIC + melatonin rats than in AIC + ADMSC and AIC + melatonin + ADMSC treated rats; in all cases, P < 0.001. The numbers of CD14+, CD74+, CD68+, MIP+, Cox‐2+, substance P+, cells and protein expression of IL‐6, IL‐12, RANTES, TNF‐α, NF‐κB, MMP‐9, iNOS (i.e. inflammatory biomarkers), glycosaminoglycan level, expression of oxidized protein, and protein expression of reactive oxygen species (NOX‐1, NOX‐2, NOX‐4) in the bladder tissue exhibited an identical pattern compared with that of hematuria among the five groups (all P < 0.0001). The integrity of epithelial layer and area of collagen deposition displayed an opposite pattern compared to that of hematuria among all groups (P < 0.0001). The cellular expressions of antioxidants (GR, GPx, HO‐1, NQO 1) showed a significant progressive increase form controls to AIC + melatonin + ADMSC (all P < 0.0001). Combined regimen of melatonin and ADMSC was superior to either alone in protecting against CYP‐induced AIC.     

Effect of human umbilical cord blood‐derived mesenchymal stem cells in a cirrhotic rat model

Background/Aim: Cirrhosis is a long‐term consequence of chronic hepatic injury and no effective therapy is currently available for this disease. Recent reports have shown that the mesenchymal stem cells (MSCs) have the capacity to differentiate into hepatocytes, and umbilical cord blood is a rich source of MSCs. Hence, we investigated the effect of infusing of human umbilical cord blood‐derived MSCs (HMSCs) in carbon tetrachloride (CCl₄)‐induced cirrhosis in a rat model. Methods: The effect of HMSCs on cirrhosis was evaluated using haematoxylin and eosin and Masson's trichrome staining. To evaluate cirrhosis‐related factors, we measured protein and mRNA expression of transforming growth factor β₁ (TGF‐β₁), collagen type I and α‐smooth muscle actin (α‐SMA). Results: Histological findings showed that liver fibrosis in rats was alleviated by HMSCs infusion. Interestingly, CM‐DiI‐labelled HMSCs expressed the hepatocyte‐specific markers, human albumin and α‐fetoprotein. Infusion of HMSCs significantly inhibited TGF‐β₁, collagen type I and α‐SMA expressions in CCl₄‐induced cirrhotic rats. Conclusion: Our results showed that HMSCs infusion could improve liver fibrosis in rats with CCl₄‐induced cirrhosis, raising the possibility for clinical use of HMSCs in the treatment of cirrhosis.     

利用共培养法诱导人胎盘间充质干细胞向成骨细胞分化

目的观察体外人足月胎盘间充质干细胞(h PMSCs)和成骨细胞共培养体系条件下成骨细胞对h PMSCs分化的影响。方法采用胶原酶消化法从人足月胎盘中分离纯化间充质干细胞(MSCs),检测细胞表面标志物、生长曲线、细胞超微结构及成骨能力并对h PMSCs进行鉴定。共培养组将成骨细胞接种于Transwell双层培养皿底层,h PMSCs接种于上层;对照组上层与底层均接种h PMSCs。对诱导后细胞进行碱性磷酸酶染色鉴定。结果胎盘分离细胞经形态、生长速度、细胞表面标志物(CD44和CD29阳性表达为99%,CD34和CD106为1%),确定为胎盘间充质干细胞;头盖骨分离细胞经碱性磷酸酶染色确定为成骨细胞。采用Transwell共培养h PMSCs和成骨细胞组碱性磷酸酶活性染色阳性率为(21.7±5.3)%,表现成骨细胞特性,对照组染色呈阴性。结论人足月胎盘含MSCs,与其他来源MSCs生物学特性相似,成骨细胞生长过程提供的微环境对h PMSCs分化为成骨细胞具有诱导促进作用。