Autologous microfragmented adipose tissue treatment of knee osteoarthritis demonstrates effectiveness in 68% of patients at 4-year follow-up.

BACKGROUND: Adipose tissue-derived stem cells are an interesting therapeutic option for early knee osteoarthritis (OA) treatment due to their high plasticity, easiness of harvesting and rapidity of administration. The aim of this study was to evaluate the medium-term effectiveness and safety of Microfragmented Autologous Fat Tissue (MFAT) injection treatment at 4-year follow-up and to investigate potential correlations among patients' pre-treatment clinical condition and clinical outcomes to identify possible predicting factors for procedure success or failure. PATIENTS AND METHODS: This is a prospective trial enrolling 46 patients with diagnosis of symptomatic knee OA and failure of previous conservative measures who underwent diagnostic arthroscopy and single autologous MFAT injection between June 2017 and July 2018. Patients were assessed with repeated clinical scoring systems at baseline, 6 months, 1 and 4 years after surgery. The evaluation included demographic characteristics, arthroscopic findings, and stem cell number from injected tissue. RESULTS: No major complications were reported during follow-up period and there was a significant increase of Lysholm knee score from baseline value of 61.7 ± 13.8 to 79.5 ± 16.9 at 4 years (p < 0.001). The WOMAC score increased from a baseline value of 66.5 ± 14.7 to 82.8 ± 15.7 at 4 years (p < 0.001) and there was a significant decrease of VAS pain score from baseline value of 6.3 ± 1.5 to 3.5 ± 2.6 at 4-year follow-up (p < 0.001). ROM improved significantly from 118.4 ± 2.6 to 122.5 ± 2.5 at 12 months (p < 0.001), but did not improve at 4 years (p > 0.05). 15 patients (32.6%) were considered treatment failures, because they required secondary surgery, further injection therapy or experienced symptoms persistence. Patient with synovitis had 75% failure rate, although synovitis did not result as a statistically significant factor influencing clinical outcome up to 4-year follow-up (p = 0.058). Age, cartilage defects severity, BMI, concomitant procedures, and stem cell number from injected MFAT did not show any significant correlation with the results. CONCLUSIONS: MFAT intra-articular injection is a safe procedure with positive improvements up to 4-year follow-up in patients with early knee OA. These findings suggest MFAT could be a minimally invasive treatment of early knee OA with durable benefits at mid-term evaluation. TRIAL REGISTRATION: IRB number ID-3522.

Decreased miR-17-92 cluster correlates with senescence features, disrupted oxidative homeostasis, and impaired therapeutic efficacy of mesenchymal stem cells.

Aging and replicative cellular senescence are associated with the reduced therapeutic potential of mesenchymal stem cells (MSCs) on a variety of diseases. This study aimed to determine the mechanism in MSC senescence and further explore a modification strategy to reverse senescence-associated cell dysfunction to improve the therapeutic efficacy of MSCs on acute liver failure (ALF). We found that the adipose tissue-derived MSCs from old mice (oAMSCs) exhibited senescence phenotypes and showed reduced therapeutic efficacy in lipopolysaccharide and D-galactosamine-induced ALF, as shown by the increased hepatic necrosis, liver histology activity index scores, serum liver function indicator levels, and inflammatory cytokine levels. The expression of miR-17-92 cluster members, especially miR-17 and miR-20a, was obviously decreased in oAMSCs and replicatively senescent AMSCs, and was consistent with the decreased oncogene c-Myc level during AMSC senescence and may mediate c-Myc stemness addiction. Further experiments revealed that c-Myc-regulated miR-17-92 expression contributed to increased p21 expression and redox system dysregulation during AMSC senescence. Furthermore, modification of AMSCs with the two key miRNAs in the miR-17-92 cluster mentioned above reversed the senescence features of oAMSCs and restored the therapeutic effect of senescent AMSCs on ALF. In conclusion, the cellular miR-17-92 cluster level is correlated with AMSC senescence and can be used both as an index for evaluating and as a modification target for improving the therapeutic potential of AMSCs.NEW & NOTEWORTHY We reported for the first time that c-Myc-regulated miR-17-92 contributed to increased p21 expression and redox system dysregulation during AMSC senescence and was associated with the reduced therapeutic effects of senescent AMSCs on ALF. Moreover, modifying the expression of the miR-17-92 cluster members, especially miR-17 and/or miR-20a, could reverse AMSC senescence. Thus, miR-17-92 cluster can be used both as an index for evaluating and as a modification strategy for improving the therapeutic potential of AMSCs.

Implantation of CPT1AM-expressing adipocytes reduces obesity and glucose intolerance in mice.

Obesity and its associated metabolic comorbidities are a rising global health and social issue, with novel therapeutic approaches urgently needed. Adipose tissue plays a key role in the regulation of energy balance and adipose tissue-derived mesenchymal stem cells (AT-MSCs) have gained great interest in cell therapy. Carnitine palmitoyltransferase 1A (CPT1A) is the gatekeeper enzyme for mitochondrial fatty acid oxidation. Here, we aimed to generate adipocytes expressing a constitutively active CPT1A form (CPT1AM) that can improve the obese phenotype in mice after their implantation. AT-MSCs were differentiated into mature adipocytes, subjected to lentivirus-mediated expression of CPT1AM or the GFP control, and subcutaneously implanted into mice fed a high-fat diet (HFD). CPT1AM-implanted mice showed lower body weight, hepatic steatosis and serum insulin and cholesterol levels alongside improved glucose tolerance. HFD-induced increases in adipose tissue hypertrophy, fibrosis, inflammation, endoplasmic reticulum stress and apoptosis were reduced in CPT1AM-implanted mice. In addition, the expression of mitochondrial respiratory chain complexes was enhanced in the adipose tissue of CPT1AM-implanted mice. Our results demonstrate that implantation of CPT1AM-expressing AT-MSC-derived adipocytes into HFD-fed mice improves the obese metabolic phenotype, supporting the future clinical use of this ex vivo gene therapy approach.

LNX2 involves in the role of ghrelin to promote the neuronal differentiation of adipose tissue-derived mesenchymal stem cells.

Adipose tissue-derived mesenchymal stem cells (ADSCs) have promising effects on nerve repair due to the differentiation ability to neural cells. Ghrelin has been shown to promote the neural differentiation of ADSCs. This work was designed to explore its underlying mechanism. Herein, we found high expression of LNX2 in ADSCs after neuronal differentiation. Knockdown of LNX2 might block neuronal differentiation of ADSCs, as evidenced by the decreased number of neural-like cells and dendrites per cell, and the reduced expressions of neural markers (including ß-Tubulin III, Nestin, and MAP2). We also demonstrated that LNX2 silencing suppressed the nuclear translocation of ß-catenin in differentiated ADSCs. Luciferase reporter assay indicated that LNX2 inhibited wnt/ß-catenin pathway by reducing its transcriptional activity. In addition, results showed that LNX2 expression was increased by ghrelin, and its inhibition diminished the effects of ghrelin on neuronal differentiation. Altogether, the results suggest that LNX2 is involved in the role of ghrelin to facilitate neuronal differentiation of ADSCs.

Biological nerve conduit model with de-epithelialized human amniotic membrane and adipose-derived mesenchymal stem cell sheet for repair of peripheral nerve defects.

In this study, a biological conduit, consisting of an adipocyte-derived mesenchymal stem cell (AdMSCs) sheet and amniotic membrane (AM), was designed for the reconstruction of peripheral nerve defects. To evaluate the effect of the produced conduit on neural regeneration, a 10-mm sciatic nerve defect was created in rats, and experiments were carried out on six groups, i.e., sham control group (SC), negative control group (NC), nerve autograft group (NG), the biological conduit (AdMSCs + AM) group, the commercial PGA tube conduit (PGA) group, and the conduit only consisting of AM (AM) group. The effects of different nerve repair methods on the peripheral nerve and gastrocnemius muscle were evaluated by functional, histological, and immunohistochemical tests. When the number of myelinated axons was compared between the groups of AdMSCs + AM and PGA, it was higher in the AdMSCs + AM group (p < 0.05). The percentage of gastrocnemius collagen bundle area of AdMSCs + AM group was found to be statistically lower than the PGA group (p < 0.05). The muscle fiber diameter of AdMSCs + AM group was lower than that of the NG group, but significantly higher than that of the PGA group and the AM group (p < 0.001). Muscle weight index was significantly higher in the AdMSCs + AM group compared to the PGA group (p < 0.05). It was observed that nerve regeneration was faster in the AdMSCs + AM group, and there was an earlier improvement in pin-prick score and sciatic functional index compared to the PGA group and the AM group. In conclusion, the biological conduit prepared from the AdMSCs sheet and AM is regarded as a new biological conduit that can be used as an alternative treatment method to nerve autograft in clinical applications.

Schizophyllan promotes osteogenic differentiation of human adipose tissue-derived mesenchymal stem cells in vitro.

BACKGROUND: Bioactive polysaccharides are a promising way for bone disease prevention with high efficiency. Schizophyllan (SPG) is a polysaccharide derived from a species of fungus with anticancer, antitumor, and anti-inflammatory effects. In the present study, for the first time, the cell proliferation, osteogenic markers, mineral deposition, and osteogenic gene expression of human adipose tissue-derived mesenchymal stem cells (hADMSCs) grown on SPG were evaluated by in vitro assays. METHODS AND RESULTS: The cytotoxicity of SPG was measured using the MTT assay and acridine orange staining. Differentiation of hADMSCs was assessed using alkaline phosphatase (ALP) activity test, cellular calcium content assay, and mineralized matrix staining. To this end, Alizarin red S, von Kossa staining, and the expression of bone-specific markers, including ALP, Runx2, and osteonectin, were used by real-time RT-PCR over a 2-week period. According to the results, SPG at 10 µg/ml concentration was determined as the optimal dosage for differentiation studies. The results of osteogenic differentiation tests showed that compared to the control groups in vitro, SPG enhanced the osteogenic markers and mineralization as well as upregulation of the expression of bone specific genes in differentiated hADMSCs during differentiation. CONCLUSIONS: The results revealed that SPG could be applied as effective factor for osteogenic differentiation in the future. The current study provides insights into the hADMSC-based treatment and introduces promising therapeutic material for individuals who suffer from bone defects and injuries.

NAMPT encapsulated by extracellular vesicles from young adipose-derived mesenchymal stem cells treated tendinopathy in a "One-Stone-Two-Birds" manner.

BACKGROUND: Tendinopathy is the leading sports-related injury and will cause severe weakness and tenderness. Effective therapy for tendinopathy remains limited, and extracellular vesicles (EVs) derived from adipose tissue-derived mesenchymal stem cells (ADMSCs) have demonstrated great potential in tendinopathy treatment; however, the influence of aging status on EV treatment has not been previously described. RESULTS: In this study, it was found that ADMSCs derived from old mice (ADMSCold) demonstrated remarkable cellular senescence and impaired NAD+ metabolism compared with ADMSCs derived from young mice (ADMSCyoung). Lower NAMPT contents were detected in both ADMSCold and its secreted EVs (ADMSCold-EVs). Advanced animal experiments demonstrated that ADMSCyoung-EVs, but not ADMSCold-EVs, alleviated the pathological structural, functional and biomechanical properties in tendinopathy mice. Mechanistic analyses demonstrated that ADMSCyoung-EVs improved cell viability and relieved cellular senescence of tenocytes through the NAMPT/SIRT1/PPARγ/PGC-1α pathway. ADMSCyoung-EVs, but not ADMSCold-EVs, promoted phagocytosis and M2 polarization in macrophages through the NAMPT/SIRT1/Nf-κb p65/NLRP3 pathway. The macrophage/tenocyte crosstalk in tendinopathy was influenced by ADMSCyoung-EV treatment and thus it demonstrated "One-Stone-Two-Birds" effects in tendinopathy treatment. CONCLUSIONS: This study demonstrates an effective novel therapy for tendinopathy and uncovers the influence of donor age on curative effects by clarifying the detailed biological mechanism.

Age and synovitis affect the results of the treatment of knee osteoarthritis with Microfragmented Autologous Fat Tissue.

PURPOSE: This study aims to assess the effectiveness of Microfragmented Autologous Fat Tissue (MFAT) treatment for knee osteoarthritis and to investigate whether patients' pre-treatment clinical condition, such as synovitis, correlates with clinical outcomes, to identify potential predicting factors for the success or failure of the treatment. METHODS: In this prospective Cohort Study Level II multicentric trial, consecutive patients with a diagnosis of early/mild osteoarthritis and failure of previous conservative measures were enrolled to undergo diagnostic arthroscopy and a single MFAT injection. Patients were assessed with repeated scoring systems at baseline, 6 months, and 12 months after surgery. The demographic features, the arthroscopic findings, the immunophenotype of injected tissue and the histologic examination of synovia of failed patients were analyzed. RESULTS: Data from 91 patients showed a significant improvement in Lysholm, WOMAC scores at 1-year follow-up (p < 0.001). A significant decrease in VAS score was observed, while a significant improvement of measured flexion angle was registered at 1 year (p < 0.001). No major complications were reported. Age and synovitis were identified as significant factors influencing the clinical outcome (p < 0.05). Body mass index, previous or concomitant procedures, and specific cartilage defects had no influence. The mean number of injected adipose tissue-derived mesenchymal stem cells seem not to correlate with the clinical outcome. CONCLUSION: MFAT is effective in reducing pain when used with a single dose injection in early/mild OA of the knee, without major complications. Age over 60 and synovitis may be predictive for persistent pain at one year and should be considered before indications.

Adipose Tissue-Derived Mesenchymal Stem Cell-Derived Exosomes Promote Wound Healing and Tissue Regeneration.

Wound healing is a complex process involving cell proliferation, migration, and extracellular matrix (ECM) remodeling. Extracellular vesicles (EVs) or exosomes derived from adipose tissue-derived stem cells (ASCs) are emerging as promising alternatives to cell therapy for advanced wound healing. Hyaluronic acid (HA), a major component of the skin ECM, is widely utilized in wound dressings and dermal fillers. This study aimed to investigate the effects of ASC-derived exosomes (ASC-EXOs) on human dermal fibroblasts (HDFs) and their potential combination with HA in in vivo wound healing and dermal filler models. In HDFs, ASC-EXOs increased cell proliferation and migration. ASC-EXOs also upregulated the expression of genes involved in cell proliferation and wound healing while stimulating collagen production in HDFs. In a porcine wound healing model, topical treatment with a combination of HA and ASC-EXOs led to higher wound closure rates compared to HA alone. Histological examination showed increased re-epithelialization and collagen type III deposition in wounds treated with the combination of HA and ASC-EXOs. In a mouse dermal filler model, tissues injected with the combination of HA and ASC-EXOs exhibited thicker tissue layers, increased vascularization, enhanced infiltration of myofibroblasts, and higher levels of collagen III and collagen fiber content compared to HA alone. These findings suggest that ASC-EXOs have beneficial effects on cell proliferation, migration, and gene expression related to wound healing, and they may accelerate wound closure and promote tissue regeneration. Furthermore, the combination of HA and ASC-EXOs may enhance wound healing and tissue remodeling, indicating its potential for both clinical and regenerative aesthetic applications in skin repair and regeneration.

Therapeutic Potential of Exosomes Derived from Adipose Tissue-Sourced Mesenchymal Stem Cells in the Treatment of Neural and Retinal Diseases.

Therapeutic agents that are able to prevent or attenuate inflammation and ischemia-induced injury of neural and retinal cells could be used for the treatment of neural and retinal diseases. Exosomes derived from adipose tissue-sourced mesenchymal stem cells (AT-MSC-Exos) are extracellular vesicles that contain neurotrophins, immunoregulatory and angio-modulatory factors secreted by their parental cells. AT-MSC-Exos are enriched with bioactive molecules (microRNAs (miRNAs), enzymes, cytokines, chemokines, immunoregulatory, trophic, and growth factors), that alleviate inflammation and promote the survival of injured cells in neural and retinal tissues. Due to the nano-sized dimension and bilayer lipid envelope, AT-MSC-Exos easily bypass blood-brain and blood-retinal barriers and deliver their cargo directly into the target cells. Accordingly, a large number of experimental studies demonstrated the beneficial effects of AT-MSC-Exos in the treatment of neural and retinal diseases. By delivering neurotrophins, AT-MSC-Exos prevent apoptosis of injured neurons and retinal cells and promote neuritogenesis. AT-MSC-Exos alleviate inflammation in the injured brain, spinal cord, and retinas by delivering immunoregulatory factors in immune cells, suppressing their inflammatory properties. AT-MSC-Exos may act as biological mediators that deliver pro-angiogenic miRNAs in endothelial cells, enabling re-vascularization of ischemic neural and retinal tissues. Herewith, we summarized current knowledge about molecular mechanisms which were responsible for the beneficial effects of AT-MSC-Exos in the treatment of neural and retinal diseases, emphasizing their therapeutic potential in neurology and ophthalmology.

Synergic Effect of Early Administration of Probiotics and Adipose-Derived Mesenchymal Stem Cells on Alleviating Inflammation-Induced Chronic Neuropathic Pain in Rodents.

This study investigated the hypothesis that probiotics enhanced the therapeutic effect of adipose-derived mesenchymal stem cells (ADMSCs) on alleviating neuropathic pain (NP) due to chronic constriction injury (CCI) mainly through regulating the microbiota in rats. SD rats (n = 50) were categorized into group 1 (sham-control), group 2 (NP), group 3 (NP + probiotics (i.e., 1.5 billion C.F.U./day/rat, orally 3 h after NP procedure, followed by QOD 30 times)), group 4 (NP + ADMSCs (3.0 × 105 cells) 3 h after CCI procedure, followed by QOD six times (i.e., seven times in total, i.e., mimic a clinical setting of drug use) and group 5 (NP + probiotics + ADMSCs (3.0 × 105 cells)) and euthanized by day 60 after NP induction. By day 28 after NP induction, flow-cytometric analysis showed circulating levels of early (AN-V+/PI−) and late (AN-V+/PI+) apoptotic, and three inflammatory (CD11b-c+, Ly6G+ and MPO+) cells were lowest in group 1 and significantly progressively reduced in groups 2 to 5 (all p < 0.0001). By days 7, 14, 21, 28, and 60 after CCI, the thresholds of thermal paw withdrawal latency (PWL) and mechanical paw withdrawal threshold (PWT) were highest in group 1 and significantly progressively increased in groups 2 to 5 (all p < 0.0001). Numbers of pain-connived cells (Nav1.8+/peripherin+, p-ERK+/peripherin+, p-p38+/peripherin+ and p-p38+/NF200+) and protein expressions of inflammatory (p-NF-κB, IL-1ß, TNF-α and MMP-9), apoptotic (cleaved-caspase-3, cleaved-PARP), oxidative-stress (NOX-1, NOX-2), DNA-damaged (γ-H2AX) and MAPK-family (p-P38, p-JNK, p-ERK1/2) biomarkers as well as the protein levels of Nav.1.3, Nav.1.8, and Nav.1.9 in L4-L5 in dorsal root ganglia displayed an opposite pattern of mechanical PWT among the groups (all p < 0.0001). In conclusion, combined probiotic and ADMSC therapy was superior to merely one for alleviating CCI-induced NP mainly through suppressing inflammation and oxidative stress.

Improvement of an Effective Protocol for Directed Differentiation of Human Adipose Tissue-Derived Adult Mesenchymal Stem Cells to Corneal Endothelial Cells.

Corneal disease affects 12.5 million individuals worldwide, with 2 million new cases each year. The standard treatment consists of a corneal transplantation from a human donor; however, the worldwide demand significantly exceeds the available supply. Lamellar endothelial keratoplasty, the replacement of only the endothelial layer of the cornea, can partially solve the problem. Progressive efforts have succeeded in expanding hCECs; however, the ability to expand hCECs is still limited, and new sources of CECs are being sought. Crucial advances have been achieved by the directed differentiation of embryonic or induced pluripotent stem cells, but these cells have disadvantages, such as the use of oncogenes, and are still difficult to establish. We aimed to transfer such knowledge to obtain hCECs from adipose tissue-derived adult mesenchymal stem cells (ADSC) by modifying four previously published procedures. We present several protocols capable of the directed differentiation of human ADSCs to hCECs. In our hands, the protocol by Ali et al. was the best adapted to such differentiation in terms of efficiency, time, and financial cost; however, the protocol by Wagoner et al. was the best for CEC marker expression. Our results broaden the type of cells of autologous extraocular origin that could be employed in the clinical setting for corneal endothelial deficiency.

HATMSC Secreted Factors in the Hydrogel as a Potential Treatment for Chronic Wounds-In Vitro Study.

Mesenchymal stem cells (MSCs) can improve chronic wound healing; however, recent studies suggest that the therapeutic effect of MSCs is mediated mainly through the growth factors and cytokines secreted by these cells, referred to as the MSC secretome. To overcome difficulties related to the translation of cell therapy into clinical use such as efficacy, safety and cost, we propose a hydrogel loaded with a secretome from the recently established human adipose tissue mesenchymal stem cell line (HATMSC2) as a potential treatment for chronic wounds. Biocompatibility and biological activity of hydrogel-released HATMSC2 supernatant were investigated in vitro by assessing the proliferation and metabolic activity of human fibroblast, endothelial cells and keratinocytes. Hydrogel degradation was measured using hydroxyproline assay while protein released from the hydrogel was assessed by interleukin-8 (IL-8) and macrophage chemoattractant protein-1 (MCP-1) ELISAs. Pro-angiogenic activity of the developed treatment was assessed by tube formation assay while the presence of pro-angiogenic miRNAs in the HATMSC2 supernatant was investigated using real-time RT-PCR. The results demonstrated that the therapeutic effect of the HATMSC2-produced factors is maintained following incorporation into collagen hydrogel as confirmed by increased proliferation of skin-origin cells and improved angiogenic properties of endothelial cells. In addition, HATMSC2 supernatant revealed antimicrobial activity, and which therefore, in combination with the hydrogel has a potential to be used as advanced wound-healing dressing.

The Potential Role of Human NME1 in Neuronal Differentiation of Porcine Mesenchymal Stem Cells: Application of NB-hNME1 as a Human NME1 Suppressor.

This study aimed to investigate the effects of the human macrophage (MP) secretome in cellular xenograft rejection. The role of human nucleoside diphosphate kinase A (hNME1), from the secretome of MPs involved in the neuronal differentiation of miniature pig adipose tissue-derived mesenchymal stem cells (mp AD-MSCs), was evaluated by proteomic analysis. Herein, we first demonstrate that hNME1 strongly binds to porcine ST8 alpha-N-acetyl-neuraminide alpha-2,8-sialyltransferase 1 (pST8SIA1), which is a ganglioside GD3 synthase. When hNME1 binds with pST8SIA1, it induces degradation of pST8SIA1 in mp AD-MSCs, thereby inhibiting the expression of ganglioside GD3 followed by decreased neuronal differentiation of mp AD-MSCs. Therefore, we produced nanobodies (NBs) named NB-hNME1 that bind to hNME1 specifically, and the inhibitory effect of NB-hNME1 was evaluated for blocking the binding between hNME1 and pST8SIA1. Consequently, NB-hNME1 effectively blocked the binding of hNME1 to pST8SIA1, thereby recovering the expression of ganglioside GD3 and neuronal differentiation of mp AD-MSCs. Our findings suggest that mp AD-MSCs could be a potential candidate for use as an additive, such as an immunosuppressant, in stem cell transplantation.

Toxicological evaluation of exosomes derived from human adipose tissue-derived mesenchymal stem/stromal cells.

Several studies report that the therapeutic mechanism of action of mesenchymal stem/stromal cells (MSCs) is mainly mediated by paracrine factors that are released from MSCs such as exosomes. Exosomes are nano-sized extracellular vesicles that are transferred to target cells for cell-to-cell communication. Although MSC-derived exosomes (MSC-exosomes) are suggested as novel cell-free therapeutics for various human diseases, evaluation studies for the safety and toxicity of MSC-exosomes are limited. The purpose of our study was to evaluate the toxicological profile, including skin sensitization, photosensitization, eye and skin irritation, and acute oral toxicity using exosomes derived from human adipose tissue-derived MSCs (ASC-exosomes) in accordance with the OECD guidelines and the principles of Good Laboratory Practice. The ASC-exosomes were classified as a potential non-sensitizer in the skin sensitization test, UN GHS no category in the eye irritation test, and as a skin non-irritant in the skin irritation test, and did not induce any toxicity in the phototoxicity test or in acute oral toxicity testing. Our findings are the first to suggest that ASC-exosomes are safe for use as a topical treatment, with no adverse effects in toxicological testing, and have potential application as a therapeutic agent, cosmetic ingredient, or for other biological uses.

Osteochondral Regeneration Using Adipose Tissue-Derived Mesenchymal Stem Cells.

Osteoarthritis (OA) is a major joint disease that promotes locomotor deficiency during the middle- to old-age, with the associated disability potentially decreasing quality of life. Recently, surgical strategies to reconstruct both articular cartilage and subchondral bone for OA have been diligently investigated for restoring joint structure and function. Adipose tissue-derived mesenchymal stem cells (AT-MSCs), which maintain pluripotency and self-proliferation ability, have recently received attention as a useful tool to regenerate osteocartilage for OA. In this review, several studies were described related to AT-MSC spheroids, with scaffold and scaffold-free three-dimensional (3D) constructs produced using "mold" or "Kenzan" methods for osteochondral regeneration. First, several examples of articular cartilage regeneration using AT-MSCs were introduced. Second, studies of osteochondral regeneration (not only cartilage but also subchondral bone) using AT-MSCs were described. Third, examples were presented wherein spheroids were produced using AT-MSCs for cartilage regeneration. Fourth, osteochondral regeneration following autologous implantation of AT-MSC scaffold-free 3D constructs, fabricated using the "mold" or "Kenzan" method, was considered. Finally, prospects of osteochondral regeneration by scaffold-free 3D constructs using AT-MSC spheroids were discussed.

[Effect of basic fibroblast growth factor treatment on efficacy of adipose-derived mesenchymal stem cells in liver cirrhosis].

Objective: To observe the effect of basic fibroblast growth factor (bFGF) treatment on efficacy of adipose-derived mesenchymal stem cells (ADSCs) in cirrhotic rats. Methods: A rat model of liver cirrhosis was established via intraperitoneal injection of carbon tetrachloride (CCl(4)) for 10 weeks. Thirty SD rats were randomly divided into 3 groups (n = 10): control group served as group A, and 0.5ml of phosphate-buffered saline (PBS) was transfused into the tail vein; ADSCs single-dose transplantation group served as group B, and 1×10(6) ADSCs were transplanted into the tail vein; bFGF-treated ADSCs treatment group served as group C, and 1×10(6) bFGF-treated ADSCs were transplanted through tail vein. Liver function, pathological and cytokine changes, and the in vivo survival transformation condition of the transplanted cells were measured at one week after transplantation. F test and an independent sample t test were used. Results: bFGF treatment had significantly promoted the proliferation, differentiation and overexpression of hepatocyte growth factor (HGF) in ADSCs [ADSCs single: (2 137.16 ± 261.52) pg/ml vs. ADSCs (bFGF): (4 776.23 ± 532.44) pg/ml, P < 0.05]. The bFGF-treated ADSCs treatment group had statistically significant differences in promoting the recovery of liver function [alanine aminotransferase (ALT): ADSCs single (190.8 ± 34.98) vs. ADSCs (bFGF): (117.8 ± 35.81) pg/ml; aspartate aminotransferase (AST): ADSCs single (295.2 ± 33.71) U/L vs. ADSCs (bFGF): (183.8 ± 41.29) U/L, P ​​< 0.05). There was no statistically significant difference in serum albumin levels between the control group, ADSCs single group, and ADSCs (bFGF) group. Compared with the control group, the serum albumin level of ADSCs (bFGF) group was increased significantly (P < 0.05), and the difference between the control group and the ADSCs single transplantation group in improving liver regeneration and reducing liver damage was statistically significant (P < 0.05). Masson trichrome staining showed that the percentage of the liver fibrosis area in the bFGF-treated ADSCs treatment group was 6.78% ± 0.56%, which was significantly higher than that of the control and ADSCs single dose transplantation group (7.96% ± 0.64%) (P < 0.05). Western blot analysis showed that the expression of HGF protein in the bFGF-treated ADSCs treatment group was significantly up-regulated, while the expression of α-smooth muscle actin was significantly down-regulated, and the difference was significant in the ADSCs single transplantation group. A double fluorescent staining method showed that the numbers of stem cells implanted in the liver tissue of the bFGF-treated ADSCs treatment group were higher than that of the ADSCs single transplantation group. In-vitro cell experiments confirmed that bFGF had significantly promoted the overexpression of HGF in ADSCs. Conclusion: bFGF-treated ADSCs transplantation can significantly improve liver function and fibrosis as compared with ADSCs single-dose transplantation in cirrhotic rats.

H19/miR-30a/C8orf4 axis modulates the adipogenic differentiation process in human adipose tissue-derived mesenchymal stem cells.

The adipogenic differentiation of adipose tissue-derived mesenchymal stem cells (ADSCs) is a critical issue in many obesity-related disorders. Cytidine-cytidine-adenosine-adenosine-thymidine (CCAAT) enhancer binding protein α (CEBP-α) and peroxisome proliferator-activated receptor-γ are two important lipogenic and adipogenic transcription factors and markers in adipogenic differentiation. Noncoding RNAs participate in adipogenic differentiation. The long noncoding RNA (lncRNA) H19 is related to multiple cellular differentiation, including adipogenic differentiation; however, its function and precise molecular mechanism in human ADSCs (hADSCs) adipogenic differentiation are unclear. microRNAs that were differentially expressed in adipogenic differentiation and could be targeted by H19 were screened and selected; the regulation and interaction between H19 and miR-30a were verified. The interaction between miR-30a and predicted downstream target C8orf4 was validated. The dynamic effects of H19 and miR-30a on C8orf4 messenger RNA (mRNA) expression and protein and adipogenic differentiation were evaluated. miR-30a negatively regulated H19 with each other through direct binding. As predicted by TargetScan and verified using luciferase reporter gene assays, miR-30a directly bound to the 3'-untranslated region of C8orf4 to inhibit its expression; H19 knockdown suppressed while miR-30a inhibition promoted the mRNA expression and the protein levels of C8orf4 and adipogenic differentiation; the effect of H19 knockdown could be partially reversed by miR-30a inhibition. The lncRNA H19 serves as a competing endogenous RNA (ceRNA) for miR-30a to augment miR-30a downstream target C8orf4, therefore modulating adipogenic differentiation in hADSCs. From the perspective of lncRNA-miRNA-mRNA regulation, we provided a novel regulatory mechanism of hADSCs adipogenic differentiation.

Bone marrow macrophage M2 polarization and adipose-derived stem cells osteogenic differentiation synergistically promote rehabilitation of bone damage.

By differentiating into and the balance being regulated between M1 (pro-inflammatory) and M2 (anti-inflammatory) heterogeneous populations, macrophages play critical roles during the host immune response in various physiological contexts in both health and diseases. Besides regulating innate and adaptive immune capacity, macrophages are also decisively involved in tissue homeostasis. However, how resident macrophages are regulated after tissue damages is still far from elucidation. In the present study, we found that adipose-derived stem cells (ADSCs) apparently promote bone defect rehabilitation in vivo via skewing differentiation of bone marrow-derived macrophage (BMDMs) towards anti-inflammatory M2 macrophages. In vitro data demonstrated that although ADSCs have the potential to differentiate to osteoblasts and adipose cells by using standard tissue culture-differentiating conditions, these mesenchymal progenitors are mainly regulated to differentiate into osteoblasts with overexpressed runt-related transcription factor 2, osteoprotegerin, osterix, and downregulated receptor activator of nuclear factor κB ligand in the presence of BMDMs-conditioned medium. Whereas BMDMs are polarized toward M2 macrophages with higher levels of arginase 1 and mannose receptor, but lower levels of inducible nitric oxide synthase and tumor necrosis factor-α when cocultured with ADSCs. In short, all these findings collectively demonstrated that ADSCs and resident host cells can synergistically contribute to the bony repair through mutual regulation of their differentiation and cytokine secretion.

Immunomodulatory effects of mesenchymal stem cell-derived exosomes on experimental type-1 autoimmune diabetes.

Exosomes derived from adipose tissue-derived mesenchymal stem cells (AD-MSCs) have immunomodulatory effects of T-cell inflammatory response and reduction of clinical symptoms on streptozotocin-induced of the type-1 diabetes mellitus (T1DM). Beside control group and untreated T1DM mice, a group of T1DM mice was treated with intraperitoneal injections of characterized exosomes derived from autologous AD-MSCs. Body weight and blood glucose levels were measured during the procedure. Histopathology and immunohistochemistry were used for evaluation of pancreatic islets using hemotoxylin and eosin (H&E) staining and anti-insulin antibody. Isolated splenic mononuclear cells (MNCs) were subjected to splenocytes proliferation assay using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, immunophenotyping of regulatory T cells and cytokines. A significant increase in the levels of interleukin-4 (IL-4), IL-10, and transforming growth factor-ß, and a decrease in the levels of IL-17 and interferon-γ in concordance with the significant increase in the Treg cell ratio in splenic MNCs (P < 0.05) was shown in T1DM mice treated with AD-MSC's exosomes as compared to T1DM untreated mice. This amelioration of autoimmune reaction after treatment of T1DM mice with the AD-MSC exosomes was confirmed with a significant increase in islets using H&E staining and Immunohistochemistry analyses. As expected, body weight, blood glucose levels in a survival of T1DM mice treated with AD-MSC's exosomes were maintained stable in comparison to untreated T1DM mice. It can be concluded that AD-MSC's exosomes exert ameliorative effects on autoimmune T1DM through increasing regulatory T-cell population and their products without a change in the proliferation index of lymphocytes, which makes them more effective and practical candidates.