Development of composite functional tissue sheets using hiPSC-CMs and hADSCs to improve the cardiac function after myocardial infarction.

Human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) have been widely used in therapy of ischemic heart disease. However, there are still remaining issues that limit the therapeutic efficacy, such as immune rejection and low retention of hiPSC-CMs. Human adipose mesenchymal stromal cells (hADSCs) have been reported to be able to regulate the immune response, promote angiogenesis and promote the maturation of hiPSC-CMs. In this study, we co-cultured these two types of cells on fiber scaffold made of biodegradable poly (D,L-lactic-co-glycolic acid) (PLGA) polymer for several days to develop a composited 3D cardiac tissue sheet. As expected, the cells formed 231.00 ± 15.14 µm thickness tissue, with improved organization, alignment, ECM condition, contractile ability, and paracrine function compared to culture hiPSC-CMs only on PLGA fiber. Furthermore, the composited 3D cardiac tissue sheet significantly promoted the engraftment and survival after transplantation. The composited 3D cardiac tissue sheet also increased cardiac function, attenuated ventricular remodeling, decreased fibrosis, and enhanced angiogenesis in rat myocardial infarction model, indicating that this strategy wound be a promising therapeutic option in the clinical scenario.

Fibroblast function recovery through rejuvenation effect of nanovesicles extracted from human adipose-derived stem cells irradiated with red light.

Fibroblasts (hDFs) are widely employed for skin regeneration and the treatment of various skin disorders, yet research were rarely investigated about restoration of diminished therapeutic efficacy due to cell senescence. The application of stem cell and stem cell-derived materials, exosomes, were drawn attention for the restoration functionality of fibroblasts, but still have limitation for unintended side effect or low yield. To advance, stem cell-derived nanovesicle (NV) have developed for effective therapeutic reagents with high yield and low risk. In this study, we have developed a method using red light irradiated human adipose-derived stem cells (hADSCs) derived NV (R-NVs) for enhancing the therapeutic efficacy and rejuvenating hDFs. Through red light irradiation, we were able to significantly increase the content of stemness factors and angiogenic biomolecules in R-NVs. Treatment with these R-NVs was found to enhance the migration ability and leading to rejuvenation of old hDFs to levels similar to those of young hDFs. In subsequent in vivo experiments, the treatment of old hDFs with R-NVs demonstrated a superior skin wound healing effect, surpassing that of young hDFs. In summary, this study successfully induced rejuvenation and leading to increased therapeutic efficacy to R-NVs treated old hDFs previously considered as biowaste.

Attenuation of experimental osteoarthritis with human adipose-derived mesenchymal stem cell therapy: inhibition of the pyroptosis in chondrocytes.

AIM: To explore the role and mechanism of human adipose-derived mesenchymal stem cells (hAD-MSCs) in the treatment of osteoarthritis (OA). METHODS: OA hulth model of Sprague Dawley (SD) rats and 20 ng/ml TNF-α treated chondrocytes were used as models of OA in vivo and in vitro, respectively. hAD-MSCs were administrated in the articular cavity by injection in vivo and co-cultured with chondrocytes using transwell in vitro. Haematoxylin and eosin staining and Safranin-O/Fast green staining were performed to detect tissue destruction and histopathology. Scanning electron microscopy and transmission electron microscopy were used to observe the ultrastructure of chondrocytes. The pyroptosis signaling pathway-related proteins were detected by immunohistochemistry, immunofluorescence, qRT-PCR and Western blot. And small interference technology was used to study the mechanism in depth. RESULTS: hAD-MSCs could delay the development of rat OA, improve the pathological changes of joints, inhibit the expression of NLRP3, Caspase1, GSDMD and TNFR1. In vitro, the expression of pyroptosis signal proteins in chondrocytes was significantly elevated when stimulated with TNF-α, the level of inflammatory factors such as IL-1ß, IL-18 was increased, and the cell morphology was significantly destroyed. While co-cultured with hAD-MSCs, these syndromes were reversed. Knockout of TNFR1 also returned the upregulation of pyroptosis signals which caused by TNF-α. CONCLUSION: These results demonstrated that hAD-MSCs could inhibit pyroptosis signaling pathway of chondrocytes induced by TNF-α, which have raised our understanding of the role of hAD-MSCs as promising therapy for the management of OA.

Single Cell Effects of Photobiomodulation on Mitochondrial Membrane Potential and Reactive Oxygen Species Production in Human Adipose Mesenchymal Stem Cells.

Photobiomodulation (PBM) has recently emerged in cellular therapy as a potent alternative in promoting cell proliferation, migration, and differentiation during tissue regeneration. Herein, a single-cell near-infrared (NIR) laser irradiation system (830 nm) and the image-based approaches were proposed for the investigation of the modulatory effects in mitochondrial membrane potential (ΔΨm), reactive oxygen species (ROS), and vesicle transport in single living human adipose mesenchymal stem cells (hADSCs). The irradiated-hADSCs were then stained with 2',7'-dichlorodihydrofluorescein diacetate (H2DCFDA) and Rhodamine 123 (Rh123) to represent the ΔΨm and ROS production, respectively, with irradiation in the range of 2.5-10 (J/cm2), where time series of bright-field images were obtained to determine the vesicle transport phenomena. Present results showed that a fluence of 5 J/cm2 of PBM significantly enhanced the ΔΨm, ROS, and vesicle transport phenomena compared to the control group (0 J/cm2) after 30 min PBM treatment. These findings demonstrate the efficacy and use of PBM in regulating ΔΨm, ROS, and vesicle transport, which have potential in cell proliferation, migration, and differentiation in cell-based therapy.

Effect of biomolecules derived from human platelet-rich plasma on the ex vivo expansion of human adipose-derived mesenchymal stem cells for clinical applications.

Mesenchymal stem cells are a tool in cell therapies but demand a large cell number per treatment, for that, suitable culture media is required which contains fetal bovine serum (FBS). However, for cell-based therapy applications, the use of FBS is problematic. Several alternatives to FBS have been explored, including human derivatives from platelet-rich plasma (hD-PRP). Although various studies have evaluated the impact of hD-PRP on MSC proliferation and differentiation, few of them have assessed their influence on processes, such as metabolism and gene expression. Here, we cultured human adipose-derived MSCs (hAD-MSCs) in media supplemented with either 10% hD-PRP (hD-PRP-SM) or 10% FBS (FBS-SM) in order to characterize them and evaluate the effect of hD-PRP on cell metabolism, gene expression of associated regenerative factors, as well as chromosome stability during cell expansion. We found that hAD-MSCs cultured in hD-PRP-SM have a greater cell elongation but express similar surface markers; in addition, hD-PRP-SM promoted a significant osteogenic differentiation in the absence of differentiation medium and increased the growth rate, maintaining chromosomal stability. In terms of cell metabolic profile, hAD-MSC behavior did not reveal any differences between both culture conditions. Conversely, significant differences in collagen I and angiopoietin 2 expression were observed between both conditions. The present results suggest that hD-PRP may influence hAD-MSC behavior.

A preliminary study on the development of a novel biomatrix by decellularization of bovine spinal meninges for tissue engineering applications.

Here, we aim at developing a novel biomatrix from decellularized bovine spinal meninges for tissue engineering and regenerative medicine applications. Within this concept, the bovine spinal meninges were decellularized using 1% Triton X-100 for 48 h, and residual nuclear content was determined with double-strand DNA content analysis and agarose gel electrophoresis. The major matrix components such as sulfated GAGs and collagen before and after the decellularization process were analyzed with DMMB, hydroxyproline assay and SDS-PAGE. Subsequently, the native bovine spinal meninges (nBSM) and decellularized BSM (dBSM) were physiochemically characterized via ATR-FTIR spectroscopy, TGA, DMA and tensile strength test. The dsDNA content in the nBSM was 153.39 ± 53.93 ng/mg dry weight, versus in the dBSM was 39.47 ± 4.93 ng/mg (n = 3) dry weight and DNA fragments of more than 200 bp in length were not detected in the dBSM by agarose gel electrophoresis. The sulfated GAGs contents for nBSM and dBSM were observed to be 10.87 ± 1.2 and 11.42 ± 2.01 µg/mg dry weight, respectively. The maximum strength of dBSM in dry and wet conditions was found to be 19.67 ± 0.21 MPa and 13.97 ± 0.17 MPa, while nBSM (dry) was found to be 26.26 ± 0.28 MPa. MTT, SEM, and histology results exhibited that the cells attached to the surface of dBSM, and proliferated on the dBSM. In conclusion, the in vitro preliminary study has demonstrated that the dBSM might be a proper and new bioscaffold for tissue engineering and regenerative medicine applications.

Aberrant proliferation and differentiation of glycogen storage disease type Ib mesenchymal stem cells.

Glycogen storage disease type Ib (GSD-Ib) is caused by mutations of the glucose-6-phosphate transporter (G6PT) and characterized by disrupted glucose homeostasis, neutropenia, and neutrophil dysfunction. To investigate the role of G6PT in human adipose-derived mesenchymal stem cells (hMSCs), the G6PT gene was mutated by CRISPR/Cas9 technology and single cell-derived G6PT-/- hMSCs were established. G6PT-/- hMSCs have significantly increased cell proliferation but impaired adipogenesis and osteogenesis. These phenotypes are associated with two mechanisms: i) metabolic reprogramming in G6PT-/- hMSCs causing a metabolic shift toward glycolysis rather than oxidative phosphorylation and ii) increased cyclooxygenase-2-derived prostaglandin E2 secretion in G6PT-/- hMSCs. This study demonstrates that G6PT is essential for proliferation and differentiation of MSCs, providing important insights into the GSD-Ib phenotypes.

Regulation of PPARγ and CIDEC expression by adenovirus 36 in adipocyte differentiation.

This study is to investigate the role of adenovirus 36 (Ad36) in regulating expression of peroxisome proliferator-activated receptor γ (PPARγ) and cell death-inducing DFFA-like effector c (CIDEC) in Ad36-induced adipocyte differentiation. Human adipose-derived mesenchymal stem cells (hAMSCs) were isolated and cultured, and then infected with Ad36. Ad36-induced adipocytes were identified using quantitative real-time PCR and Oil red O staining. The expression levels of PPARγ and CIDEC in Ad36-induced adipocytes were determined by quantitative real-time PCR and Western blot analysis. Glucose uptake and intracellular triglyceride content were also determined in these induced cells. Our results from the Oil red O staining showed that Ad36 induced the differentiation of hAMSCs into human adipocytes in vitro. Moreover, the medium glucose concentration was significantly decreased, while the intracellular triglyceride content was significantly increased, in the Ad36-induced adipocytes, compared with the control group. Furthermore, our results showed that, the mRNA and protein expression levels of PPARγ and CIDEC were significantly upregulated in Ad36-induced adipocytes, in a time-dependent manner. On the other hand, compared with the control group, the CIDEC expression was downregulated when the Ad36-induced adipocytes were treated with the PPARγ inhibitor, GW9662. Ad36 could upregulate the expression level of CIDEC through increasing PPARγ expression during the adipocyte differentiation process.