Enhanced potent immunosuppression of intracellular adipose tissue-derived stem cell extract by priming with three-dimensional spheroid formation.

Immunomodulatory properties of mesenchymal stem cells are widely studied, supporting the use of MSCs as cell-based therapy in immunological diseases. This study aims to generate cell-free MSC extract and improves their immunomodulatory potential. Intracellular extracts were prepared from adipose-derived stem cells (ADSC) spheroid via a freeze-thawing method. The immunomodulatory capacities of ADSC spheroid extracts were investigated in vitro, including lymphocyte proliferation, T regulatory cell expansion, and macrophage assays. A comparative study was conducted with ADSC monolayer extract. The key immunomodulatory mediators presented in ADSC extract were identified. The results revealed that ADSC spheroid extract could suppress lymphocyte activation while enhancing T regulatory cell expansion. Immunomodulatory molecules such as COX-2, TSG-6, and TGF-ß1 were upregulated in ADSC priming via spheroid culture. Selective inhibition of COX-2 abrogates the effect of ADSC extract on inducing T regulatory cell expansion. Thus, ADSC spheroid extract gains high efficacy in regulating the immune responses which are associated in part by COX-2 generation. Furthermore, ADSC spheroid extract possessed a potent anti-inflammation by manipulation of TNF-α production from LPS-activated macrophage. Our current study has highlighted the opportunity of using cell-free extracts from adipose tissue-derived mesenchymal stem cells spheroid as novel immunomodulators for the treatment of immunological-associated diseases.

Application of human platelet lysate in chondrocyte expansion promotes chondrogenic phenotype and slows senescence progression via BMP-TAK1-p38 pathway.

Osteoarthritis (OA) is one of the most common musculoskeletal degenerative. OA treatments are aiming to slow down disease progression; however, lack of cartilage regeneration efficacy. Autologous chondrocyte implantation (ACI) is a promising cartilage-regeneration strategy that uses human articular chondrocytes (HACs) as cellular materials. However, the unreadiness of HACs from prolonged expansion, cellular senescence, and chondrogenic dedifferentiation occurred during conventional expansion, thus, minimizing the clinical efficacy of ACI. We aimed to examine the effects of a human platelet lysate (HPL) as an alternative human-derived HAC medium supplement to overcome the limitations of conventional expansion, and to explain the mechanism underlying the effects of HPL. During passages 2-4 (P2-P4), HPL significantly increased HAC proliferation capacities and upregulated chondrogenic markers. Simultaneously, HPL significantly reduced HAC senescence compared with conventional condition. HACs treated with LDN193189 exhibited a reduction in proliferation capacity and chondrogenic marker expression, whereas the HAC senescence increased slightly. These findings indicated involvement of BMP-2 signaling transduction in the growth-assistive, anti-senescent, and chondrogenic-inductive properties of HPL, which demonstrated its beneficial effects for application as HAC medium supplement to overcome current expansion limitations. Finally, our findings support the roles of platelets in platelet-rich plasma as a promising treatment for patients with OA.

Annexin A2 Improves the Osteogenic Differentiation of Mesenchymal Stem Cells Exposed to High-Glucose Conditions through Lessening the Senescence.

Osteoporosis is frequently found in chronic diabetic patients, and it results in an increased risk of bone fractures occurring. The underlying mechanism of osteoporosis in diabetic patients is still largely unknown. Annexin A2 (ANXA2), a family of calcium-binding proteins, has been reported to be involved in many biological process including bone remodeling. This study aimed to investigate the role of ANXA2 in mesenchymal stem cells (MSCs) during in vitro osteoinduction under high-glucose concentrations. Osteogenic gene expression, calcium deposition, and cellular senescence were determined. The high-glucose conditions reduced the osteogenic differentiation potential of the MSCs along with the lower expression of ANXA2. Moreover, the high-glucose conditions increased the cellular senescence of the MSCs as determined by senescence-associated ß-galactosidase staining and the expression of p16, p21, and p53 genes. The addition of recombinant ANXA2 could recover the glucose-induced deterioration of the osteogenic differentiation of the MSCs and ameliorate the glucose-induced cellular senescence of the MSCs. A Western blot analysis revealed an increase in p53 and phosphorylated p53 (Ser 15), which was decreased by recombinant ANXA2 in MSC osteoblastic differentiation under high-glucose conditions. Our study suggested that the alteration of ANXA2 in high-glucose conditions may be one of the plausible factors in the deterioration of bones in diabetic patients by triggering cellular senescence.

Bone marrow-mesenchymal stem cell-derived extracellular vesicles affect proliferation and apoptosis of leukemia cells in vitro.

Mesenchymal stem cells (MSCs) have been proposed to have potential for tissue engineering and cell therapy due to their multilineage differentiation potential and ability to secrete numerous paracrine factors, including extracellular vesicles (EVs). Increasing evidence has demonstrated that MSC-derived EVs (MSC-EVs) are able to induce the repair of tissue damage and regulate the immune system. However, their role in cancer development is still unclear. Reports have suggested that whether MSC-EVs have an inhibitory or promoting effect on cancer is dependent on the type of cancer. In this study, the role of MSC-EVs in the regulation of leukemic cell growth in vitro was investigated. The EVs were collected from conditioned media of MSCs by ultrafiltration using a 10 kDa molecular weight cutoff (MWCO) filter. The isolated MSC-EVs were comprised of microvesicles and exosomes, as examined by the size of vesicles and exosomal proteins, CD81 and flotillin-1. Cell proliferation, cell cycle status, apoptosis, and gene expression were examined in the leukemic cell lines NB4 and K562 after treatment with MSC-EVs. Suppression of cell proliferation and induction of apoptosis was observed. Gene expression analysis revealed differential expression of apoptotic-related genes in NB4 and K562. MSC-EVs increased the expression of BID and BAX and decreased expression of BCL2, indicating the induction of intrinsic apoptosis in NB4. In contrast, MSC-EVs increased the expression of the death receptor gene TRAILR2 and cell cycle regulator genes P21 and CCNE2 in K562. In conclusion, MSC-EVs partially induce leukemic cell apoptosis, and thus may have potential for the development of supportive therapies for leukemia.

Overexpression of anti-fibrotic factors ameliorates anti-fibrotic properties of Wharton's jelly derived mesenchymal stem cells under oxidative damage.

Transplantation with Wharton's jelly derived mesenchymal stem cells (WJ-MSCs) showed great benefits for restoring myocardial function. However, the outcome of WJ-MSCs transplantation was unsuccessful due to multiple factors including oxidative damage. The presence of oxidative stress due to myocardium injury influences fibrous tissue formation, which causes disability of cardiac muscle. Hepatocyte growth factor (HGF), insulin-like growth factor (IGF1), and sonic hedgehog (SHH) are well-known master regulators in anti-fibrosis when secreted by WJ-MSCs. They showed a beneficial role in the recovery of cardiac fibrosis after WJ-MSCs transplantation. This study hypothesizes whether the reduction of the anti-fibrosis property in WJ-MSCs from oxidative damage can be recovered by overexpression of the HGF, IGF1, or SHH gene. Overexpression was attained by transfection of WJ-MSCs with pCMV3-HGF, pCMV3-IGF1, or pCMV3-SHH followed by H2O2 exposure and co-culturing with cardiac fibroblasts. Myofibroblast specific markers comprised of alpha-smooth muscle actin (α-SMA) and collagen type 1 (COL1) were evaluated. The WJ-MSCs treated with H2O2 influenced the expression of myofibroblastic markers, whereas the overexpression of HGF, IGF1 or SHH reduced myofibroblastic formation. These results indicate that the oxidative stress impaired anti-fibrotic property of WJ-MSCs, leads to an increase of myofibroblasts. Overexpression of anti-fibrotic genes restored the endogenous HGF, IGF1, and SHH alleviating improvement of cardiac function.

Inhibitory effect of oxidative damage on cardiomyocyte differentiation from Wharton's jelly-derived mesenchymal stem cells.

Ischemic heart diseases are a serious health problem worldwide. The transplantation of mesenchymal stem cells (MSCs) has been investigated in numerous clinical trials on various other diseases due to the self-renewal capacity of these cells and their potential to differentiate into a variety of cell types. The presence of excess reactive oxygen species in injured myocardium causes cardiac dysfunction and leads to inefficient repair of the heart. The poor outcomes of stem cell transplantation have been suggested to result from residual oxidative damage affecting the transplanted cells. The aim of the present study was to compare the effects of hydrogen peroxide (H2O2) on Wharton's jelly-derived MSCs (WJ-MSCs) and bone marrow-derived MSCs (BM-MSCs) in vitro, in order to provide information useful for the future selection of MSC types for cardiac differentiation and transplantation. H2O2 at concentrations of 200, 500 and 1,000 µM was applied to WJ-MSCs and BM-MSCs under cardiogenic differentiation conditions. The morphology of MSCs treated with H2O2 was similar to that of untreated cells, whereas the cell density decreased in direct association with the dose of H2O2. Cardiac differentiation markers were then evaluated by immunofluorescence analysis of GATA4 and cardiac troponin T (cTnT). The fluorescence intensity levels of the two markers were identified to be diminished by increasing doses of H2O2 from 500 to 1,000 µM. The expression levels of homeobox protein Nkx2.5, cTnT and cardiac α-actin were also examined, and were identified to be low in the WJ-MSCs treated with 1,000 µM H2O2, which was similar to the findings observed in BM-MSCs. These results suggested that oxidative stress affects cardiomyocyte differentiation via the downregulation of cardiac genes and cardiac proteins. Furthermore, it should be noted that there was a marked difference in the effect depending on the source of MSCs. This evidence provided supportive information for the use of stem cells in transplantation.

Cell type of origin influences iPSC generation and differentiation to cells of the hematoendothelial lineage.

The use of induced pluripotent stem cells (iPSCs) as a source of cells for cell-based therapy in regenerative medicine is hampered by the limited efficiency and safety of the reprogramming procedure and the low efficiency of iPSC differentiation to specialized cell types. Evidence suggests that iPSCs retain an epigenetic memory of their parental cells with a possible influence on their differentiation capacity in vitro. We reprogramme three cell types, namely human umbilical cord vein endothelial cells (HUVECs), endothelial progenitor cells (EPCs) and human dermal fibroblasts (HDFs), to iPSCs and compare their hematoendothelial differentiation capacity. HUVECs and EPCs were at least two-fold more efficient in iPSC reprogramming than HDFs. Both HUVEC- and EPC-derived iPSCs exhibited high potentiality toward endothelial cell differentiation compared with HDF-derived iPSCs. However, only HUVEC-derived iPSCs showed efficient differentiation to hematopoietic stem/progenitor cells. Examination of DNA methylation at promoters of hematopoietic and endothelial genes revealed evidence for the existence of epigenetic memory at the endothelial genes but not the hematopoietic genes in iPSCs derived from HUVECs and EPCs indicating that epigenetic memory involves an endothelial differentiation bias. Our findings suggest that endothelial cells and EPCs are better sources for iPSC derivation regarding their reprogramming efficiency and that the somatic cell type used for iPSC generation toward specific cell lineage differentiation is of importance.

Osteoporosis: the current status of mesenchymal stem cell-based therapy.

Osteoporosis, or bone loss, is a progressive, systemic skeletal disease that affects millions of people worldwide. Osteoporosis is generally age related, and it is underdiagnosed because it remains asymptomatic for several years until the development of fractures that confine daily life activities, particularly in elderly people. Most patients with osteoporotic fractures become bedridden and are in a life-threatening state. The consequences of fracture can be devastating, leading to substantial morbidity and mortality of the patients. The normal physiologic process of bone remodeling involves a balance between bone resorption and bone formation during early adulthood. In osteoporosis, this process becomes imbalanced, resulting in gradual losses of bone mass and density due to enhanced bone resorption and/or inadequate bone formation. Several growth factors underlying age-related osteoporosis and their signaling pathways have been identified, such as osteoprotegerin (OPG)/receptor activator of nuclear factor B (RANK)/RANK ligand (RANKL), bone morphogenetic protein (BMP), wingless-type MMTV integration site family (Wnt) proteins and signaling through parathyroid hormone receptors. In addition, the pathogenesis of osteoporosis has been connected to genetics. The current treatment of osteoporosis predominantly consists of antiresorptive and anabolic agents; however, the serious adverse effects of using these drugs are of concern. Cell-based replacement therapy via the use of mesenchymal stem cells (MSCs) may become one of the strategies for osteoporosis treatment in the future.