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.

The Use of Human Platelet Lysate as a Coating Substance for Adipose-Derived Stem Cell Expansion.

BACKGROUND: Large-scale production of mesenchymal stromal cells is essential for sufficient therapeutic doses in regenerative medicine. However, long-term cultivation encounters limited cell growth and cellular aging. Therefore, an alternative cell culture approach that promotes proliferation and attenuates cell senescence is required. Human platelet lysate (HPL) is a potent supplement for in vitro cell expansion. Applying HPL as a coating material can potentially improve mesenchymal stromal cell cultures. METHOD: To examine the capacity of HPL, it was used to pre-coat a tissue culture plate for in vitro adipose-derived mesenchymal stromal cell expansion. Alterations in biological features of adipose-derived stem cells (ADSCs) were investigated, including cell adhesion assays, cell proliferation, population doubling time, and cellular senescence. RESULTS: ADSCs cultured on HPL-coated plates significantly increased cell adhesion rate, shortened population doubling time, and stimulated cell growth. The senescent cells were significantly decreased in ADSCs cultured in an HPL-coated plate, and the expression levels of senescence-associated genes, including p16, p21, and p53, were downregulated. Furthermore, Western blotting analysis revealed that HPL was enriched with fibronectin and vitronectin, essential cell adhesive proteins. CONCLUSIONS: HPL was effectively used as a coating material for ADSC expansions. Cellular cultivation on the HPL coating is an alternative approach for producing mesenchymal stromal cells.

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.

P-cresol and Indoxyl Sulfate Impair Osteogenic Differentiation by Triggering Mesenchymal Stem Cell Senescence.

Chronic kidney disease (CKD) patients obtained high levels of uremic toxins progressively develop several complications including bone fractures. Protein-bound uremic toxins especially p-cresol and indoxyl sulfate are hardly eliminated due to their high molecular weight. Thus, the abnormality of bone in CKD patient could be potentially resulted from the accumulation of uremic toxins. To determine whether protein-bound uremic toxins have an impact on osteogenesis, mesenchymal stem cells were treated with either p-cresol or indoxyl sulfate under in vitro osteogenic differentiation. The effects of uremic toxins on MSC-osteoblastic differentiation were investigated by evaluation of bone phenotype. The results demonstrated that p-cresol and indoxyl sulfate down-regulated the transcriptional level of collagen type I, deceased alkaline phosphatase activity, and impaired mineralization of MSC-osteoblastic cells. Furthermore, p-cresol and indoxyl sulfate gradually increased senescence-associated beta-galactosidase positive cells while upregulated the expression of p21 which participate in senescent process. Our findings clearly revealed that the presence of uremic toxins dose-dependently influenced a gradual deterioration of osteogenesis. The effects partially mediate through the activation of senescence-associated gene lead to the impairment of osteogenesis. Therefore, the management of cellular senescence triggered by uremic toxins could be considered as an alternative therapeutic approach to prevent bone abnormality in CKD patients.

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.