The effect of exogenous ciliary neurotrophic factor on cell cycle and neural differentiation markers of in vitro model cells: New insights for future therapeutic approaches.

Retinoblastoma is known as childhood rare malignancy of the retina. Ciliary neurotrophic factor (CNTF) was previously found to reduce degeneration and promote retina survival. This work investigated the effects of CNTF supplementation on in-vitro model cells including retinoblastoma (Y79) and adipose-derived mesenchymal stem cells (AMSCs) viability, proliferation, gene expression and cell cycle. A drop of viability was detected in Y79 treated with CNTF in a dose-dependent manner (P < .05). However, the proliferation of AMSCs was increased at lower concentrations of CNTF (5 ng/mL), but declined in higher doses (50 and 100 ng/mL). The BrdU assay confirmed the MTT assay results. Cell cycle was arrested in both Y79 and AMSCs in the G0/G1 phase by CNTF treatment. A considerable down-regulation of Bcl2, CycD1 and N-Myc genes expression (P < .05) inversely, P15 and P21 genes up-regulation in treated Y79 cells was observed. Besides, stemness genes' transcription was reduced in AMSCs (P < .05), and levels of neuronal-specific markers such as neuron-specific enolase (NSE) and neuronal nuclei (NeuN) were increased (P < .05). The findings of this study suggest a promising potential of CNTF in terms of arresting Y79 retinoblastoma cells, and differentiation-inducing to AMSCs, which could be valuable for managing future innovative treatments targeting retinoblastoma. SIGNIFICANCE OF THE STUDY: We demonstrate that CNTF has the potential to reduce proliferation of Y79 cells and induce the cell cycle arrest of them. Also, down-regulation of oncogenes (such as N-Myc) while up-regulation of tumour suppressor genes (such as P21) was detected by exposure of Y79 cells to CNTF. Furthermore, we observed the cell cycle arrest, reduction of stemness gene and up-regulation of neural differentiation markers in AMSCs treated with CNTF. These results support the probable promising effects of CNTF for controlling retinoblastoma.

Rat cortex and hippocampus-derived soluble factors for the induction of adipose-derived mesenchymal stem cells into neuron-like cells.

To simulate brain microenvironment, adipose-derived mesenchymal stem cells (AMSC) were induced to differentiate to neuronal-like cells in rat cortex and hippocampus medium (Cox + Hip). First, isolated AMSC were characterized by flow cytometer and the capacity of adipogenesis and osteogenesis. After induction in rat cortex and hippocampus conditioned medium, the cell morphological change was examined and neural marker proteins (ß-Ш-Tubulin, NSE, Nissl body) expression was detected by immunofluorescence staining. A variety of synaptic marker proteins, including GAP43, SHANK2, SHANK3 and Bassoon body, were detected. ELISA was used to measure brain derived neurotrophic factor (BDNF) and nerve growth factor (NGF) secretion at different time-points. AMSCs positively expressed CD13, CD44 and CD90 and could differentiate into osteoblasts or adipocytes. After induction in Cox + Hip medium for 14 days, cells had a typical neuronal perikaryal appearance, which was suggestive of neuronal differentiation. After 14 days of Cox + Hip treatment, the percentage of cells expressing ß-Ⅲ-Tubulin, NSE and Nissl was 53.9 ± 0.8%, 51.3 ± 1.7% and 16.4 ± 2.1%, respectively. Expression of GAP43, SHANK2, SHANK3 and Bassoon body was detected, indicating synapse formation after treatment in Cox + Hip medium. Differentiated AMSCs secreted neurotrophic factors NGF and BDNF. Thus rat cortex and hippocampus-derived soluble factors can induce AMSCs to a neuronal-like phenotype, suggesting that AMSCs have a dual role in supplementing newborn neurons and secreting neurotrophic factors, and therefore could be help as a potential treatment for nervous system diseases.

Enhanced In Vitro Recapitulation of In Vivo Liver Regeneration by Co-Culturing Hepatocyte Organoids with Adipose-Derived Mesenchymal Stem Cells, Alleviating Steatosis and Apoptosis in Acute Alcoholic Liver Injury.

Hepatocyte organoids (HOs) have superior hepatic functions to cholangiocyte-derived organoids but suffer from shorter lifespans. To counteract this, we co-cultured pig HOs with adipose-derived mesenchymal stem cells (A-MSCs) and performed transcriptome analysis. The results revealed that A-MSCs enhanced the collagen synthesis pathways, which are crucial for maintaining the three-dimensional structure and extracellular matrix synthesis of the organoids. A-MSCs also increased the expression of liver progenitor cell markers (KRT7, SPP1, LGR5+, and TERT). To explore HOs as a liver disease model, we exposed them to alcohol to create an alcoholic liver injury (ALI) model. The co-culture of HOs with A-MSCs inhibited the apoptosis of hepatocytes and reduced lipid accumulation of HOs. Furthermore, varying ethanol concentrations (0-400 mM) and single-versus-daily exposure to HOs showed that daily exposure significantly increased the level of PLIN2, a lipid storage marker, while decreasing CYP2E1 and increasing CYP1A2 levels, suggesting that CYP1A2 may play a critical role in alcohol detoxification during short-term exposure. Moreover, daily alcohol exposure led to excessive lipid accumulation and nuclear fragmentation in HOs cultured alone. These findings indicate that HOs mimic in vivo liver regeneration, establishing them as a valuable model for studying liver diseases, such as ALI.

CD73+ adipose-derived stem cells reduce scar formation through PLOD1.

Background: Reducing cutaneous scar formation is important for assessing the success of skin wound healing. Although it is generally accepted that adipose-derived mesenchymal stem cells (AMSCs) have substantial therapeutic potential, efforts are continuously made to improve the outcome of AMSC therapy. Post-transcriptional suppression of procollagen-lysine 1, 2-oxoglutarate 5-dioxygenase 1 (PLOD1) in AMSCs has been shown to greatly reduce scar formation during skin wound healing, likely through modulating macrophage polarization. In the present study, we tested whether a CD73+ subpopulation of AMSCs could reduce scar formation compared with CD73- AMSCs. Methods: The gene profile of CD73+ versus CD73- AMSCs was obtained from a validated public database, GSE167219. AMSCs were isolated from adipose tissue surrounding the groin of mice, after which CD73+ versus CD73- AMSCs were sorted using flow cytometry. PLOD1 levels were determined in CD73+ versus CD73- AMSCs. Then, PLOD1 in CD73- AMSCs was depleted by a short-hair interfering RNA against PLOD1 (sh-PLOD1), while PLOD1 in CD73+ AMSCs was increased by expression of a PLOD1 transgene. A blade was used to induce a skin injury on the middle back of the mice. Either CD73+ AMSCs or CD73+ PLOD1 AMSCs or CD73- AMSCs or CD73- sh-PLOD1 AMSCs were intravenously transplanted into the injured region of the mice. Fibrosis and the underlying mechanisms were investigated. Co-immunoprecipitation was performed to evaluate interaction between CD73 and PLOD1. Results: CD73+ AMSCs expressed significantly lower levels of PLOD1, a potent stimulator of fibrosis, compared with CD73- AMSCs. Transplantation of CD73+ AMSCs generated significantly reduced fibrosis at the skin injury site compared with CD73- AMSCs. However, expression of PLOD1 in CD73+ AMSCs abolished its advantageous effects on fibrosis reduction, while depletion of PLOD1 in CD73- AMSCs improved the outcome of fibrosis to the levels of transplantation of CD73+ AMSCs. Co-immunoprecipitation showed no direct protein interaction between CD73 and PLOD1. Conclusions: CD73+ AMSCs are a subgroup of AMSCs with better therapeutic effects on wound healing, and can inhibit scar formation through reduced PLOD1 in an indirect manner.

Posttranscriptional control of PLOD1 in adipose-derived stem cells regulates scar formation through altering macrophage polarization.

BACKGROUND: The level of cutaneous scar formation is a critical parameter to determine the success of skin wound healing. Adipose-derived mesenchymal stem cells (AMSCs) have been applied to improve treatment of cutaneous injury with the purpose of reducing scar formation. METHODS: The levels of procollagen-lysine 1,2-oxoglutarate 5-dioxygenase 1 (PLOD1) were assessed at scar sites. Then, PLOD1 in AMSCs was depleted by either expression of a PLOD1-specific short-hair interfering RNA (shPLOD1) or by expression of microRNA-449 (miR-449) that targets and suppresses protein translation of PLOD1 through 3 prime untranslated region (3'-UTR) interfering. For induction of skin injury, a blade cut of 1.5-cm long and 2-mm thick was made on the middle back of the mice. Transplantation of either AMSCs-shPLOD1 or AMSCs-miR-449 into the injured region of the mice was performed via tail vein injection. The fibrosis as well as underlying mechanisms were assessed. RESULTS: The AMSCs expressed high levels of PLOD1, a potent stimulator of fibrosis. We knocked down PLOD1 in AMSCs by expression of either shPLOD1 or miR-449. Transplantation of either AMSCs-shPLOD1 or AMSCs-miR-449 significantly reduced the fibrotic process in the injured region of the mice to a similar degree. Mechanistically, transplantation of either AMSCs-shPLOD1 or AMSCs-miR-449 shifted macrophage polarization from M2 to M1-like and reduced both reactive oxygen species (ROS) and activation of myofibroblasts from fibroblasts. CONCLUSIONS: Suppression of PLOD1 levels in AMSCs either directly by shPLOD1 or indirectly by miR-449 may substantially improve the anti-fibrotic potential of AMSCs during wound healing, likely through altering macrophage polarization.

Hydrostatic pressure induces osteogenic differentiation of adipose-derived mesenchymal stem cells through increasing lncRNA-PAGBC.

Induced osteogenesis of adipose-derived mesenchymal stem cells (AMSCs) has been used to facilitate bone regeneration. Specifically, hydrostatic pressure (HP) has been implicated as a key regulator of AMSC differentiation, whereas the mechanisms that underlie the effects of HP on osteogenesis of AMSCs are not fully understood. Long noncoding RNAs (lncRNAs) are emerging regulators for osteogenic differentiation from AMSCs. In the current study, we found that lncRNA-PAGBC was a specific lncRNA that significantly upregulated during osteogenic differentiation of AMSCs based on published database. HP increased lncRNA-PAGBC, which is a competitive endogenous RNA (ceRNA) that binds to the osteogenesis-inhibitory microRNA, miR-133b, to regulate osteogenic differentiation of AMSCs. Moreover, a key osteogenesis-trigger gene, runt-related transcription factor 2 (RUNX2), was identified as a target gene for miR-133b. Suppression of RUNX2 by miR-133b caused impaired osteogenic differentiation of AMSCs. Furthermore, lncRNA-PAGBC overexpression upregulated, whereas lncRNA-PAGBC silencing decreased the expression of RUNX2 through miR-133b. Together, these data suggest that HP induces osteogenic differentiation of AMSCs through increasing lncRNA-PAGBC.

Culture and properties of adipose-derived mesenchymal stem cells: characteristics in vitro and immunosuppression in vivo.

OBJECTIVE: To compare the two sources of adipose and bone marrow derived mesenchymal stem cells (BMSCs and AMSCs) in immune regulation and to evaluate the therapeutic effects of AMSCs on Con A induced hepatitis and the possible mechanism involved in it. METHODS: We isolated bone marrow and adipose derived mesenchymal stem cells respectively and compared their differences on T lymphocyte activation, proliferation and suppression. We also test the anti-apoptosis ability of AMSCs on LO2 cell line. The effects of intravenous infusion of AMSCs on liver damage were also tested and we detected donor AMSCs in liver of recipient and their effects on the activity of intrahepatic NKT cells. RESULTS: BMSCs and AMSCs were similar in cell phenotype and the difference existed only in the expression of CD106. The results showed that the capacity of suppressing T cells proliferation and activation was weakened in AMSCs. AMSCs ameliorated liver damage and this effect was time and dose dependent. We detected donor AMSCs in liver of recipient which suggested tissue damage could be a clue for AMSCs migration. We also found AMSCs suppress the activity of intrahepatic NKT cells, but this suppress effects was not restricted in liver only, but the whole body. CONCLUSION: Cell origin and abundance are decisive factors in stem cells applications and with the same premise of AMSCs and BMSCs, adipose tissue is a more promising origin source of stem cells. The immunoregulatory features of MSCs might play an important role in various MSCs cellular therapies.