Therapeutic effects of mesenchymal stem cells-conditioned medium derived from suspension cultivation or silymarin on liver failure mice.

BACKGROUND: Common treatments of liver disease failed to meet all the needs in this important medical field. It results in an urgent need for proper some new adjuvant therapies. Mesenchymal stem cells (MSCs) and their derivatives are promising tools in this regard. We aimed to compare the Silymarin, as traditional treatment with mesenchymal stem cell conditioned medium (MSC-CM), as a novel strategy, both with therapeutic potentialities in term of liver failure (LF) treatment. METHODS AND RESULTS: Mice models with liver failure were induced with CCl4 and were treated in the groups as follows: normal mice receiving DMEM-LG medium as control, LF-mice receiving DMEM-LG medium as sham, LF-mice receiving Silymarin as LF-SM, and LF-mice receiving MSC sphere CM as LF-MSC-CM. Biochemical, histopathological, molecular and protein level parameters were evaluated using blood and liver samples. Liver enzymes, MicroRNA-122 values as well as necrotic score were significantly lower in the LF-SM and LF-MSC-CM groups compared to sham. LF-SM showed significantly higher level of total antioxidant capacity and malondialdehyde than that of LF-MSC-CM groups. Sph-MSC-CM not only induced more down-regulated expression of fibrinogen-like protein 1 and receptor interacting protein kinases1 but also led to higher expression level of keratinocyte growth factor. LF-MSC-CM showed less mortality rate compared to other groups. CONCLUSIONS: Hepato-protective potentialities of Sph-MSC-CM are comparable to those of Silymarin. More inhibition of necroptosis/ necrosis and inflammation might result in rapid liver repair in case of MSC-CM administration.

Co-culture of mesenchymal stem cell spheres with hematopoietic stem cells under hypoxia: a cost-effective method to maintain self-renewal and homing marker expression.

BACKGROUND: Hematopoietic stem cell (HSC) transplantation is considered a possible treatment option capable of curing various diseases. The aim of this study was the co-culturing of mesenchymal stem cell (MSC) spheres with HSCs under hypoxic condition to enhance the proliferation, self-renewal, stemness, and homing capacities of HSCs. METHODS AND RESULTS: HSCs were expanded after being subjected to different conditions including cytokines without feeder (Cyto), co-culturing with adherent MSCs (MSC), co-culturing with adherent MSCs + hypoxia (MSC + Hyp), co-culturing with MSCs spheres (Sph-MSC), co-culturing with MSCs spheres + hypoxia (Sph-MSC + Hyp), co-culturing with MSC spheres + cytokines (Sph-MSC + Cyto). After 10 days, total nucleated cell (TNC) and CD34+/CD38- cell counts, colony-forming unit assay (CFU), long-term culture initiating cell (LTC-IC), the expression of endothelial protein C receptor (EPCR), nucleostemin (NS), nuclear factor I/X (Nfix) CXCR4, and VLA-4 were evaluated. The TNC, CD34+/CD38- cell count, CFU, and LTC-IC were higher in the Sph-MSC + Hyp and Sph-MSC + Cyto groups as compared with those of the MSC + Hyp group (P < 0.001). The expanded HSCs co-cultured with MSC spheres in combination with hypoxia expressed more EPCR, CXCR4, VLA-4, NS, and Nfix mRNA. The protein expression was also more up-regulated in the Sph-MSC + Cyto and Sph-MSC + Hyp groups. CONCLUSION: Co-culturing HSCs with MSC spheres under hypoxic condition not only leads to higher cellular yield but also increases the expression of self-renewal and homing genes. Therefore, we suggest this approach as a simple and non-expensive strategy that might improve the transplantation efficiency of HSCs.

Mesenchymal Stem Cell-based Therapy as a New Horizon for Kidney Injuries.

Today, the prevalence of kidney diseases is increasing around the world, but there has still been no effective medical treatment. The therapeutic choices are confined to supportive cares and preventive strategies. Currently, mesenchymal stem cells (MSCs)-based cell therapy was proposed for the treatment of kidney injuries. However, after the transplantation of MSCs, they are exposed to masses of cytotoxic factors involving an inflammatory cytokine storm, a nutritionally-poor hypoxic environment and oxidative stresses that finally lead to minimize the efficacy of MSCs based cell therapy. Therefore, several innovative strategies were developed in order to potentiate MSCs to withstand the unfavorable microenvironments of the injured kidney tissues and improve their therapeutic potentials. This review aims to introduce MSCs as a new modality in the treatment of renal failure. Here, we discuss the clinical trials of MSCs-based therapy in kidney diseases as well as the in vivo studies dealing with MSCs application in kidney injuries mainly from the proliferation, differentiation, migration and survival points of view. The obstacles and challenges of this new modality in kidney injuries are also discussed.

Nuclear factor erythroid-2 related factor 2 overexpressed mesenchymal stem cells transplantation, improves renal function, decreases injuries markers and increases repair markers in glycerol-induced Acute kidney injury rats.

OBJECTIVES: Recently cell therapy is a promising therapeutic modality for many types of disease including acute kidney injury (AKI). Due to the unique biological properties, mesenchymal stem cells (MSCs) are attractive cells in this regard. This study aims to transplant MSCs equipped with nuclear factor E2-related factor 2 (Nrf2) in rat experimental models of acute kidney and evaluate regeneration potential of injured kidney especially expression of injury and repaired biomarkers. MATERIALS AND METHODS: Nrf2 was overexpressed in bone marrow-derived MSCs by pcDNA.3.1 plasmid. AKI was induced using glycerol in rat models. The regenerative potential of Nrf2-overexpressed MSCs was evaluated in AKI-Induced animal models using biochemical and histological methods after transplantation. Expression of repaired genes, AQP1 and CK-18, as well as injury markers, Kim-1 and Cystatin C, was also assayed in engrafted kidney sections. RESULTS: Our results revealed that transplantation of Nrf2-overexpressed MSCs into AKI-induced rats decreased blood urea nitrogen and creatinine and ameliorated kidney regeneration throughout 14 days. Upregulation of repaired markers and downregulation of injury markers were considerable 14 days after transplantation. CONCLUSIONS: Overexpression of Nrf2 in MSCs suggests a new strategy to increase efficiency of MSC-based cell therapy in AKI.

Autophagy-Modulated Human Bone Marrow-Derived Mesenchymal Stem Cells Accelerate Liver Restoration in Mouse Models of Acute Liver Failure.

BACKGROUND: Mesenchymal stem cells (MSCs) have been recently received increasing attention for cell-based therapy, especially in regenerative medicine. However, the low survival rate of these cells restricts their therapeutic applications. It is hypothesized that autophagy might play an important role in cellular homeostasis and survival. This study aims to investigate the regenerative potentials of autophagy-modulated MSCs for the treatment of acute liver failure (ALF) in mice. METHODS: ALF was induced in mice by intraperitoneal injection of 1.5 ml/kg carbon tetrachloride. Mice were intravenously infused with MSCs, which were suppressed in their autophagy pathway. Blood and liver samples were collected at different intervals (24, 48 and 72 h) after the transplantation of MSCs. Both the liver enzymes and tissue necrosis levels were evaluated using biochemical and histopathological assessments. The survival rate of the transplanted mice was also recorded during one week. RESULTS: Biochemical and pathological results indicated that 1.5 ml/kg carbon tetrachloride induces ALF in mice. A significant reduction of liver enzymes and necrosis score were observed in autophagy-modulated MSC-transplanted mice compared to sham (with no cell therapy) after 24 h. After 72 h, liver enzymes reached their normal levels in mice transplanted with autophagy-suppressed MSCs. Interestingly, normal histology without necrosis was also observed. CONCLUSION: Autophagy suppression in MSCs ameliorates their liver regeneration potentials due to paracrine effects and might be suggested as a new strategy for the improvement of cell therapy in ALF.

Down-regulation of the autophagy gene, ATG7, protects bone marrow-derived mesenchymal stem cells from stressful conditions.

BACKGROUND: Mesenchymal stem cells (MSCs) are valuable for cell-based therapy. However, their application is limited owing to their low survival rate when exposed to stressful conditions. Autophagy, the process by which cells recycle the cytoplasm and dispose of defective organelles, is activated by stress stimuli to adapt, tolerate adverse conditions, or trigger the apoptotic machinery. This study aimed to determine whether regulation of autophagy would affect the survival of MSCs under stress conditions. METHODS: Autophagy was induced in bone marrow-derived MSCs (BM-MSCs) by rapamycin, and was inhibited via shRNA-mediated knockdown of the autophagy specific gene, ATG7. ATG7 expression in BM-MSCs was evaluated by reverse transcription polymerase chain reaction (RT-PCR), western blot, and quantitative PCR (qPCR). Cells were then exposed to harsh microenvironments, and a water-soluble tetrazolium salt (WST)-1 assay was performed to determine the cytotoxic effects of the stressful conditions on cells. RESULTS: Of 4 specific ATG7-inhibitor clones analyzed, only shRNA clone 3 decreased ATG7 expression. Under normal conditions, the induction of autophagy slightly increased the viability of MSCs while autophagy inhibition decreased their viability. However, under stressful conditions such as hypoxia, serum deprivation, and oxidative stress, the induction of autophagy resulted in cell death, while its inhibition potentiated MSCs to withstand the stress conditions. The viability of autophagy-suppressed MSCs was significantly higher than that of relevant controls (P<0.05, P<0.01 and P<0.001). CONCLUSION: Autophagy modulation in MSCs can be proposed as a new strategy to improve their survival rate in stressful microenvironments.

The Lcn2-engineered HEK-293 cells show senescence under stressful condition.

OBJECTIVES: Lipocalin2 (Lcn2) gene is highly expressed in response to various types of cellular stresses. The precise role of Lcn2 has not been fully understood yet. However, it plays a key role in controlling vital cellular processes such as proliferation, apoptosis and metabolism. Recently it was shown that Lcn2 decreases senescence and increases proliferation of mesenchymal stem cells (MSC) with finite life span under either normal or oxidative stress conditions. However, Lcn2 effects on immortal cell line with infinite proliferation are not defined completely. Materials and. MATERIALS AND METHODS: HEK-293 cells were transfected with recombinant pcDNA3.1 containing Lcn2 fragment (pcDNA3.1-Lcn2). Expression of lipocalin2 in transfected cells was evaluated by RT-PCR, real time RT-PCR, and ELISA. Different cell groups were treated with H2O2 and WST-1 assay was performed to determine their proliferation rate. Senescence was studied by ß-galactosidase and gimsa staining methods as well as evaluation of the expression of senescence-related genes by real time RT-PCR. RESULTS: Lcn2 increased cell proliferation under normal culture condition, while the proliferation slightly decreased under oxidative stress. This decrease was further found to be attributed to senescence. CONCLUSION: Our findings indicated that under harmful conditions, Lcn2 gene is responsible for the regulation of cell survival through senescence.

Positive selection of Wharton's jelly-derived CD105(+) cells by MACS technique and their subsequent cultivation under suspension culture condition: A simple, versatile culturing method to enhance the multipotentiality of mesenchymal stem cells.

OBJECTIVE: Wharton's jelly (WJ), an appropriate source of mesenchymal stem cells (MSCs), has been shown to have a wide array of therapeutic applications. However, the WJ-derived MSCs are very heterogeneous and have limited expression of pluripotency markers. Hence, improvement of their culture condition would promote the efficiency of WJ-MSCs. This study aims to employ a simple method of cultivation to obtain WJ-MSCs which express more pluripotency markers. METHODS: CD105(+) cells were separated by magnetic-associated (activated) cell sorting from umbilical cord mucous tissue. CD105(+) cells were added to Methocult medium diluted in α-minimum essential medium (α-MEM) and seeded in poly(2-hydroxyethyl methacrylate) (poly-HEMA)-coated plates for suspension culture preparation. Differentiation capacity of isolated cells was evaluated in the presence of differentiation-inducing media. The expression of pluripotency markers such as Oct3/4, Nanog, and Sox2 was also analyzed by RT-PCR and western blot techniques. Moreover, immunocytochemistry was performed to detect alpha-smooth muscle actin (antigene) (α-SMA) protein. RESULTS: WJ-MSCs grew homogeneously and formed colonies when cultured under suspension culture conditions (Non-adhesive WJ-MSCs). They maintained their growth ability in both adherent and suspension cultures for several passages. Non-adhesive WJ-MSCs expressed Oct3/4, Nanog, and Sox2 both at transcriptional and translational levels in comparison to those cultured in conventional adherent cultures. They also expressed α-SMA protein. DISCUSSION: In this study, we isolated WJ-MSCs using a slightly modified culture condition. Our simple non-genetic method resulted in a homogeneous population of WJ-MSCs, which highly expressed pluripotency markers. CONCLUSION: In the future, more multipotent WJ-MSCs can be harnessed as a non-embryonic source of MSCs in MSC-based cell therapy.