Antioxidants cause rapid expansion of human adipose-derived mesenchymal stem cells via CDK and CDK inhibitor regulation.

BACKGROUND: Antioxidants have been shown to enhance the proliferation of adipose-derived mesenchymal stem cells (ADMSCs) in vitro, although the detailed mechanism(s) and potential side effects are not fully understood. RESULTS: During log-phase growth, exposure to ImF-A resulted in a higher percentage of ADMSCs in the S phase of the cell cycle and a smaller percentage in G0/G1 phase. This resulted in a significantly reduced cell-doubling time and increased number of cells in the antioxidant-supplemented cultures compared with those supplemented with FGF-2 alone, an approximately 225% higher cell density after 7 days. Western blotting showed that the levels of the CDK inhibitors p21 and p27 decreased after ImF-A treatment, whereas CDK2, CDK4, and CDC2 levels clearly increased. In addition, ImF-A resulted in significant reduction in the expression of CD29, CD90, and CD105, whereas relative telomere length, osteogenesis, adipogenesis, and chondrogenesis were enhanced. The results were similar for ADMSCs treated with antioxidants and those under hypoxic conditions. CONCLUSION: Antioxidant treatment promotes entry of ADMSCs into the S phase by suppressing cyclin-dependent kinase inhibitors and results in rapid cell proliferation similar to that observed under hypoxic conditions.

Butylidenephthalide suppresses human telomerase reverse transcriptase (TERT) in human glioblastomas.

BACKGROUND: Telomerase is widely expressed in most human cancers, but is almost undetectable in normal somatic cells and is therefore a potential drug target. Using the human telomerase promoter platform, the naturally occurring compound butylidenephthalide (BP) was selected for subsequent investigation of antitumor activity in vitro and in vivo. METHODS: We treated human glioblastoma cells with BP and found a dose-dependent decrease in human telomerase reverse transcriptase (hTERT) mRNA expression and a concomitant increase in p16 and p21 expression. Because c-Myc and Sp1 are involved in transcriptional regulation of hTERT, the effect of BP on c-Myc and Sp1 expression was examined. RESULTS: Using electrophoretic mobility shift assays and western blotting, we showed that BP represses hTERT transcriptional activity via downregulation of Sp1 expression. Using the telomerase repeat amplification protocol, an association between BP concentration and suppression of telomerase activity, induction of human glioblastoma senescence, and inhibition of cellular proliferation was identified. This was supported by a mouse xenograft model, in which BP repressed telomerase and inhibited tumor proliferation, resulting in tumor senescence. Overexpression of hTERT restored telomerase activity in human glioblastoma cells and overcame replicative senescence. CONCLUSIONS: These findings suggest that BP inhibits proliferation and induces senescence in human glioblastomas by downregulating hTERT expression and consequently telomerase activity. This is the first study to describe regulation of telomerase activity by BP in human glioblastomas.

Protective effects of low-molecular-weight components of adipose stem cell-derived conditioned medium on dry eye syndrome in mice.

The present study demonstrated the protective effects of low-molecular-weight adipose-derived stem cell-conditioned medium (LADSC-CM) in a mouse model of dry eye syndrome. Mice subjected to desiccating stress and benzalkonium chloride had decreased tear secretion, impaired corneal epithelial tight junction with microvilli, and decreased conjunctival goblet cells. Topical application of adipose-derived stem cell-conditioned medium (ADSC-CM) stimulated lacrimal tear secretion, preserved tight junction and microvilli of the corneal epithelium, and increased the density of goblet cells and MUC16 expression in the conjunctiva. The low-molecular-weight fractions (< 10 kDa and < 3 kDa) of ADSC-CM (LADSC-CM) provided better protections than the > 10 kDa or > 3 kDa fractions of ADSC-CM. In the in vitro study, desiccation for 10 min or hyperosmolarity (490 osmols) for 24 h caused decreased viability of human corneal epithelial cells, which were reversed by LADSC-CM. The active ingredients in the LADSC-CM were lipophobic and stable after heating and lyophilization. Our study demonstrated that LADSC-CM had beneficial effects on experimental dry eye. It is worthy of further exploration for the active ingredient(s) and the mechanism.

Pulsed electromagnetic fields accelerate proliferation and osteogenic gene expression in human bone marrow mesenchymal stem cells during osteogenic differentiation.

Osteogenesis is a complex series of events involving the differentiation of mesenchymal stem cells to generate new bone. In this study, we examined the effect of pulsed electromagnetic fields (PEMFs) on cell proliferation, alkaline phosphatase (ALP) activity, mineralization of the extracellular matrix, and gene expression in bone marrow mesenchymal stem cells (BMMSCs) during osteogenic differentiation. Exposure of BMMSCs to PEMFs increased cell proliferation by 29.6% compared to untreated cells at day 1 of differentiation. Semi-quantitative RT-PCR indicated that PEMFs significantly altered temporal expression of osteogenesis-related genes, including a 2.7-fold increase in expression of the key osteogenesis regulatory gene cbfa1, compared to untreated controls. In addition, exposure to PEMFs significantly increased ALP expression during the early stages of osteogenesis and substantially enhanced mineralization near the midpoint of osteogenesis. These results suggest that PEMFs enhance early cell proliferation in BMMSC-mediated osteogenesis, and accelerate the osteogenesis.

Effect of pulsed electromagnetic field on the proliferation and differentiation potential of human bone marrow mesenchymal stem cells.

Pulsed electromagnetic fields (PEMFs) have been used clinically to slow down osteoporosis and accelerate the healing of bone fractures for many years. The aim of this study is to investigate the effect of PEMFs on the proliferation and differentiation potential of human bone marrow mesenchymal stem cells (BMMSC). PEMF stimulus was administered to BMMSCs for 8 h per day during culture period. The PEMF applied consisted of 4.5 ms bursts repeating at 15 Hz, and each burst contained 20 pulses. Results showed that about 59% and 40% more viable BMMSC cells were obtained in the PEMF-exposed cultures at 24 h after plating for the seeding density of 1000 and 3000 cells/cm2, respectively. Although, based on the kinetic analysis, the growth rates of BMMSC during the exponential growth phase were not significantly affected, 20-60% higher cell densities were achieved during the exponentially expanding stage. Many newly divided cells appeared from 12 to 16 h after the PEMF treatment as revealed by the cell cycle analysis. These results suggest that PEMF exposure could enhance the BMMSC cell proliferation during the exponential phase and it possibly resulted from the shortening of the lag phase. In addition, according to the cytochemical and immunofluorescence analysis performed, the PEMF-exposed BMMSC showed multi-lineage differentiation potential similar to the control group.

G-CSF enhances the therapeutic potency of stem cells transplantation in spinal cord-injured rats.

Aim: The therapeutic effects of human wisdom teeth-derived neuronal stem cell (tNSC) cotreatment with granulocyte-colony-stimulating factor (G-CSF) were evaluated for contusion-induced spinal cord injury in rats. Materials & methods: 7 days after contusion, tNSCs were transplanted to the injury site and followed by G-CSF cotreatment for 5 days. Behavioral deficits were evaluated by the Basso, Beattie and Bresnahan test. The injury site was collected for immunohistochemistry analysis. Results: The Basso, Beattie and Bresnahan test significantly improved in the cotreated group compared with the tNSCs or G-CSF single treatment groups. However, inflammation indices did not differ among the three groups. In vitro experiment demonstrated that tNSCs express both G-CSF and its relevant receptor. G-CSF enhanced tNSC proliferation and neurotrophins secretion in vitro. Conclusion: This study demonstrated that G-CSF enhances neurotrophins secretion of tNSCs, and might help improving functional recovery from spinal cord injury in rats if they were given together.

Palmitic Acid Methyl Ester Induces G2/M Arrest in Human Bone Marrow-Derived Mesenchymal Stem Cells via the p53/p21 Pathway.

Bone marrow-derived mesenchymal cells (BM-MSCs) are able to differentiate into adipocytes, which can secrete adipokines to affect BM-MSC proliferation and differentiation. Recent evidences indicated that adipocytes can secrete fatty acid metabolites, such as palmitic acid methyl ester (PAME), which is able to cause vasorelaxation and exerts anti-inflammatory effects. However, effects of PAME on BM-MSC proliferation remain unclear. The aim of this study was to investigate the effect of PAME on human BM-MSC (hBM-MSC) proliferation and its underlying molecular mechanisms. hBM-MSCs were treated with PAME for 48 h and then subjected to various analyses. The results from the present study show that PAME significantly reduced the levels of G2/M phase regulatory proteins, cyclin-dependent kinase 1 (Cdk1), and cyclin B1 and inhibited proliferation in hBM-MSCs. Moreover, the level of Mdm2 protein decreased, while the levels of p21 and p53 protein increased in the PAME-treated hBM-MSCs. However, PAME treatment did not significantly affect apoptosis/necrosis, ROS generation, and the level of Cdc25C protein. PAME also induced intracellular acidosis and increased intracellular Ca2+ levels. Cotreatment with PAME and Na+/H+ exchanger inhibitors together further reduced the intracellular pH but did not affect the PAME-induced decreases of cell proliferation and increases of the cell population at the G2/M phase. Cotreatment with PAME and a calcium chelator together inhibited the PAME-increased intracellular Ca2+ levels but did not affect the PAME-induced cell proliferation inhibition and G2/M cell cycle arrest. Moreover, the half-life of p53 protein was prolonged in the PAME-treated hBM-MSCs. Taken together, these results suggest that PAME induced p53 stabilization, which in turn increased the levels of p53/p21 proteins and decreased the levels of Cdk1/cyclin B1 proteins, thereby preventing the activation of Cdk1, and eventually caused cell cycle arrest at the G2/M phase. The findings from the present study might help get insight into the physiological roles of PAME in regulating hBM-MSC proliferation.

Investigating the mincing method for isolation of adipose-derived stem cells from pregnant women fat.

The success of stem cell application in regenerative medicine, usually require a stable source of stem or progenitor cells. Fat tissue represents a good source of stem cells because it is rich in stem cells and there are fewer ethical issues related to the use of such stem cells, unlike embryonic stem cells. Therefore, there has been increased interest in adipose-derived stem cells (ADSCs) for tissue engineering applications. Here, we aim to provide an easy processing method for isolating adult stem cells from human adipose tissue harvested from the subcutaneous fat of the abdominal wall during gynecologic surgery. We used a homogenizer to mince fat and compared the results with those obtained from the traditional cut method involving a sterile scalpel and forceps. Our results showed that our method provides another stable and quality source of stem cells that could be used in cases with a large quantity of fat. Furthermore, we found that pregnancy adipose-derived stem cells (P-ADSCs) could be maintained in vitro for extended periods with a stable population doubling and low senescence levels. P-ADSCs could also differentiate in vitro into adipogenic, osteogenic, chondrogenic, and insulin-producing cells in the presence of lineage-specific induction factors. In conclusion, like human lipoaspirates, adipose tissues obtained from pregnant women contain multipotent cells with better proliferation and showed great promise for use in both stem cell banking studies as well as in stem cell therapy.

Enhancement of Mitochondrial Transfer by Antioxidants in Human Mesenchymal Stem Cells.

Excessive reactive oxygen species is the major component of a harsh microenvironment after ischemia/reperfusion injury in human tissues. Combined treatment of N-acetyl-L-cysteine (NAC) and L-ascorbic acid 2-phosphate (AAP) promoted the growth of human mesenchymal stem cells (hMSCs) and suppressed oxidative stress-induced cell death by enhancing mitochondrial integrity and function in vitro. In this study, we aimed to determine whether NAC and AAP (termed MCA) could enhance the therapeutic potential of hMSCs. We established a coculture system consisting of MCA-treated and H2O2-treated hMSCs and investigated the role of tunneling nanotubes (TNTs) in the exchange of mitochondria between the 2 cell populations. The consequences of mitochondria exchange were assessed by fluorescence confocal microscopy and flow cytometry. The results showed that MCA could increase the mitochondrial mass, respiratory capacity, and numbers of TNTs in hMSCs. The "energized" mitochondria were transferred to the injured hMSCs via TNTs, the oxidative stress was decreased, and the mitochondrial membrane potential of the H2O2-treated hMSCs was stabilized. The transfer of mitochondria decreased the expression of S616-phosphorylated dynamin-related protein 1, a protein that dictates the fragmentation/fission of mitochondria. Concurrently, MCA also enhanced mitophagy in the coculture system, implicating that damaged mitochondria were eliminated in order to maintain cell physiology.

Synergistic protection of N-acetylcysteine and ascorbic acid 2-phosphate on human mesenchymal stem cells against mitoptosis, necroptosis and apoptosis.

Human mesenchymal stem cells (hMSCs) contribute to ischemic tissue repair, regeneration, and possess ability to self-renew. However, poor viability of transplanted hMSCs within ischemic tissues has limited its therapeutic efficiency. Therefore, it is urgent to explore new method to improve the viability of the grafted cells. By using a systematic analysis, we reveal the mechanism of synergistic protection of N-acetylcysteine (NAC) and ascorbic acid 2-phosphate (AAP) on hMSCs that were under H2O2-induced oxidative stress. The combined treatment of NAC and AAP (NAC/AAP) reduces reactive oxygen species (ROS) generation, stabilizes mitochondrial membrane potential and decreases mitochondrial fission/fragmentation due to oxidative stress. Mitochondrial fission/fragmentation is a major prologue of mitoptosis. NAC/AAP prevents apoptotic cell death via decreasing the activation of BAX, increasing the expression of BCL2, and reducing cytochrome c release from mitochondria that might lead to the activation of caspase cascade. Stabilization of mitochondria also prevents the release of AIF, and its nuclear translocation which may activate necroptosis via H2AX pathway. The decreasing of mitoptosis is further studied by MicroP image analysis, and is associated with decreased activation of Drp1. In conclusion, NAC/AAP protects mitochondria from H2O2-induced oxidative stress and rescues hMSCs from mitoptosis, necroptosis and apoptosis.

Cells and materials for liver tissue engineering.

Liver transplantation is currently the most efficacious treatment for end-stage liver diseases. However, one main problem with liver transplantation is the limited number of donor organs that are available. Therefore, liver tissue engineering based on cell transplantation that combines materials to mimic the liver is under investigation with the goal of restoring normal liver functions. Tissue engineering aims to mimic the interactions among cells with a scaffold. Particular materials or a matrix serve as a scaffold and provide a three-dimensional environment for cell proliferation and interaction. Moreover, the scaffold plays a role in regulating cell maturation and function via these interactions. In cultures of hepatic lineage cells, regulation of cell proliferation and specific function using biocompatible synthetic, biodegradable bioderived matrices, protein-coated materials, surface-modified nanofibers, and decellularized biomatrix has been demonstrated. Furthermore, beneficial effects of addition of growth factor cocktails to a flow bioreactor or coculture system on cell viability and function have been observed. In addition, a system for growing stem cells, liver progenitor cells, and primary hepatocytes for transplantation into animal models was developed, which produces hepatic lineage cells that are functional and that show long-term proliferation following transplantation. The major limitation of cells proliferated with matrix-based transplantation systems is the high initial cell loss and dysfunction, which may be due to the absence of blood flow and the changes in nutrients. Thus, the development of vascular-like scaffold structures, the formation of functional bile ducts, and the maintenance of complex metabolic functions remain as major problems in hepatic tissue engineering and will need to be addressed to enable further advances toward clinical applications.

Functional cells cultured on microcarriers for use in regenerative medicine research.

Microcarriers have been successfully used for many years for growing anchorage-dependent cells and as a means of delivering cells for tissue repair. When cultured on microcarriers, the number of anchorage-dependent cells, including primary cells, can easily be scaled up and controlled to generate the quantities of cells necessary for therapeutic applications. Recently, stem cell technology has been recognized as a powerful tool in regenerative medicine, but adequate numbers of stem cells that retain their differentiation potential are still difficult to obtain. For anchorage-dependent stem cells, however, microcarrier-based suspension culture using various types of microcarriers has proven to be a good alternative for effective ex vivo expansion. In this article, we review studies reporting the expansion, differentiation, or transplantation of functional anchorage-dependent cells that were expanded with the microcarrier culture system. Thus, the implementation of technological advances in biodegradable microcarriers, the bead-to-bead transfer process, and appropriate stem cell media may soon foster the ability to produce the numbers of stem cells necessary for cell-based therapies and/or tissue engineering.

Transplantation of human Wharton's Jelly-derived stem cells alleviates chemically induced liver fibrosis in rats.

There is currently no effective treatment method available for liver fibrosis. We therefore evaluated the use of Wharton's jelly stem cells (WJSCs; the major umbilical cord stem cell population) to treat chemically induced liver fibrosis via intraperitoneal injection of thioacetamide. WJSCs were transplanted into liver-damaged rats via the portal vein and the treatment was evaluated by assessing serum biochemistry and histopathology. Transplanted WJSCs were distributed in the fibrotic area and around blood vessels, and hepatic recovery was accelerated. Serum prothrombin time significantly recovered, and serum albumin also improved at 21 days posttransplantation; collagen accumulation also decreased at 14 days. Thus, human WJSCs promoted recovery after chronic liver damage. Using immunohistochemical analyses, we determined that transplanted WJSCs produce albumin, hepatocyte growth factor (HGF), and metalloproteinase (MMP) after transplantation to chemically injured liver, indicating that WJSC may help to decrease liver collagen and thus may be useful for treating liver fibrosis.

Mesenchymal stem cells facilitate recovery from chemically induced liver damage and decrease liver fibrosis.

AIMS: To investigate the feasibility and mechanism of liver damage repair using human bone marrow mesenchymal stem cells (hBMMSCs), we investigated the potential for hBMMSCs in recovery from liver damage, including fibrotic liver repair, using the CCl(4)-induced model for liver damage in the rat. MAIN METHODS: Rats were injected with 0.5 ml/kg CCl(4) to induce liver damage and progressive liver fibrosis. hBMMSCs labeled with GFP were injected into the rats through the portal vein. KEY FINDINGS: After one day of transplantation, GFP-labeled cells were found around the liver lobules, the hepatic blood vessels, and the edge of the liver lobes. Biochemical and histopathological analyses showed significantly increased recovery from liver damage in the transplanted group. In addition, transplanted hBMMSCs express matrix metalloproteinases (MMP), and liver fibrosis was significantly decreased. The degree of fibrosis reduction paralleled the number of hBMMSCs observed in liver sections. SIGNIFICANCE: Our data suggest that hBMMSCs may facilitate recovery from chronic liver damage and may decrease liver fibrosis. Therefore, hBMMSCs are a potential option for treatment of liver cirrhosis.