Immunomodulative effects of mesenchymal stem cells derived from human embryonic stem cells in vivo and in vitro.

OBJECTIVE: Human embryonic stem cells (hESCs) have recently been reported as an unlimited source of mesenchymal stem cells (MSCs). The present study not only provides an identical and clinically compliant MSC source derived from hESCs (hESC-MSCs), but also describes the immunomodulative effects of hESC-MSCs in vitro and in vivo for a carbon tetrachloride (CCl(4))-induced liver inflammation model. METHODS: Undifferentiated hESCs were treated with Rho-associated kinase (ROCK) inhibitor and induced to fibroblast-looking cells. These cells were tested for their surface markers and multilineage differentiation capability. Further more, we analyzed their immune characteristics by mixed lymphocyte reactions (MLRs) and animal experiments. RESULTS: hESC-MSCs show a homogenous fibroblastic morphology that resembles bone marrow-derived MSCs (BM-MSCs). The cell markers and differentiation potential of hESC-MSCs are also similar to those of BM-MSCs. Unlike their original cells, hESC-MSCs possess poor immunogenicity and can survive and be engrafted into a xenogenic immunocompetent environment. CONCLUSIONS: The hESC-MSCs demonstrate strong inhibitory effects on lymphocyte proliferation in vitro and anti-inflammatory infiltration properties in vivo. This study offers information essential to the applications of hESC-MSC-based therapies and evidence for the therapeutic mechanisms of action.

Promotion of self-renewal of embryonic stem cells by midkine.

AIM: To characterize the expression and function of midkine (MK) in an in vitro embryonic stem cell (ESC) culture system. METHODS: To investigate the potential roles of MK, the expression of MK in ESCs was evaluated by RT-PCR and immunocytochemistry. The effects of MK on the self-renewal of ESCs were measured using alkaline phosphatase assays, immunocytochemistry, RT-PCR and colony-forming assays. The mechanism of the growth-promoting effect of MK in mESCs was assessed by cell cycle analysis and Western blot analysis. RESULTS: MK is expressed in mouse embryonic stem cells (mESCs), human embryonic stem cells (hESCs) and mouse embryonic fibroblasts (MEFs). MK promotes proliferation and self-renewal of mESCs both in feeder and feeder free culture systems. It also promotes self-renewal and proliferation of hESCs. Further study showed that MK promotes the growth of mESCs by inhibiting apoptosis while accelerating the progression toward the S phase, and enhances mESC self-renewal through PI3K/Akt signaling pathway. CONCLUSION: MK plays profound roles in ESCs. MK/PTPzeta signaling pathway is a novel pathway in the signal network maintaining pluripotency of ESCs. The results extend our knowledge on pluripotency control of ESCs and the relationship between ESCs and cancers.

The effect of extracellular calcium and inorganic phosphate on the growth and osteogenic differentiation of mesenchymal stem cells in vitro: implication for bone tissue engineering.

The aim of this study is to demonstrate the effect of extracellular calcium ion (Ca2+) and inorganic phosphate (Pi) concentrations on the growth and differentiation of bone-marrow-derived mesenchymal stem cells (MSCs), which is essential to understand the interaction between calcium phosphate ceramic (CPC) scaffolds and seeded cells during the construction of tissue-engineered bones. MSCs were separated from rabbits and cultured in media with different concentrations of Ca2+ and Pi supplements. Their proliferation, apoptosis, mineralization and osteogenic differentiation were determined by the MTT assay, TUNEL assay, Vonkossa stain and RT-PCR examination. A two-way ANOVA calculation with comparisons of estimated marginal means by LSD was used for statistical analysis. Results showed that the optimal extracellular Ca2+ and Pi concentrations for the cells to proliferate and differentiate were 1.8 mM and 0.09 mM, respectively, which are the concentrations supplied in many commonly used culture media such as DMEM and alpha-MEM. Cell proliferation and differentiation decreased significantly with greater or lower concentrations of the Pi supplement. Greater Pi concentrations also led to significant cell apoptosis. Greater Ca2+ concentrations did not change cell proliferation but significantly inhibited cell differentiation. In addition, greater Ca2+ concentrations could significantly enhance cell mineralization. In conclusion, extracellular Ca2+ and Pi significantly influence the growth and osteogenic differentiation of MSCs. It is important to take the cellular effect of Ca2+ and Pi into consideration when designing or constructing scaffolds for bone tissue engineering with CPC.

Different effects of nanophase and conventional hydroxyapatite thin films on attachment, proliferation and osteogenic differentiation of bone marrow derived mesenchymal stem cells.

UNLABELLED: It is suggested that nanophase hydroxyapatite (nHAP) might have advantages over conventional hydroxyapatite (cHAP) as a biomaterial for bone regeneration. To be a satisfactory candidate for bone tissue engineering, it is important to support the growth and differentiation of bone marrow-derived mesenchymal stem cells (BMSCs). The purpose of this study is to determine whether nHAP as cell growth substrata could give better support for attachment, proliferation and osteogenic differentiation of BMSCs than cHAP. MATERIALS AND METHODS: nHAP and cHAP films were prepared as the substrata for the cell growth. BMSCs obtained from rabbit were seeded on the films. Attachment, proliferation and osteogenic differentiation of BMSCs on the two kinds of films were evaluated. RESULTS: Cell attachment ratio on nHAP films was significantly higher than that on cHAP films. Doubling time on nHAP films was significantly shorter than that on cHAP films. Amount of total proteins detected from cells cultured on nHAP films was significantly higher than that on cHAP films. However, alkaline phosphatase activity and osteocalcin content of the two groups showed no significant difference. CONCLUSIONS: nHAP films favored cell attachment and proliferation but not osteogenic differentiation of BMSCs compared with cHAP films.

Dynamical influence of Cordyceps sinensis on the activity of hepatic insulinase of experimental liver cirrhosis.

BACKGROUND: Cordeceps sinensis (CS) is a herb which can inhibit the liver fibrosis. Hyperinsulinemia is common in liver cirrhosis patients. The activity of insulin degrading enzyme could reflect the metabolism of insulin. This study was to detect the dynamical effects and mechanisms of CS on the activity of hepatic insulinase in CCl4 induced liver cirrhosis in rats. METHODS: Rats were randomly allocated into three groups: normal group, model group and CS group. The rats in the normal group were sacrificed at the beginning of experiment, and the other two groups were sacrificed randomly at the end of the third, sixth and ninth weeks. Blood and tissue specimens were taken. Biochemical assays were used to determine the changes of alanine transaminase (ALT), albumin levels in serum. And radioimmunological assays were used to determine the changes of hyaluronic acid (HA), insulin levels in serum and the activity of hepatic insulinase. RESULTS: No significant differences were seen in the serum levels of ALT, albumin, HA between the CS group and the model group at the third and sixth weeks (P>0.05). The serum levels of ALT, HA in the CS group were lower than those in the model group at the ninth week (P<0.05), but the serum level of albumin in the CS group was higher than that in the model group at the ninth week (P<0.05). No significant differences were observed in the serum levels of insulin and the activity of hepatic insulinase between the CS and model groups at the third week and the normal group (P>0.05). The serum levels of insulin in the CS and model groups at the sixth and ninth weeks were higher than those in the normal group (P<0.05). But the activity of hepatic insulinase was lower than that in the normal group (P<0.05 or P<0.01). No significant differences were found in the serum levels of insulin and the activity of hepatic insulinase between the CS and model groups at the third, sixth and ninth weeks (P>0.05). CONCLUSIONS: CS may decrease the damage to hepatocyte by CCl4, and inhibit hepatic fibrogenesis. Six weeks after CCl4 administration, the activity of hepatic insulinase began decreasing. CS could not inhibit the decrease of the activity of hepatic insulinase.

Inhibitive effect of cordyceps sinensis on experimental hepatic fibrosis and its possible mechanism.

AIM: To investigate the inhibitive effect and its possible mechanism of Cordyceps Sinensis (CS) on CCl(4)-plus ethanol-induced hepatic fibrogenesis in experimental rats. METHODS: Rats were randomly allocated into a normal control group, a model control group and a CS group. The latter two groups were administered with CCl(4) and ethanol solution at the beginning of the experiment to induce hepatic fibrosis. The CS group was also treated with CS 10 days after the beginning of CCl(4) and ethanol administration. All control groups were given corresponding placebo at the same time. At the end of the 9th week, rats in each group were humanely sacrificed. Blood and tissue specimens were taken. Biochemical, radioimmunological, immunohistochemical and molecular biological examinations were used to determine the level change of ALT, AST, HA, LN content in serum and TGFbeta(1), PDGF, collagen I and III expression in tissue at either protein or mRNA level or both of them. RESULTS: As compared with the model control group, serum ALT, AST, HA, and LN content levels were markedly dropped in CS group (86.0+/-34.4 vs 224.3+/-178.9, 146.7+/-60.2 vs 272.6+/-130.1, 202.0+/-79.3 vs 316.5+/-94.1 and 50.4+/-3.0 vs 59.7+/-9.8, respectively, P<0.05). Tissue expression of TGFbeta(1) and its mRNA, collagen I mRNA were also markedly decreased (0.2+/-0.14 vs 1.73+/-1.40, 1.68+/-0.47 vs 3.17+/-1.17, 1.10+/-0.84 vs 2.64+/-1.40, respectively, P<0.05). More dramatical drop could be seen in PDGF expression (0.87+/-0.43 vs 1.91+/-0.74, P<0.01). Although there was no statistical significance, it was still strongly suggested that collagen III mRNA expression was also decreased in CS group as compared with model control group (0.36+/-0.27 vs 0.95+/-0.65, P=0.0615). In this experiment, no significant change could be found in PDGF mRNA expression between two groups (0.35+/-0.34 vs 0.70+/-0.46, P>0.05). CONCLUSION: Cordyceps sinensis could inhibit hepatic fibrogenesis derived from chronic liver injury, retard the development of cirrhosis, and notably ameliorate the liver function. Its possible mechanism involves inhibiting TGFbeta(1) expression, and thereby, down regulating PDGF expression, preventing HSC activation and deposition of procollagen I and III.

[Influence of Cordyceps sinensis on pancreatic islet beta cells in rats with experimental liver fibrogenesis].

OBJECTIVE: To investigate the effect of Cordyceps sinensis (CS) on pancreatic islet B cells of experimental hepatic fibrogenesis rats. METHODS: Rats were randomly allocated into three groups: normal group, model group and CS group. The rats in the latter two groups were administered with CCl(4) solution to induce liver fibrosis, the CS group was also treated with CS 10 days after the beginning of CCl(4) administration. Rats in normal group were sacrificed at the beginning of the experiment, while the rats in the other two groups were sacrificed randomly at the end of the third and sixth weeks. The rats' islets were isolated and cultured in vitro, then the basal insulin level of the islets and the serum level of insulin were determined by radioimmunological assay. RESULTS: It seemed no change that the levels of serum insulin and basal insulin between the model group and the normal group at the third week. But at the sixth week, both insulin levels in the model group were higher than those in the normal group (52.6 mU/L2.5 mU/L vs 23.7 mU/L 2.3 mU/L, q=13.01, p<0.05; 52.94muU/ml 13.12muU/ml vs 35.16muU/ml 5.64muU/ml, q=10.06, p<0.01). No significant change could be seen in the serum levels of insulin between the CS group and the model group at the third and sixth weeks. But the basal insulin levels in the CS group were apparently higher than those in the model group at the third and sixth weeks (156.63muU/ml 6.57muU/ml vs 39.64muU/ml 3.95muU/ml, q=66.94, p<0.001; 140.44muU/ml 38.53muU/ml vs 52.94muU/ml 13.12muU/ml, q=12.98, p<0.01). CONCLUSION: Cordyceps sinensis can increase the basal insulin level of the islets in CCl(4)-induced liver fibrosis rats.