Ex vivo expansion of circulating CD34(+) cells enhances the regenerative effect on rat liver cirrhosis.

Ex vivo expansion of autologous cells is indispensable for cell transplantation therapy of patients with liver cirrhosis. The aim of this study was to investigate the efficacy of human ex vivo-expanded CD34(+) cells for treatment of cirrhotic rat liver. Recipient rats were intraperitoneally injected with CCl4 twice weekly for 3 weeks before administration of CD34(+) cells. CCl4 was then re-administered twice weekly for 3 more weeks, and the rats were sacrificed. Saline, nonexpanded or expanded CD34(+) cells were injected via the spleen. After 7 days, CD34(+) cells were effectively expanded in a serum-free culture medium. Expanded CD34(+) cells were also increasingly positive for cell surface markers of VE-cadherin, VEGF receptor-2, and Tie-2. The expression of proangiogenic growth factors and adhesion molecules in expanded CD34(+) cells increased compared with nonexpanded CD34(+) cells. Expanded CD34(+) cell transplantation reduced liver fibrosis, with a decrease of αSMA(+) cells. Assessments of hepatocyte and sinusoidal endothelial cell proliferative activity indicated the superior potency of expanded CD34(+) cells over non-expanded CD34(+) cells. The inhibition of integrin αvß3 and αvß5 disturbed the engraftment of transplanted CD34(+) cells and aggravated liver fibrosis. These findings suggest that expanded CD34(+) cells enhanced the preventive efficacy of cell transplantation in a cirrhotic model.

Prevention of liver fibrosis and liver reconstitution of DMN-treated rat liver by transplanted EPCs.

BACKGROUND: Using the dimethylnitrosamine (DMN) rat model of induced fibrosis, we investigated whether transfer of in vitro-expanded endothelial progenitor cells (EPCs) could reconstitute liver tissue and protect against liver fibrosis. MATERIALS AND METHODS: Low-density, adherent, rat bone marrow-derived mononuclear cells were cultured for one week in medium supporting the growth of chemokine (C-X-C motif) receptor 4 (CXCR4)-positive EPCs that were used for transplantation. Test rats were treated with weekly intraperitoneal injections of DMN over a period of 4 weeks. During that period, the rats were also transplanted weekly with in vivo-expanded EPCs. RESULTS: Transplanted CXCR4-positive expanded EPCs entered around the portal tracts, fibrous septa and hepatic sinusoids, locations at which stromal cell-derived factor 1 (SDF-1), a ligand attracting CXCR4-positive cells, was expressed nearby. In EPC-transplanted rats, we observed suppression of liver fibrogenesis, reduced deposition of type I collagen and fibronectin, fewer α-smooth muscle actin-positive cells and lower expression of transforming growth factor (TGF)-ß. The expression of growth factors promoting hepatic regeneration (hepatocyte growth factor, transforming growth factor-α (TGF-α), epidermal growth factor and vascular endothelial growth factor) was significantly increased in EPC-transplanted rats, resulting in hepatocyte proliferation. Immunohistochemical analyses of eNOS and isolectin B4 demonstrated that the livers of EPC-transplanted animals had markedly increased vascular density, suggesting reconstitution of sinusoidal blood vessels with endothelium. Liver function tests of transaminase, total bilirubin, total protein and albumin demonstrated that normal levels were maintained in EPC-transplanted rats. CONCLUSIONS: EPC transplantation effectively promotes the remodelling of tissues damaged by liver fibrosis; it can also reconstitute sinusoids in chronic liver injury.

Human peripheral blood CD34-positive cells enhance therapeutic regeneration of chronically injured liver in nude rats.

We investigated whether transplantation of purified human peripheral blood CD34(+) cells could reduce established liver fibrosis and up-regulate therapeutic regeneration. Human peripheral blood CD34(+) cells were isolated from total mononuclear cells of healthy volunteers by magnetic cell sorting. Recipient nude rats were injected intraperitoneally with carbon tetrachloride (CCl(4)) twice weekly for 3 weeks before single administration of CD34(+) cells. CCl(4) was then re-administered twice weekly for 3 more weeks, and the nude rats were sacrificed. Saline (control group), 1 × 10(5) (low-dose group), 5 × 10(5) (middle-dose group), or 2 × 10(6) (high-dose group) CD34(+) cells/kg body weight were intrasplenically transplanted after CCl(4) treatment for 3 weeks. Reverse transcriptase-polymerase chain reaction analysis of the freshly isolated CD34(+) cells revealed the expression of CD31, keratin19, α-smooth muscle actin (α-SMA), and epithelial growth factor, but not other liver related markers. The transplanted cells differentiated into vascular and sinusoidal endothelial cells, and vascular smooth muscle cells. CD34(+) cell transplantation reduced liver fibrosis in a dose-dependent fashion, with decreased collagen type-I and α-SMA-positive cells after 6 weeks of CCl(4) treatment by Mallory's Azan and immunohistochemical staining. Gelatin zymography showed that the expression levels of active matrix metalloproteinase-2 and -9 in CD34(+) cell transplanted livers were significantly stronger than those in saline-infused livers. In recipients of high-doses of CD34(+) cells, the number of PCNA-positive hepatocyte increased 6 weeks after CCl(4) treatment compared with saline-infused livers. We conclude that human peripheral blood CD34(+) cell transplantation halts established liver fibrosis and promotes hepatic regeneration in CCl(4)-induced chronic liver injury.