growth of autologous cells is indispensable for cell transplantation therapy of individuals with liver cirrhosis. individuals. We tested the preventive effectiveness of these expanded cells and 4SC-202 nonexpanded PB-CD34+ cells in the treatment of carbon tetrachloride (CCl4)-induced cirrhotic liver. Results Characterization of expanded MGC20461 G-CSF-mobilized PB-CD34+ cells After 7 days in tradition expanded G-CSF-mobilized PB-CD34+ cells restored vasculogenic potential of new PB-CD34+ cells. (a) PB-CD34+ cells were characterized by circulation cytometric analysis. PB-CD34+ cells were also progressively positive for cell surface markers of VE-cadherin … Cell proliferation was analyzed using circulation cytometry and western blotting. Expanded PB-CD34+ cells were compared with nonexpanded (new) PB-CD34+ cells. The percentage of the cell populace in the G0/G1 phase in the fresh versus expanded PB-CD34+ cells was 79.8 versus 52.6% 14.4 versus 42.4% in S phase and 5.8 versus 5.0% in G2/M phase (Number 1b). The manifestation level of proliferating cell nuclear antigen (PCNA) was upregulated in expanded PB-CD34+ cells (Number 1c). The primitive EPC-colony forming models (CFUs) and definitive EPC-CFUs were counted separately (Number 1d). After 20 days in tradition the number of EPC-CFUs per dish of expanded PB-CD34+ cells was significantly greater than that of new PB-CD34+ cells (primitive EPC-CFUs: new 4 expanded 9.8 definitive EPC-CFUs: fresh 12.7 expanded 28.3 Number 1e). The RT-PCR of expanded PB-CD34+ cells exposed the manifestation of human specific genes for was not detected (Number 2a). To clarify the paracrine effects of transplanted cells we measured the mRNA manifestation of various growth factors and proangiogenic factors in new and expanded PB-CD34+ cells using real-time PCR. The mRNA manifestation levels 4SC-202 of in expanded PB-CD34+ cells were significantly higher than those in new PB-CD34+ cells (Number 2a ? b).b). In contrast the expression level of in expanded PB-CD34+ cells was significantly lower than that in new PB-CD34+ cells (Number 2b). Number 2 Characterization of expanded G-CSF-mobilized PB-CD34+ cells and was not observed. (b) The mRNA manifestation levels … Transplanted expanded PB-CD34+ cells differentiated into vascular and sinusoidal endothelial cells and vascular clean muscle cells Human being CD31-positive endothelial cells derived from transplanted expanded PB-CD34+ cells were located near the vessels within 4SC-202 the fibrous septa and along the hepatic sinusoids of CCl4-treated livers (Number 2c). Moreover we observed human being SM1-positive vascular clean muscle mass cells. Human vascular clean muscle cells derived from expanded PB-CD34+ cells were located in the vasculature within the periportal areas (Number 2c). However the transplanted expanded PB-CD34+ cells did not differentiate into human being keratin19-positive bile ductular epithelial cells human being albumin-positive hepatocytes or human being AFP-positive cells (data not demonstrated). We did not detect any human being cells in saline-infused livers treated with CCl4 (Number 2c). Transplantation of expanded PB-CD34+ cells prevented the progression of liver fibrosis inside a dose-dependent manner Reduction of liver fibrosis by transplantation of expanded PB-CD34+ cells was shown by Mallory’s Azan histologic staining (Number 3a) and by immunohistochemical analysis for αSMA (Number 3c) in CCl4-treated livers. Semi-quantitative analysis indicated the relative extent of the fibrotic area was significantly reduced in a dose-dependent manner for transplanted new PB-CD34+ cells and expanded PB-CD34+ cells (saline 8.7 new low-dose (Lo) group 7 4SC-202 new high-dose (Hi) group 5.5 expanded Lo group 6.3 expanded Hi group 4.5 Number 3b). However there was no significant difference in liver fibrosis between new PB-CD34+ cell transplantation and expanded PB-CD34+ cell transplantation. The number of αSMA-positive cells in the liver transplanted with new or expanded PB-CD34+ cells was fewer than that in nontreated liver (Number 3c). These inhibitory effects were observed ubiquitously throughout the liver. Real-time PCR showed that the manifestation of mRNAs was significantly decreased inside a dose-dependent manner in new and expanded PB-CD34+ cell-transplanted livers compared to nontreated livers 4SC-202 with the exception of new Lo PB-CD34+ cell-transplanted livers (Number 3d). Number 3 Transplantation of expanded PB-CD34+ cells prevented the progression 4SC-202 of liver fibrosis inside a dose-dependent manner. (a) Fibrosis was less notable in expanded PB-CD34+.