Human liver stem cell‐derived microvesicles accelerate hepatic regeneration in hepatectomized rats

Abstract

Several studies indicate that adult stem cells may improve the recovery from acute tissue injury. It has been suggested that they may contribute to tissue regeneration by the release of paracrine factors promoting proliferation of tissue resident cells. However, the factors involved remain unknown. In the present study we found that microvesicles (MVs) derived from human liver stem cells (HLSC) induced in vitro proliferation and apoptosis resistance of human and rat hepatocytes. These effects required internalization of MVs in the hepatocytes by an α4-integrin-dependent mechanism. However, MVs pre-treated with RNase, even if internalized, were unable to induce hepatocyte proliferation and apoptosis resistance, suggesting an RNA-dependent effect. Microarray analysis and quantitative RT-PCR demonstrated that MVs were shuttling a specific subset of cellular mRNA, such as mRNA associated in the control of transcription, translation, proliferation and apoptosis. When administered in vivo, MVs accelerated the morphological and functional recovery of liver in a model of 70% hepatectomy in rats. This effect was associated with increase in hepatocyte proliferation and was abolished by RNase pre-treatment of MVs. Using human AGO2, as a reporter gene present in MVs, we found the expression of human AGO2 mRNA and protein in the liver of hepatectomized rats treated with MVs. These data suggested a translation of the MV shuttled mRNA into hepatocytes of treated rats. In conclusion, these results suggest that MVs derived from HLSC may activate a proliferative program in remnant hepatocytes after hepatectomy by a horizontal transfer of specific mRNA subsets.

Role of Lefty in the anti tumor activity of human adult liver stem cells

“…In addition, HLSC are known to be positive for several embryonic factors, including nanog, Oct-4, Sox2 and SSEA-4. 21 However, at variance of embryonic stem cells, HLSC are not pluripotent and do not form teratoma, but they are only multipotent as they differentiate in hepatocytes, osteocytes, adipocytes, endothelial and islet-like cells under specific culture conditions. 18 In the present study, we found that the CM of HLSC inhibited proliferation and enhanced apoptosis of HepG2 in vitro and induced HepG2 tumor regression in vivo.…”

“…(Cambrex Bio Science, Verviers, Belgium) cultured in minimum essential medium/endothelial cell basal medium-1 (a-MEM/EBM) (3:1) (Cambrex Bio Science) supplemented with L-glutamine (5 mM), Hepes (12 mM, pH 7.4), penicillin (50 IU/ml), streptomycin (50 mg/ml) (all from Sigma-Aldrich, St Louis, MO, USA), fetal bovine serum (FBS, 10%) as previously described. 18,21 The expanded cells were transferred to a T-75 flask and analyzed when they approached confluence.…”

“…Cells were characterized by FACS analysis for the expression of mesenchymal stem cell markers, and in the case of HLSC also for the expression of tissuespecific markers, including a-fetoprotein and human albumin and of resident stem cell markers, such as vimentin and nestin, as previously described. 18,21 Moreover, HLSC were negative for the oval cell markers CD34, CD117 and cytokeratin 19 and express the embryonic stem cell markers nanog, Oct4, SOX2 and SSEA-4. 18,21 Both the cell types were able to undergo osteogenic, adipogenic and chondrogenic differentiation when cultured in the appropriate differentiative media.…”

“…18,21 Moreover, HLSC were negative for the oval cell markers CD34, CD117 and cytokeratin 19 and express the embryonic stem cell markers nanog, Oct4, SOX2 and SSEA-4. 18,21 Both the cell types were able to undergo osteogenic, adipogenic and chondrogenic differentiation when cultured in the appropriate differentiative media. 18,22 HLSC were also able to differentiate in mature hepatocytes.…”

“…Immunohistochemistry for detection of proliferation and apoptosis was performed using the anti-PCNA monoclonal antibody (Santa Cruz Biotechnology) or TUNEL, respectively, as previously described. 21 …”