Enhanced hepatic differentiation of human cord blood-derived mesenchymal stem cells using activin A-based protocol

Abstract

Recent studies have shown that mesenchymal stem cells (MSCs), which are isolated from various sources such as bone marrow, cord blood, adipose tissue, amniotic fluid, and the placenta, could differentiate into hepatocyte-like cells in vitro. However, these in vitro differentiation protocols are not practical for clinical usage, as more than 28 days is required to differentiate MSCs into hepatic lineages and the differentiation efficiency is low. In addition, the in vivo hepatic differentiation of MSCs and therapeutic efficacy of MSC administration in chronic liver injury models remains controversial. The goal of the present study was to improve and modify the hepatic differentiation protocol of cord blood-derived MSC (CBMSC), based on the current knowledge of liver ontogeny. Also, we investigated the therapeutic effects of systemic CBMSC infusion in cirrhotic rat model.MSCs were isolated from human cord blood using the standard plastic adherence method, and its surface phenotype and multi-lineage differentiation potential were examined. Isolated CBMSCs were differentiated into hepatic lineages using the activin A-based protocol or previously published two-step protocol. The activin A-based protocol was modified from the two-step protocol to reflect cytokine expression during embryonic hepatogenesis (activin A plus fibroblast growth factor (FGF)-4, followed by a combination of hepatocyte growth factor, FGF-4, oncostatin M and dexamethasone). Differentiated hepatocyte-like cells were examined for hepatocyte-specific markers, such as alpha-fetoprotein (AFP) and albumin. Ex vivo expanded CBMSCs were infused in Wistar rats with thioacetamide-induced chronic liver injury. Biochemical markers, liver fibrosis and engraftment of CBMSCs were assessed 1 or 4 weeks after cell or placebo infusion.After induction under each protocol, cuboidal morphology, which is characteristic

of hepatocytes, was observed. These hepatocyte-like cells, which were induced by the two-step protocol, did not express mRNA of hepatocyte-specific AFP and albumin at 4 weeks of induction. However, using the activin A-based protocol, CBMSCs (five among 12 cell lines from different donors) revealed expression of AFP, albumin and cytokeratin 19 as early as at 2 weeks of induction. Four weeks after CBMSC infusion into rats with chronic liver injury, there was no difference in serological markers of liver injury and area of liver fibrosis between the cell and placebo infusion groups. Infused CBMSCs were detected in the perivascular and fibrous region of the liver and did not acquire mature hepatic phenotypes.CBMSCs have the potential to differentiate into hepatic lineages in vitro and activin A is hereby essential to promote differentiation of CBMSCs towards functional hepatocyte-like cells. However, improvement of liver function and liver fibrosis after infusion of CBMSCs could not be found in our experimental cirrhotic rat model.