Differentiation of Human Peripheral Blood Mononuclear Cells and Cd14+ Monocytes Into Hepatocyte-Like Cells Obtained From Liver Cirrhosis Patients
European Journal of Molecular & Clinical Medicine,
2020, Volume 7, Issue 11, Pages 4121-4133
Abstract
The current study has benefit of the potentiality of peripheral blood monocytes (MONs) and adherent peripheral blood mononuclear cells (MNCs) to differentiate into various progenitor cells according to growth factors added. Aim: the aim of this work was to investigate and compare the efficiency of PB monocytes (MONs), and adherent mononuclear cells (MNCs) obtained from cirrhotic patients to expand and differentiate into functioning hepatocyte-like cells in vitro. Material and methods: 20 patients suffering from chronic hepatitis and liver cirrhosis were included in the study. MNCs were isolated from PB using Ficoll Hypaque. MONs (CD14+), and adherent MNCs (contain CD34+Cs) were separately cultured for28 days in presence and absence of recombinant human hepatic growth factor (rh-HGF). The culture media were changed every 7 days and culture supernatants were harvested, allocated, and stored at – 80C0. Hepatocyte-like cells were examined for morphology, function, and hepatocyte markers by: a) estimation of albumin secretion, urea production, and LDH release in the culture supernatants, b) qRT-PCR for cultured cells at end points of cultures to detect albumin gene expression, and c) detection of CK 18 and Hep Par-1 proteins in cultured cells using immunohistochemical technique.Results: Data obtained revealed: Monocyte % as detected by differential leukocyte count and flowcytometer showed highly significant increase in suspensions prepared using human Pan Monocyte isolation kit as compared to Ficoll separated MNCs, suspensions. Albumin, urea, and LDH showed highly significant differences between culture supernatants containing rh-HGF and those of control cultures depended on time points of estimations. Late specific liver markers; Hep par-1 and CK18 by immunocytochemistry showed increase in adherent MNCs cultured with rh-HGF compared to CD14+ MONs. Albumin gene expression increased significantly in differentiated cells derived from MNCs cultured with rh-HGF compared to CD14+ MONs at end point of culture as detected by qRT-PCR. Conclusion: The obtained results showed successful in-vitro generation of functioning hepatocyte-like cells from easily accessible source of cells in presence of rh-HGF. So innovative regenerative cellular therapy indicates the possibility of autologous cell transfusion after hepatic differentiation for treatment of CLD and provide a promising alternative treatment for end stage liver disease.
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