EGF and HB-EGF enhance the proliferation of programmable cells of monocytic origin (PCMO) through activation of MEK/ERK signaling and improve differentiation of PCMO-derived hepatocyte-like cells

EGF and HB-EGF enhanced cell proliferation of PCMOs as demonstrated by increased expression of cycle control genes (ABL, ANAPC2, CDC2, CDK4, CDK6), phosphorylation of the retinoblastoma protein, and increased PCMO cell numbers after stimulation with EGF or HB-EGF. EGF also raised the number of monocytes expressing the proliferation marker Ki67. PCMOs expressed the EGF receptors EGFR (ERBB1) and ERBB3, and expression of both increased during PCMO generation. Phosphoimmunoblotting of PCMOs indicated that both EGF and HB-EGF activated MEK-1/2 and ERK1/2 in a concentration-dependent fashion with the effect of EGF being more prominent. EGF treatment further decreased expression of p47phox and increased that of Nanog indicating enhanced dedifferentiation and pluripotency, respectively. Treatment with both EGF and HB-EGF resulted in NeoHepatocytes with improved functional parameters.The results suggested that the addition of EGF or HB-EGF to PCMO differentiation medium superactivates MEK/ERK signaling which then increases both PCMO proliferation, number, and functional differentiation of PCMO-derived NeoHepatocytes.Although hepatocyte transplantation is a therapeutic option for end-stage liver diseases, cell material is scarce due to a critical shortage of liver tissues and the lack of protocols that allow maintaining the differentiated hepatocyte phenotype in culture for more than a week. Thus, generation of hepatocyte-like cells from stem cells or stem cell-like cells may represent a promising alternative [1]. One such cell type with inherent stem cell-like features is the human peripheral blood monocyte [2-5]. By initially inducing a process of dedifferentiation we have generated from these cells a more plastic derivative termed “programmable cell of monocytic origin” (PCMO). PCMOs are prone to acquire functional activities of hepatocyte-like cells (NeoHepatocytes) upon stimulation with appropriate differentiation media in vitro[2,3], and in vivo following transplantati