New Approaches in the Differentiation of Human Emb

Authors: Iman Saramipoor Behbahan Yuyou Duan Alexander Lam Shiva Khoobyari Xiaocui Ma Tijess P Ahuja Mark A Zern

Publish Date: 2011/02/19

Volume: 7, Issue: 3, Pages: 748-759

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Abstract

Orthotropic liver transplantation is the only established treatment for endstage liver diseases Utilization of hepatocyte transplantation and bioartificial liver devices as alternative therapeutic approaches requires an unlimited source of hepatocytes Stem cells especially embryonic stem cells possessing the ability to produce functional hepatocytes for clinical applications and drug development may provide the answer to this problem New discoveries in the mechanisms of liver development and the emergence of induced pluripotent stem cells in 2006 have provided novel insights into hepatocyte differentiation and the use of stem cells for therapeutic applications This review is aimed towards providing scientists and physicians with the latest advancements in this rapidly progressing fieldOrthotropic liver transplantation is the only established treatment for endstage liver disease However because of the shortage of viable livers available for transplant many patients die while remaining on the extensive waiting list and many more are never added to the list Utilization of hepatocyte transplantation and bioartificial liver devices have been proposed as alternative therapeutic approaches to this problem 1 2 These two approaches however require an unlimited source of hepatocytes and human primary hepatocytes provide the most desirable solution for cell therapies Yet the utilization of primary hepatocytes in therapy has been hindered by their slow growth loss of function and dedifferentiation in vitro 3 Stem cells possessing the ability to produce functional hepatocytes for clinical applications and drug development may provide the answer to this problem As yet it is not clear which stem cell type will be the most effective in forming lines that will be effective in regenerative medicine Hepatocyteslike cells have previously been derived from embryonic stem cells ESC bone marrow stem cells adipose tissue and mesenchymal cells as well as multipotent progenitor cells in the human umbilical cord 4 5 6 7 8 9 10 New discoveries in the mechanisms of liver development and the emergence of induced pluriplotent stem cells in 2006 have provided novel insights into hepatocyte differentiation and the use of stem cells for therapeutic applications This review is aimed towards providing scientists and physicians with the latest advancements in this rapidly progressing fieldDuring embryogenesis the differentiation of progenitor cells into fully mature hepatocytes depends on the initiation of complex pathways by numerous signals released from adjacent cells Variations in timing and concentration of the cell signals are both necessary for the regulation of specific transcription factors that ultimately orchestrate this transition into mature cells With the elucidation of the intricate mechanisms of liver development through recent discoveries in mouse zebrafish and chicken embryos reviewed by SiTayeb K et al 2010 11 Lemaigre FP 2009 12 new protocols of hepatocyte differentiation have begun to mimic the development of hepatocytes in vivoThe newly specified bipotent hepatic cells in embryos are known as hepatoblasts which express albumin and transthyretin and are capable of becoming either hepatocyte or biliary epithelial cells 25 Fig 1b After specification the liver diverticulum forms from the primitive gut at day 22 of human embryonic age Proliferation of these hepatoblasts then leads to the production of a pseudostratified epitheliumlike tissue called the hepatic bud 12 26 Afterwards the columnar hepatoblasts go through an epithelialmesenchymal transition while invading into the septum transversum 11 Fig 1a A complex transcriptional network consisting of factors such as GATA6 27 prospero homeobox protein 1 Prox1 28 Tbox transcription factor 3 Tbx3 29 hepatocyte nuclear factor 6 HNF6 30 and HNF4 31 control these events 12 Fig 1bThe development of the embryonic liver results in three distinct cell populations which differ in their cell markers and induction signals Hepatocytecommitted cells are recognized by extensive expression of albumin and AFP Cholangiocytecommitted cells are distinguished by their cytokeratin CK19 expression The cell population expressing both hepatic and biliary markers is the bipotent hepatoblast 11 12 32 Oncostatin M OCM which is expressed in hematopoietic cells in the fetal liver 33 along with glucocorticoids 32 34 suppress the differentiation toward the hematopoietic lineage and promote the differentiation of the liver progenitor cells towards hepatocytes in vitro 11 32 35 Meanwhile some mitogenic factors from sinusoidal endothelial cells such as hepatocyte growth factor HGF and interleukin 16 IL16 acting via the vascular endothelial growth factor VEGF receptors enhance hepatocyte proliferation 11 HGF inhibits the progression of the cells into the cholangiocytic lineage by the blocking of notch signaling This enhancement of hepatocyte specification appears to be stimulated by insulin Gradually during fetal hepatic maturation the number of bipotent hepatoblasts decreases and the number of mature cells expressing specific hepatic or biliary markers increases 11 During this process AFP is highly expressed in early embryonic and fetal liver development then the production of AFP decreases later in maturation and is markedly lower in adulthood 32 36 Finally whole liver maturation occurs after birth by a combination of growth factors secreted by surrounding nonparenchymal liver cells such as HGF FGF4 and epidermal growth factor EGF 11In the injured liver a heterogeneous population of progenitor cells in or adjacent to the canal of Hering become activated 37 Farber first described them as “small oval cells with scant lightly basophilic cytoplasm and pale bluestaining nuclei” with a high nuclear/cytoplasmic ratio 38 “Oval cells” are recognized as bipotent hepatoblasts possessing the ability to differentiate into either hepatocytes or biliary epithelial cells and express both hepatocyte AFP albumin and cholangiocyte CK 7 CK 19 OV6 markers 39 In addition activation of stem cell genes such as ckit 40 CD34 41 flt 3 receptor 42 and leukemia inhibitory factor LIF 43 has been reported during oval cell propagation It is suggested that oval cells produce ductlike structures in the periportal regions and expand into the liver acinus after several days clusters of small basophilic hepatocytes and mature bile ducts form from these ductlike structures 44Previously bipotent hepatoblasts were recognized as the only human hepatic stem cell Recently adult hepatic stem cells were characterized by their multipotency and selfrenewal capacity Hepatic stem cells and hepatoblasts are now considered the multipotent progenitor populations in human liver The ductal plates of fetal and neonatal livers and the canals of Hering in pediatric and adult livers are proposed to be their niche 45 46 Both cell populations express albumin CK8 CK18 and CK19 Sonic and Indian Hedgehog proteins telomerase and CD133 and they are negative for hematopoietic markers CD45 CD38 CD14 and glycophorin A hepatic stellate cell markers desmin αsmooth muscle actin and CD146 and endothelial cell markers VEGFr CD31 and van Willebrand’s factor 45 46 47 The hepatic stem cell population which is thought to be the precursors of hepatoblasts express epithelial cell adhesion molecule EpCAM neural cell adhesion molecule NCAM CK19 as well as ± albumin and are also negative for AFP Hepatoblasts are characterized by the expression of the combination of EpCAM intercellular cell adhesion molecule ICAM1 CK19 albumin++ and AFP++ 45 47 Fig 1c

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