Abstract
Human embryonic stem cells (hESCs) are being utilized in diverse areas of studies such as development and disease modeling, cell replacement therapy, or drug toxicity testing because of their potential to be differentiated into any cell type in the body. The directed differentiation of hESCs into hepatocytes could provide an invaluable source of liver cells for various liver-based applications. Therefore, several protocols have been established in the past for hESC-hepatocyte differentiation based on the knowledge of signaling pathways and growth factors involved in different stages of embryonic hepatogenesis. Although successful derivation of hepatocytes has been achieved through these protocols, the efficiency is not always ideal. Herein, we have tested several combinations of published protocols, for example, growth factor vs. small molecule and different time durations of treatment for definitive endoderm (DE) induction and further hepatocyte differentiation to develop an efficient DE induction and hepatocyte differentiation in a highly reproducible manner based on the stage-specific marker expression and functional analysis.
Highlights
The human embryonic stem cells, isolated from the inner cell mass of the blastocyst stage of embryo, have the potential to be differentiated into all three germ layers
The human embryonic stem cells (hESCs)-hepatocyte differentiation protocols are based on the knowledge of liver development principles gained from various model organisms
We have first focused on identifying a protocol for the definitive endoderm (DE) induction of hESCs which could result in highly homogenous DE cell population with least or no remaining pluripotent cells
Summary
The human embryonic stem cells (hESCs), isolated from the inner cell mass of the blastocyst stage of embryo, have the potential to be differentiated into all three germ layers. Differentiation of hESCs into hepatocytes could be utilized in such studies due to its role in drug detoxification or conditions such as liver failure. Several previous studies have suggested the role of Activin/Nodal, Wnt, BMP, and FGF signaling pathways for the induction of definitive endoderm (DE) from hESCs which further gives rise to many endoderm-derived tissues including liver, pancreas, small intestine, and lungs [2,3,4]. The use of Activin A alone or combined with Wnt3a has been utilized most often for the induction of DE as reported in several previous studies [5] These protocols result in a significant proportion of cells still expressing pluripotency markers along with DE-specific markers suggesting a heterogeneous cell population which could hamper further hepatic differentiation efficiency [6].
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