Differentiation of human embryonic stem cells into hepatocytes in 2D and 3D culture systems in vitro

Abstract

Human embryonic stem cells (hESCs) have enormous potential as a source of cells for cell replacement therapies and as a model for early human development. In this study we examined the differentiating potential of hESCs into hepatocytes in two-and three-dimensional (2D and 3D) culture systems. Embryoid bodies (EBs) were inserted into a collagen scaffold 3D culture system or cultured on collagen-coated dishes and stimulated with exogenous growth factors to induce hepatic histogenesis. Immunofluorescence analysis revealed the expression of albumin (ALB) and cytokeratin-18 (CK-18). The differentiated cells in 2D and 3D culture system displayed several characteristics of hepatocytes, including expression of transthyretin, α-1-antitrypsin, cytokeratin 8, 18, 19, tryptophan-2,3-dioxygenase, tyrosine aminotransferase, glucose-6-phosphatase (G6P), cytochrome P450 subunits 7a1 and secretion of alpha-fetoprotein (AFP) and ALB and production of urea. In 3D culture, ALB and G6P were detected earlier and higher levels of urea and AFP were produced, when compared with 2D culture. Electron microscopy of differentiated hESCs showed hepatocyte-like ultrastructure, including glycogon granules, welldeveloped Golgi apparatuses, rough and smooth endoplasmic reticuli and intercellular canaliculi. The differentiation of hESCs into hepatocyte-like cells within 3D collagen scaffolds containing exogenous growth factors, gives rise to cells displaying morphological features, gene expression patterns and metabolic activities characteristic of hepatocytes and may provide a source of differentiated cells for treatment of liver diseases.

Expression of Tight Junction Components in Hepatocyte-Like Cells Differentiated from Human Embryonic Stem Cells

“…The liver is a good objective for studying tissue stem cell differentiation into tumor tissue cells in vitro [55], since several stem cells lines can differentiate and regenerate into functional liver cells [59] , and effective hepatic differentiation protocols have already been established [49][50][51][52][53]60]. It has also been demonstrated that the in vitro produced hepatocyte-like cells transplanted into patients can differentiate into functional liver cells [6].…”

“…For the current study, HUES1 and HUES9 as human embryonic stem cell lines were used. There are a number of models, based on which human embryonic stem cell lines are differentiated into hepatic direction [51,53,60]. The differentiation protocol applied in our study is straight forward and characterized with well-defined differentiation stages [49,50].…”

Status of tissue engineering and regenerative medicine in Iran and related advanced tools: Bioreactors and scaffolds

“…It was not until 2000-2010, however, that one began to see more and more researches in this emerging field. This is when many of the pioneers in TE research began to make their mark [15][16][17][18]. It is important to consider that the focus of Iranian researchers was on engineering bone and nerve tissue, by an integrated use of stem cells and biomaterial scaffolds.…”

“…Results revealed that the differentiated cells give rise to cells displaying morphological features, gene expression patterns, and metabolic activities characteristic of hepatocytes. Authors proposed that hESCs may provide a source of differentiated cells for treatment of liver diseases [15].…”

Dual Gene Expression in Embryoid Bodies Derived from Human Induced Pluripotent Stem Cells Using Episomal Vectors

“…[9][10][11] The 3D structure of EBs promotes differentiation more efficiently than cells in 2D and is proposed to be the mechanism underlying this phenomenon. 12 Sequential stimulation with growth factors and introduction of growth factors promotes human iPS cells to produce hepatocyte-like cells. [13][14][15][16] EBs that express the genes necessary for differentiation would be expected to differentiate more efficiently.…”