Human embryonic and rat adult stem cells with primitive endoderm-like phenotype can be fated to definitive endoderm, and finally hepatocyte-like cells

Abstract

Stem cell-derived hepatocytes may be an alternative cell source to treat liver diseases or to be used for pharmacological purposes. We developed a protocol that mimics mammalian liver development, to differentiate cells with pluripotent characteristics to hepatocyte-like cells. The protocol supports the stepwise differentiation of human embryonic stem cells (ESC) to cells with characteristics of primitive streak (PS)/mesendoderm (ME)/definitive endoderm (DE), hepatoblasts, and finally cells with phenotypic and functional characteristics of hepatocytes. Remarkably, the same protocol can also differentiate rat multipotent adult progenitor cells (rMAPCs) to hepatocyte-like cells, even though rMAPC are isolated clonally from cultured rat bone marrow (BM) and have characteristics of primitive endoderm cells. A fraction of rMAPCs can be fated to cells expressing genes consistent with a PS/ME/DE phenotype, preceding the acquisition of phenotypic and functional characteristics of hepatocytes. Although the hepatocyte-like progeny derived from both cell types is mixed, between 10-20% of cells are developmentally consistent with late fetal hepatocytes that have attained synthetic, storage and detoxifying functions near those of adult hepatocytes. This differentiation protocol will be useful for generating hepatocyte-like cells from rodent and human stem cells, and to gain insight into the early stages of liver development.

Figure 1. Overview of liver embryogenesis on which liver differentiation protocol is based.

Genes specifically expressed at different steps during liver development are shown, in italics, under each step of lineage commitment. Cytokines used as well as other culture conditions are shown.

Figure 2. Sequential expression of genes found at different stages of endoderm and hepatic specification.

Quantitative RT-qPCR was used to evaluate gene expression in (A) hESC-H9 and (B) rMAPC-1 during the 20 day differentiation process. Shown are relative expression values as compared to d0 on days 6, 10, 14 and 20 for genes in early liver development (n>3)(a) and relative expression to fetal hepatocytes (depicted as percentages and in comparison with mature hepatocytes) for more mature liver-specific genes (b).

Figure 3. Immunofluorescence assessment of transcription factors, structural proteins and hepatoblast/hepatocyte specific proteins in hESC and rMAPC-1 and their progeny.

[A] hESC-H9 and rMAPC-1 day 0, 6, 20. [B] Electron microscopy of d20- progeny of hESC-HSF6 and rMAPC-1. Panels A–C: Differentiated hESC-HSF6 show characteristics of hepatocyte-like cells. Shown are polarized hepatocyte-like cells with desmosomes (C, black arrow), bile canaliculi (A and B, white arrow), mitochondria (A–C, black asterisk) and rough endoplasmatic reticulum (A–C, black arrowhead). Panels D–I: d20 rMAPC-1 progeny have characteristics of both hepatocyte-like cells (D–F) and bile duct-like cells (G–I). Hepatocyte like cells (D–F): Some colony forming epithelial cells (D) contained many cytoplasmatic organelles as RER cisternae (E, arrow) and mitochondriae (E, arrow head), and formed with each other intercellular bile canalicular structures lined by microvilli (F, arrow) and sealed by junctional complexes (F, arrow head), even gap junctions were observed (F inset, arrow head). Bile duct-like cells (G–I): In addition, some epithelial cells arranged in layers and tubules presented at their apical pole many short microvilli (G and H, arrow) while their basal pole was surrounded by basement membranes (G, arrow head). The lateral membranes formed many interdigitations (H, arrow heads and I, arrow). In the cytoplasm a moderate amount of bundles of cytokeratin filaments (I, arrow heads) became obvious. Inset in I shows a bundle of cytokeratin filaments (arrow head) and a desmosome (dashed arrow).

Figure 4. hESC-H9 and rMAPC-1 progeny display functional properties of hepatocytes on d20 (n >3).

The following percentages are functional capacity of hESC- and rMAPC progeny compared to mature human and rat hepatocytes, respectively. [A] Albumin secretion (ng/mL/48 h), hESC 2.6%, rMAPC 0.6%. [B] Storage of glycogen (nmol glucose/mg protein), hESC 632%, rMAPC 59.8%. [C] Spontaneous (hESC 4.0%, rMAPC 4.3%) and NH₄HCO₃-stimulated urea production (hESC 7.7% (+94% induction), rMAPC 5.5% (+26% induction)) [D] Baseline (hESC 2.8%, rMAPC 0.8%) and induced cytochrome P450 activity (hESC 11.1% (+303% induction), rMAPC 1.4% (+69% induction)). 500 µM phenobarbital was used for induction of CYP3A4, while 10 µM omeprazole was used to induce Cyp1a2). [E] Glutathion S-transferase activity (nmol/min/mg protein), hESC 80.0%, rMAPC 27.0%.

Figure 5. Time course analysis of PS/ME/DE and visceral endoderm gene expression during differentiation of rMAPC and hESC.

[A] Quantitative RT-PCR was used to evaluate the time point of maximal expression of transcripts of genes expressed in PS/ME/DE (Eomes, Gsc, Mixl1) and visceral endoderm genes (Hnf4a, Ttr, Afp) in rMAPC and hESC. Shown are mean DeltaCT values (n≥3). [B] Immunofluorescence assessment of rMAPC-1 progeny by staining for Mixl1 (green)/Sox7 (red) on d0, d6 without cytokines (- step1) and d6 with cytokines (+ step1) (Activin-A/Wnt3a).