Establishment of novel detection system for embryonic stem cell-derived hepatocyte-like cells based on nongenetic manipulation with indocyanine green.

Hepatocytes derived from embryonic stem cells (ESCs) are expected to be useful for basic research and clinical applications. However, in several studies, genetic methods used to detect and obtain them are difficult and pose major safety problems. Therefore, in this study, we established a novel detection system for hepatocytes by using indocyanine green (ICG), which is selectively taken up by hepatocytes, based on nongenetic manipulation. ICG has maximum light absorption near 780 nm, and it fluoresces between 800 and 900 nm. Making use of these properties, we developed flow cytometry equipped with an excitation lazer of 785 nm and specific bandpass filters and successfully detected ESC-derived ICG-positive cells that were periodic acid-Schiff positive and expressed hepatocyte phenotypic mRNAs. These results demonstrate that this detection system based on nongenetic manipulation with ICG will lead to isolate hepatocytes generated from ESCs and provide the appropriate levels of stability, quality, and safety required for cell source for cell-based therapy and pharmaceutical studies such as toxicology.

In vitro transdifferentiation of HepG2 cells to pancreatic-like cells by CCl4, D-galactosamine, and ZnCl2.

OBJECTIVE: The objective of the study was to induce transdifferentiation of human hepatoma HepG2 cells into pancreatic-like cells without direct genetic intervention. METHODS: HepG2 cells were transfected with plasmids for the hepatocyte marker protein green fluorescent protein (albumin-GFP) and the pancreatic cell marker Discosoma spp red fluorescent protein (elastase-DsRed) to create FAE-HepG2 cells. Fluorescent marker expression was used to monitor in vitro transdifferentiation stimulated 100 mM CCl4, 2 mM D-galactosamine, or 200 µM ZnCl2. Concentrations were selected for optimal cell survival rate. Transdifferentiation was also characterized by immunohistochemical detection of amylase, glucagon, and insulin and by polymerase change reaction analysis of amylase and insulin mRNA production. RESULTS: Control cells expressed albumin-GFP but no elastase-DsRed. By 30 days of culture, all 3 agents induced expression of pancreatic-like cell marker elastase-DsRed. ZnCl2 was the most effective as most cells expressed elastase-DsRed in the absence of simultaneous expression of albumin-GFP. For CCl4 and D-galactosamine, elastase-DsRed was expressed in the same cells as albumin-GFP. Cells treated by each agent also expressed amylase, insulin, and glucagon proteins and mRNAs. CONCLUSIONS: Without direct genetic intervention, select low small molecules can induce in vitro transformation of hepatoma cells into pancreatic-like cells.

Lanford medium induces high quality hepatic lineage cell differentiation directly from mouse embryonic stem cell-derived mesendoderm.

To establish an effective induction method for hepatic differentiation using serum-free media, the effects of activin in serum-containing and serum-free conditions on embryoid body (EB) induction into mesendoderm were investigated by Western blot analysis and real-time reverse transcription-polymerase chain reaction (RT-PCR) as a first step. The expression of P-smad2 and mesendodermal markers was markedly enhanced by 100ng/ml activin under serum-free conditions but were inhibited or masked under serum-containing conditions. Next, serum-free Lanford medium was used to attempt the direct induction of activin-treated EBs expressing mesendodermal markers into hepatic lineage cells and this induction was compared to that induced using Iscove's Modified Dulbecco's medium containing 20% fetal bovine serum. Once immersed in the Lanford medium, EBs began to show typical hepatic features by day 17, including Alb, AFP, TTR, and AAT expression detected by RT-PCR, and ALB, AFP, and CK18 expression detected by immunostaining. On day 22, these cells were of high quality characterized by the expression of metabolizing enzymes, including Ugt1a1, Slcola4, cyp3a11, cyp2b10, and cyp7a1 detected by real-time PCR, a 50-fold greater cyp3A11 response than control to 100muM dexamethasone stimulation, specific cellular uptake of indocyanine green, and glycogen storage in the cytoplasm. These results indicate that this simple two-step induction method under serum-free conditions induces hepatic lineage cells with high quality directly from mouse embryonic stem (ES) cell-derived mesendoderm.

Hepatocyte differentiation from human ES cells using the simple embryoid body formation method and the staged-additional cocktail.

To induce hepatocytes from human embryonic stem (hES) cells easily and effectively, a simple suspension culture method that separates ES colonies with a scraper and transfers them into newly developed, nonadherent MPC (2-methacryloyloxyethyl phosphorylcholine) plates, and the staged-additional cocktail method, including growth factors, cytokines, and Lanford serum-free medium, were developed and evaluated mainly by morphological analysis. The formed embryoid bodies (EBs) showed compact cellular agglomeration until day 4 and later formed coeloms in their interior. RT-PCR (reverse transcriptase-polymerase chain reaction) analysis showed that they are gene markers of the three germ layers. Mesenchymal cells with rough endoplasmic reticulum (rER) and extracellular matrix (ECM), and without junctions, were recognized in the interior of the EBs by transmission electron microscopy (TEM) in addition to epithelial cells. When they were stimulated by the staged-additional cocktail, they expressed albumin-positive immunoreactivity, indocyanine green (ICG) uptake, and typical ultrastructures of the hepatocytes, including bile canaliculi. These results indicate that these combined methods promote EB formation and hepatocyte differentiation from hES cells.