In vitro differentiation of embryonic stem cells into hepatocyte-like cells identified by cellular uptake of indocyanine green.
OBJECTIVE
Embryonic stem (ES) cells have a pluripotent ability to differentiate into a variety of cell lineages in vitro. We have recently found the emergence of cell clusters that show the cellular uptake of indocyanine green (ICG) in the culture of differentiated ES cells. ICG is clinically used as a test substance to evaluate liver function because it is eliminated exclusively by hepatocytes. The aim of the present study was to investigate the hepatic characteristics of ICG-stained cells.


METHODS
Embryoid bodies (EBs), formed by a 5-day hanging drop culture of ES cells, were allowed to outgrow in the placed culture. Gene expression of hepatocyte markers was analyzed by reverse transcriptase-polymerase chain reaction, and albumin production was examined immunohistochemically. Morphology and cellular components were investigated by electron microscopy. ICG-stained cells were further transplanted into the portal vein of mice.


RESULTS
ICG-stained cells appeared around 14 days of the EB culture and formed distinct three-dimensional structures. They were immunoreactive to albumin and expressed mRNAs such as albumin, alpha-fetoprotein, transthyretin, hepatocyte nuclear factor 3 beta, alpha-1-antitrypsin, tryptophan-2,3-dioxygenase, urea cycle enzyme, gluconeogenic enzyme, and liver-specific organic anion transporter-1. An ultrastructural analysis revealed a well-developed system of organelles such as mitochondria, lysosomes, Golgi apparatus, and rough and smooth endoplasmic reticulum. The transplantation of ICG-positive cells into the portal vein resulted in the incorporation into mice livers, where they were morphologically indistinguishable from neighboring hepatocytes.


CONCLUSIONS
ES cell-derived ICG-positive cells possess characteristics of hepatocytes, and ICG-staining is a useful marker to identify differentiated hepatocytes from EBs in vitro.
Yamada
Takatsugu
T
Division of Developmental Biology, Department of Parasitology, Nara Medical University, 840 Shijo-cho, Kashihara, Nara 634-8521, Japan. highnet@nmu-gw.naramed-u.ac.jp
Yoshikawa
Masahide
M
Kanda
Seiji
S
Kato
Yoko
Y
Nakajima
Yoshiyuki
Y
Ishizaka
Shigeaki
S
Tsunoda
Yukio
Y
D000418:Albumins / Q000378:metabolism; D000818:Animals; D002448:Cell Adhesion / Q000502:physiology; D002449:Cell Aggregation / Q000502:physiology; D018929:Cell Culture Techniques / Q000379:methods*; D002454:Cell Differentiation / Q000502:physiology*; D002478:Cells, Cultured; D004396:Coloring Agents; D004622:Embryo, Mammalian / Q000378:metabolism* / Q000648:ultrastructure; D015870:Gene Expression / Q000502:physiology; D006085:Graft Survival / Q000502:physiology; D022781:Hepatocytes / Q000378:metabolism* / Q000648:ultrastructure; D007150:Immunohistochemistry; D007208:Indocyanine Green; D008297:Male; D051379:Mice; D008854:Microscopy, Electron; D012333:RNA, Messenger / Q000378:metabolism; D033581:Stem Cell Transplantation; D013234:Stem Cells / Q000378:metabolism* / Q000648:ultrastructure; D016378:Tissue Transplantation
D000418:Albumins; D004396:Coloring Agents; D012333:RNA, Messenger; D007208:Indocyanine Green