Comparative atomic force and scanning electron microscopy: an investigation of structural differentiation of hepatic stellate cells.
The molecular mechanism leading to the transdifferentiation of hepatic stellate cells (HSC) into myofibroblast-like cells following liver injury is not well understood. The state of cultured rat HSCs was determined using primarily fluorescence microscopy (UV), immunofluorescence (IF) (Glial fibrillary acidic protein (GFAP), Desmin, alpha-smooth muscle actin (alpha-SMA), F-actin) and immunocytochemistry (ICC) (GFAP, Desmin, alpha-SMA, Fibulin-2). Additionally, tapping-mode atomic force microscopy (TM-AFM) and field-emission scanning electron microscopy (FE-SEM) with low-resistivity indium-tin-oxide (ITO) thin-film were performed to observe the micro-morphological character of cells during HSC differentiation. Quiescent HSCs changed to the activated state were identified via UV, IF, and ICC observations. Normal rat HSCs (NHSCs) and thioacetamide-induced rat HSCs (THSCs) were demonstrated to be UV(-), GFAP(+), Desmin(+), alpha-SMA(+) and Fibulin-2(-). After F-actin staining, lamellipodia and filopodia were found in both NHSCs and THSCs, but membrane ruffles were only seen in THSCs. The micro-structures of lamellipodia and filopodia in both NHSCs and THSCs were confirmed using FE-SEM and TM-AFM with ITO; in contrast, the micro-projection was not found. Moreover, "aerial root" structures were observed for the first time in the filopodia of THSCs using TM-AFM. These results reveal that HSC transdifferentiation to a myofibroblastic-like cell (activated HSC) from thioacetamide-induced rat HSC induces extensive changes in the cytoskeleton.
Chang
Kai-Ting
KT
Institute of Biotechnology, National Dong Hwa University, 974 Hualien, Taiwan.
Tsai
May-Jywan
MJ
Cheng
Ya-Ting
YT
Chen
Jyun-Jlang
JJ
Hsia
Ray-Hom
RH
Lo
Yu-Shiu
YS
Ma
Yuan-Ron
YR
Weng
Ching-Feng
CF
D000818:Animals; D054338:Cell Transdifferentiation; D002478:Cells, Cultured; D005455:Fluorescent Antibody Technique; D055166:Hepatic Stellate Cells / Q000648:ultrastructure*; D007150:Immunohistochemistry; D008297:Male; D018625:Microscopy, Atomic Force; D008855:Microscopy, Electron, Scanning; D032446:Myoblasts / Q000166:cytology*; D051381:Rats; D017207:Rats, Sprague-Dawley