In vitro generation of myofibroblasts-like cells from liver epithelial progenitor cells of rhesus monkey (Macaca mulatta)

Abstract

The origin of the myofibroblast, the primary effector cell of liver fibrosis, is still elusive. Here, we report that fluorescence-activated cell sorting purified E-cad + rhesus monkey liver epithelial progenitor cells (mLEPCs) may serve as a potential source for liver myofibroblasts. Adult mLEPCs colonies were cultured in medium containing 2 ng/ml transforming growth factor β (TGF-β) and 10% fetal bovine serum (FBS) to induce differentiation. Phenotypic changes of cells were analyzed by morphological observation, immunostaining, and reverse transcription-polymerase chain reaction (RT-PCR). After cultured with TGF-β and FBS, some cells in adult mLEPCs colonies converted to fibroblasts-like cells. Immunostaining showed that fibroblasts-like cells had acquired the expression of mesenchymal cell marker vimentin but lost the expression of epithelial cell marker CK8. Fibroblasts-like cells were maintained in culture for up to 40 passages. RT-PCR analysis revealed that fibroblasts-like cells had acquired the expression of mesenchymal genes (snail, PAI-1, and collagen I) and lost the expression of epithelial specific genes (E-cad, ZO-1, CK18, and occludin). In addition, more than 60% of fibroblasts-like cells expressed myofibroblastic-related proteins such as αSMA, vimentin, and N-cad, which were not presented in mLEPCs. Furthermore, increased cell motility was also detected in these fibroblasts-like cells by time-lapse video observation. Our results demonstrate that hepatic epithelial progenitor cells, mLEPCs, transform to myofibroblast-like cells via epithelial-mesenchymal transition. This finding will facilitate understanding of the origin of myofibroblasts in liver fibrosis.

Migration of Mononuclear Cells Expressing β-Actin Through the Adventitia into Media and Intima in Coronary Arteriogenesis and Venogenesis in Ischemic Myocardium

“…21) β-MNCs are different from SMC progenitor cells or fibroblasts which do not express β-actin and CD 31 . 31) It was reported that resident stem cells/progenitor cells are present in adventitia and can differentiate into SMCs and ECs.…”

“…β-MNCs expressed CD 34 , a marker for bone marrow mesenchymal cells 17) ( Figure 2D to 2D-1), but not CD 31 , a marker for ECs, 17) vimentin, a marker for SMCs and fibroblasts/myofibroblasts, 18,19) α-SMA, a marker for the contractile phenotype of SMCs and fibroblasts/myofibroblasts, 18,19) SM-1, a specific marker for matured SMC, 20) nor N-cadherin, a specific marker for myofibroblasts (Table II). 21) Migration of β-MNCs through the adventitia into the media to become SMCs: At 1-2 weeks, β-MNCs attached to or wrapped around pre-existing small arterioles ( Figures 3B, 3C). In larger arterioles or arteries, β-MNCs with an amoebic configuration migrated through the adventitia into the media ( Figure 3D).…”

“…β-MNCs express CD 34 , a nonspecific marker for bone marrow origin, 17) but not CD 31 , a marker for ECs, 17,29) vimentin, a marker for SMCs and myofibloblasts, 30) α-SMA, a marker for contractile phenotype of SMCs and fibroblasts/myofibroblasts, [19][20][21] SM-1, a marker for matured SMCs, 20) or N-cadherin, a marker for fibroblasts and myofibrolasts. 21) β-MNCs are different from SMC progenitor cells or fibroblasts which do not express β-actin and CD 31 .…”

“…The cells in the interstitial space were stained for cell identity of β-SMCs because it was difficult to identify a given cell in the vessel wall using two slices: β-actin (monoclonal anti-β-actin clone AC-15, mouse ascites fluid, Sigma Co, St Louis, USA), which is considered to be a cytoplasmic actin and a marker of the synthetic phenotype of vascular smooth muscle cells (SMCs) in vitro, 16) CD 34 (Epitomics Inc., Burlingame, USA) for bone marrow mesenchymal cells, 17) α-smooth muscle actin (α-SMA; Sigma Co.) for contractile phenotype of vascular SMCs; vimentin (Dako Epos, Glostrup, Denmark) for SMCs, fibroblasts and myofibroblasts; 18,19) vimentin (Dako Epos) for VSMCs; 18,19) smooth muscle myosin heavy chain -1 (SM-1; Abcam Co, Cambridge, MA, USA), a specific marker for matured SMCs; 20) N-cadherin, a specific marker for myofibroblasts (Epitomics Inc.); 21) and CD 31 (Abgent Co, San Diego, USA) for endothelial cell (ECs). 17) Figure 1.…”

β-Actin-Positive Mononuclear Cells Participate in Coronary Microvascular Medial Hyperplasia by Migrating Through Adventitia into Media, With Special Reference to Microvessel Angina

“…Therefore, adjacent slices were immunostained for cell markers using antibodies. The cells in the interstitial space were stained for cell identity of β-SMCs because it was difficult to identify a given cell in the vessel wall using two slices: β-actin (rabbit monoclonal anti-human β-actin antibody, clone AC-15, Sigma Co, St Louis, USA), which is considered to be a cytoplasmic actin and a marker of the synthetic phenotype of vascular smooth muscle cells in vitro; 25) CD 34 (rabbit monoclonal anti-human CD 34 antibody, clone EP 373Y, Epitomics Inc., Burlingame, CA, USA) for bone marrow mesenchymal cells; 26) α-smooth muscle actin (α-SMA; mouse anti-human smooth muscle action, clone/ Klon 1A4, Code No M0851, Dako Epos, Glostrup, Denmark) for contractile phenotype of vascular smooth muscle cells (VSMCs), fibroblasts and myofibroblasts; 27,28) vimentin (rabbit anti-human vimentin antibody, clone Vim 3B4, Code No U7034, Dako Epos) for VSMCs; 27,28) smooth muscle myosin heavy chain -1 (SM-1; mouse monoclonal anti-human smooth muscle myosin heavy chain-1 antibody, clone 3F8, Abcam Co, Cambridge, MA, USA), a specific marker for matured SMCs; 29) N-cadherin, a specific marker for myofibroblasts (rabbit monoclonal anti-human N-cadherin antibody, clone ESR 1792Y, Epitomics Inc); 30) and CD 31 (rabbit monoclonal anti-human CD 31 antibody/PECAM-1, clone EP 3093, Abgent, San Diego, CA, USA) for endothelial cell (ECs).…”