Spatio-temporal control of hepatic stellate cell-endothelial cell interactions for reconstruction of liver sinusoids in vitro.

Vascularization of engineered tissues in vitro remains a major challenge in liver tissue engineering. Liver microvessels, termed liver sinusoids, have highly specialized structures, and recapturing these sinusoidal structures is essential for reconstruction of functional liver tissue in vitro. Liver sinusoids are composed of hepatocytes, hepatic stellate cells (HSCs), and endothelial cells (ECs). Direct HSC-EC contacts are increasingly recognized for their roles in EC capillary morphogenesis. However, the hypothetical role of HSC-EC contacts in morphogenesis remains unclear in hepatocyte-HSC-EC triculture. In the present study, we first determined the effects of direct HSC-EC contacts on EC capillary morphogenesis using a hepatocyte-HSC-EC triculture model where HSC behavior was spatially controlled to achieve HSC-mediated proximal layers of hepatocytes and ECs. EC capillary morphogenesis was induced by overlaying Matrigel on an EC layer. Direct HSC-EC contacts inhibited EC capillary morphogenesis, suggesting that the HSC-EC contacts may be an important factor in capillary formation. We next tested the hypothesis that, in addition to spatial control, temporal control of HSC behavior is also important in achieving capillary morphogenesis in the triculture. ECs responded to the induction of capillary morphogenesis before the formation of direct HSC-EC contacts, while the ECs remained to form monolayers when capillary morphogenesis was induced after the HSC-EC contacts were established. When capillary morphogenesis was successfully achieved in the triculture, HSCs tended to preferably localize near the preformed capillary-like structures, resulting in the reconstruction of liver sinusoidal structures. In these structures, hepatocyte maturation was induced. Our findings indicate that control, both spatial and temporal, of HSC behavior is a key engineering strategy for the vascularization of engineered liver tissue in vitro.

[1]  Kazuo Tanishita,et al.  Hepatic stellate cell-mediated three-dimensional hepatocyte and endothelial cell triculture model. , 2011, Tissue engineering. Part A.

[2]  R. Gutiérrez,et al.  Pericytes. Morphofunction, interactions and pathology in a quiescent and activated mesenchymal cell niche. , 2009, Histology and histopathology.

[3]  K. Shakesheff,et al.  A simple method for the simultaneous isolation of stellate cells and hepatocytes from rat liver tissue , 2003, Molecular and Cellular Biochemistry.

[4]  Y. Sakai,et al.  Spontaneous formation of stably-attached and 3D-organized hepatocyte aggregates on oxygen-permeable polydimethylsiloxane membranes having 3D microstructures , 2010, Biomedical microdevices.

[5]  Kazuo Tanishita,et al.  Reconstruction of 3D stacked‐up structures by rat small hepatocytes on microporous membranes , 2005, FASEB journal : official publication of the Federation of American Societies for Experimental Biology.

[6]  L. Griffith,et al.  Tissue Engineering--Current Challenges and Expanding Opportunities , 2002, Science.

[7]  C. Betsholtz,et al.  Endothelial/Pericyte Interactions , 2005, Circulation research.

[8]  Jennifer R. Hurley,et al.  Complex temporal regulation of capillary morphogenesis by fibroblasts. , 2010, American journal of physiology. Cell physiology.

[9]  Amitava Das,et al.  Platelet-derived growth factor signaling through ephrin-b2 regulates hepatic vascular structure and function. , 2008, Gastroenterology.

[10]  A. Hall,et al.  Rac regulates endothelial morphogenesis and capillary assembly. , 2002, Molecular biology of the cell.

[11]  Y. Yamaoka,et al.  Morphological changes induced by extracellular matrix are correlated with maturation of rat small hepatocytes , 2002, Journal of cellular biochemistry.

[12]  T. Mizuguchi,et al.  Reconstruction of hepatic organoid by rat small hepatocytes and hepatic nonparenchymal cells , 1999, Hepatology.

[13]  Sangeeta N Bhatia,et al.  Micromechanical control of cell–cell interactions , 2007, Proceedings of the National Academy of Sciences.

[14]  A. Palmetshofer,et al.  Contact-dependent inhibition of angiogenesis by cardiac fibroblasts in three-dimensional fibrin gels in vitro: implications for microvascular network remodeling and coronary collateral formation , 1998, Cell and Tissue Research.

[15]  Yasuhiko Tabata,et al.  Formation of hepatocyte spheroids with structural polarity and functional bile canaliculi using nanopillar sheets. , 2010, Tissue engineering. Part A.

[16]  M. Shibuya,et al.  The Overexpression of PKCδ Is Involved in Vascular Endothelial Growth Factor-Resistant Apoptosis in Cultured Primary Sinusoidal Endothelial Cells , 2001 .

[17]  J. Iredale,et al.  Sinusoidal remodeling and angiogenesis: A new function for the liver‐specific pericyte? , 2007, Hepatology.

[18]  K. Shakesheff,et al.  Long-term culture of functional liver tissue: three-dimensional coculture of primary hepatocytes and stellate cells. , 2003, Tissue engineering.

[19]  L. Griffith,et al.  Transport‐mediated angiogenesis in 3D epithelial coculture , 2009, FASEB journal : official publication of the Federation of American Societies for Experimental Biology.

[20]  S. Friedman.  Hepatic stellate cells: protean, multifunctional, and enigmatic cells of the liver. , 2008, Physiological reviews.

[21]  Masayuki Yamato,et al.  Engineering functional two- and three-dimensional liver systems in vivo using hepatic tissue sheets , 2007, Nature Medicine.

[22]  P. Sheard,et al.  Role of hepatic stellate cell/hepatocyte interaction and activation of hepatic stellate cells in the early phase of liver regeneration in the rat. , 2004, Journal of hepatology.

[23]  C. Hellerbrand,et al.  Hepatic stellate cells display a functional vascular smooth muscle cell phenotype in a three-dimensional co-culture model with endothelial cells. , 2008, Differentiation; research in biological diversity.

[24]  Antonio Martinez‐Hern Andez,et al.  The extracellular matrix in hepatic regeneration , 1995, FASEB journal : official publication of the Federation of American Societies for Experimental Biology.

[25]  M. Yarmush,et al.  Amino acid‐mediated heterotypic interaction governs performance of a hepatic tissue model , 2009, FASEB journal : official publication of the Federation of American Societies for Experimental Biology.

[26]  K. Wake.  Hepatic stellate cells: Three-dimensional structure, localization, heterogeneity and development , 2006, Proceedings of the Japan Academy. Series B, Physical and biological sciences.

[27]  Timothy O'Brien,et al.  Bone marrow-derived mesenchymal stem cells promote angiogenic processes in a time- and dose-dependent manner in vitro. , 2009, Tissue engineering. Part A.