Engineering hepatocyte functional fate through growth factor dynamics: the role of cell morphologic priming.

We have reported previously that cellular stimulation induced by variable mechanochemical properties of the extracellular microenvironment can significantly alter liver-specific function in cultured hepatocytes (Semler et al., Biotech Bioeng 69:359-369, 2000). Cell activation via time-invariant presentation of biochemical growth factors was found to either enhance or repress cellular differentiation of cultured hepatocytes depending on the mechanical properties of the underlying substrate. In this work, we investigated the effects of dynamic growth factor stimulation on the cell growth and differentiation behavior of hepatocytes cultured on either compliant or rigid substrates. Specifically, hepatotrophic growth factors (epidermal and hepatocyte) were either temporally added or withdrawn from hepatocyte cultures on Matrigel that was crosslinked to yield differential degrees of mechanical compliance. We determined that the functional responsiveness of hepatocytes to fluctuations in GF stimulation is substrate specific but only in conditions in which the initial mechanochemical environment induced significant cell morphogenesis. Our studies indicate that in conditions under which hepatocytes adopted a "rounded" phenotype, they exhibited increased levels of differentiated function upon soluble stimulation and markedly decreased function upon the depletion of GF stimulation. In contrast, hepatocytes that assumed a "spread" phenotype exhibited slightly increased function upon the depletion of GF stimulation. By examining the functional responsiveness of hepatocytes of differential morphology to varied fluctuations in GF activation, insights into the ability of cell shape to "prime" hepatocyte behavior in dynamic microenvironments were elucidated. We report on the possibility of uncoupling and, thus, selectively manipulating, the concerted contributions of GF-induced cellular activation and substrate- and GF-induced cell morphogenesis toward induction of cell function.

[1]  R Langer,et al.  Selective cell transplantation using bioabsorbable artificial polymers as matrices. , 1988, Journal of pediatric surgery.

[2]  C. I. Pogson,et al.  The mitogenic response to EGF of rat hepatocytes cultured on laminin-rich gels (EHS) is blocked downstream of receptor tyrosine-phosphorylation. , 1996, Biochemical and biophysical research communications.

[3]  K. Ishimura,et al.  Importance of cell aggregation for expression of liver functions and regeneration demonstrated with primary cultured hepatocytes , 1993, Journal of cellular physiology.

[4]  T. Nakamura,et al.  Upregulation of molecular motor‐encoding genes during hepatocyte growth factor‐and epidermal growth factor‐induced cell motility , 1996, Journal of cellular physiology.

[5]  A. Horwitz,et al.  Adhesion-growth factor interactions during differentiation: an integrated biological response. , 1996, Developmental biology.

[6]  R Langer,et al.  Localized delivery of epidermal growth factor improves the survival of transplanted hepatocytes , 1996, Biotechnology and bioengineering.

[7]  Daniel I. C. Wang,et al.  Engineering cell shape and function. , 1994, Science.

[8]  A. Wells,et al.  Tumor invasion: role of growth factor-induced cell motility. , 2000, Advances in cancer research.

[9]  M. Opas.  Expression of the differentiated phenotype by epithelial cells in vitro is regulated by both biochemistry and mechanics of the substratum. , 1989, Developmental biology.

[10]  D. Stolz,et al.  Comparative effects of hepatocyte growth factor and epidermal growth factor on motility, morphology, mitogenesis, and signal transduction of primary rat hepatocytes , 1994, Journal of cellular biochemistry.

[11]  W. Saltzman,et al.  Growth versus Function in the Three‐Dimensional Culture of Single and Aggregated Hepatocytes within Collagen Gels , 1993, Biotechnology progress.

[12]  R Langer,et al.  Switching from differentiation to growth in hepatocytes: Control by extracellular matrix , 1992, Journal of cellular physiology.

[13]  D. Jackson,et al.  The extracellular matrix coordinately modulates liver transcription factors and hepatocyte morphology , 1991, Molecular and cellular biology.

[14]  P. Leung,et al.  Hepatocyte growth factor promotes in vitro scattering and morphogenesis of human cervical carcinoma cells. , 2000, Gynecologic oncology.

[15]  R. Ezzell,et al.  Hepatocyte Aggregation and Reorganization of EHS Matrix Gel , 1997 .

[16]  T. Borg,et al.  Modulation of Heart Fibroblast Migration and Collagen Gel Contraction by IGF-I , 2000, Cell adhesion and communication.

[17]  M L Yarmush,et al.  Culture matrix configuration and composition in the maintenance of hepatocyte polarity and function. , 1996, Biomaterials.

[18]  T. Borg,et al.  Beta 1 integrin-mediated collagen gel contraction is stimulated by PDGF. , 1990, Experimental cell research.

[19]  G. Michalopoulos,et al.  Liver Regeneration , 1997, Science.

[20]  A. Ben-Ze'ev,et al.  Cell-cell and cell-matrix interactions differentially regulate the expression of hepatic and cytoskeletal genes in primary cultures of rat hepatocytes. , 1988, Proceedings of the National Academy of Sciences of the United States of America.

[21]  P. Moghe,et al.  Mechanochemical manipulation of hepatocyte aggregation can selectively induce or repress liver-specific function. , 2000, Biotechnology and bioengineering.

[22]  D E Ingber,et al.  Convergence of integrin and growth factor receptor signaling pathways within the focal adhesion complex. , 1995, Molecular biology of the cell.

[23]  D. Mischoulon,et al.  Cell-extracellular matrix interactions can regulate the switch between growth and differentiation in rat hepatocytes: reciprocal expression of C/EBP alpha and immediate-early growth response transcription factors , 1994, Molecular and cellular biology.

[24]  G. Wilding,et al.  DNA fluorometric assay in 96-well tissue culture plates using Hoechst 33258 after cell lysis by freezing in distilled water. , 1990, Analytical biochemistry.

[25]  K. Matsumoto,et al.  Cell density-dependent regulation of albumin synthesis and DNA synthesis in rat hepatocytes by hepatocyte growth factor. , 1992, Journal of biochemistry.

[26]  S. Farmer,et al.  Effects of Extracellular Matrix on Hepatocyte Growth and Gene Expression: Implications for Hepatic Regeneration and the Repair of Liver Injury , 1990, Seminars in liver disease.

[27]  D E Ingber,et al.  Cellular tensegrity: exploring how mechanical changes in the cytoskeleton regulate cell growth, migration, and tissue pattern during morphogenesis. , 1994, International review of cytology.

[28]  M. Becich,et al.  Comparative analysis of mitogenic and morphogenic effects of HGF and EGF on rat and human hepatocytes maintained in collagen gels , 1993, Journal of cellular physiology.

[29]  D E Ingber,et al.  Fibronectin controls capillary endothelial cell growth by modulating cell shape. , 1990, Proceedings of the National Academy of Sciences of the United States of America.

[30]  K. Ogawa,et al.  Hepatocytic cells form bile duct-like structures within a three-dimensional collagen gel matrix. , 1996, Experimental cell research.

[31]  J. Muschler,et al.  Integrin alpha subunit ratios, cytoplasmic domains, and growth factor synergy regulate muscle proliferation and differentiation , 1996, The Journal of cell biology.

[32]  E. Sage,et al.  Contraction of fibrillar type I collagen by endothelial cells: A study in vitro , 1996, Journal of cellular biochemistry.

[33]  T. Nakamura,et al.  Structure and function of hepatocyte growth factor. , 1990, Progress in growth factor research.