Adipogenesis of murine embryonic stem cells in a three-dimensional culture system using electrospun polymer scaffolds.

A mechanistic understanding of adipose tissue differentiation is critical for the treatment and prevention of obesity and type 2 diabetes. Conventional in vitro models of adipogenesis are preadipocytes or freshly isolated adipocytes grown in two-dimensional (2D) cultures. Optimal results using in vitro tissue culture models can be expected only when adipocyte models closely resemble adipose tissue in vivo. Thus the design of an in vitro three-dimensional (3D) model which faithfully mimics the in vivo environment is needed to effectively study adipogenesis. Pluripotent embryonic stem (ES) cells are a self-renewing cell type that can readily be differentiated into adipocytes. In this study, a 3D culture system was developed to mimic the geometry of adipose tissue in vivo. Murine ES cells were seeded into electrospun polycaprolactone scaffolds and differentiated into adipocytes in situ by hormone induction as demonstrated using a battery of gene and protein expression markers along with the accumulation of neutral lipid droplets. Insulin-responsive Akt phosphorylation, and beta-adrenergic stimulation of cyclic AMP synthesis were demonstrated in ES cell-derived adipocytes. Morphologically, ES cell-derived adipocytes resembled native fat cells by scanning electron and phase contrast microscopy. This tissue engineered ES cell-matrix model has potential uses in drug screening and other therapeutic developments.

[1]  I. Heschel,et al.  Human preadipocytes seeded on freeze-dried collagen scaffolds investigated in vitro and in vivo. , 2001, Biomaterials.

[2]  K. O'Shea,et al.  Self-renewal vs. Differentiation of Mouse Embryonic Stem Cells1 , 2004, Biology of reproduction.

[3]  I. Mian,et al.  Tissue architecture: the ultimate regulator of breast epithelial function. , 2003, Current opinion in cell biology.

[4]  J. Vacanti,et al.  Tissue engineering : Frontiers in biotechnology , 1993 .

[5]  Markus Neubauer,et al.  Three-dimensional in vitro model of adipogenesis: comparison of culture conditions. , 2004, Tissue engineering.

[6]  P. Schultz,et al.  A role for chemistry in stem cell biology , 2004, Nature Biotechnology.

[7]  Y. Tabata,et al.  Time course of de novo adipogenesis in matrigel by gelatin microspheres incorporating basic fibroblast growth factor. , 2002, Tissue engineering.

[8]  Christopher S. Chen,et al.  Cell shape, cytoskeletal tension, and RhoA regulate stem cell lineage commitment. , 2004, Developmental cell.

[9]  B. Burgering,et al.  Expression of a constitutively activated form of protein kinase B (c-Akt) in 3T3-L1 preadipose cells causes spontaneous differentiation. , 1996, Endocrinology.

[10]  P. Grimaldi,et al.  The roles of PPARs in adipocyte differentiation. , 2001, Progress in lipid research.

[11]  T. Burdon,et al.  Oct‐4 Knockdown Induces Similar Patterns of Endoderm and Trophoblast Differentiation Markers in Human and Mouse Embryonic Stem Cells , 2004, Stem cells.

[12]  Darrell H. Reneker,et al.  Beaded nanofibers formed during electrospinning , 1999 .

[13]  Jinfei Xu,et al.  Protein Kinase B/AKT 1 Plays a Pivotal Role in Insulin-like Growth Factor-1 Receptor Signaling Induced 3T3-L1 Adipocyte Differentiation* , 2004, Journal of Biological Chemistry.

[14]  D. Loskutoff,et al.  Angiogenesis in an in vivo model of adipose tissue development , 2004, FASEB journal : official publication of the Federation of American Societies for Experimental Biology.

[15]  Seeram Ramakrishna,et al.  Stable immobilization of rat hepatocyte spheroids on galactosylated nanofiber scaffold. , 2005, Biomaterials.

[16]  Kenneth M. Yamada,et al.  Taking Cell-Matrix Adhesions to the Third Dimension , 2001, Science.

[17]  H. Sul,et al.  Understanding adipocyte differentiation. , 1998, Physiological reviews.

[18]  L. Griffith,et al.  Capturing complex 3D tissue physiology in vitro , 2006, Nature Reviews Molecular Cell Biology.

[19]  B. Ratner,et al.  The engineering of biomaterials exhibiting recognition and specificity , 1996, Journal of molecular recognition : JMR.

[20]  Y. Barde,et al.  Developmental potential of defined neural progenitors derived from mouse embryonic stem cells , 2004, Development.

[21]  K. Takenouchi,et al.  Organization of extracellular matrix components during differentiation of adipocytes in long-term culture , 2000, In Vitro Cellular & Developmental Biology - Animal.

[22]  D. L. Crandall,et al.  A Review of the Microcirculation of Adipose Tissue: Anatomic, Metabolic, and Angiogenic Perspectives , 1997, Microcirculation.

[23]  C. Dani,et al.  Differentiation of embryonic stem cells into adipocytes in vitro. , 1997, Journal of cell science.

[24]  M. Lazar,et al.  Transcriptional control of adipogenesis. , 2003, Annual review of nutrition.

[25]  J. Polak,et al.  Differentiation of osteoblasts from murine embryonic stem cells by overexpression of the transcriptional factor osterix. , 2004, Tissue engineering.

[26]  Mina J Bissell,et al.  Modeling tissue-specific signaling and organ function in three dimensions , 2003, Journal of Cell Science.

[27]  C. Patrick,et al.  Adipose tissue engineering: the future of breast and soft tissue reconstruction following tumor resection. , 2000, Seminars in surgical oncology.

[28]  R Kemler,et al.  The in vitro development of blastocyst-derived embryonic stem cell lines: formation of visceral yolk sac, blood islands and myocardium. , 1985, Journal of embryology and experimental morphology.

[29]  A. Khademhosseini,et al.  Microscale technologies for tissue engineering and biology. , 2006, Proceedings of the National Academy of Sciences of the United States of America.

[30]  Y. Ikada,et al.  De novo formation of adipose tissue by controlled release of basic fibroblast growth factor. , 2000, Tissue engineering.

[31]  J. Itskovitz‐Eldor,et al.  Differentiation of human embryonic stem cells on three-dimensional polymer scaffolds , 2003, Proceedings of the National Academy of Sciences of the United States of America.

[32]  Yubing Xie,et al.  Nanoscale modifications of PET polymer surfaces via oxygen-plasma discharge yield minimal changes in attachment and growth of mammalian epithelial and mesenchymal cells in vitro. , 2002, Journal of biomedical materials research.

[33]  D. Reneker,et al.  Nanometre diameter fibres of polymer, produced by electrospinning , 1996 .

[34]  N. Rosenzweig,et al.  Extracellular matrix substrata alter adipocyte yield and lipogenesis in primary cultures of stromal-vascular cells from human adipose , 2003, Biotechnology Letters.

[35]  S. Stice,et al.  Gene Expression Profiling of Embryonic Stem Cells Leads to Greater Understanding of Pluripotency and Early Developmental Events1 , 2004, Biology of reproduction.

[36]  J. Czyż,et al.  Embryonic stem cell differentiation: the role of extracellular factors. , 2001, Differentiation; research in biological diversity.

[37]  A. Abbott.  Cell culture: Biology's new dimension , 2003, Nature.

[38]  Douglas A Kniss,et al.  Adipose tissue model using three-dimensional cultivation of preadipocytes seeded onto fibrous polymer scaffolds. , 2005, Tissue engineering.

[39]  B. Gumbiner,et al.  Cell Adhesion: The Molecular Basis of Tissue Architecture and Morphogenesis , 1996, Cell.

[40]  M. A. D. Berardino.  Animal cloning--the route to new genomics in agriculture and medicine. , 2001 .

[41]  J Auwerx,et al.  The peroxisome proliferator activated receptors (PPARS) and their effects on lipid metabolism and adipocyte differentiation. , 1996, Biochimica et biophysica acta.

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

[43]  D. Marshak,et al.  Stem Cell Biology , 2001 .

[44]  Matsuhiko Nishizawa,et al.  Multi-channel 3-D cell culture device integrated on a silicon chip for anticancer drug sensitivity test. , 2005, Biomaterials.

[45]  P. Murray,et al.  The regulation of embryonic stem cell differentiation by leukaemia inhibitory factor (LIF). , 2001, Differentiation; research in biological diversity.

[46]  S. Mandrup,et al.  Adipocyte differentiation and leptin expression. , 1997, Annual review of cell and developmental biology.

[47]  A. Bradley,et al.  Germ-line transmission of genes introduced into cultured pluripotential cells by retroviral vector , 1986, Nature.

[48]  Christopher S. Chen,et al.  Engineering cellular microenvironments to improve cell-based drug testing. , 2002, Drug discovery today.

[49]  M. Belury,et al.  Differentiation‐dependent regulation of the cyclooxygenase cascade during adipogenesis suggests a complex role for prostaglandins , 2006, Diabetes, obesity & metabolism.

[50]  C. Patrick,et al.  Tissue engineering strategies for adipose tissue repair , 2001, The Anatomical record.

[51]  Richard Tuli,et al.  Multilineage differentiation of human mesenchymal stem cells in a three-dimensional nanofibrous scaffold. , 2005, Biomaterials.

[52]  S. Bhatia,et al.  An extracellular matrix microarray for probing cellular differentiation , 2005, Nature Methods.

[53]  Darrell H. Reneker,et al.  Nanofiber garlands of polycaprolactone by electrospinning , 2002 .

[54]  M. Wiles,et al.  Hematopoietic commitment during embryonic stem cell differentiation in culture. , 1993, Molecular and cellular biology.

[55]  Barbara D. Saatkamp,et al.  Distinct Transcriptional Profiles of Adipogenesisin Vivo and in Vitro * , 2001, The Journal of Biological Chemistry.

[56]  H. Schöler,et al.  Oct‐4: Gatekeeper in the Beginnings of Mammalian Development , 2001, Stem cells.

[57]  Ijaz Ahmed,et al.  A synthetic nanofibrillar matrix promotes in vivo-like organization and morphogenesis for cells in culture. , 2005, Biomaterials.