Three-dimensional culture for expansion and differentiation of mouse embryonic stem cells.

Differentiation of embryonic stem (ES) cells typically requires cell-cell aggregation in the form of embryoid bodies (EBs). This process is not very well controlled and final cell numbers can be limited by EB agglomeration and the inability to drive differentiation towards a desired cell type. This study compares three-dimensional (3D) fibrin culture to conventional two-dimensional (2D) suspension culture and to culture in a semisolid methylcellulose medium solution. Two types of fibrin culture were evaluated, including a PEGylated fibrin gel. PEGylation with a difunctional PEG derivative retarded fibrinogen migration during through sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) as a result of crosslinking, similarly, degradation was slowed in the PEGylated gel. ES cell proliferation was higher in both the fibrin and PEGylated fibrin gels versus 2D and methylcellulose controls. FACS analysis and real-time-PCR revealed differences in patterns of differentiation for the various culture systems. Culture in PEGylated fibrin or methylcellulose culture demonstrated features characteristic of less extensive differentiation relative to fibrin and 2D culture as evidenced by the transcription factor Oct-4. Fibrin gels showed gene and protein expression similar to that in 2D culture. Both fibrin and 2D cultures demonstrated statistically greater cell numbers positive for the vascular mesoderm marker, VE-cadherin.

[1]  E. Domany,et al.  Vascular gene expression and phenotypic correlation during differentiation of human embryonic stem cells , 2005, Developmental dynamics : an official publication of the American Association of Anatomists.

[2]  C. Shun,et al.  Derivation, characterization and differentiation of human embryonic stem cells: comparing serum-containing versus serum-free media and evidence of germ cell differentiation. , 2007, Human reproduction.

[3]  J. Hubbell,et al.  Effects of fibrinolysis on neurite growth from dorsal root ganglia cultured in two‐ and three‐dimensional fibrin gels , 1996, The Journal of comparative neurology.

[4]  W. Zhang,et al.  Modulation of hematopoietic and endothelial cell differentiation from mouse embryonic stem cells by different culture conditions. , 2005, Blood.

[5]  Yihai Cao,et al.  Embryonic Stem Cell-Derived Embryoid Bodies Development in Collagen Gels Recapitulates Sprouting Angiogenesis , 2001, Laboratory Investigation.

[6]  D. Kaufman,et al.  Multilineage Differentiation from Human Embryonic Stem Cell Lines , 2001, Stem cells.

[7]  J. Thomson,et al.  Basic FGF and suppression of BMP signaling sustain undifferentiated proliferation of human ES cells , 2005, Nature Methods.

[8]  Gordana Vunjak-Novakovic,et al.  Bioactive hydrogel scaffolds for controllable vascular differentiation of human embryonic stem cells. , 2007, Biomaterials.

[9]  J. Itskovitz‐Eldor,et al.  Three‐dimensional porous alginate scaffolds provide a conducive environment for generation of well‐vascularized embryoid bodies from human embryonic stem cells , 2004, Biotechnology and bioengineering.

[10]  Q. Kong,et al.  Comparison of two types of alginate microcapsules on stability and biocompatibility in vitro and in vivo , 2006, Biomedical materials.

[11]  L. Yao,et al.  Culture of Neural Stem Cells in Calcium Alginate Beads , 2006, Biotechnology progress.

[12]  Steve Oh,et al.  High density cultures of embryonic stem cells. , 2005, Biotechnology and bioengineering.

[13]  J. Kramer,et al.  In vitro differentiation of mouse ES cells: bone and cartilage. , 2003, Methods in enzymology.

[14]  Martin Yarmush,et al.  Alginate‐PLL microencapsulation: Effect on the differentiation of embryonic stem cells into hepatocytes , 2006, Biotechnology and bioengineering.

[15]  Robert Langer,et al.  Hyaluronic acid hydrogel for controlled self-renewal and differentiation of human embryonic stem cells , 2007, Proceedings of the National Academy of Sciences.

[16]  A. Sun,et al.  Generation of alginate-poly-l-lysine-alginate (APA) biomicrocapsules: the relationship between the membrane strength and the reaction conditions. , 1994, Artificial cells, blood substitutes, and immobilization biotechnology.

[17]  V. Nehls,et al.  The configuration of fibrin clots determines capillary morphogenesis and endothelial cell migration. , 1996, Microvascular research.

[18]  R T Tranquillo,et al.  A fibrin-based arterial media equivalent. , 2003, Journal of biomedical materials research. Part A.

[19]  Paolo A Netti,et al.  The effect of matrix composition of 3D constructs on embryonic stem cell differentiation. , 2005, Biomaterials.

[20]  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.

[21]  Jun Yamashita,et al.  Flk1-positive cells derived from embryonic stem cells serve as vascular progenitors , 2000, Nature.

[22]  R. Pedersen,et al.  Nodal inhibits differentiation of human embryonic stem cells along the neuroectodermal default pathway. , 2004, Developmental biology.

[23]  David Tweedie,et al.  Differentiation of Pluripotent Embryonic Stem Cells Into Cardiomyocytes , 2002, Circulation research.

[24]  R T Tranquillo,et al.  Fibrin as an alternative biopolymer to type-I collagen for the fabrication of a media equivalent. , 2002, Journal of biomedical materials research.

[25]  P. Robson,et al.  Inner cell mass-specific expression of a cell adhesion molecule (PECAM-1/CD31) in the mouse blastocyst. , 2001, Developmental biology.

[26]  Julia M. Polak,et al.  Differentiating embryonic stem cells: GAPDH, but neither HPRT nor beta-tubulin is suitable as an internal standard for measuring RNA levels. , 2002, Tissue engineering.

[27]  M. Mosesson,et al.  The structure and biologic activities of plasma fibronectin. , 1980, Blood.

[28]  Jiehong Liao,et al.  Encapsulation of adult human mesenchymal stem cells within collagen-agarose microenvironments. , 2005, Biotechnology and bioengineering.

[29]  P. Vos,et al.  Cell encapsulation: Promise and progress , 2003, Nature Medicine.

[30]  K. Sidhu,et al.  Differentiation of Encapsulated Embryonic Stem Cells After Transplantation , 2006, Transplantation.

[31]  K. Sasaki,et al.  Spatial Distribution and Initial Changes of SSEA-1 and Other Cell Adhesion-related Molecules on Mouse Embryonic Stem Cells Before and During Differentiation , 2004, The journal of histochemistry and cytochemistry : official journal of the Histochemistry Society.

[32]  Krishnendu Roy,et al.  Biomimetic three-dimensional cultures significantly increase hematopoietic differentiation efficacy of embryonic stem cells. , 2005, Tissue engineering.

[33]  R. McKay,et al.  Toward xeno-free culture of human embryonic stem cells. , 2006, The international journal of biochemistry & cell biology.

[34]  Angelique M. Nelson,et al.  Self-renewal of human embryonic stem cells requires insulin-like growth factor-1 receptor and ERBB 2 receptor signaling , 2007 .

[35]  D. Choi,et al.  In Vitro Differentiation of Mouse Embryonic Stem Cells: Enrichment of Endodermal Cells in the Embryoid Body , 2005, Stem cells.

[36]  G. Keller,et al.  In vitro differentiation of embryonic stem cells. , 1995, Current opinion in cell biology.

[37]  H. Niwa,et al.  Oct-3/4 Maintains the Proliferative Embryonic Stem Cell State via Specific Binding to a Variant Octamer Sequence in the Regulatory Region of the UTF1 Locus , 2005, Molecular and Cellular Biology.

[38]  D. Davidson,et al.  Central roles of alpha5beta1 integrin and fibronectin in vascular development in mouse embryos and embryoid bodies. , 2002, Arteriosclerosis, thrombosis, and vascular biology.

[39]  E. Schacht,et al.  Gelatin-based microcarriers as embryonic stem cell delivery system in bone tissue engineering: an in-vitro study. , 2007, Biomacromolecules.

[40]  Wei Wang,et al.  Proliferation and Differentiation of Mouse Embryonic Stem Cells in APA Microcapsule: A Model for Studying the Interaction between Stem Cells and Their Niche , 2006, Biotechnology progress.

[41]  Aggregation of embryonic stem cells induces Nanog repression and primitive endoderm differentiation , 2004, Journal of Cell Science.

[42]  J. Itskovitz‐Eldor,et al.  Controlled, Scalable Embryonic Stem Cell Differentiation Culture , 2004, Stem cells.

[43]  A. Smith,et al.  Self-renewal of pluripotent embryonic stem cells is mediated via activation of STAT3. , 1998, Genes & development.

[44]  Heli Skottman,et al.  Culture conditions for human embryonic stem cells. , 2006, Reproduction.

[45]  K. Guan,et al.  Differentiation plasticity of chondrocytes derived from mouse embryonic stem cells , 2002, Journal of Cell Science.

[46]  M. Schuldiner,et al.  Differentiation of Human Embryonic Stem Cells into Embryoid Bodies Comprising the Three Embryonic Germ Layers , 1999 .

[47]  D. Wilson,et al.  Erythropoiesis and vasculogenesis in embryoid bodies lacking visceral yolk sac endoderm. , 1996, Blood.

[48]  Allan Bradley,et al.  Targeted disruption of the c-src proto-oncogene leads to osteopetrosis in mice , 1991, Cell.

[49]  D. Lauffenburger,et al.  Leukemia inhibitory factor (LIF) concentration modulates embryonic stem cell self-renewal and differentiation independently of proliferation. , 2000, Biotechnology and bioengineering.

[50]  J. Hubbell,et al.  Effects of fibrin micromorphology on neurite growth from dorsal root ganglia cultured in three-dimensional fibrin gels. , 1998, Journal of biomedical materials research.

[51]  F. Lim,et al.  Microencapsulated islets as bioartificial endocrine pancreas. , 1980, Science.

[52]  M. Butler,et al.  Growth inhibition in animal cell culture , 1991, Applied biochemistry and biotechnology.

[53]  J. Brash,et al.  Encapsulation of various recombinant mammalian cell types in different alginate microcapsules. , 1998, Journal of biomedical materials research.

[54]  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.

[55]  Karin Noaksson,et al.  Facilitated Expansion of Human Embryonic Stem Cells by Single‐Cell Enzymatic Dissociation , 2007, Stem cells.

[56]  A M Wobus,et al.  Embryonic stem cells as a model to study cardiac, skeletal muscle, and vascular smooth muscle cell differentiation. , 2002, Methods in molecular biology.

[57]  Gordon Keller,et al.  Development of definitive endoderm from embryonic stem cells in culture , 2004, Development.

[58]  Peter W Zandstra,et al.  Efficiency of embryoid body formation and hematopoietic development from embryonic stem cells in different culture systems. , 2002, Biotechnology and bioengineering.

[59]  Laura Suggs,et al.  A PEGylated fibrin patch for mesenchymal stem cell delivery. , 2006, Tissue engineering.