Differentiation of Embryonic Stem Cells into Cardiomyocytes in a Compliant Microfluidic System

The differentiation process of murine embryonic stem cells into cardiomyocytes was investigated with a compliant microfluidic platform which allows for versatile cell seeding arrangements, optical observation access, long-term cell viability, and programmable uniaxial cyclic stretch. Specifically, two environmental cues were examined with this platform—culture dimensions and uniaxial cyclic stretch. First, the cardiomyogenic differentiation process, assessed by a GFP reporter driven by the α-MHC promoter, was enhanced in microfluidic devices (µFDs) compared with conventional well-plates. The addition of BMP-2 neutralizing antibody reduced the enhancement observed in the µFDs and the addition of exogenous BMP-2 augmented the cardiomyogenic differentiation in well plates. Second, 24 h of uniaxial cyclic stretch at 1 Hz and 10% strain on day 9 of differentiation was found to have a negative impact on cardiomyogenic differentiation. This microfluidic platform builds upon an existing design and extends its capability to test cellular responses to mechanical strain. It provides capabilities not found in other systems for studying differentiation, such as seeding embryoid bodies in 2D or 3D in combination with cyclic strain. This study demonstrates that the microfluidic system contributes to enhanced cardiomyogenic differentiation and may be a superior platform compared with conventional well plates. In addition to studying the effect of cyclic stretch on cardiomyogenic differentiation, this compliant platform can also be applied to investigate other biological mechanisms.

[1]  R. Kamm,et al.  Cell migration into scaffolds under co-culture conditions in a microfluidic platform. , 2009, Lab on a chip.

[2]  D. Beebe,et al.  Microenvironment design considerations for cellular scale studies. , 2004, Lab on a chip.

[3]  Bernadette Ateghang,et al.  Embryonic stem cells utilize reactive oxygen species as transducers of mechanical strain‐induced cardiovascular differentiation , 2006, FASEB journal : official publication of the Federation of American Societies for Experimental Biology.

[4]  Richard T. Lee,et al.  Nuclear Shape, Mechanics, and Mechanotransduction [2008;102:1307–1318] Emerin and the Nuclear Lamina in Muscle and Cardiac Disease [2008;103:16–23] Mechanical Control of Tissue Morphogenesis , 2022 .

[5]  P. Doevendans,et al.  Human embryonic stem cell-derived cardiomyocytes survive and mature in the mouse heart and transiently improve function after myocardial infarction. , 2007, Stem cell research.

[6]  S. Smith,et al.  Selective regulation of cardiomyocyte gene expression and cardiac morphogenesis by retinoic acid , 1996, Developmental dynamics : an official publication of the American Association of Anatomists.

[7]  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.

[8]  M. Barron,et al.  Requirement for BMP and FGF signaling during cardiogenic induction in non‐precardiac mesoderm is specific, transient, and cooperative , 2000, Developmental dynamics : an official publication of the American Association of Anatomists.

[9]  G. Condorelli,et al.  Human embryonic stem cell-derived cardiomyocytes: inducing strategies. , 2006, Regenerative medicine.

[10]  S. Kudoh,et al.  Bone Morphogenetic Proteins Induce Cardiomyocyte Differentiation through the Mitogen-Activated Protein Kinase Kinase Kinase TAK1 and Cardiac Transcription Factors Csx/Nkx-2.5 and GATA-4 , 1999, Molecular and Cellular Biology.

[11]  J. Hescheler,et al.  Effects of electrical fields on cardiomyocyte differentiation of embryonic stem cells , 1999, Journal of cellular biochemistry.

[12]  Nicola Elvassore,et al.  Electrical stimulation of human embryonic stem cells: cardiac differentiation and the generation of reactive oxygen species. , 2009, Experimental cell research.

[13]  P. Rathjen,et al.  Mouse ES cells: experimental exploitation of pluripotent differentiation potential. , 2001, Current opinion in genetics & development.

[14]  C. Murry,et al.  Regenerating the heart , 2005, Nature Biotechnology.

[15]  G. Eichele,et al.  Expression of chick Tbx-2, Tbx-3, and Tbx-5 genes during early heart development: evidence for BMP2 induction of Tbx2. , 2000, Developmental biology.

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

[17]  L. Samuelson,et al.  Differentiation of Embryonic Stem (ES) Cells Using the Hanging Drop Method. , 2006, CSH protocols.

[18]  Richard T. Lee,et al.  Stem-cell therapy for cardiac disease , 2008, Nature.

[19]  Juan J de Pablo,et al.  Inhibition of human embryonic stem cell differentiation by mechanical strain , 2006, Journal of cellular physiology.

[20]  Donald E Ingber,et al.  Mechanical control of tissue and organ development , 2010, Development.

[21]  S. Oh,et al.  Use of Long-term Cultured Embryoid Bodies May Enhance Cardiomyocyte Differentiation by BMP2 , 2008, Yonsei medical journal.

[22]  Richard T. Lee,et al.  Ascorbic Acid Enhances Differentiation of Embryonic Stem Cells Into Cardiac Myocytes , 2003, Circulation.

[23]  J. D. de Pablo,et al.  TGFbeta/Activin/Nodal pathway in inhibition of human embryonic stem cell differentiation by mechanical strain. , 2008, Biophysical journal.

[24]  E. Sim,et al.  Strategies for directing the differentiation of stem cells into the cardiomyogenic lineage in vitro. , 2004, Cardiovascular research.

[25]  W. Claycomb,et al.  Effect of Mechanical Loading on Three-Dimensional Cultures of Embryonic Stem Cell-Derived Cardiomyocytes , 2008 .

[26]  David J Beebe,et al.  Diffusion dependent cell behavior in microenvironments. , 2005, Lab on a chip.

[27]  D. Beebe,et al.  Cell culture models in microfluidic systems. , 2008, Annual review of analytical chemistry.

[28]  S. Mccann,et al.  Oxytocin induces differentiation of P19 embryonic stem cells to cardiomyocytes , 2002, Proceedings of the National Academy of Sciences of the United States of America.

[29]  D. Mooney,et al.  Mechanical strain regulates endothelial cell patterning in vitro. , 2007, Tissue engineering.

[30]  J. Rana,et al.  Embryonic stem cells cultured in biodegradable scaffold repair infarcted myocardium in mice. , 2005, Sheng li xue bao : [Acta physiologica Sinica].

[31]  Darlene L. Hunt,et al.  Mechanobiology of cardiomyocyte development. , 2010, Journal of biomechanics.

[32]  A. Lassar,et al.  A role for bone morphogenetic proteins in the induction of cardiac myogenesis. , 1997, Genes & development.

[33]  J. Leor,et al.  Bioengineered Cardiac Grafts: A New Approach to Repair the Infarcted Myocardium? , 2000, Circulation.

[34]  Ali Khademhosseini,et al.  Microwell-mediated control of embryoid body size regulates embryonic stem cell fate via differential expression of WNT5a and WNT11 , 2009, Proceedings of the National Academy of Sciences.

[35]  David J Beebe,et al.  Embryonic development in the mouse is enhanced via microchannel culture. , 2004, Lab on a chip.

[36]  T. Schultheiss,et al.  Regulation of avian cardiogenesis by Fgf8 signaling. , 2002, Development.

[37]  A Ratcliffe,et al.  Scaffold-Based Three-Dimensional Human Fibroblast Culture Provides a Structural Matrix That Supports Angiogenesis in Infarcted Heart Tissue , 2001, Circulation.

[38]  J. Hescheler,et al.  Role of reactive oxygen species and phosphatidylinositol 3‐kinase in cardiomyocyte differentiation of embryonic stem cells , 2000, FEBS letters.

[39]  Zack Z Wang,et al.  Extrinsic regulation of cardiomyocyte differentiation of embryonic stem cells , 2008, Journal of cellular biochemistry.