Exploring the regulation of human neural precursor cell differentiation using arrays of signaling microenvironments

Cells of a developing embryo integrate a complex array of local and long‐range signals that act in concert with cell‐intrinsic determinants to influence developmental decisions. To systematically investigate the effects of molecular microenvironments on cell fate decisions, we developed an experimental method based on parallel exposure of cells to diverse combinations of extracellular signals followed by quantitative, multi‐parameter analysis of cellular responses. Primary human neural precursor cells were captured and cultured on printed microenvironment arrays composed of mixtures of extracellular matrix components, morphogens, and other signaling proteins. Quantitative single cell analysis revealed striking effects of some of these signals on the extent and direction of differentiation. We found that Wnt and Notch co‐stimulation could maintain the cells in an undifferentiated‐like, proliferative state, whereas bone morphogenetic protein 4 induced an ‘indeterminate’ differentiation phenotype characterized by simultaneous expression of glial and neuronal markers. Multi‐parameter analysis of responses to conflicting signals revealed interactions more complex than previously envisaged including dominance relations that may reflect a cell‐intrinsic system for robust specification of responses in complex microenvironments.

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

[2]  R. McKay,et al.  Single factors direct the differentiation of stem cells from the fetal and adult central nervous system. , 1996, Genes & development.

[3]  Tannishtha Reya,et al.  Integration of Notch and Wnt signaling in hematopoietic stem cell maintenance , 2005, Nature Immunology.

[4]  Mark M Davis,et al.  Marked Differences in Human Melanoma Antigen-Specific T Cell Responsiveness after Vaccination Using a Functional Microarray , 2005, PLoS medicine.

[5]  David J. Anderson,et al.  Regulatory Mechanisms in Stem Cell Biology , 1997, Cell.

[6]  Fred H. Gage,et al.  Astroglia induce neurogenesis from adult neural stem cells , 2002, Nature.

[7]  D. Anderson,et al.  Stem Cells and Pattern Formation in the Nervous System The Possible versus the Actual , 2001, Neuron.

[8]  David John Adams,et al.  Functional maturation of isolated neural progenitor cells from the adult rat hippocampus , 2004, The European journal of neuroscience.

[9]  B. Barres,et al.  Up a Notch Instructing Gliogenesis , 2000, Neuron.

[10]  François Guillemot,et al.  Proneural genes and the specification of neural cell types , 2002, Nature Reviews Neuroscience.

[11]  S. Whittemore,et al.  BMP signaling initiates a neural crest differentiation program in embryonic rat CNS stem cells , 2004, Experimental Neurology.

[12]  P. Bryant,et al.  Isolation and characterization of neural progenitor cells from post‐mortem human cortex , 2003, Journal of neuroscience research.

[13]  Zhang Li-xia The Development of Neural Stem Cells , 2002 .

[14]  C. Stiles,et al.  Molecular mechanisms controlling cortical gliogenesis , 2002, Current Opinion in Neurobiology.

[15]  David J. Anderson,et al.  Alternative Neural Crest Cell Fates Are Instructively Promoted by TGFβ Superfamily Members , 1996, Cell.

[16]  R. Rodnight,et al.  Age-dependent changes in the regulation by external calcium ions of the phosphorylation of glial fibrillary acidic protein in slices of rat hippocampus. , 1995, Brain research. Developmental brain research.

[17]  M. Greenberg,et al.  Basic Helix-Loop-Helix Factors in Cortical Development , 2003, Neuron.

[18]  D. Sabatini,et al.  Microarrays of cells expressing defined cDNAs , 2001, Nature.

[19]  J. Loturco,et al.  Neuronal Differentiation of Precursors in the Neocortical Ventricular Zone Is Triggered by BMP , 1998, The Journal of Neuroscience.

[20]  N. Shah,et al.  Integration of multiple instructive cues by neural crest stem cells reveals cell-intrinsic biases in relative growth factor responsiveness. , 1997, Proceedings of the National Academy of Sciences of the United States of America.

[21]  F. Gage,et al.  The Adult Substantia Nigra Contains Progenitor Cells with Neurogenic Potential , 2002, The Journal of Neuroscience.

[22]  M. Greenberg,et al.  Neurogenin Promotes Neurogenesis and Inhibits Glial Differentiation by Independent Mechanisms , 2001, Cell.

[23]  T. Jessell,et al.  Progression from Extrinsic to Intrinsic Signaling in Cell Fate Specification A View from the Nervous System , 1999, Cell.

[24]  J. de Vellis,et al.  Transition between immature radial glia and mature astrocytes studied with a monoclonal antibody to vimentin. , 1984, Brain research.

[25]  Gord Fishell,et al.  Radial Glial Identity Is Promoted by Notch1 Signaling in the Murine Forebrain , 2000, Neuron.

[26]  V. P. Collins,et al.  An immunohistochemical study of neuropeptides and neuronal cytoskeletal proteins in the neuroepithelial component of a spontaneous murine ovarian teratoma. Primitive neuroepithelium displays immunoreactivity for neuropeptides and neuron-associated beta-tubulin isotype. , 1989, The American journal of pathology.

[27]  F. Gage,et al.  Functional neurogenesis in the adult hippocampus , 2002, Nature.

[28]  S. Morrison,et al.  Prospective identification of tumorigenic breast cancer cells , 2003, Proceedings of the National Academy of Sciences of the United States of America.

[29]  A. Frankfurter,et al.  The expression and posttranslational modification of a neuron-specific beta-tubulin isotype during chick embryogenesis. , 1990, Cell motility and the cytoskeleton.

[30]  Anne E Carpenter,et al.  RNAi living-cell microarrays for loss-of-function screens in Drosophila melanogaster cells , 2004, Nature Methods.

[31]  D. Botstein,et al.  Cluster analysis and display of genome-wide expression patterns. , 1998, Proceedings of the National Academy of Sciences of the United States of America.

[32]  W. Ge,et al.  Notch signaling promotes astrogliogenesis via direct CSL‐mediated glial gene activation , 2002, Journal of neuroscience research.

[33]  M. Mehler,et al.  Bone Morphogenetic Proteins Promote Astroglial Lineage Commitment by Mammalian Subventricular Zone Progenitor Cells , 1996, Neuron.

[34]  Gord Fishell,et al.  The role of notch in promoting glial and neural stem cell fates. , 2002, Annual review of neuroscience.

[35]  A. Frankfurter,et al.  Initial tract formation in the mouse brain , 1993, The Journal of neuroscience : the official journal of the Society for Neuroscience.

[36]  D. Sabatini,et al.  Microarrays of small molecules embedded in biodegradable polymers for use in mammalian cell-based screens. , 2004, Proceedings of the National Academy of Sciences of the United States of America.

[37]  Fred H. Gage,et al.  Cell culture: Progenitor cells from human brain after death , 2001, Nature.

[38]  Sally Temple,et al.  The development of neural stem cells , 2001, Nature.

[39]  Mark M Davis,et al.  Detection and Characterizationof Cellular Immune Responses Using Peptide–MHC Microarrays , 2003, PLoS biology.

[40]  T. Jessell Neuronal specification in the spinal cord: inductive signals and transcriptional codes , 2000, Nature Reviews Genetics.

[41]  W. Ge,et al.  Making and repairing the mammalian brain--signaling toward neurogenesis and gliogenesis. , 2003, Seminars in cell & developmental biology.

[42]  Daniel G. Anderson,et al.  Nanoliter-scale synthesis of arrayed biomaterials and application to human embryonic stem cells , 2004, Nature Biotechnology.

[43]  David J. Anderson,et al.  Glial growth factor restricts mammalian neural crest stem cells to a glial fate , 1994, Cell.

[44]  J. Alberta,et al.  Bipotent Cortical Progenitor Cells Process Conflicting Cues for Neurons and Glia in a Hierarchical Manner , 1999, The Journal of Neuroscience.

[45]  I. Bernstein,et al.  Immobilization of Notch ligand, Delta-1, is required for induction of notch signaling. , 2000, Journal of cell science.

[46]  David J Anderson,et al.  Transient Notch Activation Initiates an Irreversible Switch from Neurogenesis to Gliogenesis by Neural Crest Stem Cells , 2000, Cell.