Human embryonic stem cells (hESCs) can be directed to multiple cell lineages including the neural lineage. They thus offer a unique model to reveal molecular interactions underlying early neural specification that occurs in an experimentally inaccessible early human embryo. In our previous R01 project, we discovered, unexpectedly, that the transcription factor Pax6 is expressed uniformly and specifically in early or primitive neuroepithelial (NE) cells that are differentiated from hESCs. This is reminiscent of that of Sox1, the earliest and definitive NE transcription factor in vertebrate animals. We now show that forced expression of Pax6 results in neuroepithelial differentiation in human but not mouse ESCs whereas inhibition of Pax6 expression suppresses neuroepithelial differentiation from human but not mouse ESCs. We therefore hypothesize that Pax6 acts as a key mediator of the NE fate of hESCs and that control of human NE specification may be achieved through regulation of Pax6 expression. We will build hESC lines that conditionally express Pax6 or Sox1 and inhibit Pax6 or Sox1 expression via RNA interference (RNAi). Differentiation of these transgenic hESCs to NE using our defined system will address whether Pax6 is necessary and/or sufficient for NE specification from hESCs. This will reveal a novel role of the conserved protein Pax6 in human NE specification. We will then identify extracellular factors that regulate NE specification and determine if these factors do so by directly regulating Pax6 expression using chromatin immunoprecipitation and DNA binding assays. This will directly link extracellular factors to neuroectoderm transcription factors for the first time. Finally, we will assess whether NE specification from hESCs, especially the maintenance of the primitive state of NE and hence the plasticity to be re-patterned may be controlled by regulating Pax6 expression using our transgenic Pax6 hESC lines. If so, we will uncover a way to maintain somatic (neural) stem cells. Together, information gained from this proposal will provide us with a means of controlling the fate choice of human stem cells. It will also be instrumental to expanding the repair potential of the stem cells that are present in our brain.

Public Health Relevance

The proposed study will reveal how the transition from human embryonic stem cells to neural stem cells is regulated at the genetic and epigenetic levels. Understanding the molecular regulators in directing the fate of neural stem cells will significantly expand our ability to instigate regeneration of our aging and/or diseased brain.

Agency
National Institute of Health (NIH)
Institute
National Institute of Neurological Disorders and Stroke (NINDS)
Type
Research Project (R01)
Project #
5R01NS045926-09
Application #
8259792
Study Section
Neurogenesis and Cell Fate Study Section (NCF)
Program Officer
Sutherland, Margaret L
Project Start
2003-02-01
Project End
2014-04-30
Budget Start
2012-05-01
Budget End
2013-04-30
Support Year
9
Fiscal Year
2012
Total Cost
$318,347
Indirect Cost
$103,972
Name
University of Wisconsin Madison
Department
Pediatrics
Type
Other Domestic Higher Education
DUNS #
161202122
City
Madison
State
WI
Country
United States
Zip Code
53715
Tao, Yunlong; Zhang, Su-Chun (2016) Neural Subtype Specification from Human Pluripotent Stem Cells. Cell Stem Cell 19:573-586
Chen, Yuejun; Xiong, Man; Dong, Yi et al. (2016) Chemical Control of Grafted Human PSC-Derived Neurons in a Mouse Model of Parkinson's Disease. Cell Stem Cell 18:817-26
Huang, Cindy Tzu-Ling; Tao, Yunlong; Lu, Jianfeng et al. (2016) Time-Course Gene Expression Profiling Reveals a Novel Role of Non-Canonical WNT Signaling During Neural Induction. Sci Rep 6:32600
Lu, Jianfeng; Zhong, Xuefei; Liu, Huisheng et al. (2016) Generation of serotonin neurons from human pluripotent stem cells. Nat Biotechnol 34:89-94
Chen, Hong; Qian, Kun; Chen, Wei et al. (2015) Human-derived neural progenitors functionally replace astrocytes in adult mice. J Clin Invest 125:1033-42
Chen, Yuejun; Cao, Jingyuan; Xiong, Man et al. (2015) Engineering Human Stem Cell Lines with Inducible Gene Knockout using CRISPR/Cas9. Cell Stem Cell 17:233-44
Liu, Huisheng; Lu, Jianfeng; Chen, Hong et al. (2015) Spinal muscular atrophy patient-derived motor neurons exhibit hyperexcitability. Sci Rep 5:12189
Du, Zhong-Wei; Chen, Hong; Liu, Huisheng et al. (2015) Generation and expansion of highly pure motor neuron progenitors from human pluripotent stem cells. Nat Commun 6:6626
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Lu, Jianfeng; Bradley, Robert A; Zhang, Su-Chun (2014) Turning reactive glia into functional neurons in the brain. Cell Stem Cell 14:133-4

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