Grantee Research Project Results
2016 Progress Report: Brain MAPs
EPA Grant Number: R835737C002Subproject: this is subproject number 002 , established and managed by the Center Director under grant R835737
(EPA does not fund or establish subprojects; EPA awards and manages the overall grant for this center).
Center: Human Models for Analysis of Pathways (H MAPs) Center
Center Director: Murphy, William L
Title: Brain MAPs
Investigators: Ashton, Randolph S
Institution: University of Wisconsin - Madison
EPA Project Officer: Aja, Hayley
Project Period: December 1, 2014 through November 30, 2018 (Extended to November 30, 2019)
Project Period Covered by this Report: December 1, 2015 through November 30,2016
RFA: Organotypic Culture Models for Predictive Toxicology Center (2013) RFA Text | Recipients Lists
Research Category: Chemical Safety for Sustainability
Objective:
The Brain-MAPs project aims to develop a high throughput neurotoxicity screening platform that recapitulates the diversity of regional cell phenotypes within the human central nervous system (CNS) while remaining sensitive enough to detect toxicity towards a single phenotype. As our first objective, we are creating chemically defined, standardized protocols for differentiating human pluripotent stem cells (hPSCs) into 36 tissues that span diverse CNS regions. This CNS model is being generated in a well plate format, and RNA sequencing (RNA-seq) of each regional tissue will be used to develop a model-wide transcriptomic map. In our second objective, we are translating the CNS model to a micropatterned, microfluidic platform to simplify model derivation and enhance the tissues’ organotypic cytoarchitecture. Lastly, as our third objective, we will use CRISPR/Cas9 genome editing to create high throughput screening assays for detecting phenotype-specific neurotoxicity using automated high content imaging.
Progress Summary:
Progress in Objective 1. To create a comprehensive CNS model, we proposed to differentiate hPSCs into neural tissues of nine discrete rostrocaudal (R/C) domains, which would each be further differentiated across four dorsoventral (D/V) domains. We will use previously published neural differentiation protocols to derive neural stem cells (NSC) with forebrain, midbrain, hindbrain, and spinal cord regional phenotypes. D/V patterning protocols have been less well elucidated, so we used hPSCs differentiated to a cervical spinal cord, NSC phenotype for exploratory patterning experiments. This was conducted last year by an individual who was subsequently removed from the lab for questionable practices. Thus, we have repeated these experiments and indeed discovered discrepancies from prior results. Using our chemically defined system, we conducted a bone morphogenetic protein (BMP) and sonic hedgehog (Shh) signaling dose response experiment, both in the presence or absence of early Wnt signaling, to pattern neural progenitor subtypes of diverse D/V domains. Quantitative PCR of a transcription factor panel expressed by neural progenitors spanning the D/V axis was used to assess the extent of patterning. A heat map depiction of the QPCR results indicates our success in patterning diverse D/V progenitor phenotypes (Figure 1A and B). Also, it shows that early transient Wnt signaling is beneficial for patterning motor neuron progenitors (Nkx6.1/Olig2+) but not for other ventral, intermediate, or dorsal tissues. Immunocytochemical analysis of these progenitor cultures and their terminally differentiated counterparts will be used as a final verification of our protocol’s capability to pattern neuronal tissues from diverse D/V domains.
Progress in Objective 2. The innate morphogenesis properties possessed by hPSC-derived NSCs have been demonstrated in both 2-D well plate (Figure 1C) and 3-D organoid cultures. NSCs can execute extensive levels of CNS morphogenesis spontaneously. However, a critical limitation of current in vitro culture platforms is a lack of control over their spontaneous morphogenesis. This prevents derivation of CNS tissues in vitro that have truly organotypic tissue cytoarchitecture. Therefore, we started at the beginning of CNS morphogenesis, and set out to control neural rosette induction—an analog to embryonic neural tube formation by polarized NSCs. During 2-D NSC derivation, neural rosettes arise spontaneously with random shape and size (Figure 1C). Using micropatterned culture substrates, we discovered that regulating the NSCs’ tissue morphology enabled controlled induction of neural rosette formation (Figure 1D). By discovering an optimal tissue morphology, we now can generate forebrain through spinal cord NSC tissues with single neural rosette structures at high fidelity (Figure 1E). These tissues are analogous to slice cultures of the earliest stage of neural tube development but without D/V patterning. Moreover, we have integrated this new finding with our previously published dynamic culture substrates, which allow spatiotemporal control over the tissues’ microscale morphology. In Figure 1F, we demonstrate how use of these substrates enable the 2-D NSC tissue to undergo radial outgrowth while maintaining a polarized neuroepithelial layer and depositing a circumferential laminin-rich basement membrane. This level of controlled, biomimetic CNS morphogenesis is unprecedented, and we currently are drafting a patent on the substrate designs and a manuscript for publication.
Future Activities:
For Objective 1, we will standardize protocols for deriving diencephalic, midbrain, and rostral hindbrain tissues. Then, our D/V patterning protocol will be conducted at each R/C domain to complete our microwell plate, CNS model. RNA-seq will be used to develop an accompanying transcriptomic map. For Objective 2, we will interface the micropatterned neural tissues with microfluidics to create an array of CNS tissues containing organotypic cytoarchitecture that includes D/V patterning. For Objective 3, we will engineer an hPSC line that fluorescently reports a dopaminergic neuronal phenotype.
Journal Articles on this Report : 1 Displayed | Download in RIS Format
| Other subproject views: | All 10 publications | 4 publications in selected types | All 4 journal articles |
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| Other center views: | All 215 publications | 82 publications in selected types | All 81 journal articles |
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Knight GT, Sha J, Ashton RS. Micropatterned, clickable culture substrates enable in situ spatiotemporal control of human PSC-derived neural tissue morphology. Chemical Communications 2015;51(25):5238-5241. |
R835737 (2015) R835737 (2016) R835737C002 (2015) R835737C002 (2016) |
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Progress and Final Reports:
Original AbstractMain Center Abstract and Reports:
R835737 Human Models for Analysis of Pathways (H MAPs) Center Subprojects under this Center: (EPA does not fund or establish subprojects; EPA awards and manages the overall grant for this center).
R835737C001 Liver MAPs
R835737C002 Brain MAPs
R835737C003 Cancer MAPs: A 3D Organotypic Microfluidic Culture System to
Identify Chemicals that Impact Progression and Development of Breast Cancer
R835737C004 Vascular MAPs: Vascular and Neurovascular Tissue Models
R835737C005 Pathway Analysis Core
The perspectives, information and conclusions conveyed in research project abstracts, progress reports, final reports, journal abstracts and journal publications convey the viewpoints of the principal investigator and may not represent the views and policies of ORD and EPA. Conclusions drawn by the principal investigators have not been reviewed by the Agency.