Grantee Research Project Results
2017 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, 2016 through November 30,2017
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 in order 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 RNA-seq and 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 9 discrete rostrocaudal (R/C) domains, which would each be further differentiated across 4 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. While Sonic Hedgehog (Shh) signaling acts as a standard morphogen to pattern ventral tissues, BMP4 does not appear to act as a morphogen to pattern all classes of dorsal progenitors. Instead, more complex patterns of BMPs, TGF-s, and Wnt signaling regulate the acquisition of Class A and B dorsal progenitors (Fig. 1A). Using this knowledge, we have investigated whether activation or inhibition of these pathways can upregulate Class A and B progenitor gene expression. Quantitative PCR of a transcription factor panel expressed by dorsal neural progenitors indicates that we are making progress in identifying a regimen of growth factor and inhibitor treatments that can segregate Class A and Class B domains (Fig. 1B). 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 both dorsal domains. These will be combined with varying levels of Shh signaling to complete protocols for patterning 4 D/V domains at 9 discrete, neuraxial R/C domains.
Progress in Objective 2. We previously reported our use of micropatterned culture substrates to create an array of neural tissues recapitulating the earliest stage of neural tube development but without D/V patterning. We also integrated this new finding with our previously published dynamic culture substrates2, which allows spatiotemporal control over the tissues’ microscale morphology (Fig. 2A-C). This level of controlled, biomimetic CNS morphogenesis was unprecedented, so we submitted a provisional patent1 on the substrate design and have a manuscript in review that is available online as a pre-print5.
To explore this tissue engineering approach further, we re-designed the culture substrate to space out the tissues allowing for uninhibited radial expansion. Over the course of 11 days of culture, the tissues maintained their polarized neuroepithelial core while exhibiting neuronal (Tuj1+) differentiation at their periphery (Fig. 2D-E). Each tissue’s core became three dimensional with a noticeable central cavity surrounded by polarized neuroepithelial cells (Fig. 2F). We continue to investigate the morphogenesis of these tissues with the initial goal of demonstrating forebrain corticogenesis processes within arrayed tissues.
To impart a D/V axes within microarrayed tissues, we are collaborating with the Microscale Systems Core to develop a microfluidic well plate insert that can be used to generate sustainable morphogen gradients in situ (Fig. 3A). When placed on top of micropatterned tissues, the microfluidic insert does not interfere with their culture and can sustain a gradient for several days as demonstrated both in vitro and in silico (Fig. 3B-C). The design and media exchange protocol will be further optimized to increase the slope gradient and minimize the time delay to maximum slope. Experiments are also underway to test the effects of a Shh morphogen gradient on axial patterning within microarrayed tissues.
Progress in Objective 3: Our efforts in Objective 3 have been split between generating a reporter line for dopaminergic neuron differentiation and collaborating with the Pathway Analysis Core to demonstrate inference of gene regulatory networks from tissue-wide RNA-seq data. The dopaminergic neuron reporter line will be used in the BRAIN MAPs platform to conduct preliminary screens for toxins that induce Parkinsonian symptoms. CRISPR/Cas9 technology was used to create a double transgenic hPSC line that fluorescently reports acquisition of a TH+/Pitx3+ definitive dopaminergic neuron phenotype. To date, we have verified accurate reporting of Tyrosine Hydroxylase (TH) expression, and experiments for verification of Pitx3 reporting are underway (Fig. 4A).
In the Brain MAPs platform, computational analysis of transcriptomic RNA-seq data from a spectrum of CNS tissues will be used to identify signaling pathways affected during toxin screens. Using RNA-seq data from a spectrum of hindbrain neural progenitor cultures, the Pathway Analysis Core has identified two putative regulatory networks for HoxA5 and Pou3F2 transcription factors both known for significant roles in R/C patterning and neural differentiation respectively. We have identified suitable RNAi sequences for both of these regulators, and are now conducting experiments to validate the predicted gene regulatory networks (Fig. 4B).
Future Activities:
Planned Activities for Year 4. 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 use our engineered dopaminergic reporter line to conduct a screen of the Paraquat neurotoxin. Transcriptome RNA-seq data will be used to create a gene regulatory landscape of the BRAIN MAPs platform and identify pathways perturbed by the Paraquat toxin.
Journal Articles on this Report : 3 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 |
Type | Citation | ||
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Knight GT, Lundin BF, Iyer N, Ashton LMT, Sethares WA, Willett RM, Ashton RS. Engineering induction of singular neural rosette emergence within jPSC-derived tissues. eLIFE 2018;7:e37549 (23 pp.). |
R835737 (2017) R835737 (Final) R835737C002 (2017) |
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Lemke KA, Aghayee A, Ashton RS. Deriving, regenerating, and engineering CNS tissues using human pluripotent stem cells. Current Opinion in Biotechnology 2017;47:36-42. |
R835737 (2017) R835737C002 (2017) |
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Marti-Figueroa CR, Ashton RS. The case for applying tissue engineering methodologies to instruct human organoid morphogenesis. Acta Biomaterialia 2017;54:35-44. |
R835737 (2017) R835737C002 (2017) |
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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.
Project Research Results
4 journal articles for this subproject
Main Center: R835737
215 publications for this center
81 journal articles for this center