Modeling Neurodevelopmental Disorders in Human Pluripotent Stem Cell-Derived Neurons and Organoids

Human cellular models play a central and essential role in  functional genomic research in Intellectual and Developmental Disabilities (IDDs).
Lack of experimental access to the developing human brain precludes direct study of how dysregulation of brain development and function contributes to IDDs. Furthermore, most IDD liability is genetically complex, not resolvable to a single genetic cause or deleterious monogenic variant, and therefore not possible to recapitulate in animal models.  
A critical advantage of human PSC-derived neuronal models is that they faithfully recapitulate the complex human genetics underlying disease. While animal models can successfully model some diseases, they are frequently misrepresentative of human neuropsychiatric syndromes in particular, due to significant differences between humans and rodents, both in the protein coding and non-coding sequences of the genome, and in many aspects of brain development.
Human models capture the genetic complexity of individual patient genetics in disease-relevant models of neurodevelopment. These can be exploited to test the interplay between a suspected pathogenic variant and the patient’s genetic background, by engineering isogenic lines with versus without correction of the gene variant, by introduction of the variant into a wild-type line with a different genetic background, or by testing whether manipulation of the activity of a gene phenocopies or can rescue the effect of a deleterious variant.
These models also provide a renewable, sharable resource for defining cell- and organoid-resolvable phenotypic contributors to disease, which can be used for targeted or high throughput chemical and molecular screening to identify suppressors. These are potentially actionable for the development of small molecule therapies that can target and correct the disrupted aspect of cellular or molecular function underlying dysfunction.
We work collaboratively with investigators associated with Washington University’s Intellectual and Developmental Disabilities Research Center (IDDRC) to model neurodevelopmental disorders in subject-derived stem cell and organoid models.
Recent examples of these cellular modeling projects include:

Meganathan K., Prakasam R., Gontarz P., Zhang B., Baldridge D., Bonni A., Urano F., Huettner J.E., Constantino J.N., Kroll K.L. Alterations in neuronal physiology, development and function associated with a common duplication of chromosome 15 involving CHRNA7. BioRxiv (2020), forthcoming.

Lewis, E.M.A., Meganathan, K., Baldridge, D., Gontarz, P., Zhang, B., Bonni, A., Constantino, J.N. and Kroll, K.L. Cellular and molecular characterization of multiplex autism in human induced pluripotent stem cell-derived neurons. Molecular Autism 10, 51 (2019), doi:10.1186/s13229-019-0306-0. PMC6936127

Meganathan, K, Lewis, E.M.A., Gontarz, P., Liu, S., Stanley, E.G., Elefanty, A.G., Huettner, J.E., Zhang, B., and Kroll, K.L.. Regulatory networks specifying cortical interneurons from human embryonic stem cells reveal roles for CHD2 in interneuron development. Proceedings of the National Academy of Science, 2017; 114(52):E11180–E11189. PMC5748186.

Additional information about these approaches is below:

Modeling neurodevelopmental disorders with patient-specific induced pluripotent stem cell (iPSC)-derived neurons.

Top: Schematic depicting several stages of human neural development. the 3 week embryo (left, ~1.5mm) has a future brain and spinal cord. From 5 weeks (top: 5 mm) to 15 weeks (right: 140 mm) the darker pink forebrain or telencephalon becomes patterned. 

Inset at far right shows a schematized coronal section through the 15-week telencephalon along the plane shown in red, highlighting dorsoventral patterning of the telencephalon into the cortex and ganglionic eminences (MGE/LGE). The green ventricular zone (VZ) of the cortex expresses the marker gene PAX6 and gives rise to excitatory projection neurons. The blue MGE expresses a different marker (NKX2-1) and produces inhibitory cortical interneurons.

Disrupted or imbalanced development or function of either or both of these neuronal types can cause neurodevelopmental disorders, including autism and epilepsy.

Bottom: Patient-derived somatic cells (e.g. skin, blood) can be converted into induced pluripotent stem cells (iPSCs) and differentiated into cell types and organoids relevant to neurodevelopmental disorders, including cortical excitatory and inhibitory neurons and cerebral organoids. These patient-derived models are used to determine cellular, molecular and functional neuronal abnormalities that contribute to the patient’s phenotype and to screen for chemical and molecular inhibitors of these abnormal processes that may be developed into potential interventions. 

Layers of the developing cortex are abbreviated as follows: VZ = ventricular zone; SVZ = subventricular zone; CP, SP, IZ = cortical plate, subplate, intermediate zone. Cortical radial glial neural progenitors in the ventricular zone express the marker PAX6 and differentiate into glutamatergic excitatory neurons. The MGE and LGE are demarcated by expression of NKX2–1 or ISL1/SP8 and differentiate predominantly into cortical interneurons and striatal projection neurons, respectively. Adapted from Lewis and Kroll, Epigenomics 10(2), 219-31.

Cellular Models Unit of the Intellectual and Developmental Disabilities Research Center-Missions, Operations, and Deliverables, relationship to Cross-IDDRC Cellular Models Group
Graphical view of phenotyping approaches
Detailed view of phenotyping workflow and assays
Examples of phenotyping assays and data

Examples of several cellular phenotyping assays used by the modeling unit.

Comparisons were made between iPSC-derived neurons derived from an ASD affected proband (PB) and from several non-affected individuals (C1-C3; C1/2 are unrelated to the PB, while C3 is the unaffected mother).

(A) Neurite extension is quantified after plating and differentiation of neuroids and co-immunocytochemistry (ICC) with MAP2 and SOX2

(B) Synaptic puncta are quantified in plated co-cultures of cortical excitatory (cEX) and cortical inhibitory (cIN) neurons, after ICC for VGLUT and VGAT

(C) Migration of cINs into the cEX neuroid is measured by co-culturing cIN and cEX neuroids in apposition; neuroids express Synapsin-RFP and –GFP, respectively. PB-derived neurons exhibit reduced neurite extension, migration, and generation of VGAT-expressing synaptic puncta.

(D-E) Altered action potentials (D) and responses to choline and acetylcholine (ACh) in PB-derived neurons. PB carries a duplication in the CHRNA7 gene, which modulates choline responses.

(F) A luciferase reporter measures calcium leakage in response to ER stress. PB derived neurons exhibit elevated ER stress.

(G) Identification of a selective small molecule suppressor of the PB neuron ER stress phenotype. PB-derived neurons were treated with inhibitors of ER stress that either suppress the unfolded protein response (TUDCA, PBA) or that antagonize ryanodine receptors to restore calcium homeostasis after ER stress (JTV-519, Dantrolene). JTV-519 selectively suppresses the ER stress phenotype.