Differentially labeled neurons making synapses (defined as overlap between green synapsin I puncta and magenta postsynaptic SHANK2 puncta) with unlabeled neurons in vitro.

Differentially labeled neurons making synapses
Neurons making synapses (defined as overlap between green synapsin I puncta and magenta postsynaptic SHANK2 puncta) with unlabeled neurons in vitro.

Courtesy: Kirill Zaslavsky

iPS cell-derived human neurons form synapses in a dish. (A) Schematic representation of synapse visualization. (B) iPS cell-derived neurons were labelled by immunocytochemistry with the indicated antibodies. (C) Inspection of a high-magnificaiton image reveals synapses, indicated by overlap of SYN1 and PSD95 signals along a MAP2-positive dendrite (scale bar = 5 micrometers).
iPS cell-derived human neurons form synapses in a dish
(A) Schematic representation of synapse visualization. (B) iPS cell-derived neurons were labelled by immunocytochemistry with the indicated antibodies. (C) Inspection of a high-magnificaiton image reveals synapses, indicated by overlap of SYN1 and PSD95 signals along a MAP2-positive dendrite (scale bar = 5 micrometers).
Courtesy: Joel Ross
Morphology of a single iPS cell-derived neuron stained with MAP2 (green)
iPS cell-derived neuron
Morphology of a single iPS cell-derived neuron stained with MAP2
Courtesy: Rebecca Mok

iPS Cells to Study Human Disease

 

Induced Pluripotent Stem (iPS) cells can make any cell type of the body and are generated by inducing skin cells to change into stem cells in culture. This cell induction is called “reprogramming” and is performed by delivering a combination of four genes that normally control the stem cell state. We made mouse and human iPS cells using these four genes, and developed a novel EOS lentivirus vector that signals when the skin cells have become iPS cells. This technology is in use to study mechanisms of human disease by inducing patient skin cells to become iPS cells, and then differentiating the iPS cells into the affected cell type for in vitro tests.

 

Rett syndrome and autism spectrum disorder

Our primary interest for modeling human disease using iPS cell technology is aimed toward Rett Syndrome (RTT). It is caused by defects in the X-linked MECP2 gene that specifically affects girls with devastatingly early onset leading to profound developmental challenges and autistic behavior. RTT is an outstanding candidate for disease studies using iPS cells because it is not ethically possible to obtain neurons from the brains of these individuals. To study the defects in neurons with RTT disease, we generated mouse and human iPS cells using skin cells from RTT knockout mice, RTT patients and healthy controls. We differentiated these iPS cells into neurons to identify abnormal functions of RTT neurons in culture with the Salter lab and rescued the RTT neuron abnormalities by delivery of a normal MECP2 gene. Overall, these studies determine the functional defects in RTT nerve cells derived from patients to reveal mechanisms of disease. Together with the Scherer lab we generated iPS cells from subjects with autism spectrum disorders (ASD) and knocked out ASD associated genes using CRISPR-Cas9 genome editing. We are currently differentiating these lines into neurons for phenotyping using multi-electrode arrays, and examining the neural circuitry changes with the Martinez lab at Western University. Importantly, these iPS cells can now be used to screen for new patient-specific drugs to treat RTT and ASD.

The RTT and ASD studies are funded by CIHR, Colonel Harland Sanders Rett Syndrome Research Fund, Simons Foundation Autism Research Initiative, and McLaughlin Centre. The ASD research was initiated with funds from the NIH and the Stem Cell Network.

 

Williams Beuren syndrome and Cardiomyopathy

Williams Beuren syndrome (WBS) is caused by a deletion that removes 26 genes from Chromosome 7q11.23. The major effects are cardiovascular malformations due to heterozygous deletion of the ELASTIN gene resulting in stenosis that must be surgically corrected. Deletion of other genes within the critical region leads to neurodevelopmental consequences including intellectual disability. Together with the Mital lab, we reprogrammed skin cells from WBS patients and differentiated them into vascular smooth muscle cells. In comparison to healthy controls, the WBS cells over-proliferated and were less effective at differentiating into mature functional cells. Preliminary studies suggested that the approved cancer drug rapamycin may be repurposed to partially correct the vascular defects, and we are currently using the iPS cell derived smooth muscle cells for drug testing and screening. We are expanding these studies to include cells with ELASTIN mutations or with duplications of the WBS critical region. These WBS studies were funded by CIHR.

The Mital lab is also discovering genetic variants in patients with Cardiomyopathies (CMP). We generate iPSC from the patients or introduce patient-specific variants into healthy control iPSC. To model CMP, we differentiate the stem cells into beating ventricular cardiomyocytes in vitro and the Mital lab phenotypes them using xCELLigence contractility and other assays. Variant specific phenotypes will then be screened for rescue by pre-approved targeted drugs. These CMP studies are funded by the Ted Rogers Centre for Heart Research.

 

Healthy controls for disease modeling and gene editing

The Personal genome Project Canada (PGP-C) has shown that all healthy individuals harbour heterozygous pathogenic genetic variants, suggesting that all healthy control cell lines have a genetic burden of predisposing mutations. We have generated iPSC from 3 individuals in the PGP-C and shown that they efficiently differentiate in vitro into cells from all three germ layers. Functional phenotyping has been completed on many of these cell types including cortical neurons, cardiomyocytes and hepatocytes. We will make these lines available for the stem cell community as a resource, and develop guidelines to identify the most appropriate healthy control lines to use for tissue specific disease modeling or gene-editing studies. This work is funded by the McLaughlin Centre, Ted Rogers Centre for Heart Research and the POND network.

Selected Publications

  1. Kinnear C., R. Agrawal, C. Loo, A. Pahnke, D.C. Rodrigues, T. Thompson, O. Akinrinade, S. Ahadian, F. Keeley, M. Radisic, S. Mital* and J. Ellis*. 2020 May. Everolimus rescues the phenotype of elastin insufficiency in patient iPSC-derived vascular smooth muscle cellsATVB 40: 1325-39.
  2. Mulder J., S. Sharmin, T. Chow, D.C. Rodrigues, M. Hildebrandt, R. D’Cruz, I. Rogers, J. Ellis and N.D. Rosenblum. 2020 Mar. Generation of Kidney Organoid Tissue from Infant- and Pediatric-derived Urinary Induced Pluripotent Stem CellsPediatric Research 87:647-55.
  3. Ross, P.J.+, W. Zhang+, R.S.F. Mok, K. Zaslavsky, E. Deneault, L. D’Abate, D.C. Rodrigues, R.K.C. Yuen, M. Faheem, A. Piekna, W. Wei, P. Pasceri, R.J. Landa, A. Nagy, B. Varga, M.W. Salter, S.W. Scherer and J. Ellis. 2020 Jan. Synaptic Dysfunction in Human Neurons with Autism-Associated Deletions in PTCHD1-AS. Biological Psychiatry 87:139-149.
  4. Zaslavsky K. +, W.-B. Zhang+, F, McCready, D.C. Rodrigues, E. Deneault, C. Loo, M. Zhao, P.J. Ross, J. El Hajjar, A. Romm, T. Thompson, A. Piekna, W. Wei, Z. Wang, S. Khattak, M. Mufteev, P. Pasceri, S.W. Scherer, M.W. Salter* and J. Ellis*. 2019 Apr. SHANK2 mutations associated with Autism Spectrum Disorder cause hyperconnectivity of human neurons.  Nature Neuroscience 22:556-564.
  5. Sengar A.S, H. Li, Wenbo Zhang, C. Leung, A.K. Ramani, N.M. Saw, Y. Wang, Y.S. Tu, S.W. Scherer, J. Ellis, M. Brudno, Z. Jia and M.W. Salter.  2019 Dec. Control of long-term synaptic potentiation and learning by alternative splicing of the NMDA receptor subunit GluN1. Cell Reports 29:4285-94e5.
  6. Hildebrandt M.R.+, M.S. Reuter+, W. Wei, N. Tayebi, J. Liu, S. Sharmin, J. Mulder, S. Lesperance, P.M. Bauer, R. Mok, C. Kinnear, A. Piekna, A. Romm, J. Howe, P. Pasceri, G. Meng, M. Rozycki, D.C. Rodrigues, E.C. Martinez, M. Szego, J.C. Zuniga-Pflucker, M.K. Anderson, S.A. Prescott, N.D. Rosenblum, B.M. Kamath, S. Mital, S.W. Scherer* and J. Ellis*. 2019 Dec. Precision health resource of control iPSC lines for versatile multi-lineage differentiation. Stem Cell Reports 13:1126-41.
  7. Reuter MS, Walker S, Thiruvahindrapuram B, Whitney J, Cohn I, Sondheimer N, Yuen RKC, Trost B, Paton TA, Pereira SL, Herbrick JA, Wintle RF, Merino D, Howe J, MacDonald JR, Lu C, Nalpathamkalam T, Sung WWL, Wang Z, Patel RV, Pellecchia G, Wei J, Strug LJ, Bell S, Kellam B, Mahtani MM, Bassett AS, Bombard Y, Weksberg R, Shuman C, Cohn RD, Stavropoulos DJ, Bowdin S, Hildrebrandt MR, Wei W, Romm A, Pasceri P, Ellis J, Ray P, Meyn MS, Monfared N, Hosseini SM, Joseph-George AM, Keeley FW, Cook RA, Fiume M, Lee HC, Marshall CR, Davies J, Hazell A, Buchanan JA, Szego MJ, Scherer SW. 2018 Feb. The Personal Genome Project Canada: findings from whole genome sequences of the inaugural 56 participants.  Canadian Medical Association Journal. Feb 5;190(5):E126-E136.
  8. Zhang WB, Ross PJ, Tu Y, Wang Y, Beggs S, Sengar AS, Ellis J, Salter MW. 2016 Apr. Fyn Kinase regulates GluN2B subunit-dominant NMDA receptors in human induced pluripotent stem cell-derived neuronsScience Reports. 4;6:23837.
  9. Khattak S, Brimble E, Zhang W, Zaslavsky K, Strong E, Ross PJ, Hendry J, Mital S, Salter MW, Osborne LR, Ellis J. 2015 Nov. Human induced pluripotent stem cell derived neurons as a model for Williams-Beuren syndrome. Molecular Brain. 8;8(1):77.
  10. Djuric U, Cheung AY, Zhang W, Mok RS, Lai W, Piekna A, Hendry JA, Ross PJ, Pasceri P, Kim DS, Salter MW, Ellis J. 2015 Jan. MECP2e1 isoform mutation affects the form and function of neurons derived from Rett syndrome patient iPS cellsNeurobiology of Disease. 30;76C:37-45.
  11. Kim DS, Ross PJ, Zaslavsky K, Ellis J. (2014) Optimizing neuronal differentiation from induced pluripotent stem cells to model ASD. Frontiers in Cellular Neuroscience. Apr 11;8:109.
  12. Kinnear C, Chang WY , Khattak S, Hinek A, Thompson T, Rodrigues DC, Kennedy K, Mahmut N, Pasceri P, Stanford WL, Ellis* J, Mital* S. (2013) Modeling and rescue of the vascular phenotype of Williams-Beuren syndrome in patient induced-pluripotent stem cells. *Equal corresponding authors. Stem Cells Translational Medicine. 2:2-15.
  13. Cheung AYL, Horvath L, Carrel L,  Ellis J. (2012) X chromosome inactivation in Rett Syndrome induced pluripotent stem cells. Frontiers in Molecular Psychiatry. 3:24:1-16.
  14. Farra N+, W Zhang+, P Pasceri, JH Eubanks, MW Salter*  Ellis, J*. (2012) Rett Syndrome induced pluripotent stem cell derived neurons reveal novel neurophysiology alterations. +Equal first authors. *Equal corresponding authors. Molecular Psychiatry. 17:1261-71.
  15. Cheung A.Y.L., Horvath L, Grafodatskaya D, Pasceri P, Weksberg R, Hotta A, Carrel L, Ellis J. (2011) Isolation of MECP2-null Rett Syndrome patient hiPS cells and isogenic controls through X chromosome inactivationHuman Molecular Genetics. 20:2103-15.
  16. Kattman SJ., Witty A, Gagliardi M, Dubois N, Niapour M, Hotta A, Ellis J, Keller G. (2011) Stage specific optimization of Activin/Nodal and BMP4 signaling promotes efficient cardiovascular differentiation of mouse and human pluripotent stem cell lines. Cell Stem Cell. 8:228-240.
  17. Ross PJ. and Ellis J. (2010) Modeling complex neuropsychiatric disease with induced pluripotent stem cells. F1000 Biology Reports. 2:84.
  18. Ellis J*, Baum C, Benvenisty N, Mostoslavsky G, Okano H, Stanford WL, Porteus M, Sadelain M. (2010) Benefits of utilizing gene-modified iPS cells for clinical applications. *Corresponding author. Cell Stem Cell. 7:429-30.
  19. Rastegar M., Hotta A, Pasceri P, Makarem M, Cheung AYL, Elliott S, Park KJ, Adachi M, Jones FS, Clarke ID, Dirks P and Ellis J. (2009) MECP2 isoform-specific vectors with regulated expression for Rett Syndrome gene therapy. PLoS ONE. 4(8): e6810.
  20. Hotta A, Cheung A, Farra N, Garcha K, Chang WY, Pasceri P, Stanford WL, Ellis J. (2009) EOS lentiviral vector selection system for human induced pluripotent stem cells. Nature Protocols. 4:1828-44.
  21. Belmonte JCI, Ellis J, Hochedlinger K, Yamanaka S. (2009) Induced pluripotent stem cells and reprogramming: seeing the science through the hype.  Nature Reviews Genetics. 10:878-83.
  22. Hotta A, Cheung AY, Farra N, Vijayaragavan K, Séguin CA, Draper JS, Pasceri P, Maksakova IA, Mager DL, Rossant J, Bhatia M, Ellis J. (2009) Isolation of human iPS cells using EOS lentiviral vectors to select for pluripotency. Nature Methods. 6:370-376.
  23. Ellis J, Bruneau BG, Keller G, Lemischka IR, Nagy A, Rossant J, Srivastava D, Zandstra PW, Stanford WL. (2009) Alternative induced pluripotent stem cell characterization criteria for in vitro applications. Cell Stem Cell. 4:198-199.