Post on 07-Oct-2020
http://www.bu.edu/dbin/stemcells/http://www.mostoslavskylab.com/
Gustavo Mostoslavsky, MD PhDAssociate ProfessorSection of GastroenterologyDepartment of Medicine
Co-Director Center for Regenerative Medicine (CReM)Boston University School of Medicine
July 13th, 20192019 CJD Foundation Family ConferenceWashington DC
Modeling Creutzfeldt-Jakob Disease Using Human iPSC-Derived Neurons
www.bumc.bu.edu/stemcells
Takahashi and Yamanaka, Cell 2006
Differentiated skin fibroblasts
Pluripotent
iPS cells
Oct4 Sox2Klf4
cMyc
Induced Pluripotent Stem (iPS) Cells - Reprogramming
The Right Partners
Alice AnaneFounder and President CJD Foundation Israel
David Harris, PhDChair of BiochemistryBUSM
The Project
Characterize E200K PrP from these human iPSC-derived neurons in terms of its expression levels, biochemical properties, cellular localization and trafficking, and infectivity. Compare the transcriptional profiles of these neurons to discover gene networks underlying the pathogenicity of mutant PrP
To establish a CJD E200K-specific iPSC library and test their differentiation towards cortical neurons
Determine how expression of E200K PrP in iPSC-derived neurons affects synapse number, morphology, connectivity, and electrophysiological properties. Test the effects of compounds that suppress PrPSc conversion or PrPSc-induced synaptotoxicsignaling.
2 4 5 6 9 10 14 16 17
22 24 13 32 11 33 18 12 3 20
Mother Father (49) Aunt
Grandfather
25
Granduncle
29 (95)
41
40 (96)
42
The E200K iPSC Tree
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An E200K iPSC library
Gianotti-Sommer et al, 2012Park and Mostoslavsky, 2018
iPSC already reprogrammed
CJD2 (CJD2-1, CJD2-2, CJD2-3)CJD3 (CJD3-1, CJD3-2, CJD3-3)CJD4 (CJD4-1, CJD4-2, CJD4-3)CJD5 (CJD5-1, CJD5-2, CJD5-3)CJD6 (CJD6-1, CJD6-2, CJD6-3)CJD29 (CJD29-1, CJD29-2, CJD29-3)CJD40 (CJD40-1, CJD40-2, CJD40-3)
iPSC undergoing reprogramming
CJD8CJD10CJD11CJD12CJD14CJD15CJD16CJD41CJD42
CJD4
NPC
iPSC Differentiation into Cortical Neurons
Shi et al, Livesey Lab, Nature 2012
Neural induction
Day 0 Day 12 Day 100
D12: Replatecells on laminin
Neural Differentiation
20ng/ml FGF2 4 days 20ng/mL BDNF
20ng/mL GDNF
D37: Dissociate to single cells and
freeze
hiPSC NeuronNeural progenitor cell
Neural induction medium:- Neural maintenance media- SB431542- B27-RA- Dorsomorphin
Neuro-Diff medium:- DMEM:F12- Insulin- 1x N2- 1x B27- GlutaMax- BME- Ascorbic Acid- Neurobasal
Day 37
NESTIN DAPI TUJ1 DAPI
Neural rosettes
iPSC Differentiation into Cortical Neurons
Pax6/Nestin/ DAPIPax6 Nestin DAPI
iPSC Differentiation into Cortical Neurons
iPSC Differentiation into Cortical NeuronsCJD 3-1 D65– young neurons
Action potential
0.2 nA
Tuj1: Neuron specific tubulinVGlut1: Glutamate transporter in membrane of synaptic vesiclesMAP2: microtubule-associated protein 2,dendritic marker Satb2: Upper layers &layer 5 neurons
Vglut1/Tuj1/DAPIVglut1/Tuj1/DAPITuj1 Vglut1
MAP2/Satb2/DAPIMAP2Satb2Patch clamp
iPSC Differentiation into Cortical Neurons
Vglut1/Tuj1/DAPITuj1 Vglut1
MAP2/Satb2/DAPIMAP2Satb2
CJD 40-1 D95– mature neurons
Tuj1: Neuron specific tubulinVGlut1: Glutamate transporter in membrane of synaptic vesiclesMAP2: microtubule-associated protein 2,dendritic marker Satb2: Upper layers &layer 5 neurons
iPSC Differentiation into Cortical Neurons
DAPI/Tuj1/Vglut1
D95
BU-1
CJD-3.1 CJD-6.1
CJD-29.1 CJD-40.1
iPSC-derived Neurons Express PrP
CJD 40-1
CJD3-1BU1 CJD6-1
PrP
CJD29-1 CJD40-1
D95
BU-1 CJD3-1 CJD29-1 CJD40-1 CJD6-1
Western blot PrP
Can we detect PrPSc in CJD-specific iPSC-derived Neurons?
Non PK PK 5ug/ml, 10min
N2a ScN2a N2a ScN2a
PrP/Thioflavin S(Waxman and Giasson, 2011; Le TTN, 2014; Aulic et al 2014; Morales et al, 2016)
Can we detect PrPSc in CJD-specific iPSC-derived Neurons?
BU1
Non PK
PK 5ug 10min
BU3 CJD 3 CJD 6 CJD 29
BU1 BU3 CJD 3 CJD 6 CJD 29
PrP/Thioflavin SiPSC-neurons Day 95
Phalloidin/PrP
Dendrite Morphology and NumberiPSC-neurons Day 95
BU-1 CJD-3.1 CJD-6.1
CJD-29.1 CJD-40.1BU-3
Dendritic Spines Morphology and NumberA dendritic spine is a small membranous protrusion from a neuron's dendrite that typically receives input from a single axon at the synapse.
DAPI/Phalloidin
PhalloidinPhalloidin
Dendritic spine morphologyCJD3-1 Day 105
Conclusions
• Successfully reprogrammed several iPSC lines to generate the first of its kind library of E200K CJD-specific iPSC
• Successfully differentiated the iPSC into cortical neurons in vitro, showing expression of appropriate neural markers and we have started to characterize their functional read-out (action potentials)
• Differentiated neurons showed robust expression of the prion PrP protein and most relevant to the disease model, we were able to show clear difference between normal and CJD-derived neurons which showed presence of the mutant PrP scrappie (PrPsc)
• Experiments to define the morphological and molecular consequences of PrPsc in the neurons are ongoing
• Neural toxicity caused by the PrPsc will serve as a bona fide platform for testing novel compundspreviously identified in mouse models to prevent both, prion scrappie formation and neural toxicity.
Acknowledgements
Funding
NIH 1R211NS111499-01NIH NCI 1R01CA175727-01A1NIH NHLBI R24HL123828-01 NIH NHLBI 5U01HL107443-04
The CJD FoundationThe Garry Buttermann, IV Memorial GrantThe Chuck Fear Memorial GrantThe Cheryl Molloy Memorial GrantThe Jeffrey A. Smith Memorial GrantThe Susie Curry Memorial Grant
Aldana Gojanovich, PhDDar Heinze, MD PhDWhitney ManhartMaggie BakerAditya MithalAndrew McCracken
Seonmi Park, MS
Mostoslavsky Lab Harris Lab
Nhat Le, PhDRobert Mercer, PhDBei Wu
Seonmi Park Aldana Gojanovich Nhat Le
Alice AnaneFounder and President CJD Foundation Israel