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