Neuroprotective and Regenerative Stem Cell

Therapies in a Pig Stroke Model

Franklin D. WestRegenerative Bioscience Center

University of Georgia

Financial Disclosure

• No financials to disclose

• Second leading cause of death globally

• Number one cause of long term disability in US

• Every 40 seconds someone has a stroke

• Every 4 minutes someone dies of stroke

Mozaffarian et al. Circulation. 2015.

www.educatehealth.ca

Stroke: A Global Killer

Over 700 treatments have gone to clinical trial for stroke and only two have been FDA approved:

▪Tissue Plasminogen Activator (tPA)▪Thrombectomy

•Limited window of treatment•No regenerative potential•<25% of patients can receive

tPA and/or thrombectomy

Peter McMeekin, et al. European Stroke Journal, 2017. 2(4): p. 319-326; Sharma, V.K., et al. Stroke Res Treat, 2010; Lapchak, P.A., Transl Stroke Res, 2010; Serbest, G., et al. FASEB J, 2006.

Limited Treatments for Stroke

An assessment of failed treatments by the Stem Cell Therapies as an Emerging Paradigm in Stroke (STEPS) group has resulted in the identifying 2 major needs of the research community:

1) a regenerative cell therapy that will not only protect cells from ischemic injury but replace the lost and damaged tissues

2) a translational animal model more similar to humans for testing treatments

Stem Cell Therapies as an Emerging Paradigm in Stroke (STEPS): bridging basic and clinical science for cellular and neurogenic factor therapy in treating stroke. Stroke, 2009. Savitz, S.I., et al., Stem Cell Therapy as an Emerging Paradigm for Stroke (STEPS) II. Stroke, 2011.

Why Do All the Drugs Fail?

Induced Pluripotent Stem Cell-Derived Neural Stem Cells (iNSC) to Treat Neural Injury

Induced Pluripotent Stem Cell-Derived Neural Stem Cells (iNSC) to Treat Neural Injury

iPSC-NSCs Express SOX1 and Nestin

SOX1 Nestin

Merge

Differentiated iPSC-NSCs Express βIII-Tubulin and MAP2 Neuron Markers

βIII Tubulin MAP2

Merge

Differentiated iPSC-NSCs Express the GFAP Astrocyte Marker

GFAP Merge

Differentiated iPSC-NSCs Express the O4 Oligodendrocyte Marker

MergeO4

An assessment of failed treatments by the Stem Cell Therapies as an Emerging Paradigm in Stroke (STEPS) group has resulted in the identifying 2 major needs of the research community:

1) a regenerative cell therapy that will not only protect cells from ischemic injury but replace the lost and damaged tissues

2) a translational animal model more similar to humans for testing treatments

Stem Cell Therapies as an Emerging Paradigm in Stroke (STEPS): bridging basic and clinical science for cellular and neurogenic factor therapy in treating stroke. Stroke, 2009. Savitz, S.I., et al., Stem Cell Therapy as an Emerging Paradigm for Stroke (STEPS) II. Stroke, 2011.

Why Do All the Drugs Fail?

1.Size

2.Lissencephalic Vs Gyrencephalic

3.White Matter

Stem Cell Therapies as an Emerging Paradigm in Stroke (STEPS): bridging basic and clinical science for cellular and neurogenic factor therapy in treating stroke. Stroke, 2009; Nakamura, M., et al. J Neurosurg Pediatr, 2009; Kuluz, J.W., et al. Stroke, 2007; Tanaka, Y., et al. Stroke, 2008; Recommendations for standards regarding preclinical neuroprotective and restorative drug development. Stroke; DeFelipe, J. Frontiers in Neuroanatomy, 2011.

Differences in the Rodent Brain Relative to Human Brain

T2-FLAIR DWI ADC

An assessment of failed treatments by the Stem Cell Therapies as an Emerging Paradigm in Stroke (STEPS) group has resulted in the identifying 2 major needs of the research community:

1) a regenerative cell therapy that will not only protect cells from ischemic injury but replace the lost and damaged tissues

2) a translational animal model more similar to humans for testing treatments

Stem Cell Therapies as an Emerging Paradigm in Stroke (STEPS): bridging basic and clinical science for cellular and neurogenic factor therapy in treating stroke. Stroke, 2009. Savitz, S.I., et al., Stem Cell Therapy as an Emerging Paradigm for Stroke (STEPS) II. Stroke, 2011.

Why Do All the Drugs Fail?

Overarching Hypothesis

Transplanted iNSCs will produce regenerative

trophic factors and undergo differentiation

leading to cellular, tissue and functional

recovery in a pig model of ischemic stroke.

Emily Baker

Experimental Design

Tissue Collection

Sample Collection:

1. Magnetic Resonance Imaging (MRI)2. Immunohistochemistry

MRI

10x106

DWI and ADC Maps Confirm Ischemic Stroke

T2-FLAIR DWI ADC

iNSC Treatment Leads to Slowed Tissue Atrophy

Tis

su

e V

olu

me

Ch

an

ge

(% o

f C

on

tra

late

ral

He

mis

ph

ere

)

24hr 1wk 4wk 12wk-20

-10

0

10

20 Non-TreatediNSC Treated

***** *

*#

24hr 1wk

4wk 12wk

T2 FLAIR

indicates significance from contralateral hemisphere# indicates significance between treatment groups

Normal

iNSC Treatment Leads to Improved White Matter Integrity

indicates significance from 24 hours, within treatmentiN

SC

Tre

ate

dN

on

-Tre

ate

d

Ch

an

ge in

FA

24hr 1wk 4wk 12wk0

50

100

150

200

250

300

350

400 Non-Treated

iNSC Treated

*Normal

iNSC Treatment Leads to Recovery in NAA C

han

ge in

NA

A

24hr 1wk 4wk 12wk0

2

4

6

8

10Non-Treated

iNSC Treated

*#

#

*

No

n-T

reat

ed

iNSC

Tre

ate

dindicates significance between treatment groups

# indicates significance from 24 hours, within treatment

Normal

iNSC Treatment Leads to Recovery in Choline

Ch

an

ge in

Ch

o

24hr 1wk 4wk 12wk0

2

4

6

8

10Non-Treated

iNSC Treated

* *

No

n-T

reat

ed

iNSC

Tre

ate

dindicates significance between treatment groups

Normal

iNSC Treatment Leads to Recovery in Creatine

Ch

an

ge in

Cr

24hr 1wk 4wk 12wk0

5

10

15Non-Treated

iNSC Treated

*

No

n-T

reat

ed

iNSC

Tre

ate

dindicates significance between treatment groups

Normal

iNSC Treatment Improves Cerebral Blood Perfusion

% o

f C

on

tra

late

ral

He

mis

ph

ere

24hr 1wk 4wk 12wk0

50

100

150

200 Non-TreatediNSC Treated

*

Mean Transit Time%

of

Co

ntr

ala

tera

l H

em

isp

he

re

24hr 1wk 4wk 12wk0

50

100

150

200 Non-TreatediNSC Treated

*

Time to Peak

iNSC

Tre

ate

dN

on

-Tre

ate

d

indicates significance between treatment groups

Normal

Normal

iNSC Treatment Reduces Neuron Loss at the Lesion Border

A

DNormal iNSC TreatedNon-Treated

NeuNNeuN CBA

Ne

uN

B

Neu

N+

cel

ls/m

m2

Normal Non-Treated iNSC Treated0

50

100

150

200

250

300

350

*

D

iNSC Treatment Reduces Microglia Activation at the Lesion Border

A

% Ib

a1+

Are

a

Normal Non-Treated iNSC Treated0

5

10

15

20

25 # #

#

H

F G HE

Iba1

F GNormal iNSC TreatedNon-Treated

iNSC Treatment Promotes Endogenous NeuroblastProliferation and Migration to the Lesion Border

DC

X+

cells

/mm

2

Normal Non-Treated iNSC Treated0

5

10

15

20

***

D

A B CNormal Non-Treated iNSC Treated

DC

X

Transplanted iNSCs Differentiate into NeuronsHNA

NeuN HNA NeuN DAPI

Transplanted iNSCs Differentiate into Neurons

HNA NeuN HNA NeuN DAPI

Transplanted iNSCs Differentiate into Oligodendrocytes

HNA Olig2 HNA Olig2 DAPI

Transplanted iNSCs Differentiate Primarily into Neurons

% C

olo

calizati

on

wit

h H

NA

NeuN Olig20

20

40

60

80

100

ConclusionsiNSC treatment promotes brain tissue recovery through:

– Improved white matter integrity

– Improved neurometabolism

– Enhanced cerebral perfusion

– Neuroprotective

– Promoted neurogenesis

– Cell replacement

– Immunomodulatory

Funding Sources:NIH-NINDS, DoD, Bill and Melinda Gates Foundation, USDA, ArunA Biomedical, REM, BIRC

Collaborators:Simon Platt, DVMElizabeth Howerth, DVM/PhDKylee Jo Duberstein, PhDSteve Stice, PhDDavid Hess, MDQun Zhao, PhDHea Jin Park, PhD

West Laboratory:Emily Baker, PhDVivian Lau, DVM,MSHarrison Grace, PhDHolly Kinder, PhDErin KaiserKelly ScheulinElizabeth WatersSoo Shin Sydney Sneed