iPS derived neural stem cells for

Amyotrophic Lateral Sclerosis

Giacomo P. Comi

DEPT Neurological Sciences, University of Milan, Italy

ARISLA, September 2014

ALS

Amyotrophic lateral

sclerosis (ALS) is a

devastating neurological

disease involving

selective degeneration of

motor neurons without

effective therapy.

Therapeutic approaches

Pharmacology

Gene therapy Cell therapy

• Production

• Biodistribuition/Safety

•Targeting

• Immunology

• Routes of administration

• Functional evaluation

• Screening

• Mechanism of action

• Optimization

• Culture

• Migration

• Survival

Vectors • Gene transfer

• Exon skipping

• Readthrough

• Antisense

Cell therapy for ALS

Issues to be fully addressed for clinical

applications of cell therapy

Identification of the most efficient cell type

A feasible administering protocol

A precise definition of therapeutic mechanisms

Cell populations Astrocytes Lymphocytes T

Microglia Neural stem cells (NSC)

Summary of clinical trials involving

stemcells and gene therapy for the

treatment of ALS. Schematic shows

the regions affected by ALS and

highlights the current and

developing therapies to target these

regions.

MATURE CELLS

SELF-RENEWAL

EGF/FGF-2

EGF/FGF-2

Neurospheres Dissociate SVZ

Neurons

Astrocytes Oligodendrocytes

Isolate SVZ

Treatment of SOD1-G93A transgenic mouse with a

neural stem cell subpopulation double positive

for Lewis X and the chemokine receptor CXCR4

(LeX1+CXCR4+) can modify the disease

progression

HB9-GFP mice

Neurospheres NSCs Cholinergic precursors

RA+Shh+

Heparin+GFs

LeX

Engraftment into SOD1 spinal cord

Rotarod test Survival

Corti et al., Brain 2007

Intraspinal transplanted NSCs (both mouse and human SVZ cell line) can slow

both the onset and the progression of clinical signs and prolong survival in

SOD1 G93A mice.

Neural Stem Cells in humans

Neuralstem

NSC

Human fetal spinal cord (at

8 weeks)

Spinal cord injections

Phase I complete, Phase II

current

Enhanced survival of

spinalmotor neurons in rats

Cedars-Sinai

hNPC releasing GDNF

Human fetal cortex (at 8-15

weeks)

Spinal cord injections

Preclinical

GDNF secretion in vivo

enhances survival of spinal

motor neurons in rats

Vescovi

NSC

Human fetal (unspecifed)

Spinal cord injections

Phase I current

N/A

Induced

pluripotent stem

cells (iPSCs)

Induced Pluripotent

Stem Cells (iPSCs)-

derived NSCs

The retroviral−mediated introduction of

stem cells factors can reprogram murine

fibroblasts into a pluripotent state giving

rise to iPSCs

Pluripotency, self renewal

Nobel Prize

Biopsy

Generate neural stem cells,

neurons, motor neurons, and

other relevant neuroectodermal

cell populations

AIMS: iPSCs for motor neuron diseases

Create patient and disease-

specific stem cell lines (ALS,

SMA, SMARD1) using a non-

viral method

In vitro and in vivo studies

iPSCs from human ALS fibroblasts

ALS-iPSCs express pluripotency

markers and present a typical iPSC

morphology

MN death pathogenesis

Ferraiuolo et al, Molecular pathways of motor neuron

injury in amyotrophic lateral sclerosis. Nat Rev Neurol.

2011

Autonomous motor neuron degeneration

Autonomous motor neuron degeneration,

Human ALS iPSC MN studies

Motor neurons SOD1A4V Decreased survival and morphology Kiskinis 2014

Motor neurons SOD1A4V Intrinsic membrane hyper excitability Wainger 2014

Modified from Richard , Maragakis 2014

Muscle

Motor neuron

Astrocyte

Microglia Toxic

Signal ?

Activation

signal?

Schwann cells

Inter neuron

T lymphocytes

Mutant SOD1 (SOD1G93A)

molecules within other cells

promote degeneration

Toxic

Signal?

Activation

signal?

Cell extrinsic disease mechanisms

Activated

macrophages

iPSCs as a cell source of human

neural stem cells (NSCs) for

transplantation

Isolation of neural stem cells

from wild-type iPSCs

High ALDH activity: high staminality

positivity for VLA4: ability to migrate

These cells display a neural stem/precursors phenotype

ALDHhiSSClo VLA4+ NSCs are able to

differentiate in vitro in motor neurons

Minimally invasive cell

transplantation protocols

Extravascular deposition of erythrocyte-derived hemoglobin

and erythrocyte extravasation in the spinal cord of ALS subjects.

Transplantation protocol

Human ALDHhi-SSClo-

VLA4+ GFP-transduced

NSCs

Each group had the same

number of males and

females. The

immunosupressor FK506

was administered i.p. at

1.0 mg/kg body weight

was administered to all

animal groups for the

entire length of the

experiment.

Cell engraftment after intrathecal injection

Donor cells were

detected in spinal cord

Cell engraftment after systemic injection

Donor cells were

detected in spinal cord

Systemic transplanted cells reach different targets Brain: cortical and subcortical frontal lobe

Skeletal Muscle

SOD1G93A mice lifespan after

intrathecal NSCs administration

Untreated: 143+/-6

Treated: 153+/- 5

Treated vs Untreated P =0.0135

SOD1G93A mice lifespan after

systemic NSCs administration

Untreated: 148 d ± 7

Treated: 171 d ± 6

Treated vs Untreated P = 0.0006

NSCs transplantation effects

NSCs transplantation

protects endogenous

MNs and axons

number

Cell intrinsic/extrinsic disease mechanisms

Selected NSCs produce neurotrophins

AL

DH

hiS

SC

loV

LA

4+

NS

Cs

...

A Small Molecule Screen in Stem-Cell-Derived Motor Neurons Identifies a Kinase

Inhibitor as a Candidate Therapeutic for ALS

Cell Stem Cell, Volume 12, Issue 6, 2013, 713 – 726 Lee Rubin lab

•

A new type of stem-cell-based motor

neuron screen was performed

Kenpaullone increases the survival of

wild-type and ALS motor neurons

Kenpaullone’s activities result from dual

inhibition of GSK-3 and HGK kinases

Potential value of preclinical testing

using human ALS motor neurons is

shown

Glycogen synthase kinase 3 (GSK-3)

IGF2

NSCs transplantation effects

Spinal cord analysis: NSCs transplantation reduces microglial infiltration and astrogliosis

Cell extrinsic disease mechanisms

SOD1 G93A astrocytes

express TRPV1 (transient

receptor potential vanilloid

subfamily member-1)

Cell extrinsic disease mechanisms:

NSC acts on SOD1 G93A astrocytes trough

a TRPV1-mediated mechanism

NSC-conditioned medium reduced viability of SOD1G93A astrocytes, while non-conditioned

medium (Ctrl) is uneffective.

Blocking TRPV1 with the selective antagonists iodoresiniferatoxin (I-RTX, 10 nM) or capsazepine

(CZP, 1 mM) decreased the cytotoxicity on astrocytes

The toxic effect of NSC was present on SOD1 astrocytes only, while non-transgenic mouse

astrocytes and on ALDHhiSSCloVLA4+ NSC-derived astrocytes were unaffected.

Conclusions Minimally invasive injection of iPSC-derived NSCs in SOD1 G93A

ALS mouse model results in consistent engraftment of cells

Transplantation is associated with an improvement of the disease

phenotype

iPSC-derived NSCs can modulate different aspects of ALS pathogenesis

through the inflammation reduction, reduced astrocitosis and production

of trophic factors

This strategy can point out novel therapeutic targets/strategies

iPSCs represents a promising avenue for

effective cell-based treatment for ALS and

other neurodegenerative diseases

Future directions

To generate other ALS patient-specific iPSC lines and neurons to

investigate disease specific features of affected cells

To optimize the population to be transplanted in order to increase

survival, migration, engraftment and differentiation potential in ALS

mouse model.

To investigate iPSC-derived NCS in other ALS disease models to derive

conclusions translatable to human ALS

Neural Stem Cell Lab

Stefania Corti

Monica Nizzardo

Chiara Simone

Federica Rizzo

Sabrina Salani

Monica Bucchia

Valeria Parente

Sara Dametti

Agnese Ramirez

Giulietta Riboldi

Chiara Zanetta

Irene Faravelli

Thanks

Dino Ferrari Centre for the Study and Therapy of Neuromuscular and

Neurodegenerative Disorders

Neuroscience Section,

Department of Pathophysiology and Transplantation,

University of Milan,

Neurology Unit, IRCCS Foundation Ca’ Granda Ospedale Maggiore Policlinico, Italy

Director: Nereo Bresolin

Deputy Director: Giacomo P. Comi