First in human:Irv Weissman,

Stanford University

The case for making human

mouse chimeras to understand

the function of human tissue stem

cells from normal or genetically

diseased donors

Blood-Forming Stem Cells

Ludwig Center at Stanford University

IN VIVO

VERITAS

1 Development

2 Maintenance

3 Malignant

transformation

Engraftment of human fetal blood-forming

tissues in the SCID mouse

Bruno Peault

Ethics and politics in 1986-88

, Mike McCune, Reiko Namikawa, Ann Tsukamoto Irv Weissman

Cancer Free Stem Cell Grafts

Improves Survival • Purified HSC show 3-fold higher survival vs non-purified MPB

• Stage 4 metastatic patients – failed all other therapies

~7%

~22%

~33%

Mueller,A. et al. 2011

ChemoRes

Stanford HSC

Stanford MPB

P< .02

P<.01

• Donor blood-forming and immune system

• Induce permanent transplant tolerance

• Reverse genetic autoimmune disease

Healthy Donor Transplant

Healthy Donor

Recipient

MPB or bone marrow

Pure stem cells

• Dependent on

Immunosuppressants

• Risk of Infections

(fungus, bacteria)

Recipient

Recipient

Recipient

T cells

Graft vs Host Disease Healthy

RADIATION DAMAGE OR THERAPY

GvH No GvH

Shizuru, Beilhack, Weissman

HSC Transplants In The Future

• Limited or no use of radiation or cytotoxic

drugs

• Targeted removal of host HSC, T cells,

and NK cells with Mabs

• In the far future [>5-10 years], ES or iPS

derived HSC will be cotransplanted with

other tissue stem cells from the same

donor line

Print

02/16/99 1

Can Tissue Stem Cells Transdifferentiate?

Pluripotent stem cell

CNS-SC

Neurons Oligo-

dendrocytes

Astrocytes

HSC

Blood

PSC

Trans-differentiate

Differentiate

OR

Dedifferentiate

Cardiac

or

Skeletal

or

Lung

Trans-differentiate

NO! NO!

Human CNS-SC Neurosphere Cells Engrafted, Migrated &

Differentiated Into Neurons, Astrocytes and Oligodendrocytes

In Vitro Expansion

Transplantation

Dopaminergic

Neuron

Astrocytes Neurons Oligodendrocytes

Cerebellum

Cerebral Cortex

Immunodeficient mouse brain

Uchida et al

x

Human cells derived from HuCNS-SC transplant (brown)

RMS

Migrating as

chain of neuroblasts

b-tubulin III/ Human nuclei

Proliferation at neurogeneic site

SVZ

BrdU/Human Nuclei

Site appropriate differentiation

Into Granule Neurons in the olfactory bulb

HuCNS-SC Engraft, Migrate & Mature

SC121

Olig2

MBP

Animal Studies: Production of Function Myelin

MBP

Oligodendrocyte

differentiation – new

functional myelin

Myelin enhanced nerve

conduction

MRI used to detect human

myelination in animals

Uchida, et al. 2012

16-20 dense lines

• Progressive and fatal X-linked hypomyelination disorder due

to mutations of the proteolipid protein gene, PLP1

• No current therapy - regenerating oligodendrocyte

population alone could produce clinical benefit

• Recognized as appropriate human target for proof-of-

concept of donor-derived myelination in non-inflammatory

setting

• Diagnosis readily confirmed affording opportunity for early

intervention

• MRI measures of de novo myelination available as potential

surrogate markers of engraftment and function

Hypomyelination Disease:

Pelizaeus-Merzbacher Disease (PMD)

Well-tolerated intervention with a favorable safety profile

Signs of clinical changes detected suggest a departure from

the natural history of disease

MRI findings consistent with de novo myelination in the

transplanted frontal lobe regions in all four subjects

Phase I strongly supports a controlled Phase II trial in PMD

MRI data in a human hypomyelination disorder suggests

potential applicability to other myelination disorders, such as

select forms of cerebral palsy, spinal cord injury, transverse

myelitis, and multiple sclerosis

Phase I PMD: Summary of results

Pre 1 2 3 4 5 6 8 14 12

8

10

12

14

Weeks Post Injury

Open Field

BBB Score

(0-18)

* * * * *

HuCNS-SC (n=11)

Control (n=15)

Repeated Measures ANOVA p<0.01

* individual time points p<0.05

16

HuCNS-SC: Spinal Cord Injury Preclinical Efficacy

30day Delay DT

Treatment

Loss of

Gained

Function

Contusion site

A. Anderson UC, Irvine

Extensive engraftment with CNS SC,

local deposit with MSC; both DT sens

• Favorable interim safety and feasibility

• AIS A (n=3) cohort enrolled

• No adverse events attributed to HuCNS-SC

• Observed changes in light touch sensation support safety

• Quantitative Perception Testing shows segmental gains

• No evidence of electrophysiological loss

• AIS B cohort enrolling; AIS B dosed Sept 2012

Phase I/II SCI: Interim Results

Subject ASIA Grade ASIA Spinal

Level

Months Post

Txp

001 A T8 17

002 A T9 15

003 A T4 14

normal

impaired

absent

Subject 3:

Light touch examination

Jun 28 12 (6m Post-TX) Dec 12 11 (Pre-TX)

© StemCells, Inc. 2012

HuCNS-SC Clinical Trial Summary

Study N Cell Dose(s) Location Current Follow-up

Lysosomal

Storage

Disease (NCL) 6 500 to 1000 x 106

BRAIN:

Frontal lobe

Parietal lobe

Lateral ventricle

> 5 years

(n=3)

Myelin Disorder

(PMD) 4 300 x 106

BRAIN:

Frontal lobe > 2 years

(n=4)

Spinal Cord

Injury 4/12 20 x 106

SPINAL CORD

thoracic

intramedullary

AIS A > 1 year

(n=3)

AIS B 4 months (n=1)

Retinal

Degeneration

(dry AMD)

2/16 0.2 to 1 x 106 EYE:

subretinal space 2 to 4 months

(n=2)

Lessons learned to date • Choice of animal models on target

• Animal models have been very good predictors; to date

recapitulating in humans what we have seen in animal models

• Safety profile show no concerns to date

• Metric for calculating dose choices seem to be working

• Small uncontrolled studies on patients

• Preliminary evidence of HuCNS-SC biological activity in

transplanted patients reflect what we have seen in animal models

to date

• Accumulating long-term safety (1-5yrs), sustained biological

effects

It’s the cells, stu…

The human neuron-murine brain

mouse • Several mutations allow early neural precursors,

perhaps radial glia, to proliferate, but neuron

lineage progeny die or disappear: ligase 4 dko,

xrcc4 dko, etc.

• Will same stage human neural stem/progenitors

engraft in fetal life and make mouse or human

nervous systems, if they work at all?

• Bioethics: The American Journal of Bioethics,

2007.Thinking About the Human Neuron

Mouse.Henry T. Greely; Mildred K. Cho; Linda

F. Hogle; Debra M. Satz

WHICH OF THE FOLLOWING

NEURAL DISEASES DO YOU

NOT WANT TO CURE? • Lysosomal storage diseases(Battens,

Gauchers, etc)

• Brain cancers

• Spinal cord contusion with demyelination

• Stroke

• ALS

• Parkinsons

• Alzheimers

• Huntington’s

• Cerebral palsy

• Others I forgot since medical school

Single –cell

embryo ~ Day 6 ~ Day 3-4

Embryonic

Stem Cell

Totipotent

Epiblast

Stem Cell

Pluripotent

First tissue stem

cells

Embryo – Fetal Transition

Somatic / adult stem

cell Multipotent

Cord blood &

placenta

Hematopoietic (HSC)

&

Mesenchymal (MSC)

Other tissue

stem cells

Reprogrammed iPS Pluripotent

Phase I Study:

HuCNS-SC derived myelination

Diffusion MRI shows HuCNS-SC-derived myelination in all PMD subjects: 1

year post-transplant and 3 months post-withdrawal of immunosuppression

Gupta, et al. 2012

Control

ROIs

Transplant

ROI

Subject 3: EPT and dSSEP (T7 Left)

Dec 12 ‘11

Pre-txp Jun 28 ‘12

6m post-txp

© StemCells, Inc. 2012

• Purified, expandable & cryopreservable

• Self-renewing, non-tumorigenic

• Allogeneic – homologous use into

brain, spinal cord or eye

• In vivo – CNS restricted

• survive & migrate

• regulated by host

• differentiate site specific

• Mechanism-of-action: multi-faceted

• Cell replacement with proper type:

oligodendrocytes, neurons, astrocytes

• Neuroprotection of host cells: trophic

effects by secreted factors

HuCNS-SC cell attributes and clinical translation

Neurons Astrocytes Oligodendrocytes

Subject 1

(16m)

Tracheostomy and gastrostomy at baseline. Remained neurologically stable, but was noted to have reduced nightly

continuous positive airway pressure (CPAP) at 12 months. Increased FA by MRI.

Subject 2

(42m)

Developed improved truncal support and the ability to take steps with assistance.

Began speaking audible single words and the ability to follow two-step commands.

Increased FA by MRI.

Subject 3

(14m)

Tracheostomy and gastrostomy at baseline. Nightly CPAP dependency reduced.

Developed upper extremity antigravity strength and to take some solid foods by mouth.

Greatest increase in FA by MRI; but comparable to “control” regions.

Subject 4

(66m)

Developed improved truncal support; progressed from the use of a walker with significant support to walking with minimal assistance.

Developed the ability for self-feeding and to follow two-steps commands. Increased FA by MRI.

Clinical and Radiological Observations in PMD

Patients Post Transplant

Study Outcome

Animal

(thoracic SCI mice)

Locomotor improvement dependent on

presence of human cells

Remyelination & new neurons

Human – Phase I/II

Thoracic injury

chronic

12 mo. interim results: AIS-A

No safety concerns to date

Observed changes in light touch sensation

supporting safety

No evidence of electrophysiological loss

Clinical signs of sensory gains

Spinal Cord Injury

• Swissmedic authorized

• 12 patients with thoracic injury (tSCI)

• Injury level: T2-T11

• Chronic injury stage: 3-12 months post-injury

• Broad range of injuries: AIS A, B, C

• Significant cell dose: 20 million cells

• Safety and preliminary efficacy

• Clinical

• electrophysiological and

• QoL endpoints

Phase I/II Thoracic SCI Study

Study Outcome

Animal

(RCS rat)

Preservation of visual acuity

Neuroprotection of host cone photoreceptors

Human

Phase I/II

Dry AMD

TBD

Enrolling patients

Case Study: Retinal

Degeneration

Preservation of visual acuity

# c

on

es/1

00m

m

McGill et al,

2012

• Geographic Atrophy (dry) age-related macular degeneration

• Open-label; dose-escalation (200,000 and 1 million cells)

• Cohort I: 8 subject ≤ 20/400 – dosing in progress

• Cohort II: 8 subjects 20/100- 20/200

• Objectives: Safety and preliminary efficacy

Phase I/II AMD Study ClinicalTrials.gov Identifier: NCT01632527

Dry AMD Wet AMD

MRI: NCL Brain

Normal brain 6 year-old with NCL

Mouse Model that Mimics Human NCL

PPT 1-/-

CA1

Normal (NOD-scid)

CA1

NCL mouse brain: CA1 region only 8% of neurons were detected compare to normal

Neuroprotection Through Myelination

Oligodendrocyte Myelin sheath

with 20 continuous dense wrapping

Process

Myelinated Axons

Myelinating human MBP+

Olig2+ oligodendrocgtyes

in shiverer mouse brain

Olig2/SC121/MBP

Contused Spinal Cord Injury

Improved motor function

Remyelinated damaged axons

Axon

Axon

Immuno-EM: human myelin sheath

Human myelin sheath

with multi-layers dense wrapping

Oligodendrocyte death or

aberrant myelin

production leads to

myelin-associated

diseases

CNS-SC were

transplanted into: - oligodendroctye-mutant

shiverer mouse brain

- the injured spinal cord of

NOD-scid

HuCNS-SC Protects Neurons in Batten Mouse

Hippocampus

Not transplanted Low Dose

Transplanted

High Dose

Transplanted

% o

f N

orm

al

0

20

40

60

8 %

33 %

57 %

p<0.001 p<0.001

n=6 n=9 n=6

Capela, Uchida

HuCNS-SC Restores Motor

Function in Spinal Cord

Injured Mice

Pre 1 2 3 4 5 6 8 14 16 12 Post

DT

8

10

12

14

Weeks Post Injury

Open F

ield

BB

B S

core

(0

-18)

* * * * *

DT

Treatment

Restored

Motor

Function

Lost

HuCNS-SC (n=11)

Control (n=15)

Repeated Measures ANOVA p<0.01

* individual time points P<0.05

Aileen Anderson, Brian Cummings,Nobuko Uchida et al

Testing Biological Activity of Human Cells in Animal Models: Xenogenic Transplant

Immunodeficient mice: NOD-scid