First in human:irv Weissman, Stanford University

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1 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

2 Blood-Forming Stem Cells 1 Development Ludwig Center at 2 Maintenance Stanford University IN VIVO VERITAS 3 Malignant transformation

3 Engraftment of human fetal blood-forming tissues in the SCID mouse Ethics and politics in runo Peault, Mike McCune, Reiko Namikawa, Ann Tsukamoto

4 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 P<.02 P<.01 Stanford HSC ChemoRes Stanford MPB ~33% ~22% ~7% Mueller,A. et al. 2011

5 Healthy Donor Transplant Healthy Donor Recipient Recipient T cells MPB or bone marrow RADIATION DAMAGE OR THERAPY Pure stem cells Dependent on Immunosuppressants Risk of Infections (fungus, bacteria) GvH Recipient Graft vs Host Disease Recipient Healthy No GvH Donor blood-forming and immune system Induce permanent transplant tolerance Reverse genetic autoimmune disease Shizuru, Beilhack, Weissman

6 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

7 Can Tissue Stem Cells Transdifferentiate? OR Dedifferentiate PSC Pluripotent stem cell Differentiate CNS-SC Trans-differentiate HSC Trans-differentiate Oligodendrocytes 1 Astrocytes NO! Neurons Blood NO! Cardiac or Skeletal or Lung Print 02/16/99

8 Human CNS-SC Neurosphere Cells Engrafted, Migrated & Differentiated Into Neurons, Astrocytes and Oligodendrocytes chida et al In Vitro Expansion Cerebral Cortex Transplantation Dopaminergic Neuron x Cerebellum Immunodeficient mouse brain Neurons Astrocytes Oligodendrocytes

9 HuCNS-SC Engraft, Migrate & Mature Human cells derived from HuCNS-SC transplant (brown) Proliferation at neurogeneic site Site appropriate differentiation Into Granule Neurons in the olfactory bulb SVZ Migrating as chain of neuroblasts RMS BrdU/Human Nuclei b-tubulin III/ Human nuclei

10 Animal Studies: Production of Function Myelin Uchida, et al SC121 Olig2 MBP Oligodendrocyte differentiation new functional myelin Myelin enhanced nerve conduction MRI used to detect human myelination in animals MBP dense lines

11 Hypomyelination Disease: Pelizaeus-Merzbacher Disease (PMD) 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-ofconcept 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

12 Phase I PMD: Summary of results 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

13 HuCNS-SC: Spinal Cord Injury Preclinical Efficacy Contusion site Open Field BBB Score (0-18) * * * * * HuCNS-SC (n=11) Control (n=15) 30day Delay DT Treatment Loss of Gained Function Repeated Measures ANOVA p<0.01 * individual time points p<0.05 Pre Weeks Post Injury 16 A. Anderson UC, Irvine

14 Extensive engraftment with CNS SC, local deposit with MSC; both DT sens

15 Phase I/II SCI: Interim Results 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 Subject ASIA Grade ASIA Spinal Level Months Post Txp 001 A T A T A T4 14

16 Subject 3: Light touch examination Dec (Pre-TX) Jun (6m Post-TX) normal impaired absent StemCells, Inc. 2012

17 HuCNS-SC Clinical Trial Summary Study N Cell Dose(s) Location Current Follow-up Lysosomal Storage Disease (NCL) to 1000 x 10 6 Frontal lobe Parietal lobe BRAIN: Lateral ventricle > 5 years (n=3) Myelin Disorder (PMD) x 10 6 BRAIN: Frontal lobe > 2 years (n=4) Spinal Cord Injury 4/12 20 x 10 6 SPINAL CORD thoracic intramedullary AIS A > 1 year (n=3) AIS B 4 months (n=1) Retinal Degeneration (dry AMD) 2/ to 1 x 10 6 EYE: subretinal space 2 to 4 months (n=2)

18 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

19 It s the cells, stu

20 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

21 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

22 Embryo Fetal Transition Single cell embryo ~ Day 3-4 ~ Day 6 First tissue stem cells Embryonic Epiblast Stem Cell Stem Cell Totipotent Pluripotent Somatic / adult stem cell Multipotent Cord blood & placenta Hematopoietic (HSC) & Mesenchymal (MSC) Other tissue stem cells Reprogrammed ips Pluripotent

23 Phase I Study: HuCNS-SC derived myelination Gupta, et al Control ROIs Transplant ROI Diffusion MRI shows HuCNS-SC-derived myelination in all PMD subjects: 1 year post-transplant and 3 months post-withdrawal of immunosuppression

24 Subject 3: EPT and dssep (T7 Left) Dec Pre-txp Jun m post-txp StemCells, Inc. 2012

25 HuCNS-SC cell attributes and clinical translation 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 Neurons Astrocytes Oligodendrocytes

26 Clinical and Radiological Observations in PMD Patients Post Transplant Subject 1 (16m) Subject 2 (42m) Subject 3 (14m) Subject 4 (66m) 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. 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. 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. 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.

27 Spinal Cord Injury Study Animal (thoracic SCI mice) Outcome 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

28 Phase I/II Thoracic SCI Study 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

29 Case Study: Retinal Degeneration Study Animal (RCS rat) Outcome Preservation of visual acuity Neuroprotection of host cone photoreceptors Human Phase I/II Dry AMD TBD Enrolling patients

30 # cones/100mm Preservation of visual acuity McGill et al, 2012

31 Phase I/II AMD Study ClinicalTrials.gov Identifier: NCT 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 Dry AMD Wet AMD

32 MRI: NCL Brain Normal brain 6 year-old with NCL

33 Mouse Model that Mimics Human NCL PPT 1-/- Normal (NOD-scid) CA1 CA1 NCL mouse brain: CA1 region only 8% of neurons were detected compare to normal

34 Neuroprotection Through Myelination Process Oligodendrocyte Myelinated Axons Myelin sheath with 20 continuous dense wrapping Contused Spinal Cord Injury Improved Myelinating motor human function MBP+ Remyelinated Olig2+ oligodendrocgtyes damaged axons in shiverer mouse brain Oligodendrocyte death or aberrant Immuno-EM: myelin human myelin sheath production Olig2/SC121/MBP leads to Axon myelin-associated diseases CNS-SC were transplanted into: - oligodendroctye-mutant shiverer mouse Axon brain - the injured spinal cord of NOD-scid Human myelin sheath with multi-layers dense wrapping

35 % of Normal HuCNS-SC Protects Neurons in Batten Mouse Hippocampus Capela, Uchida Not transplanted 8 % p<0.001 Low Dose Transplanted 33 % p<0.001 High Dose Transplanted 57 % n=6 n=9 n=6

36 Open Field BBB Score (0-18) HuCNS-SC Restores Motor Function in Spinal Cord 14 Injured Mice * * * * * DT Treatment Restored Motor Function Lost 8 HuCNS-SC (n=11) Control (n=15) Pre Post Weeks Post Injury DT Aileen Anderson, Brian Cummings,Nobuko Uchida et al Repeated Measures ANOVA p<0.01 * individual time points P<0.05

37 Testing Biological Activity of Human Cells in Animal Models: Xenogenic Transplant Immunodeficient mice: NOD-scid