"Safety Considerations for the Clinical Application of Human Embryonic Stem Cells" 4/4/2008

1. Safety Considerations for the Clinical
Application of Human Embryonic Stem Cells
April 2008

2. General Considerations

3. Concerns
• Genetic stability of hES cells and their differentiated derivatives.
• Karyotype analysis can miss point mutations or chromosomal exchange.
• Cell growth requirements and growth rate are important variables for measuring genetic stability
• More rapid growth rate can signal problems.
• Reduced stringency for cell growth can signal problems.
• Factors that accentuate this risk:
• Scale-up to produce the numbers of cells needed for clinical administration.
• Culture/purification processes.
• Feeder-free culture of hES.
• Differentiation Signals are not present in adult tissues to instruct ES cells despite initial
optimism that this was the case.
• Pre-differentiation of cells will be needed.
• Accentuates the need for eliminating undifferentiated ES cells from the population.
• Long-term follow-up critical to assess tumorigenic potential.
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4. Safety Study Requirements
• Proof of principle/efficacy animal studies provide critical safety data.
• Should mimic route and site of delivery planned clinically.
• Start with syngeneic models if possible as this avoids the issues associated with immune
rejection of transplanted cells.
• Confirm results with the human cells transplanted to immunodeficient/immunosuppressed
recipients.
• Appropriate identification methods should be established so that the end disposition
of transplanted cells can be determined.
• Survival or non-survival of transplanted cells needs to be ascertained to assess safety.
• Characterization of graft elements or determination that endogenous self-repair
mechanisms are being stimulated.
• Route of cell delivery important.
• Systemic route of delivery vs. direct injection at the site of repair.
• Systemic delivery poses unique risks and requirements for the distribution of cells and
whether the cells get trapped in the lungs, heart, liver, spleen or brain.
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5. Stimulation of Endogenous Repair Activities
• Cells do not survive long-term.
• Highly dependent on immune reaction.
• Immune reaction varies depending on species combination
being studied.
• Should be tested in multiple species combinations to safely
make sure cells do not survive.
• Effects more likely to be due to mechanisms independent of
the cell type used.
• Benefits should be demonstrated in comparison to control cells.
• There should be benefits unique to the therapeutic cell being
used.
• Ectopic survival still needs to be assessed.
• There may be cell survival outside the target tissue.
• Can be a problem if cells distribute to a site more conducive to
growth (e. g. cerebral spinal space)
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6. Graft Dependent Repair
• Graft can be analyzed in detail.
• Benefit vs presence of undesired cell types can be
assessed.
• Benefits can be correlated to cell survival.
• Efficacy studies give significant safety data.
• Graft can be assessed for risk of overgrowth.
• Consistency of cell preparations can be better
assessed.
• Fewer unknowns when cells survive.
• Site of administration and injection method more
important.
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7. Cell Survival Assessments
• Cell tracking tools can be problematic.
• Cell tags (GFP, BrdU, ß-gal, Iridium, Fe-particles or fluorescent dyes)
are all subject to significant artifact.
• Toxic to cells
• Artifactual fluorescence or enzymatic activity.
• Markers can be transferred from cell to cell due to fusion,
macrophages take up cellular debris containing tags.
• Cell survival vs cell fusion needs to be distinguished.
• Many instances of cell survival have been shown to be due to cell fusion
artifact.
• Cell survival needs to be sufficient to account for functional benefit or
benefit needs to be dependent on cell type used.
• Transplant of human cells to experimental animal species allows for
precise assessment of cell survival.
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8. Cross Species Transplants
• Immuno-permissive Model
• Systemic immunosuppression (Cyclosporin, FK-506, etc).
• Immuno-deficient recipient.
• Immunodeficient animal models vary depending on cells
being transplanted and the location of the transplant, so
the correct model needs to be selected.
• Nude Mouse and Rat models acceptable for transplant to
an immunoprivileged site.
• NOD, SCID, ß2 knockout mouse strain or equivalent
required for most cells when not transplanted to an
immunoprivileged site (must eliminate B-cell, T-cell and NK
cell activity).
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9. Cell Sourcing
• The cells to be used clinically should be derived from the
same source as the cells tested pre-clinically.
– Human cells behave differently than those from other
species.
– Require use of immune suppression or immune deficient
recipients.
• Cell shipping and storage conditions should be modeled
after what will be used clinically.
– Shipping and storage can effect cell function and survival.
– Thawing of cryopreserved cells at clinical site introduces
risk and sterility issues.
– A specially trained team needs to be utilized if cells are to
be shipped frozen and thawed before administration.
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10. Cell Manufacturing
• Cells used in pre-clinical studies should be produced by
process to be used for clinical production.
• Cell Source can affect risk of tumor or teratoma formation.
• Undifferentiated vs Differentiated ESC
• Purity of cell preparation
• Mode of culture can affect risk of tumor or teratoma formation.
• Monolayer vs cell clusters.
• Undifferentiated ES cells can linger in cell clusters.
• Less likely an issue with monolayer culture.
• Selection process
• Length of the selection process is important.
• Whether cells are cultured after selection is important.
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11. Mode of Delivery
• Injection device to be used clinically should be tested for
biocompatibility.
• Look at short term effects on cell viability.
• Look at long term effects on cell function.
• Cells can reveal deficiencies in the injection device not
revealed by passing media through the device.
• Mass effect created by the cells in suspension can drag
debris from the injection device not seen with media alone.
• Debris cause foreign body reactions at site of injection.
• Specific large animal models may be required to test
safety for human injection.
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12. Tumorigenisis and Biodistribution Studies
• Can ideally be combined with animal efficacy studies.
– Use immunodeficient of immunosuppressed host.
– Use same route of delivery and target location planned for clinical studies.
– Not always feasible
• Independent studies
– Immunodeficient host
– Cells tested using route of delivery and location to be used clinically.
– Positive control to show cells can survive in host.
– Test cells produced by GMP process
• This includes any scale-up processes
• Should be done with cells under storage and shipping conditions.
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13. Specific Example: hES-derived Retinal
Pigmented Epithelial (RPE) Cells

14. 14
RPE Cell Function
RPE
Layer
Deteriorates
in Patients
with AMD
and other
Retinal
Degenerative
Diseases
 Pigmented cells
 Allow for direct selection
 Adhere to Bruchs membrane
 Phagocytose shed
photoreceptor segments
 Produce trophic factors
hES derived RPE cells

15. RPE Cell Production
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7 – 14 days 2 months 1 month

16. RPE Cell Characterization
• RPE cells derived from hES differentiation.
• RPE cells selected based on pigmented appearance.
• Karyotype confirmed before and after expansion.
• After selection RPE cells are cultured and expanded in monolayer culture.
• Cells screened for homogeneity and pigment content.
• Cells screened for absence of hES cell markers by:
– PCR, Western Blot, Immunohistochemistry (e. g. Oct-4, Nanog, Sox-2, etc.).
– Non detectable.
• RPE screened for presence RPE markers:
– PCR, Western Blot, Immunohistochemistry (RPE65, Bestrophin, PEDF, etc)
– >95%
• RPE screened for in vitro function: phagocytosis, elastin secretion and PEDF production.
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17. RPE Survival and Integration
• Human to rat transplant
• Immunopriviliged location
• Cyclosporin Immunosuppression
• Cells survive and provide long-term benefit, >100
days.
• No evidence for cell rejection
• Cells integrate and function to prevent
photoreceptor loss
• Dose can be estimated for larger eye of a human.
• Primate studies undertaken to confirm capability
to inject projected cell dose.
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18. 18
RPE Rescue in the RCS Rat
RCS retina at P100 with
H9-RPE injection:
A: low power view of retina
showing extensive rescue.
B: high power of (b)
showing rescued
photoreceptors.
C: high power of (c)
showing non-rescue area.
Photoreceptor
Rescue in RCS Rat
by transplanted
H9-RPE cells

19. Clinical Considerations for the use of Retinal
• Limitation of Risks
• Small dose of cells required in the eye.
• Confined space.
• Focal degeneration
• No special devices required for eye injection.
• Eye is an immuno-privileged location reducing or
eliminating need for immunosuppression.
• Eye is a self-contained space, limiting issues of
cell migration.
• The target location in the eye can be easily
visualized and monitored.
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