BRMAC Meeting #27

**Human Stem Cells as Cellular Replacement Therapies for Neurological
Disorders**

### July 13-14, 2000

Hilton Hotel, Gaithersburg, MD

**PRODUCT: Discussion Questions**

Stem cells are distinct from other cellular products in that they are
intended to differentiate in order to assume their final therapeutic
phenotype. Frequently, the process of terminal phenotypic
differentiation will be initiated *ex vivo* prior to patient
administration. The capacity for stem cells to generate phenotypically
distinct cell lineages through differentiation is fundamental to their
utility but also imposes significant challenges in developing meaningful
product specifications, predicting outcomes and assessing safety. Neural
stem cells represent an example typifying the phenotypic potentiality of
stem cells. The proposed use of neural stem cells generally is to
produce functional neuronal circuitry through differentiation of
developmentally immature precursors. In contrast to their potential
therapeutic benefits, neural stem cells may also possess the ability to
develop aberrant neural connections that result in adverse clinical
effects, including seizures and inhibition or hyperactivation of
existing circuitry. These events may occur as a consequence of
unregulated neurotransmitter release, release of unintended
neurotransmitters, or differentiation into an undesired cellular
phenotype that contributes to the formation of gliotic scars. Careful
scrutiny must be given these unintended events. Current knowledge of the
molecular and cellular biology of most cellular products is unlikely to
allow analytical specifications that provide information sufficient for
precise product characterization. Accordingly, it will be necessary to
develop guiding principles that make best use of available information
in order to identify cellular characteristics that can be defined
analytically, to establish well-defined manufacturing process controls,
and to design appropriate biological assays. Collectively, these
principles will facilitate development of stem cells as safe and
effective therapies.

# 1. Human Stem Cell Sources 

Human stem cells may be obtained from a variety of sources. These
include autologous or allogeneic adult cells, allogeneic fetal cells or
tissues, as well as established allogeneic stem cell lines of embryonic
or adult origin. Stem cells may also be derived from mutation expressing
transformed cells or immortalized cell lines that are the product of
genetic engineering. The extent of information that might be reasonably
obtained through safety testing and characterization of stem cells will
vary substantially depending upon their source.

a.  What source controls are appropriate for donor tissue? Are existing
    standards for blood banking and/or organ transplantation
    appropriate? In cases where cell lines might be established or where
    culture times might be prolonged, would it be appropriate to retest
    donors for HIV to eliminate donors in the "window" period?

b.  Does the BRMAC concur with the position that records linking cell
    preparations with donors and patients should be maintained? If so,
    what records should be kept, who would have access to them, and for
    how long should they be maintained?

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c.  If there were circumstances when the cell or tissue source of origin
    is not traceable or is poorly characterized, would there be
    conditions acceptable for considering use of such stem cells in
    humans?

> d\. Does the BRMAC recommend that molecular genetic analysis be a
> component of stem cell source qualification? If so, which genetic
> markers should be evaluated (mutational analysis of parkin gene and
> -synuclein, Parkinson's disease; SOD-1, ALS; HD gene, Huntington's
> disease)? Are existing genetic tests sufficiently reliable as to be
> useful for stem cell source qualification? In situations when testing
> of stem cells derived from embryonic or fetal sources reveals
> mutations that are positively associated with neurodegenerative
> disease onset, what would be the obligations for informing donors?

e.  Are there different intrinsic safety concerns for stem cells based
    on their source of derivation (e.g., autologous adult donation,
    allogeneic adult donation, embryonic germ cells, or embryonic stem
    cells)?

f.  Does the BRMAC recommend different characterization studies for stem
    cells depending upon the procedures and sources of their derivation
    or are these issues generic to this entire product category?

## 2. Manufacturing of Stem Cells

a.  To what extent do stem cell isolation and maintenance procedures
    determine desirable cell fates and preclude undesired cell fates?

b.  What are likely to be critical manufacturing process controls?
    Examples include qualification of the source(s) from which stem
    cells are obtained, standardization of procedures used to procure,
    expand or maintain stem cells, and the development of validated
    tests that allow monitoring of stem cell product identity and
    heterogeneity.

> What evidence supports these conclusions and what further studies are
> needed to resolve these questions?

# 3. Characterization of Stem Cell Preparations and Selection of Specifications

The ability to reliably identify stem cells derived from various
cellular types is essential for assessing purity of cellular
preparations and evaluating consistency achieved in the isolation or
manufacture of stem cells. In addition, a correlation must be
established between the assessment of stem cell identity and the
outcomes obtained following their administration. Finally, unambiguous
identification of the therapeutic cell type is essential for accurate
dosing. Therefore, an explicit, detailed description of stem cell
preparations with respect to identity and purity is required.

a.  Stem cell preparations may consist of heterogeneous cell
    populations. Some may be essential to the intended effect, some
    deleterious, and some inert. Both to minimize the possibility of
    untoward reactions and to ensure consistent dosing, both purity and
    the profile of impurities should be controlled. In a complex product
    of this type, what principles should guide selection of purity
    specifications?

b.  Have specific markers been identified that permit unambiguous
    identification of cell phenotype, determination, or fate?

c.  Have specific markers been identified that are *necessary* to ensure
    that product cells will assume the correct, functional, therapeutic
    phenotype?

d.  Have specific markers been identified that are *sufficient* to
    ensure that product cells will assume the correct, functional,
    therapeutic phenotype?

e.  Have specific markers been identified that indicate that isolated
    stem cells will lead to adverse events such as ectopic tissue
    differentiation, deleterious cell fate, tumorigenesis, or some other
    undesired outcome?

f.  What approaches should be used to evaluate proposed specifications?

# 4. Potency Assays for Stem Cell Products

An ideal potency assay would guarantee that each lot of the product that
performs acceptably will have the desired clinical effect. When this
cannot be achieved, it is useful nevertheless to devise an assay for
which an acceptable result is associated reliably with desirable product
performance characteristics.

a.  What recommendations does the committee have for development of stem
    cell potency assays?

b.  At present, it is unlikely the molecular determinants that would
    ensure desirable product characteristics are known completely. Given
    the difficulty of assessing intended cellular function, are there
    surrogate measures that might be predictive for assuring intended
    bioactivity of stem cell implants? If not, what studies would
    provide this information?

c.  What degree of inter-assay variability would be considered
    acceptable?

d.  In what manner might a dosage unit of potency be expressed? Would
    such units be based on the number of total cells in a preparation or
    defined by a subset of stem cells expressing a specific marker
    predictive of differentiative phenotype (e.g. percentage of cells
    expressing neural-specific markers such as nestin, type III
    -tubulin, neuron-specific nuclear protein or tyrosine hydroxylase)
    and presumed clinical outcome?