Stem cell based therapies through tissue regeneration and repair as well as through the targeted delivery of genetic material are expected to be effective in the treatment of a wide range of medical conditions. Efforts to analyze and assess the safety of using human stem cells in the clinical setting are vitally important to this endeavor. Transplanted human stem cells are dynamic biological entities that interact intimately with and are influenced by the physiology of the recipient. Assessing human stem cell safety requires the implementation of a comprehensive strategy. Included in this global assessment are the derivation, expansion, manipulation, and characterization of human stem cell lines, as well as preclinical efficacy and toxicity testing in appropriate animal models.
Although precedents exist for the clinical use of human stem cells, there is considerable reluctance to proceed with clinical trials involving human stem cells derived from embryonic and fetal sources. This hesitancy extends to adult human stem cells of non-hematopoietic origin, even though, by contrast, their plasticity is generally considered to be lower than that of their embryo- and fetus-derived counterparts. For human stem cells to advance to the stage of clinical investigation, a virtual safety net composed of a core set of safeguards is required.
| Safeguard | Embryo | Fetus | Adult: Autologous (self) | Adult: Allogeneic (nonself) |
|---|---|---|---|---|
Screen donors
|
++ | ++ | + | ++ |
| Use controlled, standardized practices and procedures for establishing stem cell lines | ++ | ++ | ++ | ++ |
| Develop alternatives to culturing on cell-feeder layer | ++ | ++ | NA | NA |
|
++ | ++ | ++ | ++ |
Conduct preclinical animal testing
|
++ | ++ | ++ | ++ |
|
++ | ++ | ++ | ++ |
|
++ | ++ | + | + |
| Monitor patient and do long-term follow-up | ++ | ++ | ++ | ++ |
| ++ = more important; + = less important; NA = not applicable. | ||||
Safety Assurance Begins with Adequate Donor Screening
Whether human stem cells are of embryonic, fetal, or adult origin, donor sources must be carefully screened. Routine testing should be done to guard against the inadvertent transmission of infectious diseases. Additionally, pedigree assessment and molecular genetic testing appear to be warranted. This is arguably the case when human stem cells intended for transplantation are derived from an allogeneic donor and especially if the cells are obtained from a master cell bank that has been established using human embryonic stem or human embryonic germ cells.
Using Controlled, Standardized Practices and Procedures for Establishing Cultured Human Stem Cell Lines Enhances Safety
To ensure the integrity, uniformity, and reliability of human stem cell preparations intended for clinical use, it is essential to demonstrate that rigorously controlled, standardized practices and procedures are being followed in establishing and maintaining human stem cell lines in culture. Failure to standardize procedures for maintaining and expanding cells in culture could result in unintended alterations in the intrinsic properties of the cells. The initial seeding density of the cells, the frequency with which the culture medium is replenished, and the density cells are permitted to achieve before subdividing will all affect the characteristics of human stem cells maintained in culture. Altering the concentrations of supplemental growth factors and chemical substances, even switching from one supplier to another, may lead to changes in cell growth rate, expression of defining cell markers, and differentiation potential.
Alternatives to Culturing on a Feeder Layer of Animal Cells Improve Safety
An issue unique to the culturing of human embryonic stem and embryonic germ cells involves the use of mouse embryonic fibroblast feeder cells to keep the embryonic cells in a proliferating, undifferentiated condition. Transplanting into humans stem cell preparations derived from founder cells that have been in direct, intimate contact with nonhuman animal cells constitutes xenotransplantation. The principal concern of xenotransplantation is the unintended transfer of animal viruses into humans. Human embryonic stem cells maintained in the absence of direct culture on a mouse feeder cell layer–the newly developed culture conditions–are comparable to human embryonic stem cells co-cultured with mouse feeder cells.
Detailed Characterization of Human Stem Cell Populations Reinforces the Safety Net
Identifying the cells that make up an human stem cell population intended for clinical study requires identifying cells exhibiting the desired phenotype within the preparation, as well as those that do not. Parameters that will prove useful in establishing identity include 1) cell morphology, 2) expression of unique cell-surface antigens, 3) characterization of biochemical markers such as a tissue-specific enzymatic activity (e.g., enzymes that produce neurotransmitters for nerve cells), and 4) expression of genes that are unique to a particular cell type. Further, analysis of the nuclear chromosomal karyotype may be used to assess genetic stability of established human embryonic stem and embryonic germ cell lines maintained in culture for extended periods of time. DNA microarray analysis (simultaneous screening for many genes) and proteomics (protein profiling) technologies will significantly enhance stem cell characterization.
Proof of Concept, Toxicity Testing, and Evaluation of Proliferative Potential in Animal Models Are Important to the Assessment of Human Stem Cell Safety
A critical element of the safety net is the transplantation of human stem cells into animals to demonstrate that the therapy does what it is supposed to do (“proof of concept”) and to assess toxicity. Human stem cells must be transplanted into animal models of human disease. In addition to efficacy, evidence for anatomic and functional integration of transplanted human stem cells should be assessed. The migration of transplanted human stem cells to a nontarget site and subsequent differentiation into a tissue type that is inappropriate for that anatomic location could be problematic.
From the perspective of toxicology, the proliferative potential of undifferentiated human embryonic and embryonic germ cells evokes the greatest level of concern. A characteristic of human embryonic stem cells is their capacity to generate teratomas when transplanted into immunologically incompetent strains of mice. Undifferentiated embryonic stem cells are not considered as suitable for transplantation due to the risk of unregulated growth. The question that remains is, at what point during differentiation does this risk become insignificant, if ever Identifying the stage at which the risk for tumor formation is minimized will depend on whether the process of stem cell differentiation occurs only in a forward direction or is reversible.
