ReviewRisk of tumorigenicity in mesenchymal stromal cell–based therapies—Bridging scientific observations and regulatory viewpoints
Introduction
Mesenchymal stem cells/mesenchymal stromal cells (MSCs) have been studied and used for more than a decade now to treat various diseases (1). Human MSCs are most commonly isolated from the mononuclear fraction of the bone marrow (BM) or from adipose tissue. Other sources for MSCs are also used, for example, some cell preparations are isolated from placenta, amniotic fluid or periosteum 2, 3. The isolated MSCs show phenotypic heterogeneity, depending on the origin of the cells and the isolation/manufacturing techniques. According to current thinking, the cell surface proteins expressed by MSCs include cluster of differentiation (CD)105, CD73, CD44, CD90, CD71, Stro-1, CD106 CD166 and CD29. MSCs do not express hematopoietic markers such as CD45, CD14 or CD11. They also do not express co-stimulatory molecules CD80, CD86 or CD40 or adhesion molecules CD31, CD18 or CD56. In 2006, the research groups within the International Society for Stem Cells agreed on the minimal cell surface markers, considered necessary to distinguish mesenchymal stromal cells from other cell types (4).
Several researchers have described a role of MSCs in tumor formation 5, 6. Furthermore, some groups have reported that human MSCs may develop genetic instabilities and undergo a transformation process after long-term culture, as suggested for adipose-derived MSCs (7), and for BM-derived MSCs (8). In contrast, however, several authors have reported that MSCs cultivated in vitro can be expanded over multiple cell doublings without apparent loss of differentiation potential or chromosomal alterations 9, 10. In addition, long-term cultured MSCs can develop chromosomal abnormalities but without evidence of transformation potential (11). This discrepancy may be explained by reports that the cell cultures used in the studies performed by Rubio et al. (7) and Rosland et al. (8) were contaminated by malignant cells that initially grew slowly in the presence of human MSCs 12, 13.
In October 2011, the Cell Products Working Party (CPWP) arranged an expert meeting to discuss the findings of the research groups working in this area. The expert meeting was intended to obtain information and discuss the opportunities and challenges currently faced when MSCs are used as therapeutic products. The focus was on tumorigenicity as a safety concern frequently expressed by regulatory authorities, although practicalities involved in the development of MSCs as medicinal product were also more broadly discussed.
Section snippets
Manufacturing and quality aspects
Garcia et al. (12) and Torsvik et al. (13) reported that the original observations of tumor formation for isolated MSCs were the result of MSC cultures contaminated with tumor cell lines. The meeting experts therefore emphasized the importance of recommending that cell culture is performed under good manufacturing practice conditions to ensure proper segregation and control of starting and raw materials.
The discussions also highlighted some specific issues around terminology and the need for
Cells with chromosomal aberrations
Observed chromosome aberrations can be broadly divided into two categories: (i) spontaneous abnormalities and (ii) recurrent abnormalities. Spontaneous (nonrecurrent) abnormalities are heterogeneous from one batch to another and from one patient to another. To exclude products containing cells with abnormalities potentially conferring a proliferative advantage, it was suggested that a karyotyping analysis would be sufficient as a release test in the case that it is possible (frozen product). A
Analytical techniques available to assess cytogenetic abnormalities
Two groups of techniques were discussed as possible methods for testing of genetic instability: (i) conventional karyotyping (GTG-banding, G bands by trypsin with the use of Giemsa) and Spectral karyotyping (SKY) technique) and (ii) molecular cytogenetic techniques (fluorescent in situ hybridization [FISH] and comparative genomic hybridization array (CGH array) and single nucleotide polymorphism array (SNP array). Karyotyping should be used to screen cells for chromosomal abnormalities during
Nonclinical aspects
Occurrence of cell abnormalities appears to be mainly related to the manufacturing process as opposed to patient-specific factors (personal communication of the experts). It is therefore important to determine during preclinical development whether the manufacturing process leads to chromosomal abnormalities.
The nonclinical discussion focused on the in vivo tests available for tumorigenicity testing and their reliability and relevance for the intended purposes.
Experiments performed with the use
Clinical aspects
The majority of expert presentations at this workshop, including those from experts with experience with the use of human MSCs in clinical trials, focused on quality and preclinical findings that acknowledge the importance of these steps in the development of medicinal product in general and especially when evaluating risks in clinical application.
During the discussion on clinical aspects, the experts shared their experiences regarding the hypothetical risk for tumor development in patients
Discussion
The expert group reached agreement on several issues discussed. First, occurrence of recurrent cell abnormalities appears to be mainly related to the manufacturing process. In addition, some donor-related recurrent abnormalities have been detected. Thus, the culture conditions should be chosen to avoid a high proliferative rate (e.g., use of excessive amount of growth factors) because this may potentiate chromosomal abnormalities. Furthermore, the number of population doublings should be kept
Acknowledgments
The authors acknowledge the valued contribution by Dr K. LeBlanc to the expert group meeting on mesenchymal stromal cells and tumorigenicity held at European Medicines Agency (EMA) on October 7, 2011, as well as the organizational and scientific support by Lucia D'Apote and Patrick Celis, EMA scientific secretariat for the Committee of Advanced Therapies.
Disclosure of interests: The authors have no commercial, proprietary, or financial interest in the products or companies described in this
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These authors contributed equally to this work.