Given that more than 100 MSC-related clinical research protocols are listed in www.clinicaltrials.gov
and, in all probability, more than 2,000 patients have been treated with MSCs worldwide, it is not surprising that considerable variation exists in the mode and stringency of the manufacture of these cells. Many involve protocols for single arm studies with small numbers of subjects for whom the MSCs were generated in HCT processing laboratories or similar facilities. Other protocols employ current Good Manufacturing Practice (cGMP) standards in which the cells are manufactured under the highest standards of sterility, quality control and documentation. The impact of these differences is unknown, but clinical prudence and regulatory requirements in many countries mandate at the least, that the manufacturing is conducted under a good laboratory practice (GLP) standard. The cGMP standard is at a higher level and although it involves a similar stringency in the implementation of standard operating procedures (SOPs), additional requirements include a formal and independent quality assurance program.
A number of factors in the cell manufacturing process influence the nature and, probably, the function of MSCs. For example, under identical culture conditions, the prevention of cell adhesion alters the immunophenotype of the cells considerably, and possibly their biodistribution129
. Other factors include the oxygen tension, temperature, and composition of the culture medium. As discussed above, more recent protocols eschew FBS in favor of human plasma or human platelet lysate. While many regulatory agencies tolerate the presence of FBS in the culture medium of MSCs for phase I trials, later phase studies tend to require serum-free medium. It will be important to determine immunophenotypic, genotypic, and functional changes in MSCs when culture media are modified.
Although the International Society for Cellular Therapy (ISCT) has established the definition of MSCs10, 130
, release criteria were not dictated, tend to be protocol-dependent, and are determined in conjunction with regulatory agencies. It is especially important, for example, when employing allogeneic MSCs (in contrast to autologous MSCs) to ensure that B or T cell contamination is low or absent in order to eliminate the possibility of GVHD131
. A major challenge in establishing release criteria is the lack of an accepted functional assay. However, given the wide range of potential clinical effects of MSCs—from the treatment of specific tissue injury to immunosuppression for GVHD—any such assay(s) will need to be specific to the particular indication or clinical trial. Another confounding issue is that some MSC products may be distinguished from other MSC products by differences in immunophenotype or function in vitro, in part to employ unique MSC cell types for purposes of intellectual property protection. Because very few comparative studies have been done, it is difficult to assess the importance of these differences on clinical outcomes. There may also be significant differences among MSC products from different tissue sources. The most extensive comparisons have involved adipose-derived MSCs versus MSCs from bone marrow132
. The source of MSCs may influence the ability of the cells to differentiate along, for example, osteogenic, chondrogenic or myogenic lineages. Functional differences appear to exist between MSCs derived from human umbilical cord perivascular cells and those from bone marrow (personal observation of one of us, AK). Cell manufacturing protocols must therefore take into account the variability in the characteristics of the MSCs, including their proliferative and differentiative capacities.
Other factors that may influence the function and safety of MSC preparations include age and sex of the donor and the number of cell doublings necessary to arrive at the final product. To mitigate malignant transformation of human MSCs, meticulous attention must be taken to prevent cell senescence and ensure that preferably fewer than 25–30 cell doublings occur (Darwin J. Prockop, Malcolm Brenner, Willem E. Fibbe, Edwin Horwitz, Katarina LeBlanc, Donald G. Phinney, Paul J. Simmons, Luc Sensebe, and Armand Keating, submitted). Despite these measures, potential genetic instability remains a concern, hence many centers advocate the demonstration of a normal karyotype as part of the release criteria for MSCs.
Two approaches can be taken to make the MSC product available quickly: the use of allogeneic cells that have been cultured, tested, cryopreserved and ready for release and administration after thawing; or the rapid culturing of autologous MSCs by aspirating, under local anesthesia, a large volume of bone marrow (100–150mL), that will require fewer passages to achieve the desired number of cells but in medium supplemented with cytokines, such as fibroblast growth factor alpha. The latter approach could provide autologous MSCs within two weeks. Another strategy is to grow the MSCs more rapidly in bioreactors. The final approach will be dictated by the research protocol and the clinical importance of an autologous versus allogeneic source. There is a perception that trials with autologous cells may receive more rapid approval by regulatory agencies, although this is not certain and the decision might be more appropriately reached by considerations of feasibility and the underlying pathophysiology of the disease targeted.
Future Clinical Trials
Although perhaps as many as several thousand patients have been treated with MSC to date, no infusional toxicity or immediate adverse out comes have been reported, suggesting MSC infusion to be safe. However, rare adverse event and late complications of the treatment can only be detected in large cohorts of patients with long follow up. The long experience of cooperative groups such as the CIBMTR and the EBMT to collect data on patient treated with HSCT and evaluate long term patient outcome provide an excellent infrastructure that can be employed to patients treated with novel cellular therapies, such as MSC, also to avoid publication bias. In fact, a registry specific for novel cellular therapies has already been established in the EBMT and efforts to establish a similar registry are ongoing in the CIBMTR133
Efficacy of MSC treatment, however, remains to be established for most indications. Pilot trials aim at establishing safety, but comparative studies are needed to show a beneficial effect of MSC. Reproducibility of patient responses in several centers and by MSC produced in different labs is best shown in collaborative multicenter studies adhering to similar protocols for generation of MSC. Unbiased comparisons of the clinical effect of MSC derived from donors of various degrees of HLA-matching, generated in different growth media and after various periods of in vitro culturing will further be essential to optimize MSC treatment.
Protocol design for tissue regeneration with MSCs was based on the assumption that the cells differentiated into the cells of the injured organ (e.g., MSCs introduced after acute myocardial infarction differentiated into cardiomyocytes). This notion is now considered unlikely134
and has been replaced by myriad mechanisms to explain the objective improvements that have been documented in some cases135
. The presence of the MSCs at sites of injury in pre-clinical animal models generally has been transient (days rather than weeks) and paracrine mechanisms have been invoked29
. Such findings raise a number of challenging issues in the design of MSC trials in the future. First, it will be important to determine whether threshold effects occur and if an MSC dose response and/or infusion duration actually exists for a particular indication or end-point. Secondly, it will be useful to correlate biodistribution of MSCs with therapeutic response. Finally, real time imaging and tracking studies of MSCs in patients will provide an enormous impetus in moving the field forward. The most feasible imaging agent to enter human clinical trials is likely to be a form of superparamagnetic iron. It is hoped that a suitable iron formulation will be available in the near future for tracking the marked cells by magnetic resonance imaging (MRI).
Given the very high costs of conducting early phase cell therapy protocols, including those with MSCs, it is important to optimize the information obtained even from phase I clinical trials to gain a better understanding of the mechanisms by which MSCs mediate immune suppression or tissue regeneration. There is the added issue that it has frequently not been possible to garner relevant data on human MSC-immune interactions from xenogeneic models to inform the design of subsequent trials in patients.