MSCs are being increasingly used in clinical trials for treating different disease indications ranging from vascular disorders, autoimmune, cardiac, nervous, orthopaedic disorders etc [7]. Though MSCs can be obtained from various sources, the preferred ones and the mostly commonly used ones in the clinical trials are from bone marrow. Irrespective of the source of origin, all the MSCs exerts clinical relevant properties like multilineage differentiation potential, secretion of various trophic factors with angiogenic, immune regulation, tissue remodeling and other abilities [8].
In the clinical settings, MSCs are proven to be safe but the efficiency outcome is mixed for different indications. As a result, there are only very few MSC based cell therapy products available commercially. One of the main reasons that contributes to this ambiguity is the heterogeneity of the MSCs that is attributed by the innate biological variability of the donors [17, 18]. Traditionally, MSCs are isolated from single donors, culture expanded, cryopreserved and after extensive characterization are used in clinical set up. There are several disadvantages of single donor derived MSCs including the secretome profile not meeting the specifications, compromised immune regulatory properties, absence of differentiation potential to a particular lineage, batch to batch variation in specifications including potency assay etc [17–20]. Further, to obtain adequate cell therapy doses, single donor MSCs either won’t suffice or needs to be expanded for prolonged time in culture that contributes to poor proliferation potential and compromise efficiency of the final product [21].
One of the major hindering factors in cell therapy manufacturing is the heterogeneity of the product from different donors that leads to lack of reproducibility and unpredictable clinical outcome [22]. We developed a method for minimizing the heterogeneity and reproducibly producing the MSC product by pooling the MSCs from different donors in the GMP conditions. Though there are previous reports on pooling of different donors as a way to reduce heterogeneity is proposed [14, 23], there is no comprehensive study that analysed different pools with the range of characteristics like proliferation, differentiation, phenotypic marker expression, immunomodulatory potential, secretome, senenscence and tumorigenicity characters of the pooled MSCs. Our previous reports also used this pooled MSCs to prove their safety and efficiency in different preclinical and clinical settings [6, 24–26].
For this study, we isolated mono nuclear cells (MNCs) from nine different healthy, volunteer donors, expanded till P1 and cryopreserved as Master cell bank (MCBs). The MCBs were analysed for the basic characters of MSCs including the morphology, trilineage differentiation potential and phenotypic marker expression. All the MCBs met these minimum criteria and are used for pooling experiment. At the MCB level, MSCs from three single donors were mixed in equal proportions, culture expanded till P3 and cryopreserved as working cell banks (WCBs). We created three pools from nine different individual donor MSCs that are further expanded till P5 and cryopreserved as investigational medicinal product (IMPs). The IMPs were extensively characterized for their proliferation kinetics, CFU-F potential, immunosuppression abilities, secretome profile, senescence and tumorigenic property along with all the basic characteristics.
The individual donor derived MSCs and the pooled MSCs showed typical spindle shaped morphology. There is no distinction in the morphology as both the individual and pooled cells looks alike. Also pooling doesn’t compromise the MSCs characteristic phenotypic marker expression. Both the individual and pooled MSCs expressed > 90% for all the positive markers and < 1% for negative markers except for one single donor MSCs that express CD 90 as 85%. There is shortfall in the positive marker expression as per ISCT criteria [2], where the expression should be ≥ 95%, but this minimum criteria is heavily debated and amendment is suggested by many groups [27]. But interestingly, meeting other ISCT criteria of plastic adherence and trilineage potential, we are sure that of all our individual and pooled cells are MSCs.
For chracterisation of proliferation, we analysed total cell yield, PDT and CPD of individual and pooled MSCs. The individual donor MSCs vary widely in terms of total cell yield. Similar differences on proliferation kinetics has been reported by others and was attributed by age and sex of the donors [10, 28, 29]. Interestingly, the three pooled MSCs showed comparatively uniform cell yield than that of the individuals. The PDT showed an increasing trend with the passage numbers and we observed a huge heterogeneity among the individuals with some individual donor PDT at P5 is double than the average. On comparison of average doubling time of individuals to their corresponding pools, the pooled MSCs have lesser doubling time but the results are not statistically significant. The pooling has shown a definite advantage that even the pool (Pool 1) containing the slowest growing individual has the PDT similar to the other 2 pools. The CPD shows a more uniform trend among the individuals and pools, with the average CPD of individuals matching to their corresponding pools. As CPD is one of the deciding factors of MSCs therapeutic potential, all our individual and pooled MSCs are within the range of 20 CPDs [30] and these cells were grown to the maximum of five passages as the efficiency was questioned beyond this [31].
One of the other hallmark of MSCs is their ability to form colonies and are analysed by Colony forming unit- Fibroblast (CFU-F) assay. Though all the individuals’ donor MSCs showed CFU-F capability, we found a huge heterogeneity in the number of colonies formed by the individuals depending on their proliferation potential. Siegel et al reported a similar finding in the variation in CFU-F potential among the MSCs from different donors [28]. The pooling doesn’t compromised the CFU-F ability and in fact the pooled cells showed higher proliferation potential and number of colonies than the individuals though the results are not statistically significant.
Next, we evaluated the immunosuppressive capabilities of individual and pooled MSCs as this property plays a vital role in determining the efficacy of MSCs in treatments related to inflammatory and immune diseases. In the in vitro settings, immunosuppression property is measured by the ability of the MSCs to suppress the proliferation of mitogen or antigen stimulated T lymphocytes or NK cells or B cells. In our study we analysed the potential of individual and pooled MSCs in regulating the proliferation of phytohemagglutinin stimulated PBMCs (Di Nicola) at different ratios ranging from 1:1 to 1:10 of MSC to PBMCs. This ratios are well within the range as proposed by the ISCT working proposal [32] and also includes a larger concentration of MSCs to analyse the maximum effect. As expected we found a direct correlation between the MSCs and the PBMCs concentrations, with the maximum suppression obtained at 1:1 ratio. However, we observed a huge variation in the immunosuppressive abilities among the individuals in the different ratios. The % CV varies between 30 to 300% among the individuals for these ratios, with some of the individuals failed to show immunosuppression at the 1:1 and 1:10 ratio. As per the ISCT working proposal recommendations [32], these donors won’t be eligible for the treatment of immune related disorders. Interestingly, pooling of MSCs from three donors showed higher and more consistent immunosuppression with % CV of 13 to 50%. This consistency of immunosuppression occurs, even with low or week immunosuppressive effects of individual donor MSC’s pooled and cultured together. It is also noteworthy that at the highest ratio, the immunosuppression of the pools are above the average values of the corresponding individuals though the results are not significant. So pooling eventually reduced the heterogeneity among the individuals and due to some synergy that occurred by mixing the individuals, the pools showed consistent and better immunosuppressive profiles and could reduce the product variability. A similar result on pooling that decrease the intra-individual variability was reported earlier [15].
To further understand, if pooling has any distinctive benefit over the secretion of different cytokines, we analysed the set of angiogenic factors in the secretome of pools and their corresponding individuals. It is well known that MSCs secrete various angiogenic factors like VEGF, TGFβ, ANG-1, HGF, SDF-1 etc. and as a complex they are involved in neo angiogenesis and tissue regeneration [33]. From our previous study [34, 35], we selected five angiogenic factors viz., VEGF, TGFβ, ANG-1, IL-8 and SDF-1 that plays a major role in the outcome of angiogenic diseases. The individuals and the pools secreted these five factors at varying concentrations in the culture. There is a huge variation in the amount of these cytokines secreted by the individual donor MSCs. In the individual donor MSCs, on an average, the factor IL-8 was secreted in higher concentrations followed by VEGF and the least being SDF-1 with some donors doesn’t secrete SDF-1 at all. However in case of pools a more homogenous secretion pattern was observed compared to the individuals. All these five factors were secreted by the pools with VEGF being predominantly secreted and SDF-1 the least. This result is of importance when using the MSCs for treating vascular disorders since in our earlier studies we proved VEGF as a single important factor that determine the angiogenic potential of BMMSCs [34, 35]. Another advantage of pooling is that the pools have consistent secretion of growth factors as in case of pool 3 which contains two out of three donors that doesn’t secrete SDF-1 but still showed the secretion level similar to other pools.
One of the main hurdles in cell therapy is that the cells reaches replicative senescence in culture. As the senescent MSCs have decreased differentiation and immunomodulatory potentials that severely hinders the clinical outcomes of MSCs [36], it is important to analyse the percentage of senescent cells in the final cell therapy product. Since our product is the P5 MSCs, we examined the cultures for the presence of any senescent cells by using β-galactosidase staining. The percentage of senescent cells were less than 3% at P5 and even till P7 it was not more than 10% (data not shown) in both individual and pool MSCs. It was also reported that MSCs in culture start to increase in size starting from P5 [37], but in our study we never noticed any size enlargement at P5. We confirmed that pooling doesn’t have any impact on the morphology of the MSCs by inducing senescence at higher passages.
To understand if the observed effect of pooling is contributed by the presence of all the three individual donors, we did a STR analysis at the P3 and P5 stage. To our knowledge there are no previous studies that have been done to confirm the presence of individual donors in the pooled population. We found that all the three donors were present in the final pooled product but with different proportions. At P3, the representation of the individuals were uniform and this ratio skews with some individual donor MSCs dominates in the pool at P5. We don’t find any correlation between the dominant donors to the growth kinetics i.e., it is not the highest yielder or the fast proliferator that is represented in higher percentage or vice versa.
The major risk which the cell therapist are worried is the safety of stem cells with respect to their tumorigenic potential or their ability to form tumors after injection. The in vitro colony formation assay or soft agar assay is most commonly employed method to assess the tumorigenic potential of cell therapy products [38]. This method relies on the ability of transformed cells to grow in an anchorage independent manner and form colonies in the soft agar. We noticed that pooling doesn’t cause any transformation in the MSCs as observed by the absence of colonies in both the individuals and pooled MSCs. A previous report from our lab also confirmed this findings [39].