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Advantages of pooling of human bone marrow-derived mesenchymal stromal cells from different donors versus single-donor MSCs – Scientific Reports

In clinical settings, MSCs are proven to be safe, but the efficiency outcome is mixed for different indications. As a result, there are 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, attributed to the innate biological variability of the donors17,18,19. Traditionally, MSCs are isolated from single donors, culture expanded, and cryopreserved, and after extensive characterization, the cells are used in clinical settings. 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.17,20,21,22. Further, to obtain adequate cell therapy doses, single-donor MSCs either do not suffice in terms of volume or need to be expanded for a prolonged time in culture that contributes to poor proliferation potential and compromise the efficiency of the final product23.

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 outcome19,24. We developed a method for minimizing the heterogeneity and reproducibly producing an MSC product by pooling the MSCs from different donors in GMP conditions. Although previous reports mention pooling of different donors as a way to reduce heterogeneity14,25, there is no comprehensive study that has analysed different pools for characteristics like proliferation, differentiation, phenotypic marker expression, immunomodulatory potential, secretome, senenscence, and tumorigenicity of the pooled MSCs. Our study’s novelty relies on analyzing this entire range of parameters, including potency markers, between the individual and pooled MSCs. Another novelty of our study is the use of different pools comprising different biological replicates, leading to improved statistical strength of this study. Our previous reports also used this pooled MSC to prove their safety and efficiency in different preclinical and clinical settings6,26,27,28.

For this study, we isolated MSCs from nine different healthy, volunteer donors, expanded them, and banked them as MCBs at P1. For pooling, three different donor MCBs were mixed in equal proportion, expanded till P3, and banked at WCBs. The three pools created were cultured further till P5, and all further analyses were done on these cells.

The individual donor-derived MSCs and the pooled MSCs showed typical spindle-shaped morphology. There was no distinction in the morphology as both the individual and pooled cells looked similar. Further, pooling did not compromise the MSC characteristic phenotypic marker expression. Both the individual and pooled MSCs had positive marker expressions > 90%, while < 1% expression was noted for negative markers except for that in one single donor MSC that expressed CD90 at 85%. However, there was a reduced expression in the positive marker as per ISCT criteria2 as the expression should be ≥ 95%, but this minimum criteria is heavily debated, with amendments suggested by many groups29. Nevertheless, as the other ISCT criteria of plastic adherence and trilineage potential were met, of all our individual and pooled cells were indeed MSCs.

For chracterisation of proliferation, we analysed the total cell yield, PDT, and CPD of individual and pooled MSCs. Although unintentional, the donors in our study were all males between the ages of 22 and 29 years. It is worth noting that even with this small difference in the age among the donors, there was a large difference in the cell yield (CV = 90%). Similar differences on proliferation kinetics have been reported by others and was attributed to the age and sex of the donors10,30,31,32. Interestingly, the three pooled MSCs showed comparatively uniform cell yield, rather than the individual MSCs. Regarding the PDT, we observed a marked heterogeneity at P5 among the individual samples, with some individual donor PDT being double the average. Earlier studies also reported increasing doubling time in AD-MSCs with the passage number33. On comparison of average doubling time of individuals to their corresponding pools, the pooled MSCs had lesser doubling time, but the results were not statistically significant at all the passages. The pooling has shown a definite advantage in that even the pool (Pool 1) containing the slowest growing individual had a PDT similar to that of the other two pools. The CPD shows a more uniform trend among the individuals and pools, with the average CPD of individuals matching those of their corresponding pools. As CPD is one of the deciding factors of MSCs therapeutic potential, all our individual and pooled MSCs fell within the range of 20 CPDs34 and these cells were grown to the maximum of five passages as the efficiency was questionable beyond this35. For all these parameters (cell yield, PDT, and CPD), the CV for technical replicates was less than 10%, and we also observed that among these parameters the CPD showed the least intra-assay variability.

One of the other hallmarks of MSCs is their ability to form colonies. This was analysed using the CFU-F assay. Although all individual donor MSCs showed CFU-F capability, we found a large heterogeneity in the number of colonies formed by the individuals depending on their proliferation potential. Siegel et al.30 reported a similar finding in the variation in CFU-F potential among MSCs from different donors. Therefore, the pooling did not compromise the CFU-F ability; moreover, the pooled cells showed higher proliferation potential and number of colonies than the individual ones, though the results were 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 was 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 to regulate the proliferation of phytohemagglutinin-stimulated PBMCs at different ratios ranging from 1:1 to 1:10 of MSC-to-PBMCs. This ratios were well within the range as proposed by the ISCT working proposal36 and also included a larger concentration of MSCs to analyse the maximum effect. As expected, we found a direct correlation between MSC and PBMC concentrations, with the maximum suppression obtained at a 1:1 ratio. However, we observed a large variation in the immunosuppressive abilities among the individuals at the different ratios (Supplementary Table 5). The % CV varied between 30 and 300% among the individual samples, with the highest difference observed at the 1:10 ratio. The intra-assay variability was also the highest in the ratio of 1:10 and least in the highest ratio of 1:1. It is also interesting to note that, some of the individual samples failed to show immunosuppression at the 1:1 and 1:10 ratios. As per the ISCT working proposal recommendations36, these donors are not 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% at 1:2.5 and up to 50% at 1:5 ratios (Supplementary Table 6). Similar to that in individual samples, the intra-assay variability was more prominent in 1:1 and least in the 1:10 ratio. This consistency of immunosuppression occurs, even with low or week immunosuppressive individual donor MSC’s were pooled and cultured together. It is also noteworthy that at the highest ratio, the immunosuppression of the pools was above the average values of the corresponding individual samples, though the results were not significant. Thus, 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 decreases the intra-individual variability was reported earlier15.

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 neoangiogenesis and tissue regeneration37. Based on our previous studies38,39, we selected five angiogenic factors, viz., VEGF, TGFβ, ANG-1, IL-8, and SDF-1, that play major roles in the outcome of angiogenic diseases. The individual and the pooled MSCs secreted these five factors at varying concentrations in the culture. There was a large variation in the amount of these cytokines secreted by the individual donor MSCs. In the individual donor MSCs, on average, the factor IL-8 was secreted in higher concentrations followed by VEGF, with the least being SDF-1, and some donors not secreting SDF-1 at all. However, in case of pools, a more homogenous secretion pattern was observed compared to that in the individuals. All these five factors were secreted by the pooled samples, 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 BMMSCs38,39. 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 do not secrete SDF-1 but still show a secretion level similar to that of the other pools. For all these factors analyzed, the inter-assay variability was less than 10% CV for both the individuals and the pools. It is also of note that among all these assays, secretome analysis showed the least batch to batch variability.

One of the main hurdles in cell therapy is that the cells reach replicative senescence in culture. As the senescent MSCs have decreased differentiation and immunomodulatory potentials that severely hinder the clinical outcomes of using MSCs40, it is important to analyse the percentage of senescent cells in the final cell therapy product. Since our product was the P5 MSCs, we examined the cultures for the presence of any senescent cells by using β-galactosidase staining. The percentage of senescent cells was less than 3% at P5, and even till P7 it was not more than 10% (data not shown) in both individual and pooled MSCs. It was also reported that MSCs in culture start to increase in size starting from P541, but in our study we never noticed any size enlargement at P5. We confirmed that pooling does not have any impact on the morphology of the MSCs by inducing senescence at higher passages.

To understand if the observed effect of pooling was due to the presence of all the three individual donors, we performed STR analysis at the P3 and P5 stages. To our knowledge there are no previous studies that 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, but this ratio skews with some individual donor MSCs dominating in the pool at P5. We did not find any correlation between the dominant donors and the growth kinetics, i.e., it is not the highest yielder or the fast proliferator that is represented in higher percentage or vice versa. But interestingly there was a correlation between the individual donor CFU-F capacities and its final presence in the pooled population. In each of the pools, the dominant donor is the one with highest CFU-F. To have a better understanding, further studies can be planned by choosing individual donors with the same CFU-F and pooling them.

The major risk of using this as a therapy 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 products42. 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 did not 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 finding32.

Despite being a comprehensive study, there are still questions providing scope for future studies. All our analyses are conducted at P5, raising questions about whether the properties of the pooled cells change after this passage as the dominance of single donor become more and when other donors get eliminated from the pool. Also, for our pooling experiment we used only 3 donors as the tracking would be comparatively easier. We also did not explain the effect of pooling if more than 3 donors form a pool. Further, all the donors in our study were males, and the future studies must be done using all female donors and mixed donors to check the potential effects of sex on pooling. The current study can be r strengthened by analysing the global gene expression pattern of the individuals and the corresponding pools.

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