Pathogen-free, plasma-poor platelet lysate and expansion of human mesenchymal stem cells

Mesenchymal stem cells (MSC) are multipotent progenitors cells with self-replicative and differentiation abilities. Actually, MSC differentiate into several tissues of mesoderm lineage, although their potential to give rise to cells of several embryonic origin as neurons, hepatocytes or epithelial cells has also been reported [1‐3]. With their plasticity, self-renewal and immunoregulatory capacities these cells have generated great interest for clinical applications either in cell therapy aimed to induce immune tolerance or in tissue engineering [4‐11].

MSC are typically obtained from bone marrow (BM), but they can be also isolated from a variety of other human sources as adipose tissue, muscle, connective tissue, dental pulp, cord blood, placenta and amniotic fluid [12, 13]. In BM, MSC are present at a very low frequency (0.001-0.01%), thus ex-vivo expansion is an essential step to reach a number of MSC which appears appropriate for clinical applications. To sustain cell growth, most clinical-scale MSC production protocols use cocktails which contain serum of animal origin as supplement. However, these products maintain the potential risk of pathogen transmission and immunological reactions related to the different species origin. Platelet lysate (PL) contains a wide series of growth factors, thanks to which platelets (PLT) are capable to mediate tissue repair at injured sites in physio-pathological conditions; for these reasons it has been proposed as a potential supplement for MSC cultures. Various studies have demonstrated that growth factors derived from PL are able to sustain MSC growth and expansion [14‐18] and, since these observations have been reported, several efforts have been made to standardize its production. In regard to this issue, it has to be taken into account that the concentration of soluble growth factors released by PLT is highly variable among different individuals. Therefore, PLT from multiple donors should be required and included in each preparation to compensate individual variability and to obtain a more standardized and reproducible PL product. Pooling PLT obtained by whole blood-derived buffy-coats is a standardized procedure to produce pooled PLT concentrates for transfusional use. Considering that transmission of pathogens via blood transfusion is still a major threat, plasma or PLT pathogen inactivation (PI) has been introduced for routine blood component production at several sites. The innovative technology of photochemical PI utilizes a synthetic psoralen, as active compound, which specifically interacts with nucleic acids when exposed to UVA light, blocking both DNA or RNA replication. Thus, the technology shows efficacy in inactivating viruses, bacteria, protozoa and eventual residual leucocytes. Starting from these concepts, we adopted a PLT pooling procedure followed by an additional step of photochemical treatment necessary for PI to produce a plasma-poor, pathogen-free PL in a closed sterile system. The pathogen inactivated PL (PI-PL) preparations were employed to sustain the growth and the expansion of MSC from different BM samples. This preparation was named Mesengen™ by a trademark associated with the registration of the international patent application of this product (PCT/IB2012/055062).

The use of animal sera for clinical-scale MSC expansion raises concerns because of the risk to transmit prions, viruses or to induce immunological reactions in recipients following infusion and for these reasons these products are not allowed in good manufacturing practice (GMP) applied to cell therapy. Therefore, culture supplements derived from humans have been investigated in order to replace animal serum in MSC cultures. Human AB blood serum and umbilical cord blood serum have been found to provide a stimulus for MSC growth comparable to animal serum or higher with the preservation of MSC capacity to differentiate in three different mesodermal lineages [21]. Human PL is currently under investigation for its ability to sustain ex vivo MSC growth and data have been reported comparing the efficacy of PL with animal serum in promoting MSC expansion in the setting of cell-therapies [14‐16]. From these evidences appear that PL is a powerful substitute of animal serum and its use may contribute to ameliorate the safety of MSC-based products in terms of immunological side effects and xenogenic pathogen transmission. PL properties are based on the release of growth factors by PLT which play a physiological role in the wound healing process. The content of PDGF-AB, TGF-β1 and bFGF in PL may account for its efficacy in promoting MSC growth since these cells have been shown to express surface receptors for PLT derived factors [14]. Nevertheless, procedures to prepare PL are highly variable and although this human source is rigorously screened for potential infectious threats, the risk of disease transmission by human derived products still requires careful consideration. In this study, a standardized procedure to generate PL by pooling multiple units of BC-derived PLT is described. The protocol includes a step of PI by Intercept technology, which is an approved and routinely applied practice for production of PLT concentrates for transfusional use in European countries [22], and the final resuspension of the PLT product in AS with a minimum residue of plasma (20-30%), as compared to the original products. PI technology by exposure to psoralen compound and UVA light has been found effective in inactivating a broad panel of viruses and bacteria without compromising PLT hemostatic function [23] and, therefore, PI-PL may represent a safer product for ex vivo MSC expansion to generate clinical grade cells for therapeutic strategies. However, a relevant issue is that to investigate whether pathogen reduction step may affect PL in its ability to generate ex vivo MSC. Here, the results of growth, expansion, differentiation and immunosuppressive function of several MSC populations cultivated in medium supplemented with both PL and PI-PL showed comparable features and growth potential. Differences between the various preparations, even though not relevant, may depends on the individual variability in PLT growth factor content. To obtain a more standardized PL preparation, we generated PI-PL by a pooling procedure which combined 12 PLT units, starting from 2 initial pools of 6, being six the maximum number of units which may be pooled for the PI step by the Intercept technology. An additional advantage of PI-PL preparation is the very low amount of residual plasma which may prevent the occurrence of immunological adverse reactions, often related to the presence of this human component. Some authors produced a plasma-free PL in human albumin solution, called PL-HA, for in vitro expansion of MSC [24, 25]. The authors showed that MSC expanded in medium supplemented with PL-HA proliferated more extensively with respect to cultures additioned with FBS or conventional PL. In the same context, Burnouf et al. and Shih et al. [26, 27] developed a virally inactivated PL for cell cultures by a solvent detergent procedure for viral inactivation, followed by oil extraction and adsorption with activated charcoal or soybean oil extraction and centrifugation, respectively. These preparations had been tested in growing cell lines, as MG63 and SIRC, and for selection and expansion of adipose tissue MSC. Neverthless, these studies reported that PDGF-AB and VEGF were completely removed by the adsorption step with charcoal as well as bFGF was partially removed by soybean oil extraction; from this evidence we may argue that these products are not completely suitable for MSC growth due to the well-described action of these growth factors in supporting optimal MSC expansion.

In a recent work Fekete et al. [28] produced PL from pool of 4 PLT units irradiated and quarantined for at least 4 months. The authors released the PLT units for PL production only if the all four donors contributing to a PLT pool had tested again as negative for conventional transfusion-transmitted infectious diseases. This approach to guarantee a safer clinical grade PL requires a longer storage of the starting material before PL preparation and is depending on the donor comeback for control testing. The addition of PI may further contribute to the safety of PL preparation by covering also the potential risk of new emerging infectious agents. Actually, over recent years the threat of transfusion transmitted agents focuses the attention on emerging pathogens which can be present in the blood and may concern public health, as the case of West Nile Virus [29]. Testing donation for evidence of a new pathogen may be adopted when the test become available and is validated for donor screening. Therefore, pathogen reduction could be considered the prompt approach to struggle with emerging infection transmissible via blood. Cell amplification by ex-vivo cultures, in the presence of human or animal blood components, is not a process free from the potential risk of transmission of infectious disease or immunological reactions; hence, the safety of this procedure may be greatly improved by the use of a human plasma-poor standardized product, contained in lots with a known growth factor content and subjected to an effective PI process. In this setting, PI-PL preparations represent a safe plasma-poor, well-standardized platelet lysates, prepared in lots which may be characterized for growth factor content and specific functional activity; these preparations had been improved in their quality grade by adding PI step and, for these reasons, appear as ideal supplements to develop uniform and well standardized protocols for ex vivo expansion of MSC without affecting antigenic and functional characteristics of these cells, as may happen with some serum-free commercial media [30].