Inactivated Platelet Lysate Supports Proliferation and Immunomodulant Characteristics Of Mesenchymal Stromal Cells in GMP Culture Condition

: For their clinical use Mesenchymal Stromal Cells (MSCs), isolated from bone marrow (BM-MSCs) are considered Advanced Therapy Medicinal Products (ATMP) and need to be produced according to Good Manufacturing Practice (GMP). Human platelet lysate (HPL) represents a good GMP-compliant alternative to animal serum and we demonstrated that after pathogen inactivation with Psoralen , it was more efficient and safer to produce MSCs in GMP condition. In this study MSCs cultivated in FBS (FBS-MSC) or inactivated HPL (iHPL-MSC), were compared for their immunomodulant properties. In particular, the effects of MSCs on: 1)proliferation of total Lymphocytes (Ly) and on naïve T Ly subsets induced to differentiate versus Th1 and Th2 Ly; 2) the immunophenotype of different T cell subsets; 3)the cytokine release to verify Th1, Th2 and Th17 polarization were analyzed by using in vitro co-culture system. We observed that iHPL-MSCs showed the same immunomodulant properties observed in the FBS-MSC co-cultures. Although, a more efficient effect on the increase of naïve T cells and in the Th1 cytokine release related to iHPL was observed. This study confirms that iHPL, used as medium supplement, may be considered a good alternative to FBS for a GMP-compliant MSC expansion to preserve also their immunomodulant proprieties.

factors in their secretome, have been considered more than their multilineage differentiation potential for their clinical use in clinical trials in severe disease: autoimmune, chronic inflammatory and degenerative conditions [1,2].
For their clinical use MSCs isolated from bone marrow (BM-MSCs) are considered Advanced Therapy Medicinal Products (ATMP) and need to be produced according to Good Manufacturing Practice (GMP) [3,4]. Since the use of xenogeneic protein free GMP-compliant growth media is a prerequisite for clinical MSC isolation and expansion, human platelet lysate (HPL) has been efficiently substituted to fetal bovine serum (FBS) into MSC clinical manufacturing. For these reasons, it represents a good GMP-compliant alternative to animal serum for MSC clinical production confirming recent data reported in the literature [5][6][7]. As the risk of transmission of infective agents not routinely tested, or for which no tests are available remains, HPL quality and safety had to be greatly improved. We demonstrated that pathogen inactivation with Psoralen was efficient to isolate and expand safer MSCs in GMP conditions [8]. Pathogen Inactivation (PI) technology was used for the first time in 1991 [9] to treat fresh-frozen plasma and then also for platelet and red cells (RBCs) [10]. This technology efficiently remove a wide range of pathogens, with no toxicity or effect on product potency and might also prevent the transmission of unknown pathogens [11][12][13][14][15]. For this reason to make a safer preparation of HPL for GMP production we used pathogen inactivation by psoralen to make HPL safer for the production of MSCs in GMP condition [8]. We also demonstrated that HPL subject to pathogen inactivation (inactivated HPL: iHPL) was more advantageous in terms of cellular growth and stemness in MSCs isolated from bone marrow (BM-MSCs). On the base of our findings about iHPL and of literature data on a possible decrease of immunomodulant properties of MSCs cultured in HPL [16] we studied some iHPL effects on immunomodulatory properties of MSCs.
In this study BM-MSCs were isolated and expanded simultaneously in iHPL ( iHPL-MSCs) in GMP compliant conditions and in FBS (FBS-MSCs) in standard condition usually reported in the literature in immunomodulant studies [17]. In particular, we focused on the effects of FBS-MSCs and iHPL-MSCs on T lymphocytes (Ly) as investigated also in a previous paper [18]. MSCs do not express MHC class II and costimulatory molecules, such as CD40, CD80 or CD86, and different studies show that MSCs are able to inhibit or limit inflammatory responses and mitigate anti-inflammatory pathway inhibiting directly or indirectly disease-associated Th1, Th2, and Th17 cells as well as cytotoxic T lymphocytes [17][18][19][20]. The effects of MSCs cultivated with the two different supplements were analyzed by using in vitro co-culture system with Peripheral Blood Mononuclear Cells (PBMC) stimulated with Phytohemagglutinin (PHA-PBMC). In particular, the following points were analyzed: • the effect on proliferation of total Ly; • the effect on proliferation of naïve T Ly subsets induced to differentiate versus Th1 and Th2 Ly; • the immunophenotype of different T cell subsets (naïve, memory, effector, Th1 and Th2 lymphocytes);

Isolation and expansion, analysis and characterization of human MSCs
Human BM samples were obtained from healthy donors from the discarded collection bag, after filtration of BM collection performed for a familiar allogeneic hematopoietic stem cell transplantation. Anna Hospital, from a platelet pool of healthy donors and subjected to pathogen inactivation by psoralen as described in [21].
The culture was maintained at 37°C with a 5% CO 2 atmosphere. After 5-7 days, the non-adherent cells were removed, and the adherent cells were re-fed every 3-4 days. In order to expand the isolated cells, the adherent semi-confluent monolayer was detached with trypsin/EDTA 1X (Sigma-Aldrich®) for 5 minutes at 37°C and expanded for several passages until they no longer reached confluence.
Only the cells which were compliant to the International Cellular Society MSC criteria [22], and which were not senescent, were frozen in FBS with 10% dimethyl sulfoxide (DMSO, Euroclone, Pero, Mi, Italy) or in Physiological solution containing 5% of human albumin and 10% DMSO. The cells were then thawed at the moment of the experiments in this study.
BM-MSCs used for this study, were analyzed for viability, immunophenotype, differential and proliferative potential to verify that the freezing had not altered the MSCs' characteristics as described in details in a previous work [18]. The BM-MSCs isolated and expanded in FBS or in iHPL were denominated, as described above, respectively FBS-MSCs and iHPL-MSCs.

Preparation of human Peripheral Blood Mononuclear Cells (PBMC)
PBMC were separated from buffy coats by centrifugation on a Ficoll Hystopaque (Sigma-Aldrich®) density gradient. The buffy coats were obtained from the Blood Component Production and and Treponema pallidum) in accordance with Italian laws and European guidelines.

Co-culture MSCs/T cells
All co-culture experiments were performed following the same experimental design previously described [18]. Briefly, BM-MSCs were plated in 6, 24 or 96 well plates or inT-flasks (25cm 2 ) containing total PBMC from an unrelated donor (the MSCs/T cell ratio was 1:10). To trigger T lymphocytes, PBMC were stimulated with Phytohemagglutinin (PHA) (2.5 μg/mL). We also isolated T naïve cells through magnetic separation with CD45RA microbead and used a cocktail of antibodies and cytokine to trigger lymphocytes in Th1 and Th2 subsets as described in [18].

Cytofluorimentric analysis
PBMC characterization was described in details in a previous work [18]. The percentage of positive cells, calculated using the unstained cells as a negative control, was used to calculate the absolute number on the basis of the cell number counted after 5 days of co-culture.

Statistical analysis
All the data obtained in this work were analyzed by Graph PAD Prism (version 8) statistical software. All statistical tests were considered significant for a P<0.05, highly significant for p<0.001 and very highly significant for p<0.0001.

MSC characteristics
Thawed FBS-MSCs and iHPL-MSCs, grew and reached confluence within a few days. Prior to use, the cells were analysed for immunophenotype and multipotent characteristics as demonstrated in [21]. MSCs, independently from the culture condition were negative for CD45, CD34 and CD14 and HLA-DR and were positive (over 95%) for CD90 (a membrane glycoprotein, also called Thy-1), CD105 (endoglin) and CD73. CD146 (cell surface glycoprotein MUC18) was also tested and was positive in all the samples. No statistical differences were observed between the two groups in terms of both positive cell percentages and fluorescence means of the positive markers as shown in the

MSCs/T cells interaction and Proliferative assay
The 3H-thymidine incorporation data of each experiment was expressed as a mean of counts per However different modulations in proliferative activity in Th1 and Th2 induced PBMC were observed, without significant differences between FBS-MSCs and iHPL-MSCs.

T cell subsets determination
The multiparameter flow cytometric analysis allowed the identification of the following T subsets, based on the antibody combination used:  showed a significant increase of IL-6 respectively in the co-culture with FBS-MSCs and iHPL-MSCs (p=0.0402 and p=0.0469), but no statistically difference were observed in IL-17 levels. Also here, no differences were observed between FBS and iHPL-MSCs.

Discussion
Regenerative medicine is of growing interest in biomedical research and in this context, MSCs are a promising tool for cell therapies for their multipotent, bystander and immunomodulant proprieties.
For these reasons, MSCs are used for a very wide range of therapeutic applications, the majority of which are in Phase I, Phase II, or a mixture of PhaseI/II studies. Some phase III and IV are also in progress (www.clinicaltrial.gov). A variety of protocols are described for the GMP MSC production, some of these also using selected FBS, other xeno-free components or HPL or plasma. Since MSCs are considered ATMP, qualified protocols with standard and precise characteristics, large-scale quality, and relatively low-cost production need to be developed using xeno-free media for clinical-grade expansion [25,25,26]. In the last years we have been setting up the methods to isolate and expand MSCs for clinical use and we, also, demonstrated that iHPL, prepared in house from a big pool of donor platelets underwent to pathogen inactivation by psoralen, was more efficient and safer than FBS to isolate BM-MSCs in GMP condition [21,27]. As emerged from the analyses of the pluripotency markers, such as Oct-3/4 and NANOG, these proliferative and differentiative properties of the iHPL-MSCs might be linked to more immature stemness in comparison with FBS-MSCs. These observations indicated that iHPL-MSCs contain a subpopulation of multipotent stem cells, which might be the precursors of MSCs, with a more primitive phenotype than those of FBS-MSCs [8].
In this study, we tested if MSCs isolated and expanded in iHPL preserve their immunomodulant proprieties analysing their effects on T lymphocytes in comparison with MSCs isolated and expanded in FBS whose immunomodulating properties have already been described in the literature [17,18,28].
The ability to modulate the alloreactive immune response has been documented for MSCs derived from human BM; concurrently comparative studies between FBS-MSCs and HPL-MSCs were already performed and indicated that HPL, used as supplement for MSC media, supports immune modulation at least to the same extent than FBS in addition to its role during MSC isolation and expansion [29][30][31]. No comparative studies of immunomodulation were performed between FBS-MSCs and MSCs cultured in iHPL, which is even safer and more GMP compliant than HPL for MSC expansion [21]. However, from these studies, conclusions cannot be drawn on the separate roles of the different cytokines in either mediating inhibition directly or inducible inhibition because of the complex interaction of many factors.
On the base of our results we observed that iHPL preserve all the analysed immunomodulant proprieties of MSCs as well as FBS and for some effects it is also more efficient. For this reason, they may be more effective than FBS-MSCs in the control of immune-mediated pathologies, particularly GVHD. On the other hand HPL-MSCs appeared particularly useful in regenerative medicine [16]. inactivating the replication of viruses, bacteria and leukocytes in PLT concentrates [11,38]. The fact that we and other [11] found differences in immunomodulant properties of FBS-MSCs and HPL/iHPL-MSCs that, in our case, are not significant, offer an interesting clue regarding possible functional differences in MSC output and clinical applications [21].
In Conclusion, for each experiment a contingency table is given (Table 1-Summary of results) where the MSC proprieties and their effects on T-cells are considered as strong, higher or moderate and it can thus be stated that: • iHPL show a greater proliferative, differentiative and stemness potential in MSCs than FBS [8] • Both FBS-MSCs and iHPL-MSCs showed a potent immunomodulant effect on T-cells without strong significant differences between them.
• This study confirm that iHPL, used as medium supplement, may be considered a more efficient additive alternative to FBS for a GMP-compliant MSC expansion.  Table 1 summarized the results obtained in all experiments of this study. The symbols "+++", "++" and "+" or "-/+" indicate the expression or effect grade as very strong, high and moderate, statistically