Growth Factor Content in Human Sera Affects the Isolation of Mesangiogenic Progenitor Cells (MPCs) from Human Bone Marrow

Mesangiogenic Progenitor Cells (MPCs) are human bone marrow-derived multipotent cells, isolated in vitro under selective culture conditions and shown to retain both mesengenic and angiogenic potential. MPCs also co-isolated with multipotent stromal cells (MSCs) when bone marrow primary cultures were set up for clinical applications, using human serum (HS) in place of fetal bovine serum (FBS). MPC culture purity (over 95%) is strictly dependent on HS supplementation with significant batch-to-batch variability. In the present paper we screened different sources of commercially available pooled human AB type serum (PhABS) for their ability to promote MPC production under selective culture conditions. As the majority of “contaminating” cells in MPC cultures were represented by MSC-like cells, we hypothesized a role by differentiating agents present in the sera. Therefore, we tested a number of growth factors (hGF) and found that higher concentrations of FGF-2, EGF, PDGF-AB, and VEGF-A as well as lower concentration of IGF-1 give sub-optimal MPC recovery. Gene expression analysis of hGF receptors was also carried out both in MSCs and MPCs, suggesting that FGF-2, EGF, and PDGF-AB could act promoting MSC proliferation, while VEGF-A contribute to MSC-like cell contamination, triggering MPC differentiation. Here we demonstrated that managing hGF contents, together with applying specific receptors inhibitors (Erlotinib-HCl and Nintedanib), could significantly mitigate the batch-to-batch variability related to serum supplementation. These data represent a fundamental milestone in view of manufacturing MPC-based medicinal products.


INTRODUCTION
Mesenchymal stromal cells (MSCs) are multipotent adult progenitor cells isolated in vitro from a plethora of different tissues (reviewed in Murray et al., 2014) as their perivascular origin has been demonstrated and widely accepted (da Silva Meirelles et al., 2006;Crisan et al., 2008). MSC role as skeletal tissue progenitors has been investigated in a variety of studies in animal models, and cell-based therapy approaches demonstrated their beneficial effects in the regeneration of damaged skeletal tissues including bone (Watson et al., 2014), cartilage (Richardson et al., 2015), tendons (Pacini et al., 2007), and meniscus (Yu et al., 2015). For those reasons, most of the tissue engineering strategies and regenerative medicine approaches, developed to de novo skeletal tissue formation, involved MSCs (Yousefi et al., 2016). Nonetheless, it has been reported that an effective tissue remodeling response is in tight correlation with the formation of vascularized tissue (Rao and Stegemann, 2013;Hutton and Grayson, 2014). Commonly, the vascularization of the newly formed tissues in vivo has been expected driven in large part by the host response to the implant, and trigged by hypoxic condition surrounding the grafted cells (Chamberlain et al., 2015). Nonetheless, this effect could be hypothesized in acute injuries with moderate tissue loss, but in case of large implants or poor blood supply, as in non-union fracture or pseudoarthrosis (Hak et al., 2014), the vascularization of constructs would be compromised. Modular constructs obtained applying both osteoprogenitor cells, as MSCs, and endothelial cells (ECs) or their precursors (EPCs), have been investigated as an option in order to sustain vascularization of the implants, independently from the host response (Butler and Sefton, 2012). However, MSCs have been estimated to represent around 0.001-0.01% of human bone marrow mononuclear cells (hBM-MNCs) (Subbanna, 2007;Lechanteur et al., 2016), and similarly ECs and EPCs represent rare populations in their tissues of origin (Pelosi et al., 2014), thus pre-clinical and clinical applications of these cells, alone or in combination, require extensive ex vivo cell expansion to obtain therapeutic cell doses. Most widely accepted MSC isolation and expansion protocols, from bone marrow, are based on basal media supplemented with fetal bovine serum (FBS) (Haynesworth et al., 1992;Prockop, 1997). Similarly, consistent fractions of FBS should be applied also in the EC and EPC isolation (Kirton and Xu, 2010). However, all supplements of animal origin expose patients to a number of risks and represent a major obstacle to comply with good manufacturing practice (GMP) guidelines (Herberts et al., 2011). The urgent need for FBS alternatives led to consider human serum (HS) as the straightforward substitute in clinical-grade MSC production (Altaie et al., 2016).
After more than 6 years spent on MPC characterization and optimization of isolating procedures, we believe that these cells could facing a new era and possibly being involved in skeletal tissue engineering and regeneration. As MPCs demonstrated in vitro retaining both mesengenic and angiogenic potential, it reasonable hypothesizes the development of MPC-based implants in alternative to the modular constructs. The need of two or more different expanded cell populations giving a mesangiogenic potential to the engineered tissues could be overtaken applying cell populations like MPCs (Pacini and Petrini, 2014). However, the definition of a highly reproducible clinical-grade manufacturing process represents a first step in dealing with a possible application of MPCs in cell-based therapies. From the first report of mesangiogenic cells in human bone marrow cultures applying AS, a consistent variability in the MPC harvesting was reported. Only two out of every three samples cultured showed detectable MPC population coisolated with MSCs, with percentages that could vary 10-fold (Petrini et al., 2009). As a consequence, we believe that the supplementation with human sera could induce considerable culture variability, during cell manufacturing process, depending on donor sex and age as well as diseases and pharmacological treatments at the time of serum collection, especially in the autologous context. Consistent production of almost pure MPC cultures has been achieved applying commercially available PhABS. Under these selective culture conditions it was possible to obtain MPCs from all the samples, with recoveries showing very low coefficient of variation (≈10%). Conversely, culturing the same samples in FBS supplemented media led to cell products constituted by MSCs only . Nonetheless, we recently reported that isolation of MPCs at high grade of purity (over 95%) would be possible only after accurate screening of commercially available PhABS (Montali et al., 2016). In fact, a small number of tested batches produced cell products with consistent percentages of MSC-like cell population, compromising the purity of the MPC preparations. In order to define specific sera properties, allowing the optimal MPC manufacturing process, would be helpful the comparisons between PhABS with good performances and PhABS promoting MSC "contamination." Human serum contains a large and untreatable number of biological active molecules possible affecting the MPC isolation and inducing MSC proliferation (Rodrigues et al., 2010). However, recent efforts in the establishment of serum-free media for MSC in vitro expansion, demonstrated that human platelet lysate (hPL) could efficiently replace serum in culture (reviewed in Burnouf et al., 2016). This important finding narrows the choice of essential components to platelet granules content (Fekete et al., 2014). Platelets contain a lot of potent biological active molecules (Semple et al., 2011), mainly stored in the αgranules (Golebiewska and Poole, 2015), in addition to important factors involved in coagulation. These molecules include different chemokines (Semple et al., 2011) and human growth factors (hGFs), such as epidermal growth factor (EGF), basic fibroblast growth factor (FGF-2), platelet derived growth factor isoforms (PDGF-AA, -AB, and-BB), insulin-like growth factor-1 (IGF-1), vascular endothelial growth factor (VEGF), and some others (Nurden et al., 2008).
In order to define reproducible MPC manufacturing process, in the present paper we moved a first step focusing on the effect of the platelet-derived hGFs, mentioned above, on the MPC production. EGF, FGF-2, PDGFs, IGF-1, and VEGF have been reported regulating proliferation and survival of BM-derived MSCs. Thus, we hypothesize that these five hGFs or some of them could promote expansion of MSCs, during primary bone marrow cultures intended for MPC production. Here we compare six different commercial PhABS, selected on the basis of their different performance, attempting to correlate the percentage of "contaminant" MSC-like cells with the serum concentration of those hGFs.

Screening of Human Sera for MPC Isolation
The study has been performed according to the declaration of Helsinki and the sample collection protocol was approved by the ethical committee of "Azienda Ospedaliero-Universitaria Pisana". Bone marrow aspirates were obtained after written consent from 8 patients (4M/4F, median age 65), undergoing orthopedic surgery for hip replacement. Soon after femoral neck osteotomy, approximately 10 ml of bone marrow were aspirated, using a 20 ml syringe containing 500 U.I. of heparin, and promptly sent to the cell culture facility. Samples were diluted 1:4 and hBM-MNCs collected by density gradient centrifugation using Ficoll-Paque TM PREMIUM (GE Healthcare, Uppsala, Sweden). After two washes in Dulbecco's Modified Phosphate Buffer (D-PBS, LifeTechnologies, Carlsbad, USA-CA) cells were plated at 8 × 10 5 /cm 2 in hydrophobic six-well plates for suspension culture (Greiner Bio-One, Kremsmünster, Austria) and cultured in minimal essential medium supplemented with PhABS as previously described (Montali et al., 2016). Briefly, low-glucose Dulbecco's modified Eagle medium (DMEM, LifeTechnologies) was supplemented with 2 mM Glutamax R (LifeTechnologies), 100 µg/mL gentamicin (LifeTechnologies), and 10% PhABS. We tested six different commercially available PhABS (AB type and off-the-clot) from four different manufacturers (Table 1). Culture medium was changed every 48 h. After 7-8 days, plates were morphologically screened for MPCs using an inverted microscope, and cells subsequently detached by TrypLE R Select (LifeTechnologies) digestion and washed in D-PBS.

MSC Cell Culture
Duplicate hBM-MNC samples collected by density gradient centrifugation were plated at 2 × 10 5 /cm 2 in gas-treated culture plates for adherent cells. DMEM was supplemented with 2 mM Glutamax R (LifeTechnologies), 100 µg/mL gentamicin (LifeTechnologies), and 10% FBS (LifeTechnologies). Culture medium was changed after 48 h to remove non-adherent cells and refreshed twice a week.

Flow Cytometry
To identify MPCs and MSC-like cells (Keating, 2012) in MPC primary cultures freshly detached cells were incubated with anti-CD90 FITC-conjugated, anti-CD73 PE-conjugated, anti-CD31 PE/Cy7-conjugated, anti-CD18 APC-conjugated, and anti-CD45 VioBlue R -conjugated (Miltenyi Biotec, BergischGladbach, Germany) antibodies for 30 ′ at 4 • C in the dark and washed twice in MACSQuant R Running Buffer (Miltenyi Biotec). Data were acquired using MACSQuant R flow cytometer and analyzed by MACSQuantify R Analysis Software (MiltenyiBiotec). MPCs were identified as CD31 + CD18 + CD45 low CD73 neg CD90 neg events and MSCs as CD31 neg CD18 neg CD45 neg CD73 bright CD90 bright events. Statistical analysis was performed by one-way analysis of variance (ANOVA) test and Dunnett's post-test for multiple comparison. Results were expressed as mean value ± standard error (SE).

Recombinant hGF Treatments during MPC Isolation
hBM-MNCs were obtained from 4 patients (2M/2F, median age 68) and processed for MPC isolation as described above, applying two different sera batches: LZM, already screened for good performance and BIOW2 that produced cultures with consistent MSC-like cell "contamination." LZM cultures were also performed in presence of 50 ng/ml rhEGF (ThermoFisher Scientific, Waltham, MA USA), 20 ng/ml rhFGF-2 (ThermoFisher Scientific), 50 ng/ml rhPDGF-AB (PeproTech EC Ltd., London, UK) or 50 ng/ml rhVEGF (ThermoFisher). Combined treatments were also performed adding rhEGF and rhFGF-2, or rhVEGF and rhPDGF-AB. In parallel BIOW2 cultures were also supplemented with 100 ng/ml rhIGF-1 (PeproTech EC Ltd., London, UK), 30 ng/ml rhIGF-2 (PeproTech EC Ltd.) alone or in combination. The culture media were changed every 2 days restoring concentrations of the hGFs. After 7 days cells were detached by TrypLE R Select digestion and processed for flow cytometry to quantify MPC and MSC percentages in the cultures, as described above. Data were collected in duplicate and reported as median values ± standard error (SE). One-way ANOVA test, coupled with Dunnett's post-test, was applied to identify significant differences.

Inhibition of hGF Receptors, during Primary Cultures
Further six bone marrow samples (4M/2F median age 68) were processed, as described above, to obtain MPC culture in BIOW2 sera batch with or without adding 4 nM of

Inhibition of hGF Receptors, during Mesengenic Differentiation of MPCs
Erlotinib-HCl and Nintedanib were also applied during mesengenic differentiation of MPCs. Briefly, MPC culture in LZM sera batch were validated for a purity higher than 95% and re-plated at 20,000 cells/cm 2 in gas-treated 6-well plates for adherent cultures. After overnight incubation medium was replaced with StemMACS TM MSC expansion XF medium (Miltenyi Biotec), according to previously reported protocol (Montali et al., 2016). In parallel, mesengenic differentiation was performed in presence of 4 nM of Erlotinib-HCl and 100 nM of Nintedanib. Cultures were maintained changing medium twice a week and restoring inhibitor concentration. After 7 days mesengenic differentiation was evaluated by AlamarBlue R reduction assay (LifeTechnologies) as previously reported (Fazzi et al., 2011).
The "contamination" of MSC-like cells in MPC primary cultures was correlated to the concentration of growth factors in the different sera. Higher concentrations of EGF, FGF-2, PDGF-AB and VEGF-A were consistently found in BIOW1 FIGURE 2 | Gene expression analysis of growth factor receptor genes in MSCs vs. MPCs. The majority of the receptor genes resulted differentially expressed (*p < 0.05, **p < 0.01). Expression of FGFR2, PDGFRA, and EGFR was over one log higher in MSCs. Conversely, 10-fold higher IGF2R expression was evidenced in MPCs. and BIOW2 that produced cultures with higher percentages of MSC-like cells ( Table 2). FGF-2 revealed a dose-dependent correlation showing a Spearman ratio of 0.943 (p < 0.05, n = 8, Figure 3). In order to confirm the involvement of these hGFs in the poor performance of a pooled sera batch, we separately added consistent amount these factors to LZM supplemented cultures. Percentages of MSC-like cells resulted significantly increased in the rhEGF (34.3 ± 14.0%, p < 0.01, n = 4) and rhFGF-2 (39.2 ± 17.5%, p < 0.01, n = 4) treated primary cultures, respect to LZM control (4.6 ± 1.7%, n = 4, Figure 4). Combining these two hGFs produced similar results (40.5 ± 17.9%, n = 4), with no significant differences respect to cultures treated with EGF or FGF-2 separately. Conversely, adding rhVEGF (5.6 ± 1.4%, n = 4) or rhPDGF-AB (6.4 ± 0.5%, n = 4) resulted having no significant effects on MPC cultures when added separately. Combination of those hGF instead showed a mild increase in MSC-like cell percentages (11.3 ± 0.3%, p < 0.01, n = 4), resulting in cultures with purity lower than the cut-off point established for feasible MPC products.
Medium supplemented with BIOW2 was selected in order to verify if higher concentration of IGFs could be related to the good performances of feasible sera batches. A consistent reduction, around 50%, of the MSC "contamination" was reported in rhIGF-1 (17.1 ± 0.1%, p < 0.01, n = 4) and rhIGF-2 (17.7 ± 0.2%, p < 0.01, n = 4) treated cultures, alone or in combination (19.8 ± 2.8%, p < 0.01, n = 4) respect to BIOW2 control (45.8 ± 1.6%, n = 4, Figure 4). The effect of high concentration of EGF, FGF-2, VEGF and PDGFs in the poor performance of pooled sera batches, during MPC production, was also confirmed by receptor inhibition experiments. In fact, the addition of Erlotinib-HCl and Nintedanib to BIOW2 cultures resulted in the 50% reduction of the CD73 + CD90 + population (mean inhibition index: 54.2 ± 7.7%, p < 0.05, n = 6) at the primary cultures, producing suitable MPC products (>95% of purity) comparable to ones obtained applying LZM (Figure 5A). Elevated levels of IGF-1 were instead detected in the sera batches with low mesenchymal contamination (LZM, LZMF) but the inhibition of its receptors did not produce any detectable effect during primary culture ( Figure 5B).

DISCUSSION
Ex vivo expansion of bone marrow-derived multipotent cells requires supplementation of basal culture medium in order to sustain cell proliferation while maintaining cell differentiation capability (Bernardo et al., 2011). FBS-based expansion protocols have been used in the very first clinical applications of these promising cells (Le Blanc et al., 2004. However, safety concerns have been raised regarding use of supplements of animal origin in clinical-grade expansion protocols (Sensebé and Bourin, 2008;Reinhardt et al., 2011;Sensebé et al., 2011). Consequently, many efforts have been made to replace animal derived supplements and reagents with human blood derivatives as autologous serum (AS) (Stute et al., 2004), pooled human AB serum (PhABS) (Bieback et al., 2009), platelet lysate (PL) (Hemeda et al., 2014) or a combination of them (Kocaoemer et al., 2007;Muraglia et al., 2015).
In this view, during the last years our group has been focusing on the definition of a clinical grade protocol to culture bone marrow-derived multipotent cells by replacing FBS FIGURE 3 | Growth factor serum content correlated with cell composition in primary cultures. Higher concentrations of EGF, FGF-2, PDGF-AB, and VEGF-A were detected in PhABS that gave origin to cultures with higher percentages of "contaminating" MSC-like cells. Good performance in the initiation of MPC cultures at high grade of purity correlated with elevated levels of IGF-1.
with AS or PhABS. Our efforts led to the first description of mesangiogenic progenitor cells (MPCs) as previously not described cell population, co-isolated in AS-supplemented MSC cultures (Petrini et al., 2009). In parallel, a group of colleagues demonstrated the long-term efficacy and safety of this kind of cell products for the healing of atrophic pseudarthrosis of the upper limb (Giannotti et al., 2013). In this paper, Authors also hypothesized that MPCs detected in the applied cell populations, even if at low percentage (1-10%), could contribute to the long lasting healing (follow-up at 76 months) reported for the eight enrolled patients. This interesting hypothesis could be corroborated by the idea that implanted MPCs could provide efficient tissue regeneration, differentiating into early MSCs, as well as contribute to the vascularization of the engineered construct due to their demonstrated angiogenic properties (Fazzi et al., 2011). After these encouraging clinical results, we believe that manufacturing MPC-based medicinal products could provide a new promising tool in skeletal tissue engineering.
As MPCs have been reported being not-proliferating cells, the expansion is not actually permitted. Nonetheless, the frequency of MPCs has been estimated around 1% of hBM-MNCs, which is hundred to thousand times higher respect to MSCs , leading us to hypothesized cell-based therapies involving this non-expanded multipotent progenitors. The possible application of not in vitro amplified cell products carries significant advantages in terms of: (i) a reduced risk of cell transformation, (ii) reduced cellular senescence, and (iii) reduced exposition to bacterial and viral contamination, minimizing the FIGURE 4 | Managing hGF medium contents, by adding recombinant factors, affected the sera performance during MPC isolation. Adding rhEGF or rhFGF-2, alone or in combination, to the cultures supplemented with good performance serum (LZM), resulted in significantly increased MSC-like population (red bars). Under these conditions, MPC/MSC ratio was comparable to those reported applying MSC-inducing PhABS. Treating with rhVEGF or rhPDGF-AB had no effects on MPC isolation, but a combination of those two hGFs produced mild but significant increase in MSC-like counterpart. BIOW2 batch was selected as PhABS with poor performance, evaluating the effects of rhIGF-1 and rhIGF-2 during MPC isolation. The treatment with rhIGFs, either alone or in combination, produced a 50% reduction of the MSC-like population (**p < 0.01, respect to control). culture time (Schneider et al., 2010). However, this could provide limited number of cell doses forcing to investigate any possible clinical application of MPCs in the context of personalized cell therapies, which involved small-scale CBMP production similar to that described by Giannotti et al. (2013). Anyway, in the attempt to define manufacturing conditions, selective for MPCs, we observed large variability in MPC yield using AS as well as different manufacturers and batches of commercial PhABS.
In general, CBMPs represent complex biological products displaying high rate of intrinsic variability, mainly derived from two sources: (i) process starting material and (ii) process conditions (Williams et al., 2012). In particular for MPC-based product, the first mentioned source of variability could not be eliminated in the autologous context where starting materials are obtained by different patients. Moreover, even if the variability related to the production process could be significantly reduced applying automated and well-controlled manufacturing process (Liu et al., 2010;Pacini, 2014), the serum supplementation represents an inescapable source of variability. Here we showed such variability to correlate with some of the most important growth factors contained in platelet lysates (Fekete et al., 2012), whose concentrations in the serum fluctuate regardless of donors' age, sex or production method. Best performance in MPC production was obtained with serum concentrations of IGF-1 over 150 ng/ml combined to low levels of EGF (<160 pg/ml), FGF-2 (<2.0 pg/ml), PDGF-AB (<1200 pg/ml), and VEGF-A FIGURE 5 | Erlotinib-HCl in combination with Nintedanib allowed recovering quality MPC products applying sera with poor performance. (A) The simultaneous inhibition of EGFR, FGFRs, PDGFRs and VEGFRs during culture of hBM-MNCs in BIOW2 serum batch, produce a 50% reduction in the CD73 + CD90 + population, leading to a cell product comparable to once produced in LZM. (B) Conversely, the inhibition of IGF-1R exerted by Linsitinib did not affect the MPC production.
(<180 pg/ml). Screening these five growth factors could provide a simple means to select high quality HS batches for the production of MPC-based CBMPs. Moreover, data presented here also provide interesting application for Erlotinib-HCl and Nintedanib in the production of MPC in autologous serum, where any possible source of variability, related to fluctuations in hGF serum concentration, could be mitigated by receptors inhibition. This could possibly results in qualified MPC products independently by patient sex, age, etc.
Taking in consideration only hGFs as serum factors influencing MPC cultures, represents the mayor limit of this study. Serum is highly complex biological fluid composed also by many other macromolecules (i.e., cytokines and immunoglobulins), different lipids, hormones and other small molecules. Further investigations are needed to exhaustively define the essential components required to produce high quality MPC-based cell product. Anyway, here we clearly demonstrate that managing the interactions of these hGFs with their receptors could also represents a new tool in MSC production. In fact, culturing bone marrow in AS, or PhABS, and in presence of Erlotinib-HCl and Nintedanib could lead to highly purified MPCs that could act as culture initiating cells to produce more homogeneous and synchronized MSC cultures. Those cultures could be easily obtained applying the same HS, by removing receptor inhibition and supplementing with recombinant hGF cocktail including at least EGF and FGF-2, as demonstrated here. Our findings are in accordance with Yamaguchi et al. that reported comparable results between FBS and PhABS, in MSC culture, when PhABS cultures had been supplemented with 10 ng/ml of FGF-2 (Yamaguchi et al., 2002). However, the extensive literature on serum supplements in MSC cultures is very controversial. A number of authors showed no significant differences between AS and FBS (Yamamoto et al., 2003;Spees et al., 2004;Stute et al., 2004), while Shigeno and Ashton obtained greater response using AS (Shigeno and Ashton, 1995), in contrast to the extensively reported higher performances applying FBS (Koller et al., 1998;Kuznetsov et al., 2000). It is reasonable hypothesized that the origins of those controversial data, in MSC manufacturing, reside in the lack of preliminary characterization of hGF content in the HS applied. We also believe that those inconsistencies originate from restricting focus on MSCs, without taking into account the positive effects of the MPC presence within the CBMP once implanted in vivo.
HS batches rich in EGF and FGF-2 apparently worsen the production of standard monomorphic MPC cultures probably supporting the proliferation of MSC-like cells from other distinct and rare progenitors reported in the bone marrow. These mesengenic progenitors could include skeletal stem cells (SSCs), recently described as the genuine skeletal tissue stem cells (Bianco and Robey, 2015) or CD146-positive non-stem osteoprogenitor (Sacchetti et al., 2007). This hypothesis is validated by our data on growth factor receptor expression. Significantly higher levels of EGFR, FGFR2, and PDGFRA were reported in MSCs suggesting that these specific growth factors could have trophic effect on MSC-like cells (Ng et al., 2008) instead of inducing MPCs to differentiate. Conversely, VEGF-A was detected at higher concentrations in MSC-inducing sera while KDR was more expressed in MPCs, together with TGFBR2, suggesting a role for VEGF-A and possibly TGF-β in MPC differentiation toward the mesenchymal lineage. IGF-1 appeared to be acting as MPC-promoting factor as its levels were particularly high in MPC-inducing sera LZM, LZMF, and SERL, mirroring IGF receptor expression in MPCs.
In conclusion, we believe that MPCs represent also a promising alternative to hBM-MNCs as culture-initiating cells in the production of clinical grade MSCs. A purified and well-characterized progenitor cell population cultured under specific controlled conditions would significantly improve CBMP reproducibility and consequently the predictability of pre-clinical studies. MPCs are found at frequencies one to two logs higher than the other MSC progenitors described in bone marrow. Moreover, future clinical applications for CBMPs based on undifferentiated and not expanded MPCs could take advantage from their angiogenic potential that is suggestive of possible beneficial effects on neo-or re-vascularization of target tissues. A complete definition of the active growth factor cocktail for MPC efficient isolation, expansion, and differentiation is still required also in order to possibly develop specific chemically defined media (CDM), which will eliminate the biological variability related to the serum supplementation.

AUTHOR CONTRIBUTIONS
MM: Conception and design, Data collection, assembly, analysis, and interpretation. SB: Data collection, assembly, analysis, and interpretation. VC: Collection and assembly of RT-PCR data. FF: Collection and assembly of ELISA assay data. FP: Data collection. IP: Conception and design. SP: Conception and design; Data collection, assembly, analysis, and interpretation; Manuscript writing.