Effect of culture environement on mesenchymal stem cell immunomodulatory ability

Allogeneic and autologous Mesenchymal Stem Cells (MSC) did not elicit immunological response and has immunomodulatory effect in vitro and in vivo. This effect has been documented by interesting researches worldwide. Previous studies showed that inhibitory mechanisms of MSCs depends on nature of stimulus [1-5]. However the protocols of MSCs culture are different. This difference could be translated on different results regarding the mediators as well as on potency of immunomodulatory effect in both vitro and in vivo.


Introduction
Allogeneic and autologous Mesenchymal Stem Cells (MSC) did not elicit immunological response and has immunomodulatory effect in vitro and in vivo. This effect has been documented by interesting researches worldwide. Previous studies showed that inhibitory mechanisms of MSCs depends on nature of stimulus [1][2][3][4][5]. However the protocols of MSCs culture are different. This difference could be translated on different results regarding the mediators as well as on potency of immunomodulatory effect in both vitro and in vivo.
In this article we will examine and address the impact of the culture environment on immunomodulatory effect taking in account the different utilized serum and other components of cultures, duration, special condition of cultures and inhibitory mediators as well as the magnitude of immunomodulatory effect.
Studies showed that MSC derived from different origins have no difference in the immunomodulatory effect. One comparative study showed that BM-MSC had fewer inhibitory effects than other used MSC on T cell proliferation, while WJ-MSC had most prominent inhibition [15]. Others found no signifi cant differences in ability to inhibit allo-activated immune cells proliferation by MSCs derived from different sources: AM and UC-and AT-MSC [6]. This fi nding supported by fi nding that MSCs from different sources had similar partial immunomodulation on Mixed Lymphocyte Reaction (MLR) [16].
In other hand, placenta and umbilical cord from the same donor, showed a signifi cant difference in their immunosuppressive properties as assessed in MLR. The   [20].
The suppressive effect of MSC derived from severe aplastic anemia patients either at diagnosis or after immunosuppressive therapy on T-cell activation is weaker than MSC derived from normal individuals [21]. Of note, most of cases have been subjected to immunosuppressive therapy insult and subsequent change of MSC environment. Indoleamine 2,3-Dioxygenase (IDO) implicated on T cell suppression by allogeneic UC-MSCs in lupus patients. This effect induced through IFN produced predominantly by lupus CD8+ T cells. In contrast BM MSCs from patients with active lupus demonstrated defective IDO production in response to IFN and allogeneic CD8+ T cell stimulation [22]. A comparison between inhibitory effect with only single variable (autologous and allogeneic UC-MSCs versus autologous and allogeneic BM MSC) setting is needed.
In murine model, both allogenic and autologous MSC have similar inhibitory effect [1] in vitro. In contrary, origin of MSC could play a role. MSC from different origin of the same donor have different immunosuppressive effect. Where PL MSCs reduced T cell proliferation, affected the antigen presenting ability, enriched CD3(+) CD4(+) CD25(+) T regulatory cells and increased mRNA expression of FoxP3 as compared to UC MSCs [17].
Liver MSCs showed a higher expression of programmed death-ligand 1 (PD-L1) than BM MSCs, which associated with inhibition of T-cell in vitro [14].
A study showed that MSC from myelodysplastic syndromes (MDS) patients inhibit T-cell proliferation signifi cantly lower than that of normal MSC, and had different mediators expression

3-Eff ect of MSC thawing on magnitude of inhibitory eff ect
Most of published works reported post thaw cultured MSC immunosuppressive effect [7,31,32]. No difference in the suppression of lymphocyte proliferation by non-cultured postthaw CB-MSC and that undergone post-thaw culture [33].
Viability did not differ signifi cantly in MSC before freeze or after thaw, but cell health as measured by population doublings Citation: Nasef

Difference in inhibitory mediators according to ratio of MSCs
MSC elicit more important immunomodulatory function on higher ratios [3,32]

Difference in inhibitory effect mediators according to culture duration (assessment day)
There is a contradictory results regarding detected inhibitory mediators expressed by MSCs in MLR. Future studies comparing between MSC from different origins and inhibitory mediators (molecular, protein and regulatory cells) expressed by MSCs cultured for different durations; 3, 4,5, 6 and 7 days is mandatory.

Molecular inhibitory mediators
We reported the molecular expression by human MSC of TGF-B, Interleukin-10 (IL-10), Human Leukocyte Antigen-G (HLA-G), IDO and Leukemia Inhibitory Factor (LIF) in cultures with or without direct contact. However TGF-B and Il-10 were markedly increased in culture with direct contact. Expression of different genes by MSC in MLR has been reported despite using MSC from variable origins and species in different culture protocols [17,30,32,49].

Effect of infl ammatory environment
MSC immunomodulatory effect could be altered by local pro-infl ammatory environment. BM-MSC and AT-MSC cultured with pro-infl ammatory cytokines interleukin-1B (IL-1b), IL-6 and interleukin-23 (IL-23) showed suppressive ability of MSC on allogeneic T cell with signifi cant increase of TGF- and lower level of interleukin-4 (IL-4). This result is promising in cell-based therapy of degenerative, infl ammatory and autoimmune disorders [60]. Treatment of corneal stroma derived MSC by pro-infl ammatory cytokines and toll-like receptor ligands signifi cantly increased cell surface adhesion and secretion of IL-6, interleukin-8 (IL-8) and C-X-C motif chemokine 10 (CXCL-10) levels [13].
MSC supernatant has an inhibitory effect in secondary MLR, but less important than primary MLR [3]. The immunosuppression of human MSCs is higher in cultures with direct contact than in cultures with indirect contact [9,14,32,61].
The contradictory result regarding the suppressive effect of MSC in culture without direct contact [3,62], could be

Effect of radiation on MSCs inhibitory effect
Both irradiated and non-irradiated BM-MSC from healthy donors and Autoimmune Disease (AD) patients had a suppressive effect on autologous and allogeneic PBMCs. [71].
Surprisingly, MSC from AD are not compromised in their immune response. However, others found that MSC surviving irradiation changed their cytokine secretion profi le and became prematurely senescent. [72]. Effect of irradiation on MSC needs further exploration.

Effect of donor age on MSCs growth
Growth of MSC obtained from younger rats is faster and has more prominent inhibitory effect as compared with old animals. Aged AT MSC failed to induce any CD3(+)CD4(+) T cell suppression. Young MSC induced suppression was more prominent than seniors [73].
Studies are needed to study the difference in inhibitory effect between human MSC obtained from old and young individuals. Of note, MSCs obtained from bone marrow during total hip replacement is not an optimal source. As usually large number of these patients are old and many have concomitant or co-existing health problems.

Discussion and recommendation
Effect of culture environment on MSC should be studied on profound back ground However we cannot withdraw a conclusion from these variable experiments and clinical trials for the time being. We noticed that HLA-G was expressed signifi cantly only on day 3 of co-culture but not on day 6 (unpublished observation).
In addition, study of galectins 1, 9, 11 and 13 expression by BM-MSC with or without differentiation, and cultivated in three culture mediums (standard medium: alpha-MEM+FGF2, the medium (with 5% Human Platelets Lysate (HPL)), the medium (10% HPL), showed that expression of different galectins are depend on the composition of the culture medium and type of galectin [75]. This fi nding should be taken in account in future studies, as it confi rm implication of culture media in MSC changes. Recently, Sierra Parraga JM, et al. found that thawing of MSC affect adherence, viability, increased oxidative stress, and reduced mitochondrial activity, which implies reduced metabolism and survival of MSC [76].

Conclusion and future perspective
There is great need for consensus aiming at optimization of MSC-based therapeutic approaches in immunological diseases and GVHD.
Researches in agreement with consensus criteria of MSCs [77], with selection of MSC origin, specifi cation of culture conditions and duration, knowing of MSC peak inhibitory effect, elucidation of core inhibitory mediators, and their implication in different time of expansion and MSC-MLR co-culture by using a matrix assay approach [78], could help in future defi nition of most appropriate MSC origin, culture condition, MSC retrieval time and therapeutic injection for management of immunological diseases and GVHD.