The first stage of experiment included the choice of lipopolysaccharide concentration for the inducement of inflammation. The effect of a series of concentrations of LPS on LESC was assessed, however MTT assay showed no statistically significant differences between the examined groups compared to the control. Similarly Kukolj et al. [32] observed that LPS did not affect proliferation and viability of periodontal ligament stem cells and it did not change their immunophenotype and cell cycle. Kukolj et al. showed that LPS acts on differentiation potential of cells and it inhibits osteogenesis and promotes chondrogenesis and adipogenesis [32]. Cell viability may change under the influence of LPS depending on the time of incubation and its concentration [33]. Many studies suggest the application of different concentrations from very low like 0,1 µg/ml [34]; through 0,5 µg/ml [35], 1 µg/ml [33] to 10 µg/ml [33, 34] or even 24 µg/ml [36]. Moreover, the suggested incubation time which is necessary for the induction of inflammation is diverse and takes e.g. from 2 h to 24 h [33]. Some studies indicate that a proper time to induce the inflammation in mesenchymal stem cells is 24 h with 1 µg/ml of LPS concentration [37]. Other suggest increasing the concentration of LPS to 2 µg/ml, but shortening the incubation time to 6 h in morphologically similar cells like normal human skin fibroblasts (NHDF) [38]. In contrast, LPS at concentration of 0,1 µg/ml was used for the induction of inflammation in macrophages for 24 h [39]. Therefore, based on numerous literature data and our findings, the concentration selected for this experiment was 2 µg/ml of LPS.
Next, the analysis with ApoTox-Glo Triplex Assay showed that LESC viability increased after the treatment of conditioned medium from ADSC. The test showed also that the CM was not toxic to cells and as well there were no differences in cells apoptosis between groups. These results suggest that factors secreted by ADSC improve LESC’S viability. In the condition of induced inflammation, no statistically significant differences in cell viability, cytotoxicity and apoptosis were noticed under the influence of CM. These findings suggest that LPS inhibits the positive effect of ADSC’S secretome on limbal stem cells. Probably different signaling pathways were activated in the presence of LPS in the cells, not necessarily associated with the promotion of cell viability. It indicates that the addition of LPS does not significantly reduce cell activity. Probably a longer cell culture in the above-mentioned conditions could result in similar findings as in the case of cells cultured in standard conditions. However, these results show that the presence of LPS changes the activity of cellular proteases and caspase 3/7 in LECSs. Similar findings were reported by Chen and his team who investigated the effect of conditioned medium from mesenchymal stem cells derived from Wharton jelly on epithelial cells. It was shown that conditioned medium increased cells proliferation and migration by activation of stress response kinase JNK (c-Jun N-terminal Kinase) and isoform of p38 protein [40]. Farahmand et al. also confirmed these results by observing higher expression of proliferation-related genes after culture in conditioned medium [41]. Some data indicated using a conditioned medium in the therapy of e.g. arthritis. In this case, a reduction of damages in cartilage tissue was shown and an inhibition of the immune response in cells has been noted [42]. Li et al. investigated using conditioned medium as a promoter of wound healing in diabetic patients in whom this process is usually disturbed. Research showed that the use of LPS reduces the rate of migration and proliferation in keratinocytes which grown in the presence of high glucose concentration, as the disease model for analyzing the effect of type 2 diabetes on cells. It was proven that the use of CM eliminates the negative effect of LPS and also negative effect of high glucose levels [43]. Also, the high potential of using CM in the treatment of bronchopulmonary dysplasia in oxygen-induced alveolar damage model showed that MSCs support tissue with antioxidant substances which therefore can lead to new therapy [44].
Scratch wound healing assay was performed to assess the rate of cell proliferation. LESC cultured in CM compared to SM, both under standard conditions and induced inflammation, showed better proliferation rate. It was noticed that cells stimulated with LPS and cultured in CM showed lower proliferative potential compared to unstimulated cells. These results indicate inhibition of cells proliferation by LPS, which is consistent with previous findings and the literature data [43]. Zhang et al. in in vivo studies showed that injection of ADSC promoted wound healing in rabbits [45]. Zeppieri et al. presented that mesenchymal stem cells promote corneal wound healing in rats [46]. Similarly, Galindo et al. demonstrated that ADSC have a therapeutic effect on LSCD in rabbits [47].
Phenotype microarrays showed that LESC in all study groups (SM, CM, SM + LPS) had various activity. PMM7 and PMM8 arrays were coated with six replicates of the same compound but in variable concentration. Producer provides an information that these concentrations are increasing from left to right, but does not give the information about the number and the unit, so we do not really know how much of each compound is in the well. We have numbered these concentrations from 1 to 6, where 1 is the lowest concentration and 6 is the highest. Due to a lot of data on cells activity, the results are presented on heat maps what ensures clear readability. All readings are presented as the fold change compared to control which was taken as 100%. We noticed that the activity of LPS significantly impact on LESC viability. This group of cells became more sensitive for bigger concentrations of each compound what appeared in the highest spread of fold change values. Interestingly, we noticed that LESC cultured in SM with the addition of LPS were more viable in presence of IL-6, while LESC cultured in SM and CM showed neutral or toxic effect of this cytokine. The lowest viability in presence of IL-6 was noticed in CM. We assume that while ADSC secrete IL-6, the final concentration of this cytokine in one well could be high enough to act negatively on LESC [48–50]. Surprisingly, the highest toxic effect was observed at the concentration “4”. LESC showed decreased activity in response to IL-8 in all examined groups. The toxic effect was more readable in higher concentrations and the highest values were noticed in CM group, but there were no differences between SM. Similarly, IL-2 in higher concentrations showed a toxic effect on LESC viability in all groups, but in opposite, lower concentrations of IL-2 induced LESC activity what was mostly notable in CM group. We observed that INFγ exerted a neutral response of LESC. In SM and CM groups we noted slightly increasing viability in direct proportion to the INFγ concentration. LESC cultured in SM + LPS showed decreased viability in the presence of INFγ.
Phenotype microarrays brought plenty of information which could help composing supplementation of culture media to improve cells viability. These factors could be potentially cross-linked in biopolymer scaffolds adapted for carrying ADSC to strengthen the response to secreted factors. However, further studies on the influence of presented factors are needed for better understanding of variable effect on LESC activity.
The last stage included the assessment of mRNA expression and cytokine secretion of pro and anti-inflammatory cytokines in LESC due to the examined conditions. Increased expression of IL-1α and IL-1β was observed in cells cultured in CM at standard conditions compared to cells with induced inflammation (CM_LPS). IL-1α and IL-1β role is associated with the regulation of an immune response as a result of ongoing infection [51, 52]. IL-1 also acts by activating the secretion of many different cytokines and chemokines, e.g. IL-6, TNFα and IFNγ [51, 52]. IL-1 participate in the regulation of stem cell activity. In hematopoietic stem cells (HSCs), it exhibits radioprotective activity, induces their proliferation and differentiation [52]. Our findings showed no differences in the expression of IL-1α and IL-1β between cells cultured in CM and SM in both standard conditions and induced inflammation. The lower expression of both interleukins in CM_LPS group suggests that CM has a significant impact on IL-1 release during the inflammation. This activity was confirmed by ELISA detection of IL-1α. The concentration of this IL-1α in LESC’S culture medium was lower during induced inflammation under the influence of CM (CM_LPS group). Furthermore, significant difference in the secretion of IL-1α between LECSs with induced inflammation and LESC in standard conditions cultured in SM indicates that LPS has meaningful impact on IL-1α release. Finally, observed no differences in IL-1α secretion between CM, CM_LPS and SM group suggest that CM downregulates the release of IL-1α which means that ADSC secrete factors which mitigates the inflammation. Some studies suggest that an increase in interleukin-1 secretion was observed under the influence of cellular stress e.g. in a state of hypoxia, chemical or physical damage. During the apoptosis which is natural, programmed cell death IL-1α is not released by the cells [51]. Solomon et al. reported that stroma of amniotic membrane inhibits the level of IL-1α and IL-1β, after LPS stimulation, in cell culture of limbal epithelial stem cells [53]. It suggests that the use of CM simultaneously with amniotic membrane could give better therapeutic results in cornea treatment by achieving a synergistic effect. The analysis of the IL-6 mRNA expression showed decreased copy number in cells with induced inflammation cultured in CM compared to SM. ELISA also indicated a significant decrease of IL-6 secretion by LESC with induced inflammation compared to cells cultured in SM. Similarly, lower concentration of IL-6 in the same group was noticed at LESC in standard conditions. Studies indicate that IL-6 secretion increase in cells during inflammation [54, 55]. However, it was proven that MSCs naturally secrete IL-6 [56, 57]. This interleukin is responsible for the regulation of the immune response, hematopoiesis, apoptosis, proliferation and cell viability [58]. It plays important role in regulation of homeostasis in corneal limbus cells niche. IL-6 may help in wound healing of epithelial cells in vivo [59, 60]. Perhaps a significant increase in the level of this interleukin may be the result of cellular responses to unfavorable environmental conditions e.g. like LPS induced inflammation. Lower levels of IL-6 at LESC cultured in CM suggest a beneficial property of ADSC secreted factors. Probably factors present in the conditioned medium from ADSC promote the LESC regeneration. Perhaps the use of IL-6 as a supplement for cell culture could provide better proliferation. However, this issue requires further study. The analysis also revealed an increase of IFNγ mRNA expression in LESC with induced inflammation cultured in SM compared to the LECSs in standard conditions cultured in the same medium. This indicates that LPS stimulation probably caused upregulation of this gene expression. However, IFNγ was not detected by ELISA. It should be remembered that the increase in mRNA level is ahead of the protein concentration. Gene expression depends on many transcription factors which activation changes the level of mRNA in cells. Thus, when translation product is at demanded level, the DNA transcription can be suppressed. In opposite, when there is a low level of protein, gene expression can be continuously upregulated.