Preconditioning of Human Decidua Basalis Mesenchymal Stem/Stromal Cells with Glucose Increased Their Engraftment and Anti-diabetic Properties.

BACKGROUND
Mesenchymal stem/stromal cells (MSCs) from the decidua basalis (DBMSCs) of the human placenta have important functions that make them potential candidates for cellular therapy. Previously, we showed that DBMSC functions do not change significantly in a high oxidative stress environment, which was induced by hydrogen peroxide (H2O2) and immune cells. Here, we studied the consequences of glucose, another oxidative stress inducer, on the phenotypic and functional changes in DBMSCs.


METHODS
DBMSCs were exposed to a high level of glucose, and its effect on DBMSC phenotypic and functional properties was determined. DBMSC expression of oxidative stress and immune molecules after exposure to glucose were also identified.


RESULTS
Conditioning of DBMSCs with glucose improved their adhesion and invasion. Glucose also increased DBMSC expression of genes with survival, proliferation, migration, invasion, anti-inflammatory, anti-chemoattractant and antimicrobial properties. In addition, DBMSC expression of B7H4, an inhibitor of T cell proliferation was also enhanced by glucose. Interestingly, glucose modulated DBMSC expression of genes involved in insulin secretion and prevention of diabetes.


CONCLUSION
These data show the potentially beneficial effects of glucose on DBMSC functions. Preconditioning of DBMSCs with glucose may therefore be a rational strategy for increasing their therapeutic potential by enhancing their engraftment efficiency. In addition, glucose may program DBMSCs into insulin producing cells with ability to counteract inflammation and infection associated with diabetes. However, future in vitro and in vivo studies are essential to investigate the findings of this study further.


Introduction
Mesenchymal stem /stromal cells (MSCs) are isolated from many human adult organs, including the placenta [1][2][3]. MSCs have multipotent differentiation potential [1][2][3] and possess immune-modulatory properties [4][5][6][7], which are essential for tissue restoration. Therefore, MSCs are This paper is dedicated to late ''Professor Mohammad Abumaree'', who was the main architect of this study. We will continue to be inspired by his dedication and profound passion for the stem cell research at KAIMRC. May you rest in peace.
Electronic supplementary material The online version of this article (https://doi.org/10.1007/s13770-020-00239-7) contains supplementary material, which is available to authorized users. considered attractive potential therapeutic agents to treat human diseases including diabetes, hypertension, and atherosclerosis [8][9][10]. In these inflammatory diseases, the environment is characterized by high levels of oxidative stress and inflammation. Consequently, for successful use of MSCs in these diseases, MSCs must maintain their normal functional activities to repair injured tissues in hostile microenvironments associated with oxidatively stress and inflammation. MSCs that cannot resist the toxic environment, are likely to have reduced therapeutic potential [11].
Recently, we reported the isolation and phenotypic characteristics of MSCs from the maternal decidua basalis tissue (DBMSCs) of human term placenta [1]. Human pregnancy is a condition where the maternal tissues and circulation are exposed to high levels of oxidative stress. DBMSCs in the maternal tissue of decidua basalis are a vascular microenvironment (i.e. niche), and are continuously exposed to high levels of oxidative stress products in the maternal circulation [12]. As a result, DBMSCs are conditioned to resist oxidative stress, as previously reported [13]. In a recent study, we showed that DBMSCs survive the harsh oxidative environment induced by high concentrations of H 2 O 2 , and that preconditioning of DBMSCs with H 2 O 2 improved their functional activities [14]. In addition, that preconditioning of DBMSCs with H 2 O 2 modulated their expression of genes with important cellular functions [14]. We also reported that DBMSCs protect endothelial cells functions from the damaging effects of both H 2 O 2 and monocytes [15,16]. Therefore, preconditioning of DBMSCs appears to be a rational approach for increase the efficiency of stem cell therapies associated with inflammatory diseases. In this study, we examined the functional responses of DBMSCs to another oxidative stress mediator; glucose. We exposed DBMSCs to high level of glucose and their phenotypic and functional properties were then assessed. We found that DBMSCs survived the harsh environment provided by high level of glucose, and that preconditioning of DBMSCs with glucose increased important functions, including adhesion and invasion. In addition, preconditioning of DBMSCs with glucose enhanced their expression of genes associated with various cellular functions including survival, proliferation, migration, invasion, immune modulation and microbial clearance. Glucose also increased DBMSC expression of B7H4, an immune protein with ability to inhibit T cell proliferation. Finally, DBMSC expression of genes involved in insulin secretion and prevention of diabetes was also modulated by glucose. These data indicate that glucose increases functions associated with engraftment of DBMSCs, and induced beneficial phenotypic changes in DBMSCs. We conclude that DBMSCs are potential candidate for the treatment of diabetes through a mechanism involving the reduction of inflammation and the secretion of insulin to lower glucose levels. However, more studies are essential to confirm these findings in vitro and in vivo.

DBMSC proliferation and adhesion in response to glucose
DBMSC treatment groups consist of three groups as described in supplementry Table 1 and illustrated in Fig. 1A-C. The xCELLigence system (RTCA-DP version; Roche Diagnostics, Mannheim, Germany) was used to evaluate the adhesion and proliferation of HUVEC as we previously described [15][16][17][18]. Briefly, 2 9 104 DBMSCs were seeded in a complete DBMSC culture medium (as described above) containing different concentrations (25-400 mM) of glucose (Prince Care Pharma Pvt. Ltd, India) in 16-well culture plates (Catalogue number 05469813001, E-Plate 16, Roche Diagnostics). The culture plates were then placed in the xCELLigence system at 37°C in a cell culture incubator and the DBMSC cell index was then monitored. The data for cell adhesion (at 2 h) and proliferation (24-72 h) was determined as previously described [15][16][17][18]. DBMSC viability was determined by Trypan blue exclusion. Each experiment was performed in triplicate and repeated with five independent DBMSC (passage 3) preparations.

DBMSC migration in response to glucose
DBMSC migration was examined using the xCELLigence system and 16-well plates (Catalogue number 05665825001, CIM-16, Roche Diagnostics GmbH) as we described previously [15][16][17][18]. DBMSC treatment groups consist of three groups as described in supplementry Table 2 and illustrated in Fig. 3A-C. Briefly, 2 9 104 DBMSCs were seeded in the upper chamber, and the migration of cells was then monitored by the xCELLigence system [15][16][17][18]. The data were then expressed as a cell index value at 24 h. DBMSC migration with 30% FBS and without FBS served as positive and negative controls, respectively. Each experiment was performed and repeated as described above.

DBMSC invasion under the effect of glucose
We evaluated the effect of glucose on the ability of DBMSCs to invade through a monolayer of endothelial cells using the xCELLigence system. Briefly, 2 9 104 HUVEC seeded in a complete endothelial cell growth medium in a 16-well culture E-Plate (as described above) until cells reached a growth plateau (20 h). Different treatments of DBMSCs (supplementary Table 3) were then added to the monolayer of endothelial cells. At 10 h, the data for the invasion recorded and expressed as a cell index (mean ± standard error). The rate of cell invasion was determined by calculating the normalized cell index at pausing time (20 h) of HUVEC growth.

Gene expression by real-time polymerase chain reaction (RT-PCR)
DBMSC expression of 84 genes related to Human Oxidative Stress (Catalogue # PAHS-065ZD, Qiagen, Hilden, Germany) was identified using our previously published method [1,14,15,18]. Total RNA was extracted from DBMSCs pretreated with 200 mM glucose for 72 h, and cDNA was then synthesized and used in a QuantiTect Primer Assay (Qiagen, Hilden, Germany). The real-time polymerase chain reaction (RT-PCR) was performed in triplicate on the CFX96 real-time PCR detection system (BIO-RAD, Hercules, CA, USA), and the data was then analysed as previously described [1,14,15,18]. The DD -2 values were then calculated to express the results as fold changes. The relative expression of internal controls (house-keeping genes) were used as provided in the kit. Experiments were performed in triplicate and repeated three times using DBMSCs prepared from three independent placentae.

Flow cytometry
DBMSCs (1 9 105) were stained with antibodies for ICAM-1, IL-12, and B7H4 for 30 min and then flow cytometry was performed as previously described [1]. Negative controls were cells stained with FITC or PElabelled mouse IgG isotype antibody.

Statistical analysis GraphPad Prism 5 was used to analyze data using non-parametric tests
(Mann-Witney U and Kruskal-Wallis). Data were deemed statistically significant if p \ 0.05.

Glucose effect on DBMSC proliferation
DBMSCs were isolated using our established published method [1,[14][15][16] to assess the effect of glucose on their proliferation using the xCELLigence system. At 24 h, and as compared to untreated DBMSCs, the proliferation of DBMSCs unchanged at 25 mM glucose (p [ 0.05), significantly increased at 50 and 200 mM glucose (p \ 0.05), and was significantly reduced at 400 mM glucose, p \ 0.05 ( Fig. 1D). At 48 h, and as compared to untreated DBMSCs, the proliferation of DBMSCs unchanged at 50 mM glucose (p [ 0.05), significantly increased at 200 mM glucose (p \ 0.05), and significantly reduced at 25 and 400 mM glucose, p \ 0.05 (Fig. 1E). Finally, at 72 h, and as compared to untreated DBMSCs, the proliferation of DBMSCs, significantly increased at 200 mM glucose (p \ 0.05) but was significantly reduced at 25, 50 and 400 mM glucose, p \ 0.05 (Fig. 1F)

Reversibility of glucose effect on DBMSC proliferation
To evaluate the reversibility of the glucose effect on DBMSC proliferation, DBMSCs were initially cultured with 200 mM glucose for 72 h and their proliferation was then determined using the xCELLigence system. As

Glucose effect on DBMSC adhesion
To study the effects of glucose on the adhesion of DBMSCs, DBMSCs were cultured with 200 mM glucose and their adhesion was then determined using the xCELLigence system. At 2 h, and as compared to untreated DBMSCs, the adhesion of DBMSCs unchanged after treatment with 200 mM glucose, p [ 0.05 (Fig. 2). We also evaluated the reversibility of glucose effects on DBMSC adhesion. DBMSCs were initially cultured with 200 mM glucose for 72 h and their adhesion was then determined using the xCELLigence system. After 2 h, and as compared to untreated DBMSCs, and DBMSC-treated with 200 mM glucose during the experiment [200 (I)], the adhesion of DBMSCs pretreated with 200 mM glucose [200 (pre)] significantly increased, p \ 0.05 (Fig. 2).

Glucose effect on DBMSC migration
To further study the effect of glucose on DBMSC functions, the migration of DBMSCs was monitored using the xCELLigence system. At 24 h and as compared to untreated DBMSCs (DB), the migration of DBMSCs in response to 200 mM glucose [DB (To 200)] significantly increased, p \ 0.05 (Fig. 3D).
We also evaluated the reversibility of glucose effects on DBMSC migration, DBMSCs were initially cultured with 200 mM glucose for 72 h and their migration was then determined using the xCELLigence system.  (Fig. 3D). These results show the effect of glucose on DBMSC migration is irreversible.

Glucose effect on DBMSC invasion
We also evaluated the effect of glucose on DBMSC invasion through endothelial cells using the xCELLigence Real-Time Cell Analyser. In the xCELLigence Rea-Time system, increased invasion is defined as an increase in the cell index due to the infiltration of HUVEC monolayer by DBMSCs and this therefore causing detachment of HUVEC while the increased of DBMSC adhesion is

Glucose increases DBMSC expression of adhesion and anti-inflammatory markers
To evaluate the modulatory effects of glucose on DBMSC functions, a variety of immune proteins important in DBMSC functions were studied by flow cytometry and expression recorded as median fluorescence intensity. As compared to untreated DBMSCs, after incubation with

Glucose modulated the expression of genes important in DBMSC functions
The expression of oxidative stress-associated genes by DBMSCs was studied after culturing DBMSCs with 200 mM glucose for 72 h, and then analysed and assessed using the RT-PCR. Results show that glucose modulated DBMSC expression of variety of genes associated with many cellular functions as compared to untreated DBMSC as shown in Tables 1, 2, 3, 4 and 5.

Discussion
Recently, we reported the therapeutic potential of DBMSCs to treat inflammatory diseases, such as atherosclerosis and cancer [14][15][16]19]. Diabetes is another inflammatory diseases [8] where high levels of glucose (an oxidative stress mediator) cause cellular and tissue damage [20][21][22][23]. Therefore, for an effective application of DBMSCs in diabetes, it is important for DBMSCs maintain their normal reparative properties when exposed to high levels of glucose. Here, we studied the functional and phenotypic changes of DBMSCs in response to glucose. First, we examined the effect of different glucose concentrations on the survival of DBMSCs. We report that DBMSCs survive in high levels of 200 mM glucose even when cultured for long periods. DBMSCs also showed increased proliferation potential at high levels of glucose ( Fig. 1D-F), via a reversible mechanism (Fig. 1G-I). This contrasts with our previous finding in which MSCs from the chorionic villi of human placentae (pMSCs) show a reduction in their proliferation under the effect of glucose [18]. This may reflect DBMSC adaption to the elevated oxidative stress levels in normal pregnancy, as a result of their vascular microenvironment where they are directly or indirectly exposed to factors in maternal pregnant blood, which contains high level of oxidative stress mediators [24]. On the other hand, pMSCs in the chorionic vascular niche, are exposed to the fetal circulation, which contains relatively reduced levels of oxidative stress throughout normal pregnancy [13,25]. Glucose also induced DBMSC expression of genes associated with survival, anti-apoptotic [26][27][28][29][30][31][32][33][34][35][36][37], and proliferation [38][39][40] as shown in Table 1. These molecules may prevent the damaging effects of glucose on DBMSCs. However, this needs further investigation to confirm the protective roles of these molecules in the survival and proliferation of DBMSCs from glucose. In this study, preconditioning of DBMSCs with glucose enhanced their adhesion (Fig. 2) possibly via ICAM-1 (Fig. 4A). This is consistent with our previous study which showed that DBMSCs preconditioned with H 2 O 2 showed increased adhesion [14]. Other studies also support our finding, where preconditioning of hematopoietic stem cells with H 2 O 2 also increased their adhesion in vitro and in vivo [41]. Adhesion is the first important biological process required for a successful stem cell engraftment [42,43]. Migration and invasion of MSCs are other important biological processes that occur during MSC engraftment in a disease environment with high level of oxidative stress mediators [42,43]. We found that DBMSCs preconditioned with glucose improved their migration (Fig. 3D). This effect is similar to the effect of H 2 O 2 on the migration of DBMSCs [14], MSCs from the chorionic villi [44] and bone marrow [45]. DBMSCs preconditioned with glucose also improved their invasion (Fig. 3E) via a mechanism that may involve the induction of a number of genes known for their migratory [26-29, 31, 36, 46-51] and invasive properties [26-28, 47, 48], Table 1. These results demonstrate that the engraftment properties of DBMSCs can be improved by glucose pretreatment, possibly via these genes. Thus, preconditioning DBMSCs could be valuable component of cell-based therapies that must act in high oxidative stress environments. However, a future mechanistic study is necessary to confirm this further. In the pancreatic beta islets, the pro-oxidant enzymes (i.e. NOX1-5 and DUOX1-2) increase the production of the reactive oxygen specie (ROS) superoxide, which induces insulin secretion [52][53][54][55][56]. The excessive accumulation of ROS causes beta cell damage, which can be prevented by the antioxidant enzymes (i.e. GPX, CAT and SOD), which act as ROS scavengers, and therefore inhibit insulin secretion [52][53][54][55][56]. In this study, glucose induced and reduced DBMSC expression of genes with pro-oxidant [39,57,58] and anti-oxidant properties, respectively [59], Table 2. Thus, indicating that glucose may direct DBMSCs to activate pathways associated with insulin secretion. This postulate is supported by the finding that glucose also induced DBMSC expression of albumin and NOS2, which are associated with insulin secretion [32,60]. In addition, glucose also reduced DBMSC expression of PXDN, a molecule that triggers diabetes, Table 4 [61].
Generally, a basal level of ROS is required to stimulate basic cellular biological activities (i.e. proliferation, migration, and invasion). ROS is also required for insulin secretion by beta cells. As discussed above, the high level of ROS damages tissue, and consequently this is avoided by the antioxidant enzymes which are produced to scavenger ROS [62]. Glucose simultaneously induced DBMSC expression of both pro-oxidant (Table 4) and anti-oxidant genes [40,50,[63][64][65][66], Table 4. Therefore, DBMSCs may respond to glucose induction of ROS by generating antioxidants to prevent cellular damage and also to regulate insulin secretion probably by inducing the expression of UCP2 (Table 4), which has anti-insulin secretion activity [63]. In diabetes, the oxidative stress mediators generated by the high level of glucose, stimulate the recruitment of immune cells to the site of tissue injury, and this in return will intensify tissue damage [67]. One of the therapeutic strategies, is to reduce the recruitment of immune cells to the injured tissue. In this study, glucose reduced DBMSCs expression of thioredoxin (Table 4), an oxidative stress molecule that increases the recruitment of immune cells [67]. Glucose also increased the anti-inflammatory properties of DBMSCs by increasing their expression of antiinflammatory genes [26,31,34,35,63,64,[68][69][70][71][72][73][74] ( Table 3), and also by reducing their expression of proinflammatory genes including MGST3 and COX2 [75][76][77]. This finding is important, because these anti-inflammatory molecules reduce the recruitment of immune cells [70].
These results indicate that DBMSCs may function as an anti-chemoattractant agent to reduce the recruitment of immune cells to the injured tissues in inflammatory diseases. The property of DBMSCs to ameliorate inflammation is further confirmed by their enhanced expression of B7-H4 in response to glucose (Fig. 4). B7-H4 is a protein that inhibits T-cell proliferation [78], suggesting that DBMSCs may inhibit T cell proliferation, and thus reduce inflammation.
High levels of glucose also causes immune dysfunction that results in a reduction in the antimicrobial activity of the immune cells in diabetic patients, and therefore these patients are at a higher risk to bacterial infection [79]. In this study, glucose induced DBMSC expression of antibacterial genes, [71,73], Table 3. Collectively, these results suggest that DBMSCs may have the potential to treat inflammatory diseases, such as diabetes, by ameliorating inflammation and also by preventing infection associated with hyperglycaemia. This is the first study to show the beneficial effects of glucose on DBMSC functions. Preconditioning of DBMSCs with glucose may increase their therapeutic potential by enhancing their engraftment efficiency  (Fig. 5). In addition, the induction of ROS in DBMSCs by glucose may program these cells into insulin producing cells with ability to counteract inflammation and infection associated with diabetes (Fig. 5). However, future in vitro and in vivo studies are essential to investigate the findings of this study further.   Ethical statement The institutional review board (IRB) at King Abdullah International Medical Research Centre (KAIMRC), Saudi Arabia approved this study. Samples (i.e. placentae and umbilical cords) were obtained from uncomplicated human pregnancies (38-40 gestational weeks) following informed patient consent.
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