Stem cell

 STEM CELL

Abstract

It's unclear whether extracellular vesicles( EVs) from mesenchymal stem cells( MSCs) have a direct defensive effect on pancreatic islands. In addition, whether cultivating MSCs in three confines( 3D) rather of a monolayer( 2D) can induce changes in the weight of EVs that grease the polarization of macrophages into an M2 phenotype has not been delved . We sought to determine whether EVs from MSCs dressed in 3D can help inflammation and dedifferentiation in pancreatic islands and, if so, whether the defensive effect is superior to that of EVs from 2D MSCs. mortal umbilical cord blood-( hUCB-) MSCs dressed in 3D were optimized according to cell viscosity, exposure to hypoxia, and cytokine treatment grounded on the capability of the hUCB- MSC- deduced EVs to induce the M2 polarization of macrophages. Islands insulated from mortal island amyloid polypeptide( hIAPP) heterozygote transgenic mice were dressed in serum- deprived conditions with hUCB- MSC- deduced EVs. EVs deduced from 3D hUCB- MSCs had more abundant microRNAs involved in M2 polarization of macrophages and had an enhanced M2 polarization capability on macrophages, which was optimized when the 3D culture condition was cells per hunk without preconditioning with hypoxia and cytokine exposure. When islands insulated from hIAPP heterozygote transgenic mice were dressed in serum- deprived conditions with hUCB- MSC- deduced EVs, the EVs deduced from 3D hUCB- MSCs suppressed the expression of proinflammatory cytokines and caspase- 1 in pancreatic islands and increased the proportion of M2- concentrated island- resident macrophages. They bettered glucose- stimulated insulin stashing, reduced the expression of Oct4 and NGN3, and convinced the expression of Pdx1 and FoxO1. The lesser repression of IL- 1β, NLRP3 inflammasome, caspase- 1, and Oct4 and induction of Pdx1 and FoxO1 were set up in islands dressed with the EVs deduced from 3D hUCB- MSCs. In conclusion, EVs deduced from 3D hUCB- MSCs optimized for M2 polarization downgraded nonspecific inflammation and saved β- cell identity of pancreatic islands

1.INTRODUCTION

Intravenous injection of mesenchymal stem cells( MSCs) improves the function and survival of pancreatic islands and preserves β- cell identity in beast models of type 2 diabetes( 1 – 3) and pancreatic island transplantation( 4 – 6). An important medium of these benefits is the capability of MSCs to induce polarization of macrophages into the M2 subtype. In type 2 diabetes and primary island graft failure after pancreatic island transplantation, the progression of seditious responses similar as the increase in island macrophage infiltration, the polarization of macrophages into the M1 subtype, and the inflammasome activation play an essential part in the progression of β- cell failure( 7). Clinical trials on the infusion of MSCs into cases with type 2 diabetes( 8) and donors of pancreatic island autotransplantation have been conducted without severe adverse events( 9). The use of MSC- deduced extracellular vesicles( EVs) offers a promising volition to MSCs themselves by reproducing their natural function in delivering nucleic acids, proteins, and lipids to the original medium of damaged cells or apkins( 10, 11). also, EVs could drop safety enterprises because they're nonimmunogenic and not likely to beget maldifferentiation( 12, 13), which is a clinical concern of curatives using MSCs. Interestingly, it has been suggested that EVs deduced from MSCs, without fresh goods from MSCs themselves or other factors of their secretome, can ameliorate pancreatic β- cell survival and insulin perceptivity in rodents with low- cure streptozotocin and high- fat diet- convinced diabetes( 14). This indicates that the use of MSC- deduced EVs could be a new approach to delaying the progression of β- cell failure in type 2 diabetes and primary island graft failure after pancreatic island transplantation. MSCs dressed using the three- dimensional system( 3D MSCs), which more reflects an in vivo terrain, constantly parade enhancedanti-inflammatory, angiogenic, and towel reparative/ regenerative goods with bettered cell survival after transplantation( 15). similar benefits are intermediated, at least in part, by EVs from MSCs( 16). In addition to the changes in gene expression biographies similar as the expression of genes with cell stemness and migration capability in 3D MSCs, EV product increases with the three- dimensional culture system, and the performing EVs can have a weight profile that's different from 2D MSCs( 17). In a recent study, total RNA sequencing using coming- generation sequencing platforms on mortal amnion- deduced 2D and 3D MSCs revealed profound transcriptome changes, including enhanced stashing of C- C motif chemokine ligand 2( CCL2), C-X-C motif chemokine ligand 12( CXCL12), and bone morphogenetic protein 2( BMP2), which could contribute to a medium favouring polarization of macrophages into the M2 phenotype( 18). still, a former study conducted in an beast model of bleomycin- convinced lung fibrosis suggested that EVs produced from 3D MSCs didn't demonstrate enhanced immunomodulatory parcels compared with 2D MSC- deduced EVs( 19), indicating that optimization of 3D MSC- deduced EVs in each complaint model is needed. EVs from cytokine- preconditioned MSCs contain several microRNAs( miRs) that can induce macrophage polarization into the M2 subtype, in discrepancy to EVs from resting MSCs( 20, 21). still, whether the weight changes in EVs deduced from 3D MSCs include an enhanced capacity for the polarization of macrophages into the M2 phenotype has not been delved , and there has been no specific study that compared the benefits of EVs from either 2D or 3D MSCs on pancreatic islands. Although it has been suggested that intraperitoneal injection of 3D MSCs rather of 2D MSCs in a multiple low- cure streptozotocin- convinced diabetes model more attenuates seditious processes in pancreatic islands and improves glycemic control( 22), the part of 3D MSC- deduced EVs in this benefit, if any, has not been determined. thus, we delved whether EVs from MSCs dressed in 3D have direct defensive goods against inflammation and dedifferentiation on pancreatic islands and, if so, whether the defensive goods are superior to those of EVs from monolayer- dressed MSCs( 2D MSCs).

2. Materials and Methods

2.1. brutes and Cell Culture Mouse islets were isolated from heterozygous mortal islet amyloid polypeptide( hIAPP) transgenic( hIAPP/-) 8-12-week-old FVB/ N mice( Jackson Laboratory, Bar Harbor, ME, USA). The Institutional Animal Care and Use Committee of Samsung Biomedical Research Institute approved all beast experimental protocols in this study. mortal umbilical cord blood-( hUCB-) MSCs were isolated according to a reported system( 23). Umbilical cord blood( UCB) units attained from full- term deliveries were collected from the unborn placenta with the informed concurrence of the mothers and all actors handed informed concurrence for The mortal monocyte cell line THP- 1 was bought from the Korean Cell Bank( Seoul, Korea) and maintained in complete RPMI 1640 media.. Culture of 3D hUCB- MSC Places The hUCB- MSCs were grown in minimum essential medium- birth( MEM- α; Gibco, Waltham, MA, USA) supplemented with 10 fetal bovine serum( FBS; Gibco) and 1 penicillin/ streptomycin( Gibco) at 37 °C and 5 CO2 until passage 5. To gain 3D places, hUCB- MSCs at three to five passages were planted into round- bottom 96- well plates coated with poly( 2- hydroxyethyl methacrylate)( pHEMA; Sigma- Aldrich,St. Louis, MO, USA) reagent prepared in 95 ethanol. In each well containing 100 μL of MEM- α( Gibco) supplemented with 10 FBS( Gibco) and 1 penicillin/ streptomycin( Gibco),,, and MSCs well were planted and dressed at 37 °C and 5 CO2 for three days(,, and cells/ hunk appertained to as2.5 K,6.25 K, and 25 K 3D hUCB- MSCs, singly). The cell numbers were predicated on the number of seeded cells before hunk conformation. also, the 3D places were transferred into a Petri dish to gain fortified media( CM). Only one hunk was formed per well of the 96- well plate, and all 96 places were transferred into 15 mL of MEM- α( Gibco) supplemented with 10 Exo-free FBS( Gibco) and 1 penicillin/ streptomycin( Gibco) in a 100 mm fringe Petri dish. The 3D places were dressed at 37 °C and 5 CO2 for six days. This reacted in a total of 39 mL of conditioned medium per Petri dish with a total of 96 hUCB- MSC places.. sequestration and Characterization of EVs Monolayer( 2D)- dressed and 3D- dressed hUCB- MSC places were incubated in MEM- α( Gibco) supplemented with 10 exosome- depleted FBS( Gibco) for 72 h at 37 °C in a fully humidified 5 CO2 atmosphere. The CM was gathered and centrifuged at for 10 min to remove cells and debris. also, the supernatant was concentrated using an Amicon Ultra Centrifugal Filter( 100 kDa cut- off) Unit( Merck, Burlington, VT, USA). EVs were isolated from concentrated CM using a Total Exosome sequestration attack( Invitrogen, Thermo Fisher Scientific, Waltham, MA, USA). The size distribution and attention of EVs were measured by nanoparticle shadowing analysis( NTA) using a NanoSight NS300( Malvern, Worcestershire, UK). The sample was thinned to a number attention of between 108 and 109 patches per milliliter. The morphology of the EVs was characterized using transmission electron microscopy( TEM; Hitachi HT7700,HitachiLtd., Tokyo, Japan). The EVs were fixed with 4 paraformaldehyde and spotted onto a carbon- coated 300- mesh grid. also, the samples were stained with 1 uranyl acetate. subsequently, the samples were examined at100.0 keV

3. Results and Discussion

3.1. Characterization of EVs deduced from Umbilical Cord Blood MSCs To confirm the identity of the insulated hUCB- MSCs, face expression of CD90, CD44, CD73, and CD105 and the nonexpression of the negative marker blend( CD45, CD34, CD14, CD11b, CD19, and HLA- DR) were verified using inflow cytometry( Figure 1( a)). After conditioned medium was attained during 2D and 3D societies of hUCB- MSCs, hUCB- MSC- deduced EVs were insulated from each group using a polymer- grounded rush system. The mug- shaped morphology of the insulated EVs was verified in both 2D and 3D hUCB- MSC- deduced EVs When the expression of EV labels was examined using western blotting, CD63 and TSG101 were expressed, and the ER and Golgi labels CANX and GM130 weren't expressed( Figure 1( d) and Supplementary Figure S1)

4. Discussion and Conclusions

In this study, miRs involved in the M2 polarization of macrophages were more abundant in EVs  deduced from 3D hUCB- MSCs than in 2D hUCB- MSC- and 3D hUCB- MSC-  deduced EVs that  held an enhanced M2 polarization capability on THP- 1 monocytes. The defensive  goods of the 3D hUCB- MSC-  deduced EVs on hIAPP heterozygote transgenic mouse islands were more potent than 2D hUCB- MSC-  deduced EVs in terms of reducing nonspecific inflammation and conserving β- cell identity, and this was associated with a advanced proportion of island- resident macrophages with M2 polarization labels. To the stylish of our knowledge, these results are the first  substantiation that 3D hUCB- MSC-  deduced EVs have a direct defensive effect on hIAPP- expressing pancreatic islands, and the M2-  centralizing capability of the EVs might be an important contributor to  similar defensive  goods.   The lesser M2-  centralizing capability of EVs  deduced from 3D hUCB- MSCs compared to those  deduced from 2D hUCB- MSCs is a  new finding of this study. Although a recent study revealed that  mortal adipose mesenchymal stem cell-( AMSC-)  deduced EVs can directly induce the M2 polarization of macrophages through the direct effect of miRs related to M2 polarization,  similar as that of miR34a- 5p and miR 146a- 5p( 20), it has not been determined if the  situations of  similar miRs can be amplified in 3D  societies of hUCB- MSCs. In the optimized 3D culture conditions in this study, miR34a- 5p and miR 146a- 5p were more abundant in EVs  deduced from 3D hUCB- MSCs than in those  deduced from 2D hUCB- MSCs, indeed when the 2D MSCs were preconditioned by IFN- γ and TNF- α. In addition, miR127- 3p was less abundant in 3D than in 2D hUCB- MSC-  deduced EVs. miR- 34 inhibits the recap of proinflammatory cytokines by targeting Notch1, and miR- 146 targets NF- κB signaling  intercessors  similar as IRAK1 and TRAF6 to promote the expression of M2- associated genes( 20, 27, 28). These results would be applicable to the enhanced defensive  goods of 3D MSC-  deduced EVs on β- cells because macrophages are associated with inflammation in island cells and β- cell dysfunction( 7). The injection of hUCB- MSCs into a type 2 diabetes mouse model  concentrated M1 macrophages to M2 macrophages in islands( 3), reduced apoptosis of β- cells, and increased PDX- 1 and MafA expressions. The results of our study indicate that  analogous  goods can be reproduced by 3D hUCB- MSC-  deduced EVs alone, without the help of cellular  factors.   In addition to comparing miRs related to M2 polarization, we compared the protein expression biographies of 2D and 3D hUCB- MSC-  deduced EVs. Proteomic analysis of 3D hUCB- MSC-  deduced EVs( Figure 3( b)) demonstrated markedly upregulated expression of CHI3L1, IL- 6, monocyte chemoattractant protein- 1( MCP- 1), and IL- 27. CHI3L1 is an debaser of the PI3K/ AKT signaling pathways( 29) and M2  develop isolation of macrophages for Th2 inflammation( 30), IL- 6 and MCP- 1 are corrupters  of M2 mature isolation of macrophages( 31), and IL- 27 is a cytokine involved inanti-inflammatory and vulnerable-nonsupervisory functions and inhibits Th2,  ingrain lymphoid cell- 2( ILC2), and Th17 responses( 32). also, compared to 2D hUCB- MSC-  deduced EVs, there was  dropped expression of dickkopf 1( Dkk- 1), an asset of Wnt signaling( 33 – 35). Wnt/ β- catenin plays a  part in the development and function of insulin- producing β- cells( 36). These differences in protein expression biographies might have contributed to the lesser defensive  goods of 3D hUCB- MSC-  deduced EVs on island inflammation and dedifferentiation in this study.   Although the results of our study are in line with  former  exploration suggesting the superior immunomodulatory  goods of 3D MSCs over 2D MSCs( 15, 16, 18, 22), some  former studies reported  antithetical results( 19, 37). In one study, T cell- suppressive  capacities of MSCs were observed only in 2D MSCs and not in 3D MSCs; in that study, the T cell- suppressive  capacities of 3D MSCs were  incompletely restored by addition of a corticosteroid( 37). Another  former study compared the immunomodulatory energy of 2D MSC-  deduced EVs and 3D MSC-  deduced EVs in vitro and  also compared theiranti-inflammatory and antifibrotic capabilities in vivo using a bleomycin-  convinced lung fibrosis model( 19). In that study, the in vitro immunomodulatory energy of 2D MSC-  deduced EVs and 3D MSC-  deduced EVs was compared after IFN- γ stimulation. The T cell  repression capability in terms of indoleamine,3- dioxygenase  exertion after IFN- γ stimulation and macrophage phenotype in terms of phagocytosis  exertion were lower in 3D MSC  trials, indicating polarization into the M1 subtype, although they didn't directly measure M1 or M2 labels on macrophages( 19). In our study, the M2-  centralizing capability of 3D MSCs was optimized when 3D MSCs were generated with sufficient cell  figures, without hypoxia or cytokine stimulation. In 25 K 3D MSCs, cytokine stimulation reduced the M2-  centralizing capability. thus, it's possible that the immunomodulatory  parcels of 3D MSC-  deduced EVs could be lost if 3D culture conditions aren't optimized. We also used a different source of MSCs(  mortal UCB- MSCs in the current studyvs.  mortal lung towel-  deduced MSCs or bone gist-  deduced MSCs in the  former study( 19)) and different  complaint models( serum- deprived culture of  insulated pancreatic islands and cocultivation of hIAPP- producing pancreatic islands, MSCs, and macrophages). In this  environment, it's  cheering that the livery- sized 3D MSCs produced by nanopatterned culture plasticware averted β- cell death in a multiple low- cure streptozotocin-  convinced diabetes model( 22), in which vulnerable cell infiltration and progressive loss of β- cells  generally occurs. Whether 3D MSC-  deduced EVs alone without the cellular  element would have  analogous benefit should be explored in  unborn  exploration.   An important translational  eventuality in our study is the use of EVs  deduced from 3D hUCB- MSCs in the pretransplant  societies of  insulated  mortal islands before clinical island transplantation. Our  former study showed that serum  privation, as in pretransplant  societies in clinical intraportal island transplantation to avoid the use of beast-  deduced accoutrements , induces an  seditious response in  dressed hIAPP/- islands indeed without the prolonged culture with hyperglycemia( 24). This study  hypothecated that the addition of EVs  deduced from 3D hUCB- MSCs during the pretransplant culture of islands could  devaluate the  seditious response and loss of β- cell identity caused by serum  privation. In this study, 3D hUCB- MSC-  deduced EVs promoted the M2 polarization of island- resident macrophages and reduced the inflammasome activation  convinced by serum  privation in hIAPP heterozygote mouse islands. This redounded in  bettered island viability and insulin response to glucose, attenuation of proinflammatory cytokine expression, and preservation of β- cell identity. In addition to the enhanced M2 polarization capability of 3D hUCB- MSC-  deduced EVs, this  remedial effect could affect from differences in the  weight and the proteome biographies of EVs  deduced from 2D and 3D hUCB- MSCs, which include several angiogenesis- related cytokines.   Several limitations of this study should be  bandied. First, the 3D culture condition has not been optimized in  trials with primates or  mortal islands using 3D hUCB- MSC-  deduced EVs. The  attention of 3D hUCB- MSC-  deduced EVs should be optimized using  mortal islands before clinical  operation. Second, whether the results of this study can be reproduced by the systemic use of 3D hUCB- MSC-  deduced EVs in type 2 diabetes or island transplant donors remains unclear. Third, we assessed only a limited number of miRs that promoted M2 polarization of macrophages in a  former study using cytokine- pretreated 2D MSCs, rather than a systemic comparison of all miRs related to macrophage M1/ M2 polarization. Fourth, we didn't  probe whether 3D MSC-  deduced EVs could modulate vulnerable responses of T cells and other  factors of vulnerable system as well. Although we  presume that 3D MSC-  deduced EVs might be helpful for  forestallment of island graft rejection by alloimmune responses as well, the data presented in this study is  inadequate to support the  thesis.   In conclusion, we  set up that EVs  deduced from 3D hUCB- MSCs can  cover hIAPP- expressing pancreatic islands against nonspecific inflammation and dedifferentiation, and these benefits were significantly lesser than those of 2D hUCB- MSC-  deduced EVs. These results might be, at least in part, due to the enhanced M2 polarization capability of the EVs  deduced from 3D hUCB- MSCs compared to those  deduced from 2D hUCB- MSCs on island- resident macrophages