| Mesenchymal stem cells (MSCs) have been the optimal target in the development of cell based therapies, while their limited availability and massive death after transplantation remains an issue of concern in clinical application. This study described a novel effect of activated PRP (aPRP) on rat bone marrow MSCs (BM-MSCs), with the former driving a gene program which reduced apoptosis and promoted the regenerative function of the latter under a hostile microenvironment through enhancing the paracrine/autocrine factors.With reverse transcription (RT)-PCR, immunofluorescence, Western blot analysis, we showed that aPRP preconditioning alleviated the apoptosis of BM-MSCs under stress condition induced by H2O2and serum deprivation by enhancing expression of vascular endothelial growth factor (VEGF), platelet-derived growth factor (PDGF) via stimulation the platelet-derived growth factor receptor (PDGFR)/PI3K/AKT/NF-κB signaling ways. aPRP preconditioned GFP-BM-MSCs were further transplanted into rat skin6h after wound surgery occlusion. Histological and other tests were examined from0-22nd days after transplantation. Engraftment of the aPRP preconditioned BM-MSCs not only significantly attenuated self-death and wound size but also improved epithelization and blood vessel regeneration of skin via regulated the wound microenvironment.Thus, preconditioning with aPRP, which reprograms BM-MSCs to tolerate a hostile microenvironment and show better regeneration function by expression higher levels of paracrine factors through PDGFR-a/PI3K/AKT/NF-κB pass way would be a nature and safe enhancer of the effectiveness of transplantation therapy in clinic.PartⅠReduce BM-MSCs death with aPRP preconditioning in vitro under stress condition through enhance paracrine factors by up-regulated the (PDGFR)/PI3K/AKT/NF-κB signaling waysObjectiveTo explore the aPRP preconditioning on the function of BM-MSC under stress condition and study the related mechanismsMethods1. Preparation of PRP and obtaining of growth factors (GFs) concentrationsFirst, PRPs were obtained from6healthy male volunteers (47yr,49yr, and43yr old) who had not taken anti-platelet medications within1week of donation to explore the effects of PRP concentration on the survival and regeneration of rat BM-MSCs. Fur-ther, to minimize the possible influences of hormonal variation on the MSCs, we included only male and AB blood type donors. PRP was prepared as previous method [37], in brief, via double centrifugation of blood. Firstly, the blood was collected and centrifuged at1,500rpm for10min at25℃to separate the platelet-containing plasma from the red cells. Then, the platelet-containing plasma was cen-trifuged at3,000rpm for an additional10min at25℃to separate out platelet-poor plasma (PPP) and re-suspended platelet pel-lets that pooled for PRP. Finally,1part bovine thrombin stock solution (1,000U/ml; Sigma, St. Louis, MO, USA) was added to9parts PRP to activate the platelets. Each sample was incubated for1h at37℃. The resulting supernatants from the clot preparation were referred to as activated PRP (aPRP) and were stored at-80℃until use. We used the aPRP as a trophic cocktail so we didn’t separate or measure the concentrations of GFs.2. Isolation of bone marrow (BM)-MSCs and cell culturePrimary rat BM-MSCs were obtained from male Sprague-Dawley rats (250-300g SD rat,6-10weeks old) and isolated as previously described [38]. And then cells were seeded at2-3×103/cm2in culture flasks filled with12ml of low-glucose Dul-becco’s modified Eagle medium (DMEM; Gibco) with10%fetal bovine serum (FBS; Gibco) and also supplemented with100units/ml penicillin,100μg/ml streptomycin, and2m/ml-glutamine (Gibco) then incubated at370C, in5%CO2. MSCs expanded at the2rd-3th passage were identifiedand cultured for future experiments. Human aor-tic endothelial cells (HAECs) and rat GFP positive BM-MSCs were obtained from a commercial source (catalog number304-05a; HAOEC; R492K-05, cryopreserved rat MSC; Cell Applications). They were plated at4,000cells per cm2and cultured in endothelial growth medium (eGM)(211-500; Cell Applications) or DMEM that con-tained10%FBS and were trypsinized when they reached70-80%confluence. HAECS were positive for factor VIII and Dil-Ac-LDL uptake and GFP-BM-MSCs were tested well with self-renewing, multi-differentiation function and stem cell sur-face markers. They were used at1st passage or2nd-3rd passages. 3. Flow cytometry analysis (FACS) and cellular phenotypingThe adherent cells at2rd-3rd were trypsinized and centrifuged, fixed in neutralized2%paraformaldehyde solution for30minutes. The fixed cells were washed twice and re-suspended in PBS, incubated with fluorescein isothiocyanate (FITC)-labeled an-ti-rat CD34, CD45, CD29, CD44and phycoerythrin (PE)-conjugated CD90, CDllb and CD105(all from BD PharMingen, USA). Non-specific fluorescence and cell morphological features were determined by incubation with isotype-matched mouse monoclonal antibodies (BD PharMingen). Positive cells were analyzed on1x104events at least and were counted by flow cytometry with FAC scan flow cytometer (Becton Dickinson, San Jose, CA). Cellular phenotyping and multi-differentiation as well as CFU-Fs were also tested as previous studies described [39]. BM-MSCs that at1st passage were maintained in adipogenic induction medium for about14days be-fore stained with Oil Red O stain or cultured in osteogenic medium for about21days before assessed by Alizarin Red S stain. Besides, cells at1st passage in culture were also seed at a density of1x103per ml for about14days before stained with Trypan Blue to detect the CFU-F ability.4. Cell preconditioning and exposed to stress conditionsPassage2BM-MSCs were first starved in DMEM with2%FBS overnight then pre-conditioning with DMEM supplemented with various concentration of aPRP (0.1,1%,10%,20%),10%FBS or serum free medium (SFM; StemPro(?) MSC SFM, Invi-trogen) for1,3,5,7days. At each time point, half cells were first starved in DMEM for8h and then treated with different concentration of hydrogen peroxide (H2O2)(0.1,1,10,50,100μm/ml) for8h to induce stress condition, the other half were tested for normal proliferation. The effect of aPRP on the survival of MSCs was first determined by MTT based In Vitro Toxicology Assay Kit (Sigma-Aldrich). Apoptosis was assayed with a cellular dye that detected membrane alterations (phosphatidylserine flip) and stained apoptotic cells (Apoptosis’APOPercentage’ Assay Kit; Accurate Chemical&Scientific Corporation) then assayed on a colorimetric plate reader (Bio-Rad, Hercules, CA). Apoptotic cells were further stained with terminal deoxynucleotidyl transferased UTP nick-end labeling (TUNEL) assay (In Situ Cell Death Detection Kit Fluorescein and TMR red; Roche Applied Science, Indianapolis). Survival cells were excluded by Trypan blue staining. For the detection of the active form of Ser-136phosphorylated Bad and Bcl-xl, protein lysates were prepared and detected by western blot assays and confocal images.5. Preparation of Conditioned Medium (CM)MSCs were first cultured in serum free medium (SF;Gibco) with or without10%aPRP preconditioning for24h or7days (PRP/MSC and MSC) then cells were ex-posed to either normal or stress conditions (10μm/ml H2O2) for8h to prepare CM from MSCs (MSC-CM). Half medium were used to measure the secreted factors VEGF and PDGF, serum free medium and PRP were also tested for these factors. The other half was used to supplement the basal medium for the tube formation assay of HAECs. The collected culture supernatant was centrifuged at1500rpm for10min and either used immediately or stored at-20℃.6. Assays of PDGFR and CytokinesThe same cell samples were used to measure mRNA and protein expression of PDGFR-a by RT-PCR and western blot. The levels of VEGF, PDGF in culture su-pernatants were measured by RT-PCR and a Quantikine ELISA kit (R&D Systems) according to the manufacturer’s instructions (http://www.rndsystems.com/).7. In vitro Tube Formation AssayIn vitro capillary morphogenesis assay was performed in96-well plates covered with Matrigel (BD Biosciences). Matrigel (50μl;10-12mg/ml) was pipetted into culture wells and polymerized for30-60min at37OC. HAECs incubated in basal medium supplemented with MSC-CM prepared as before and then were plated at4x104per cm2in triplicate and incubated under stress conditions. The plates were photographed at6and24h. The capillary morphology was examined with a Leica TCS SP5con-focal laser scanning microscope (Leica Microsystems, Mannheim, Germany) and further quantified by measuring the percentage field occupancy of capillary projec-tions, as determined by image analysis. Six to nine photographic fields from three plates were scanned for each point.8. RNA purification, cDNA synthesis and PCRBM-MSCs at passages2were seeded at1x106per cm2on tissue culture plastic, fol-lowed by10%aPRP,10%FBS or SFM preconditioning for24h or7day separately under normal or stress condition. Total RNA was extracted using Trizol reagent (Invitrogen, Grand Island, NY) according to the manufacturer’s instructions. cDNA was prepared using the Super Script First-Strand synthesis system (Invitrogen, Grand Island, NY) using oligo-dT primer. The PCR mix consisted of13μL sterile deionized water,2.5μL Taq buffer, MgC12(final concentration2.5mM),0.28mM of dNTPs,0.4μM of each primer,1.5U of Taq DNA polymerase (5U/μL; Fermentas, Vilnius, Lithuania) and4μL of cDNA. The PCR programme for both pairs of primers was as follows:94℃for2min followed by33cycles of94℃(30s),58℃(30s) and72℃(30s) for denaturation, annealing and extension, respectively. PCR products were analyzed by electrophoresis in1.5%agarose gel and visualized by UV fluorescence after staining with ethidium bromide. The image captured using the inbuilt camera in Gel DocTM XR+Imaging System (BIO-RAD Molecular Imager, Bio-Rad Laborato-ries, Inc. California, USA) under UV light. PCR amplification was performed using the following primer sets The relative amount of each transcript in each sample was normalized to the reference gene β-actin which was considered as a loading control.9. Western blotBM-MSCs at passage2were preconditioned with10%aPRP,10%FBS and SFM for24h and7days under normal or stress condition. And then protein from the cells were extracted by cells lysis buffer (NP40Cell Lysis Buffer; life technologies) and then quantified with a Bicin Choninic Acid (BCA) protein assay kit (Thermo). The proteins were then subjected to SDS-PAGE and transferred to PVDF membrane (Millipore, Billerica,MA, USA) using a semi-dry electro blotting system. Then blocked with5%skim milk in PBS, which were substituted by goat serum when de-tecting the phosphorylated protein; after that the membranes were incubated with diluted primary antibodies, including rabbit polyclonal anti-rat antibodies to PI3ki-nase p85, phosphorylated PI3kinase p85,AKT1, Ser-473phosphorylated AKT-1, NF-κB and anti-rat antibodies to Bcl-xl, Ser-136phosphorylated Bad (1:1000dilu-tion; from EPITOMICS, Abeam, USA) and PDGF, VEGF, PDGFR-α, phospho-PDGFR-α, β-actin (1:1000dilution; all from Abeam) at40C overnight. Af-ter washing with tris-buffered saline solution containing0.1%Tween20[Tris-Buffered Saline Tween (TBST) Bio-Rad],co-incubated the membrane for1hour in a1:2000dilution of goat anti-rabbit biotin secondary antibody and in a1:2000dilution of the alternate goat anti-rabbit alkaline phosphatase secondary anti-body in10mL of WesternDotTM blocking buffer (WesternDotTM625Goat Anti-Rabbit Western Blot Kit; life technologies).After washing again using TBST, the protein bands were detected by chemiluminescence(1:2000;Santa Cruz).Protein bands were visualized using enhanced chemiluminescence (Pierce) and exposed to x-ray film. Protein levels were normalized to β-actin which was considered as a loading control relative optical density of protein bands was measured after subtract-ing the film background. β-actin was used as a protein-loading control.10. Immunocytochemistry and confocal laser scanning microscopyFor the survival factors VEGF, PDGF and PDGFR-a, AKT, NF-κB, Bad protein de-tection, cells at passage2preconditioning with10%aPRP,10%FBS, SFM for24h,7days under normal or stress condition were grown to semi-confluence on glass coverslips and fixed in3.7%buffered paraformaldehyde for15min, permeabilised with0.1%Triton X-100in PBS for30min. And then use the goat serum (Beyotime; China) to block the irrelative antigens for1h. After that cells were incubated over-night with the following primary rabbit anti-rat antibodies:PDGF, VEGF, PDGFR-a (1:100; Abcam), AKT, NF-κB, and Bad (1:100; EPITOMICS; Abcam), FITC and DyLight(?)488-conjugated goat anti-rabbit IgG(1:500; Santa Cruz) were used as the secondary antibodies and incubated cells at370C for1h. Staining of40,6-diamino-2-phenylindole (DAPI)(Sigma Aldrich) was used to visualize all nuclei. Finally, MSCs were examined with a Leica TCS SP5confocal laser scanning micro-scope. Series of optical sections (1024×1024pixels each) at intervals of0.8μm were obtained and super imposed to create a single composite image.All the detections in vitro were repeated three to four times when pretreated with20mM tyrphostin AG1295(PDGFR inhibitor; Sigma-Aldrich),30mM LY294002(PI3K inhibitor; Cell Signaling Biotechnology), and30mM SC-66(AKT inhibitor; Sigma-Aldrich), respectively for1h or pre-incubation with VEGF (MAB564), PDGF-AA (MAB1055) neutralizing antibodies (the final concentrations were10μg/ml; R&D Systems) separately or together. And all these inhibitors were firstly tested by assayed by LDH Cytotoxicity Assay (www.sciencellonline.com) to be little toxicity towards cells before we used (data not shown)Results1. aPRP preconditioning promoted cell viability and reduced cell apoptosis under stress condition2. aPRP preconditioning enhanced PDGFR-a expression by rat BM-MSC under stress condition3. aPRP preconditioning enhanced VEGF and PDGF secretion by BM-MSCs4. The paracrine factors of VEGF and PDGF contributed to anti-apoptosis effect under stress condition5. VEGF and PDGF promoted angiogenesis of HAECs under stress condition.6. aPRP preconditioning up-regulated survival genes PI3K,AKT1and activated the downstream targets of NF-κB7. The PDGFR-a/PI3K/AKT1signaling way accounted for aPRP-induced various effects on BM-MSCs under stress conditionConclusionThe nature growth factors in aPRP for preconditioning enhanced survival genes of PI3K, AKT, NF-kB expression by MSCs through PDGFR especially under stress condition which might account for the anti-apoptosis and other effects of PRP on MSCs. PartⅡReduced grafted-BM-MSCs death with aPRP preconditioning in vivo and increased wound closure, epithelization and blood vessel density by enhanced the expression of p-PI3K, p-AKTl and NF-κBMethods1. Wound model and Cell Transplantation Male Sprague-Dawley rats (250-300g,6-10weeks old) were anesthetized with xylazine (20mg/kg) and ketamine (100mg/kg). A2.0×2.0cm sized skin defect was made on the back of each rat with surgical scissors.The wounds were treated once with a total of200μl GFP-rat BM-MSCs (Cell Applications) that preconditioning with aPRP (group1) for3days, GFP-rat BM-MSCs (group2) without preconditioning by directly injected into the wounds margins or wound bed or wound skin without any treatment (group3)(n=7). Then wounds were covered with polyurethanefilms (Department of Plastic Surgery, Guangzhou General Hospital,GuangDong) which were changed every3days to measure the area of the wounds.2. Evaluation and quantification of survival of the transplanted GFP-positive BM-MSCsFor the transplanted cell survival, the wounds were sampled at1stday,3rdday,7thday,12thday,22ndday in full-thickness, including the underlying muscle. The transplanted GFP-positive cells were counted at each time point on five serial coronal sections per skin (2mm apart) using unbiased computational stereology. At the meantime, we tested the GFP signal intensity by optical in vivo imaging (Xenogen IVIS(?) Kinetic) according to the instructions(http://www.amv-europe.com/preclinical/products/in-vivo-imaging-systems/ivis-kinetic.html). Half of each specimen at7th day was used for immunohistochemistry, and the other half was used for the tissue TUNEL staining. For TUNEL Staining, the apoptotic cell death was detected using an In Situ Cell Death Detection Kit (TMR red; Roche Applied Science, Indianapolis) according to the manufacturer’s instructions. For the histological analysis, the histological specimens were fixed in formalin, embedded in paraffin, sectioned transversely at a thickness of4μm and then primary antibodies:rabbit anti-rat p-AKT1, p-PI3K, NF-κB (1:100; EPITOMICS, Abcam) were incubated overnight,4℃; horseradish peroxidase-coupled goat anti-rabbit IgG (1:1000; Santa Cruz) were used as secondary antibody and incubated with cells for2hours at room temperature followed by a light microscope (KS400, Zeiss, Munich, Germany) analysis.3. Measurement of wound size and Microscopic tissue regeneration analysisThe macroscopic wound area were quantified by processing photographs taken at various time points,1st,4th,8th,12th,16th,20th,22nd day by tracing the wound margin and calculating the pixel area relating it to the ruler using fine resolution computer mouse. The wound area was calculated as the percentage of (the initial wound area-[wound area at time]/[initial wound area]×100%). Microscopic tissue regeneration was determined on Masson’s trichrome-stained tissue sections using a light microscope observation. Morphometric analysis was performed on digital images using the imaging software Image Pro Plus x6.0(Mediacybernetics, Silver Spring, MD).4. Measurement of blood vessel density and epithelial regeneration and paracrine factorsSections collected at7thday and22ndday, each specimen was used for frozen section immunohistochemistry staining, briefly, samples were embedded in OCT compound (Tissue-Tek4583(?), Sakura Finetek USAInc, Torrance, CA) and cut into10-μm-thick sections at-22℃. To stain regenerated arterioles and epidermis and paracrine factors, sections were incubated with mouse anti-rat a-Smooth Muscle Actin(a-SMA) and VEGF;rabbit anti-rat CK5and PDGF antibodies(all from Abeam) after fixed in ice methanol for1h and permeabilised with0.1%Triton X-100in PBS for30min, following that sections were incubated in Alexa Fluor488-conjugated donkey anti-mouse and FITC-conjugated donkey anti-rabbit IgG (catalog no.A21206and no.A31571;Invitrogen) at37℃forl h. Then sections were counterstained with DAPI (Sigma Aldrich) and examined by fluorescence microscopy (Olympus, Tokyo, Japan). Ten slides were randomly selected from the middle part of each sample for analysis.Results1. aPRP preconditioning reduced grafted-BM-MSCs death in vivo2. aPRP preconditioning strengthened wound repair and skin tissue regeneration by graft-BM-MSCs3. The potential mechanism of the positive effects of aPRP preconditioning on BM-MSCs before transplantation was enhancing paracrine factors and activation of survival genes, AKT, PI3K, NF-κB.All the grafted-cells survival, skin regenerative function of MSCs by PRP preconditioning as well as potential mechanism results were also observed at1st h,2nd h,4th h,2nd,4th,6th,8th,12th,16th,18th,20th day after cells were transplanted and the surgery of wound were occurred in all the group animals (data were not shown).ConclusionTaken together, we argued that the aPRP preconditioning enhanced grafted BM-MSCs survival and skin regeneration via up-regulation of paracrine factors through PI3K/AKT/NF-κB pass ways which were interesting for the coincidence with the mechanism displayed in vitro. |
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