Mesenchymal Stem Cells Modulate Immune Responses Combined With Cyclosporine A In A Rat Renal Transplantation Model

Ⅰ. Isolation, Culturing and Identify of Rat MSCsfrom the Bone Marrow in vitroObjective: To establish immortalized rat MSC lines in vitro, to providesource for cellular transplant.Methods: One month old male Wistar rats was killed and degermed to getlong bones. Bone marrow cells were collected by flushing the longbones with DMEM medium (supplemented with 15% fetal bovineserum) and purified by density centrifuge,after Cultured the adherentcells were chosen for serial subcultivation. The third generation cellsin good growth condition was harvested and trypsinized to form cellsuspension,counted and noted the growth curve. The cell suspensionwas reserved in frozen condition supplemented with DMSO andfetal bovine serum medium. In osteocyte differentiationcondition: cells were cultured with dexamethasone,β-glycerolphosphate and ascorbate for 15 days,then stained with alkalinephosphatase(ALP). In adipocyte differentiation condition: cells werecultured with penicillin,streptomycin, transferrin,insulin;linoleic acid,dexamethasone and 3-isobutyl-1-methylxanthin for 5days,subsequently, cells were cultured for 5 days with MSC culturemedium supplemented with rapid insulin without FBS, thenconfirmed by Oil Red O staining.Results: Adherent cells were observed in cell culture flasks after theIsolated MSC cultured for 24h,nonadherent cells were removed bychanging the culture medium.The cell growth was obvious on the 3-4d,and after passage the cellsgrown rapidly to fill the floor of cell culture flasks and achievedconfluency. Purified MSC fibroblast-like cells could be harvestedafter 3 generations,and could passage for at least 10 generations.There was no obvious influence to cell growth when reserved infrozen condition. In the adipocyte differentiation, the accumulation oflipid-rich vacuoles within cells was observed on day 2 afterinduction, and the lipid was richer on the 4-5 day. In the osteocytedifferentiation, the MSCs formed aggregates or nodules andincreased their expression of alkaline phosphatase, and calciumaccumulation was evident after 3 days. The inductions wereconfirmed with staining.Conclusion: 1)MSCs are a type of stem cells with strong proliferativeactivity and sustain self-renewal at least for 10 passage. They have afeature of pluripotent and could differentiate to a variety of cell types in some condition; 2)Inoculated by a density of 200/cm~2 andtrypsinized with 0.25% trypsin for 5 min are effective for the growthand passage of Wistar rat MSCs; 3)The methods of obtaining ratMSC with density gradient centrifugation and adherence cultivationwas convenient,and could provid immortalized rat MSC lines.Significance: Our research demonstrates extensive proliferation andpluripotent of MSC, and provids a convenient harvest method forcellular transplant therapy.ⅡEstablishment of a Modified Model of Rat Renal TransplantationObjective: To establish a modified model of rat renal transplantation forhigh achievement ratio.Methods: Wistar and Lewis mice were used as the donors and therecipients. Left donor's kidney was perfused with ice-cold solution.End-to-side anatomosis of recipient's abdominal aorta to the donor'swas performed between two points. End-to-end anastomosis of bothrenal veins was performed interrupted. Urinary tract reconstructionwas accomplished by suturing the donor ureter with a bladder patchto the recipient bladder. A silk thread was remained in the operation and this silk thread was tigated later in the second day after theoperation instead of nephrectomy to reduce the strike of the extraoperation for increasing the operative achievement ratio.Result: In advance test of 30 operations on 60 rats, 21 rats were survived,the operative time of the donor's was 50±14min, and the recipient'swas 75±22min., including the warm ischemia time (about 1min), thecold ischemia time (60±11min), the whole operative time was140±30 min. Complications including anastomosis hemorrhage atarkery, rejection, renal failure, infection, thrombosis and uretalobstruction were observed. Achievement ratio is about 70%. Informal test of 36 operations on 72 rats, 34 rats were survived, theoperative time of the donor's was 35±12min, and the recipient's was65±10min. including the warm ischemia time (about 1 min), the coldischemia time (40±8min). Achievement ratio is about 95%. Onereason of the abortive cases was anastomosis of both renal veinshemorrhage, and another was thrombosis of renal artery.Conclusion: The modifiability of this model of rat renal transplantation isthe infusion of left kidney with gravity at constant pressure andprepared at prime position, and the grafted kidney was put at the leftside. Direct anastomosis is used in artery-to-artery and vein-to-vein.Urinary tract reconstruction was accomplished by suturing the donorureter with a bladder patch. Delay the nephrectomy. The whole operation is processed under the microscope. Although it is difficultin operation, the effect is satisfying.Significances: The modifiability in operation is successful to establish amodel of rat renal transplantation, and reduce the effect on the modelwith the operation.ⅢMSCs Modulate Immune Responses Combined withCyclosporine A in TransplantationObjective: To observe the effect of rat MSCs in rat renaltransplantation, study the immunological regulation and reparationof MSCs,and investigate the consideration that reduce dose ofCyclos-porine A (CsA) and even induce immune tolerance byapplication of MSCs.Methods: Rats bearing renal allografts were divided randomly into fourgroups, which was given different fore treatmen and postoperationtherapy respectively.(1)MSC monotherapy group (group 1): therecipients were engrafted respectively one week beforetransplantation, before reperfusion,one week after operation and twoweeks after operation, with MSC(1×10~7, purified from Wistar rat bone marrow cells) by intravenous injection,and without specialdrug treatment; (2)CsA therapy (group 2): CsA was injected to therecipients intraperitoneally at a dosage of 0.5 mg/kg/d from day 2after transplantation without fore treatment; (3)MSC and commondose CsA combination therapy (group 3): the recipients were treatwith MSC the same as group 1,and CsA was injected at a dosage of0.5 mg/kg/d after transplantation;(4)No therapy group (group 4): nofore treatment was given, and PBS was injected intraperitoneallyafter transplantation as control. We tested the values of S-Cr of eachanimal in four groups on day 3,5,10,15,25 and 40 aftertransplantation. Graft histology of 2 recipients from each group washarvested on day 4, the production of inflammatory mediators,including IL-1β, TNF-αand TGF-β1, was analyzed by RT-PCR.Observed survival days of other animals.Results: Group treated with CsA monotherapy (group 2) orcombination therapy (group 3) prolonged animal survivalsignificantly compared with no therapy group (group 4) (P=0.002,0.003).Indeed, animal survival of MSC monotherapy group (group 1)was prolonged compared with group 4 (P=0.001), but shortened thangroup 2 (P=0.036) and group 3(P=0.045).The recipients without anytreatment(Group 4)exhibited significantly elevated values of S-Crfrom day5 compared with group 1,2,3 (P=0.000,0.011,0.000), and the recipients in group 2 showed elevated values of S-Cr on day 5compared with group1 or group3 (P＜0.05), but there is no differenceamong group 1, group2 or group 3 on other testing days. Grafthistology shown that allografts of animals in group4 exhibitedfindings typical of severe acute rejection. Allografts of recipientswith MSC monotherapy(group1) showed findings of acute rejectionwith considerably milder severity. But allografts of recipients ingroup 2 and group 3 were markedly well preserved compared withgroup 1. The production of mRNA of all inflammatory mediators weanalyzed, including IL-1β, TNF-αand TGF-β1, was significantlydecreased in the allografts treated with a therapy of CsA (group 2) ascompared with all other groups (P＜0.01). MSC monotherapy (group1) or combination therapy (group 3) decreased production of someinflammatory mediators mildly;Conclusion: MSCs can downregulate the immune response, reduce theproduction of some inflammatory mediators, preserve the graftfunction in the initial stage after transplantation and prolong theanimal survival. But this effect was weaker than CsA therapy.Moreover, MSCs therapy combined with low-dose CsA, couldprotect the graft function, but can not prolong the animal survivaltime compared with CsA monotherapy.Significance: Our results suggest that down the downregulation of MSCs in immune response in vivo is limited, and indicate a potentialinteraction between MSC and CsA activities,which impact theimmune suppression. Therefore, previously considered reducing doseof CsA by application of MSCs need to be carefully investigated.