Clinical And Basic Study On The Safety Of Intravenous Transplantation Of Human Umbilical Cord Mesenchymal Stem Cells In The Treatment Of Advanced Retinitis Pigmentosa

Retinitis pigmentosa(RP)is a progressive hereditary retinal degeneration disease,which is mainly characterized with night blindness and progressive visual field defect.The global prevalence rate of RP is about 1/3000–1/7000[1].The major pathological mechanism of RP is the progressive apoptosis of photoreceptors caused by primary genetic defects or secondary to retinal pigment epithelium degeneration[2-4].Currently,there is no effective treatment for RP.Recent studies report that RP is regarded as a chronic inflammatory disease,and the inflammatory and immune process play an important role in the progression of retina degeneration[5],which provides new ideas for the treatment of RP.Mesenchymal stem cells(MSCs)are a subpopulation of stem cells that originate in the mesoderm of embryonic development and can be derived from a variety of tissues including bone marrow,umbilical cord,fat and dental pulp etc.They are characterized by self-renewal,multiple differentiation,immune regulation,anti-inflammatory and neurotrophic.Among them,mesenchymal stem cells form umbilical cord(UCMSCs)have lower immunogenicity and more advantages[6-8].At present,MSCs transplantation has been applied to several clinical trials for the treatment of retinal degeneration diseases such as wet age-related macular degeneration(wet-AMD),Stargardt’s disease,diabetic retinopathy,and retinitis pigmentosa etc.[9-11].Good safety and tolerability were observed in these studies with no subjects suffered immune rejection,infection or other serious adverse reactions,and some therapeutic effects were also observed.Local transplantation such as through vitreous cavity,subretinal,preretinal and subtenon’s transplantation is a popular way for retinal degeneration[12,13].Similar to other studies[14,15],our previous experiments had proved that intravitreal or subretinal transplanted BMSCs was safe,and the transplanted BMSCs could differentiate into retinal neurons and photoreceptor cells,and secrete various cytokines to delay the process of retinal degeneration.However,some researchers have observed that BMSCs transplantation in eyes may cause serious complications,such as proliferation of fibrous tissue and epiretinal membrane around the transplantation site,and even tractive retinal detachment[16,17].Intravenous transplantation,a convenient,low-cost and non-invasive approach,has been widely applied in animal and clinical research.As the retinal microenvironment of the advanced RP patients is severely damaged,appearing ischemia and hypoxia,it is hardly to nutritionally support the locally transplanted cells or provide a good living environment.For retinal degenerative diseases,Wang et al.[18]had reported that the intravenous transplanted BMSCs could migrate to the retina and protect the neurons and blood vessels of retina in Royal College Surgery(RCS)rats.And our previous study had also shown that intravenous infusion of BMSCs in patients diagnosed with non-proliferative diabetic retinopathy was safe,no adverse reactions or side effects were observed,and the patients’visual function had been improved[19].Compared with BMSCs,the gaining of hUCMSCs is safer and more convenient rather than obtaining from the donors’bone marrow,and can avoid the pathogenic genes carried by patients.At the same time,as a kind of allogeneic MSCs,hUCMSCs intravenous transplantation can avoid the immune rejection caused when retinal transplantation.Therefore,hUCMSCs intravenous transplantation may be a more suitable treatment for RP patients.And until now,there has no clinical research about hUCMSCs intravenous transplantation in the treatment of advanced RP patients been reported.However,recent studies have shown that intravenous infusion MSCs may cause serious vascular obstruction.Most of the transplanted cells were blocked in the lung,which may resulting in an increased risk of complications such as pulmonary embolism,and seriously threaten the safety of subjects[20-22].Althouth the mechanism of the vascular obstruction is not clear,the size of MSCs is thought to be a key factor for that[23].Although we have ensured the safety of all subjects in our previous clinical trial,with the increasing number of subjects and the times of administrations,there has been an underlyingsafety risk of vascular obstruction with the intravenous infusion of MSCs.Therefore,we need to optimize MSCs transplantation strategies to further improve the safety of intravenous transplantation.It is reported that the mechanism of MSCs is,on the one hand,the paracrine effect,which means the immunoregulation and neurotrophic effects mediated by the cytokines secreted by MSCs[24-26].On the other hand,it is the proliferation and differentiation of the cells transplanted to the target organs,so as to repair or replace damaged tissues[27,28].Nevetherless,increasingly evidence had shown that the majority of MSCs were blocked in the lung early after intravenous infusion,so that they could not migrate to the target organs[20,22].Therefore,the paracrine effect is considered to be the main mechanism of MSCs intravenous transplantation in the treatment of retinal degenerative diseases[29].Based on the previous studies of our laboratory,we hypothesize that intravenous infusion of human umbilical cord mesenchymal stem cells(hUCMSCs)is safe and effective in the treatment of RP,and the small hUCMSCs(S-hUCMSCs,<20μm)can further improve the safety of intravenous transplantation and paly the similar protective effects as hUCMSCs,and the main mechanism of the efficacy is immunoregulation and neurotrophy mediated by paracrine effects.This study is composed of two parts:Part One:Study on the safety and efficacy of hUCMSCs intravenous infusion in patients with advanced retinitis pigmentosa.1.This study was approved by the Ethics Committee of the Southwest Hospital of Army Military Medical University(Approval Document No.44,2014).According to the inclusion and exclusion criteria,32 patients diagnosed with advanced retinitis pigmentosa were involved in this study.2.After completion of the pre-treatment evaluation,all 32 subjects were intravenously infused with one dose of 1×10~8hUCMSCs and were followed up for 12 months.No serious local or systemic adverse effects occurred in the whole follow-up.Most patients improved their best corrected visual acuity(BCVA)in the first 3 months.The proportions of patients with improved or maintained BCVA were 96.9%,95.3%,93.8%,95.4%,90.6%and 90.6%at the 1st,2nd,3rd,6th,9th,and 12th month follow-up,respectively.Most of the patients(81.3%)maintained or improved their visual acuities for 12 months.The average National Eye Institute 25-Item Visual Function Questionnaire(NEI＿VQF-25)scores were significantly improved at the third month.The average visual field sensitivity and flash visual evoked potential(FVEP)showed no significant difference.These results indicated the intravenous infusion of hUCMSCs was safe for advanced RP patients.Most of the patients improved or maintained their visual functions in a long term.The life qualities were improved significantly in the first 3 months.Part Two:Study on the safety and efficacy of S-UCMSCs(<20μm in diameter)intravenous infusion in RCS rats.1.Small hUCMSCs(S-hUCMSCs,<20μm in diameter)were obtained through filtered hUCMSCs with Celltrics(?)10μm(Sysmex,Japan)nylon cell filter with an average diameter of 8.636±2.256μm.By comparison,it was found that S-hUCMSCs and hUCMSCs expressed the same surface antigens and both of them could be induced to differentiate into adipogenic,osteogenic and chondrogenic cells under specific culture conditions.Transcriptome sequencing showed that S-hUCMSCs and hUCMSCs shared the similar gene expression profiles,suggesting that S-hUCMSCs and hUCMSCs were the same type of cells.And the cell cycle detection showed that there were more S-hUCMSCs in the S phase and G2/M phase,and the proliferation index(PI)of S-hUCMSCs was higher than that of hUCMSCs,which revealed that the S-hUCMSCs was more proliferative than hUCMSCs.2.The hUCMSCs were transfected with lentivirus expressing Green Fluorescent Protein(GFP)before transplantation.RCS rats were intravenously infused with one dose of 1×10~6S-hUCMSCs or hUCMSCs via the tail vein,and a large number of GFP positive cells were observed in the lungs of both groups 1 h later.12 h and 24 h after transplantation,there were significantly fewer S-hUCMSCs than hUCMSCs entrapped in the lungs,and then no GPF positive cells were found any more in the lungs of all experimental groups at 72 h.The results suggested that S-hUCMSCs intravenous infusion could reduce the risk of pulmonary vascular obstruction and improve the safety.In addition,no GFP positive cells were found in the retina after intravenous transplantation,indicating that the cells did not homing to the retina.3.RCS rats at P30 were intravenous infused with with one dose of 1×10~6S-hUCMSCs and hUCMSCs via tail vein,and PBS injection and untreated groups were regarded as controls.FERG tests showed that the average amplitude/latency(A/L)ratio of the b wave of the S-h UCMSC and h UCMSC groups was significantly higher than that of the PBS and untreated groups at 7 d after cell infusion.And 7 and 14d after transplantation,the ONL thickness of the S-h UCMSC and h UCMSC groups was significantly thicker than those in the control groups.These results suggested that hUCMSCs can delay retinal degeneration and protect visual function.The level of IL-6 in the serum of RCS rats in the S-hUCMSCs and hUCMSCs groups was lower than that in the control groups at 7 d,and the level of IL-10tended to increase at 7 and 14 d after transplantation.Meanwhile,the levels of brain-derived neurotrophic factor(BDNF),ciliary neurophic factor(CTNF)and basic fibroblast growth factor(b FGF)in the two transplanted groups were significantly upregulated at 14 d or 28 d after treatment,and the difference between S-hUCMSCs and hUCMSCs group was not significant.These revealed that S-hUCMSCs and hUCMSCs have similar protective effects on visual function and photoreceptors by regulating expression levels of inflammatory and neurotrophic factors in RCS rats.4.Microglia is the main immune cell in the retina.After activated by lipopolysaccharide(LPS)for 24 h,the BV2 microglia was indirectly co-cultured with S-hUCMSCs and hUCMSCs based on Transwell co-cultured system for 12 h,24 h and 48 h.And the BV2microglia that treated with LPS only or untreated were regarded as control.The TUNEL staining showed that after co-cultured with BV2 microglia for 24 h and 48 h,the proportion of apoptotic microglia in S-hUCMSCs and hUCMSCs groups was significantly higher than that in LPS group and untreated group.Meanwhile,the ELISA assay showed the expression levels of IL-1β,IL-6 and TNF-αsecreted by BV2 microglia when co-cultured with S-hUCMSCs and hUCMSCs were significantly lower than those in LPS group for 24 h and 48 h,which demonstrated that S-hUCMSCs and hUCMSCs could promote the apoptosis of microglia and inhibit the secretion of inflammatory factors by microglia through paracrine effect.In addition,compared with hUCMSCs,S-hUCMSCs displayed no significant advantage in regard to the therapeutic effect.Based on the above results,the following conclusions are drawn:1.Intravenous infusion of hUCMSCs in advanced RP patients is safe with no adverse reactions or complications occurred.2.The life quality of RP patients was significantly improved 3 months after hUCMSCs intravenous infusion,and the visual function of 81.3%patients had been improved or maintained until 12 months after transplantation.3.The biological characteristics of S-hUCMSCs(<20μm in diameter)and hUCMSCs are similar,and the S-UCMSCs are more proliferative than UCMSCs.4.The number of S-UCMSCs blocked in lungs after intravenous infusion is fewer than that of UCMSCs,which reduced the risk of vascular obstruction and improved the safety of transpalantation.Meanwhile the S-UCMSCs and hUCMSCs could similarly protect the retinal structure and function through immunoregulation and neurotrophic effects.5.The S-hUCMSCs and hUCMSCs indirectly co-cultured with BV2 microglia in vitro could promote microglia apoptosis and inhibit the secretion of inflammatory factors through paracrine mechanism.