Empirical Study On Transplantation Of Human Amniotic-derived Mesenchymal Stem Cells Into Brain Injured Rats

Background:Brain damage is one of the diseases that pose great threat to people's health.Presently no satisfying effect has been seen in treating the loss of nerve function brought by the suffering of severe brain damage.Yet studies on stem cells bring hope for such patients.Treating brain damage by transplanting stem cells can improve the nerve function of the host.The possible meachanism is that the endocrine effect of exogenous stem cells in hosts and the paracrine effect of nerve cells in the organism itself can bring local neurotrophic factors together,improve the living environment of nerve cells and make it easier to transform from exogenous stem cells into nerve cells and have synaptic connections with them.While the possibility is expected to do more research.Currently there are three ways to transplant stem cells which are different in improving nerve function,namely, tranplanting through the original brain injured area,circulation of blood-cerebrospinal fluid,as well as blood circulation,all of which are relatively effective in improving the nerve function yet no definite conclusion can show which way is the best.Also, no relevant systemic report has ever been seen in comparing their exact treating effect. Thus the author will make further discussion about this and offer some experimental references for treating brain damage.In general,the traditional practice for studying stem cell transplantation in treating brain damage is to sacrifice animals and make them as samples for histopathologic observation.Therefore,how to make observation in vivo about the survival of exogenous stem cells is the difficulty as well as priority for doing research on stem cell transplantation.Through 1.5Tmagnetic resonance, some researchers successfully made observation in vivo about the effect of transplanting labelled mouse embryonic stem cell in vitro in treating brain injured rats, while few reports have been released to study the application of transplantation of human amniotic membrane-derived stem cells(human AD-MSCs) as well as tracking them in vivo for observing their survival in hosts.In this essay,the author treats injured rats by lablelling human AD-MSCs in vitro and the survival as well as immigration of labelled cells are traced through 3.0T nuclear magnetic resonance Previous research shows that NGF play a key role in nerve cells growth differentiation as well as the maintenance of their function.A number of studies have been done to prove that NGF can induce stem cells to transform into neuron-like cells and brain damage can be treated through transplanting NGF and exogenous stem cells which can relatively improve the nerve function of the hosts.However,how to ensure long maintenance of effective concentration of NGF in brain injured area is critical to treating brain damage by combining NGF with stem cells since NGF is weak to pass blood-brain barrier,the administration way of medicine in peripheral vascular is difficult to enter the brain injured area and NGF is hard to maitain effective concentration for a long time due to short half-life.The author will observe the situation of NGF nanoparticles by means of quantum dots(QDs) after entering the brain injured area.The treatment effect turns out to be quite positive.After tranplanting exogenous stem cells into the organism,further study need to be done about the effect to host.This essay successfully discovers the effect of transplanting stem cells to brain injured rats and finds no neoplasm with the assistance of muclear magnetic resonance after blood routine and tumor marker examination from collected blood.Based on the above study,this essay is going to explore in such matters as tracking in vitro of human for treating brain damage,the inserting ways of NGF as well as the safety issue so as to provide evidence for clinical treatment of brain damage.Objective:â… .To investigates the method of in vitro isolation,culture and purification of MSCs.â…¡.To explore the feasibility and necessary conditions for labelling AD-MSCs with QDs-NGF-NPsas well as Fe₃O₄ nanoparticles and also explores the feasibility of the survival and migration of human AD-MSCs into brain injured rats by 3.0T magnetic resonance.â…¢.To analyse improving effect of rats' ethology and nerve function by transplanting AD-MSCs with QDs-NGF.â…£.To explore the safety of treating brain damage through transplanting MSCs.Methods:â… .Isolated culture and identification of AD-MSCsUnder the sterile condition,AD-MSCs were obtained from normal placenta after abdominal delivery.Firstly,after being rinsed with physiological saline,they were digested with 0.25%parenzyme at ordinary temperature for 30 minutes and digested with DMEM/F12 solution that contained 1.0g/L collagenase for one hour at 37℃.Then they were filtered with 150 wells filter,processed into unicell suspension cultured in DMEM/F 12 nutritive medium that contained 10%fetal bovine serum (FBS).After adding basic fibroblast growth factors(bFGF),cell culture was undertaken in incubators at 37℃with volume fraction of 5%CO2 and saturation humidity.Three days later,nutritive medium change was done and the unattached cells were removed.In the following research,the removal was done every three to five days.When the cells reached 80%to 90%confluence,serial subcultivation was undertaken in the proportion of 1:2 with the concentration of 1×10⁶cells/ml.The expression of immunophenotype for MSCs from the fourth to the sixth generation such as CD29,CD44,HLA-ABC were detected by flow cytometer.The third generation AD-MSCs were chosen,and the cultured cells were induced to differentiate into cellula nervosa with the assistance of 20μg/L bFGF and 30μmol/L Retinoic acid(RA).In eight days and thirteen days before and after the induction, immunocytochemical staining was undertaken to nestin,neuronspecific enolase(NSE) and glial fibrillary acidic protein(GFAP)â…¡.The induction and differentiation of AD-MSCs labelled by QDs-NGF-NPs and Fe₃O₄ nanoparticlesWith DMEM/F12 as their culture solution that contained 10%FBS and 20μg/L When the cells reached 80%confluence,the cells were inoculated into 96-hole culture plate for 200μl per hole.Glass slide were put onto the plates.When the slides were coated with cells,QDs-NGF-NPs and Fe₃O₄ nanoparticles at different concentration were added into the nutrient medium which were divided into experimental and control groups.Groups only with Fe₃O₄ nanoparticles were classified into experimental group and control group.The control group had no Fe₃O₄ nanoparticles nutrient medium while experimental group were further divided into four sub-groups at final concentration of 20,30,40μg/L,80μg/ml respectively.Then we added poly-1-lysine at concentration of 1.5μg/ml in each group.After culturing for 24 hours,the nutrient medium was changed into medium without NGF and NPs and continue to culture for 3w and were given Prussian blue staining in the.corresponding time,The labelled groups were divided into blank control group(equal amount of physiological saline in it and pure QDs,in which the final concentration of QDs were 20,40 and 60μg/L;The experimental group was group with QDs-NGF-NPs which was added woth QDs at concentration of 20,40,and 60μg/L respectively.According to the experimental results,a combined labelled group between QDs-NGF -NPs and Fe₃O₄ nanoparticles was made which was further divided into experimental group and control group.Group with QDs-NGF-NPs were divided into three sub-groups(with concentration of 20,40,60μg/L),and group with Fe₃O₄ nanoparticles were divided into 3 sub-groups(with concentration of 20μg/ml,30μg/ml and 80μg/ml).Then they were added into Poly L lysine with the final concentration of 1.5μg/ml.Group without QDs-NGF-NPs at the same conresponding final concentration were divided into control groups for comparision.Normal control group without no QDs-NGF-NPs and Fe₃O₄ nanoparticles were changed nutrient medium for nutrient solution after being cultured for 24h.Being culturing for another 24h,48h,72h,each hole was added 20ul(5g/L)MTT solution and continued to incubate for 4 hours.At last,the supernatant was removed and 100ulDMSO was used to terminate reaction.After shaking slightly,the absorbance value(A) were measured at the wavelength of 490mm.The cell survival in normal control groups were 100% while survival in other groups was to be calculated according to the following formula: cell survival=(experimental groups A/control groups A)×100%.That is to say,the larger the A value was,the better survival rate was.The distribution of QDs among the cells was observed under fluorescent microscope.Then Prussian blue staining was applied at 12h,36h,1w and 3w so that how much Fe₃O₄ nanoparticles had entered into cells could be observed.Identification of cell differentiation:20μg/L bFGF and 30μmol/L RA were used to induce AD-MSCs to differentiate into cellula nervosa in both labelled group with QDs-NGF-NPs and combined group with QDs-NGF-NPs and Fe₃O₄ nanoparticles. Then after 8days and 13 days,they were fixed by 4%paraformaldehyde for 30 minutes and washed by PBS.Peroxidase blocking solution was used to block endogenous peroxidase and nonimmune animal serum.Then neuron-specific enolase (NSE),nestin,monoclonal antibody(1:100dilution) of glial fibrillary acidic protein (GFAP) were added to the cells which were incubated for 1 hour at ordinary temperature and washed with PBS.After that,cells were incubated for 10 minutes and washed.At last they were incubated for another 10 minutes and washed while adding streptavidin-peroxydase solution which were developed color with DAB solution and fixed with neutral gum.â…¢.Study on therapeutic effect of different transplantation methods and establishment of brain injured modelsAbout 80 wistar rats were chosen to establish brain injured models by the Feeney's free falling weight method.The necessary injury devices were made up of backboard,fixed bracket,vertical lead-screw,20g counterweight and polythene knocking conus with the diameter of 4mm and 2.5mm high.After being undertaken peritoneal injection with 10%chloral hydrate(3ml/kg),their heads were fixed in stereotaxic apparatus,the skin and hair of their heads were cut and sterilized Then the part of skin and periosteum were cut a round shape along the centerline with diameter of 5 mm,3mm behind anterior fontanel and 2mm on the left side.Then,the weight was vertically dropped the from 30 mm high,making it stroke the conus so that the rats could be damaged moderately.At last,the cut was sutured.Those brain injured models were divided into control group(n=20)and treatment group(n=60)which was further divided into 3 groups(n=20),that is group A(vein transplantation),groupB(ventricle transplantation) and group C(brain injured area transplantation).The degree of neural injury was evaluated with pegged beam walking test method.4 rats from each group were killed 1d,4d,1d,2w,3w later separately and NSE as well as nestin and GFAP immunohistochemistry staining were done to those dead rats in order to observe the survival,migration as well as differentiation of AD-MSCs.We would choose the appropriate way to transplant.â…£.The transplantation,in vivo labelling,evaluation of neuroethology,and immunehistochemical detection of AD-MSCsThe rats were divided into control group and experimental group.In control group,all of the 20 models were transplanted with Feridex physiologic saline at the same concentration that replaced AD-MSCs.40 models were divided into two experimental groups(n=20).In groupA,the third generation AD-MSCs was transplanted into the original brain injured area.In group B,the third generation AD-MSCs labelled with QDs-NGF-NPs and Feridex for 24 hours.Then 10μl physiologic saline that contained AD-MSCs(with concentration of 2.0×107/μl)was injected into and around the injured area.Each injection should be 3mm deep and remained in the injected place for about 5min.After this,bone wax was used to seal bone window incision off and periost were sutured.SIEMEMS 3.0 Trio Tim I-class MR scanner was used to performT1WI,T2WI,T2^* Wias well as SWI sequence scanning.Scan parameters were T1WI:TR/TE = 430/12ms,T2WI:TR/TE= 5990/98ms with thickness of 2.0mm,FOV10cm×10cm and matrix 288×224 and so on.The neurological scores was graded in fixed time after establishing the models. Then those models were sacrificed to observe the existence of iron nano-particles with Prussian staining 7days,14days and 21 days later respecttively.The expression of nestin and NSE and GFAP were observed with immune histochmistry method.â…¤.Safety detectionBefore establishing the models and After that for 2w and 3w,0.5ml blood was drawn from vena caudalis.But heparin(15U/kg) should be injected before hemospasia to ensure whole body heparinization.Blood routine examination and 12 tumor markers should be detected together in order to observe and evaluate the availability and safety of tranplanting AD-MSCs labelled by QDs-NGF-NPs and Feridex.Results:The cells suspension solution extracted by enzymic digestion from AD-MSCs was cultured in vitro.It was soon found that some cells became adhering to the wall as early as 10 hours later.Being cultured for another day or two days,the adhering cells increased steadily and filled the bottom in various forms.Most of them were long fusiform while some of them were polygon or in irregular forms.In general, they appeared to be spindle cells,similar to fibroblast cells.After cell passage through total change of digestion solution,cells growth accelerated and spreaded the flasks bottom all over 3 to 6 days later with some cell fusion.Most of them were long fusiform cells,distributing in stripy and spiral shape.Cells in the third generation proliferated actively and could cover the bottom of the flasks during that stage yet cells in the sixth to eighth generation became aging in flat shape proliferatng slowly or even ceasing to do so.When it comes to the 9th or 10th generation cells,this phenomenon became even more obvious.According to flow cytometry,cells in the third and fourth generation turned out that: cells extracted from AD-MSCs could express antigens CD29,CD44 and HLA-ABC which were the same with those of MSCs,indicating those were MSCs.Under the cultivation in vitro,AD-MSCs jointly induced by 20μg/L bFGF and 30μmol/ LRetinoic acid(RA) could express nestin and NSE,indicating that AD-MSCs could differentiate into neuron-like cellsResultl from Prussian staining toAD-MSCs lablelled by Feridex indicated as follows:cell labeling rate in group with Fe₃O₄ nanoparticles at the final concentration of 20μg/L was 60%,yet cell labelling rate in group with Fe₃O₄ nanoparticles at the final concentration of 30,40,80μg/L was 100%.That is to say,the higher the concentration was,the more Fe₃O₄ particles entered.And Fe₃O₄ particles from each groups reached the highest amount 36h later and they decreased when it was 1w and 3w later.We could see those Fe₃O₄ particles stained by Prussian method scattered in kytoplasm.Labelled group with QDs-NGF-NPs(concentration was less than40μg/L) and combined group between QDs-NGF-NPs(concentration was less than40μg/L) as well as Feridex(concentration was less than30μg/ml) were cultured for 6h.Under fluorescent microscope,red fluorescent light could be observed radiating from cell membrane kytoplasm.Meanwhile,the amount of QDs increased as time went by.The light reached its peak 32h later and it didn't fade away 2w later.After being stained,a lot of blue-stained Fe₃O₄ particles could be detected in the Fe₃O₄ particles of the combined group.In the two groups,some cells expressed as NSE or nestin.No cells were detected to express GFAP.Balance beam walking test was adopted to those groups one day after transplantation.But there was not significant differences between control group 5.42±0.98,group A 5.02±1.98,group B 5.54±1.34 and group C 5.12±1.04(P＞0.05). There was statistic significance between control group 5.11±1.62,group A 5.15±1.02,group B 5.51±2.04 and group C 4.55±1.02,between group C and control group,beteen group A and group B(P＜0.05).The rest of other groups were not seen any significant difference(P＞0.05).Scores were graded 13 days after the operation,there was significant diffrences between control group 5.11±1.92,groupA 5.01±0.92,groupB 4.86±0.87 and group C 2.66±1.72,between group C and other groups,between group B,group A and the control group(P＜0.05).But there was no significant differences between group A and the control group(P＞0.05).No change in the above results after raising those rats for 3 weeks.In group B with intracerebroventricular injection indicated that few cells in the injured region expressed as NSE and GFAP,though some cells in the ventricular walls expressed as NSE and GFAP.While in group C,a lot of cells expressed as NSE and GFAP indicating that most of the transplanted AD-MSCs managed to grow and survive in that area.As for group A.nothing was observed to express as NSE and GFAP.Shortly after tranaplantating AD-MSCs labelled with feridex,the control group and group B were undertaken T1,T2,T2^*G scaning,and low signal could be found in the transplanted area in which T2^*GRE was the most sensitive one.In the MR scanning 1d,7d,14d and 21d later,nothing could be found in the control group while low signal could be observed in group B.Particularly,the signal of feridex could be detected in the transplanted area 14d and 21d later while group A appeared to be nonvisulized under MR scaning.According to the score of nerve function,no significant differences in statistics could be found in the first 7 days after transplantation.Yet 8 days later,the scores in group A and group B became lower than that in control group,thus there was statistic significance while group B had differences compared with group A and it remained so for 14 days.The Prussian staining showed that some blue stained iron praticles existed along the injection point and the color became lighter and lighter as time went by and eventually spread to surrounding part.Immunohistochemical staining results showed that there was no expression of nestin,NSE and GFAP in the control group while there were such apparent expression in the two experimental groups with more expression in group A (p＜0.05).Before establishing the models and after that for 2w and 3w,0.5ml blood was drawn from vena caudalis.And blood routine examination and 12 tumor markers showed that everything was normal.Conclusion:MSCs can be obtained from human amniotic membranes by enzyme digestion.Induced by bFGF and RA,they can differentiate into neuron-like cells,fully demonstrating that human amniotic-derived MSCs posess multi-differentiating potentialHuman AD- MSCs can be successfully labelled in vitro by QDs-NGF-NPs and Feridex.QDs-NGF-NPs with concentration of less than 40μg/L and Feridex with concentration of less than 30μg/ml have no impact to the growth and differentiation of MSCs.Transplanting AD-MSCs from the injured area is the best way to treat brain injured rats which shows better improvement of the nerve function of rats compared with those of transplanting via tail veins or via intracerebroventricular.NGF-NPs can effectively enter AD-MSCs and improve the nerve function of brain injured rats.It is feasible and convenient to observe the survival and differentiation of transplanted AD-MSCs with 3.0T nuclear magnetic resonance analyser and Feridex as labelled marker.After treating brain injured rats through transplanting AD-MSCs labelled with QDs-NGF-NPs and Feridex the detection to blood routine examination and 12 tumor markers turn out to be normal,all of which indicate that this treatment was safe.