| Retinal degeneration(RD)is a common kind of blinding eye diseases,leading to a progressive death of photoreceptor caused by disorder of retinal pigment epithelium(RPE)cells or photoreceptor cells.Currently,the methods used to cure RD include gene therapy,cell replacement therapy,nutritional therapy,artificial retinal prosthesis,drug therapy and etc.Cell replacement therapy has its peculiarity of handle ability and easily evaluate,many scientist throw themselves into the lap of cell replacement therapy.As a result,cell replacement is a hot spot of research in the world.Currently,the most resources of transplanted cells include retinal precursor stem cell,ciliary body stem cell,iris pigmented epithelial cell,embryonic stem cell(ESC),bone marrow stem cell and induced pluripotent stem cell(iPSC).Tissue-specific retinal precursor cell(RPC)could survive in suspension culture and attachment culture in vitro.After transplanting into the subretinal of animal models of RD,the tissue-specific retinal precursor cell could delay the degeneration and improve the function of retina.However,the source of this kind cell is limited when used for clinical application.iPSC has strong ability of proliferation and differentiation,which could be induced by mature cells derived from patients,and has little immune rejection problem.iPSC could improve the retina function when transplanted into the subretinal space of the animal models of RD.However,low efficiency increases the cost and difficulty when used for treating patients.Currently,it has been approved by the Food and Drug Administration that RPE cells derived from hESC could be used for performing phase I/II clinical trials for treatment of dry age-related macular degeneration(AMD)and Stargardt disease.And the results showed that the cells derived from hESC could improve or maintain the function of patients without abnormal proliferation in 22 months of follow-up.Thus,we choose hESC as our research object.In our research,retinal precursor cells-derived from hESC was used as seed cells to explore its affects to the retina of animal model of RD.This study would be divided into three parts:The first part of the research was to detect the characteristics and proliferation of hESC.The second part of the research was to explore the methods of inducing the hESC into retinal precursor cell.Using immunocytochemistry(ICC),flow cytometry(FCM)and other methods to detect the differentiated cells during periods of differentiation.Then we search cells of appropriate period using as seed cells.As a prospect cell of clinical transformation,ESC had the risk of forming tumorigenicity because of its unlimited proliferation capacity.Stage-specific embryonic antigens(SSEA)is the specific marker of ESC,and it is stage-specific embryonic antigens 4(SSEA4)in human.To decrease the risk of tumorigenicity of hESC,fluorescence-activated cell sorting(FACS)was used to exclude the SSEA4-positive cells.The third part of this research was to exclude the SSEA4-positive cells in the retinal precursor cells derived from hESC using FACS.Subsequently,we transplanted the mixed cells into the subretinal space of RCS(royal college of surgeons)rats to assess the effective results.At the same time,we also transplanted the mixed cells into the groin of severe combined immune deficiency(SCID)mice to assess the safety.These results would lay a foundation for clinical transformation of hESC.Part ? The Culture and Identification of Human Embryonic Stem Cell and the Formation of Embryonic BodyObjectiveTo assess the characteristics and proliferation of hESC;to explore an appropriate method of forming Embryonic Body(EB).MethodsImmunocytochemistry,flow cytometry,cell cycle and increasing curves were used to identify the characteristics and proliferation of hESC;cell counting was used to describe multiplication curve.EB was formed with three methods,such as suspension culture,hang-drop culture and AggreWellTM400 plate culture,three methods were compared to select an advantage one.Results1.In vitro,hESC appeared small size,large nucleus,large nuclear-cytoplasmic ratio.Colony boundary was clear,and the bright center was a cluster of cells.2.The results of FCM showed that rate of SSEA4 positive cells of P42,P47 and P52 was 97.88±2.21%,98.04±1.85% and 98.00±2.02%.There was no statistically significant difference(P<0.05).The results of ICC showed that the rate of SSEA4 positive cells of P42,P47 and P52 was 97.22±2.11%,96.37±3.55% and 96.33±3.44%.There was no statistically significant difference(P<0.05).3.The percentage of Ki67-positive cells was respectively 98.50±3.01% and 99.60±1.80% detected by FCM and ICC.Cell cycle test results showed that 66.24±3.27% cells were in the proliferation of cell cycle phase(G2/M and S phases).The cell proliferation curve of hESC showed that cell proliferation reached maximum and then plateaus after the cells were plated.And the cell number increase more than 30 fold.4.Using the hang drop method,the number of small EB,middle EB and big EB was 0.33±0.58,37.33±0.58 and 0.67±0.58,respectively.The difference of the number between small EB and middle EB was statistically significant(P<0.001).The difference of the number between big EB and middle EB was statistically significant(P<0.001).Using the suspension method,the number of small EB,middle EB and big EB was 323.33±25.17,348.33±25.66 and 373.68±15.14,respectively.The difference of the number between small EB and middle EB was not statistically significant(P>0.05).The difference of the number between small EB and middle EB was not statistically significant(P>0.05).Using the AggreWellTM400 plate,the number of small EB,middle EB and big EB was 74.33±5.86,906.68±60.28 and 66.33±5.51.The difference of the number between small EB and middle EB was statistically significant(P<0.001).The difference of the number between big EB and middle EB was statistically significant(P<0.001).The number of EB was 38.33±0.58,1045.33±62.32 and 1047.33±51.39 respectively when using hang drop method,suspension culture method and Aggre WellTM400 plate method.The difference of EB between hang drop method and Aggre WellTM400 plate method was statistically significant(P<0.001).The difference of EB between suspension method and AggreWellTM400 plate method was not statistically significant(P>0.05).Conclusions1.H1 in our laboratory had stability of proliferation.The culture method used could maintain the proliferation of algebra.2.There are many methods to form EB.The number of EB was large with suspension culture,but the size of EB was differed;the size of EB was uniform with hang-drop culture,but the number of EB limited;the number of EB was large and the size of EB was uniform with AggreWellTM400 plate culture method.Part t? Inducing Human Embryonic Stem Cell into Retinal Precursor CellsObjectiveTo explore the method of inducing hESC into retinal precursor cell.MethodsFirstly,hESC was cultured to form EB.Then EB were cultured in suspension culture for 3 days,and subsequently,cultured in six-well plates with retinal precursor cell culture adding cytokines,such as IGF-1,Noggin and DKK-1.Then T3 and taurine were added into the retinal precursor culture.Cellular morphology and markers were identified at the 0th day,10 th day,the 20 th day,the 30 th day and the 40 th day of the culture.Results1.With the time of differentiation,the proliferation ability gradually declined.Results of cell cycle of hESC showed that the rate of cell number of G0/G1 phase,S phase and G2/M phase was 40.81±4.44%,36.25±3.91% and 22.95±3.21%,respectively.At the 10 th day of differentiation,the rate of cell number of G0/G1 phase,S phase and G2/M phase was 67.49±2.75%,19.38±3.25% and 13.12±5.08%,respectively.At the 20 th day of differentiation,the rate of cell number of G0/G1 phase,S phase and G2/M phase was 80.70±1.59%,9.43±0.73% and 9.87±0.97%,respectively.At the 30 th day of differentiation,the rate of cell number of G0/G1 phase,S phase and G2/M phase was 81.53±2.76%,6.23±1.54% and 12.24±4.31%,respectively.At the 40 th day of differentiation,the rate of cell number of G0/G1 phase,S phase and G2/M phase was 88.89±1.45%,5.74±1.20% and 5.37±1.16%,respectively.The difference was statistically significant(P<0.001).2.The rate of Ki67-positive cells was 98.70±0.92% detected by FCM and ICC in hESC.At the 10 th,20th,30 th and 40 th day of differentiation,the rate of Ki67-positive cells was 86.40±5.54%,44.23±3.29%,15.10±4.32%,7.87±1.69%,respectively.The differentiation was statistically significant(P<0.05).Detected with flow cytometry,the rate of Ki67-positive cells of H1 was 98.70±0.92%.at the 10,20,30 and 40 day of differentiation,the rate of Ki67-positive cells was 86.40±5.54%,44.23±3.29%,15.10±4.32%,7.87±1.69%,respectively.Detected with ICC,at the 0,10,20,30 and 40 day of the differentiation,the rate of Ki67-positive cells was 97.70±0.92%,91.40±3.63%,42.90±2.52%,13.77±2.28% and 7.20±2.39%,respectively.The difference between two time points was statistically significant.3.The rate of SSEA4-positive cells was 98.8±1.015% in hESC.The rate was respectively 48.8±4.204%,7.640±1.134%,2.213±0.799% and 0.933±0.435% at the day of 10,20,30 and 40 of differentiation analyzed by flow cytometry.And the differentiation was statistically significant(P<0.05).4.Using flow cytometry,at day 0,10,20,30 and 40 of the differentiation,the rate of Pax6-positive cells was 0.63±0.16%,34.73±2.10%,45.63±2.94%,30.97±2.12%,12.23±2.79%,respectively;the rate of Sox2-positive cells was 0.40±0.27%,49.07±4.51%,69.47±4.58%,17.20±2.82%,11.10±1.99%,respectively;the rate of Rax-positive cells was 0.31±0.18%,66.70±6.98%,46.77±6.69%,11.33±2.53%,9.70±1.87%,respectively;the rate of Nestin-positive cells was 0.61±0.26%,82.10±6.86%,69.20±5.47%,23.00±6.06%,6.90±2.46%,respectively.Using ICC,at day 0,10,20,30 and 40 of the differentiation,the rate of Pax6-positive cells was 0%,35.73±5.10%,44.69±4.38%,28.96±3.51%,11.69±3.58%,respectively;the rate of Sox2-positive cells was 0%,50.88±3.42%,67.68±3.52%,18.34±3.12%,12.29±2.03%,respectively;the rate of Rax-positive cells was 0%,65.68±7.15%,48.03±6.58%,10.24±2.47%,10.05±2.03%,respectively;the rate of Nestin-positive cells was 0%,80.12±7.03%,67.20±5.64%,22.35±5.87%,7.09±2.74%,respectively.Compared with the time point before,the differences were statistically significant(P<0.05)?5.At the 10 th day of differentiation,a small amount of Otx2 was detected,and at the 20 th day of differentiation,the expression of Otx2 reached top,65.40±5.57%,then decreased gradually.At the 20 th day of differentiation,the expression of Crx was detected,8.17±1.48%,then increased gradually.At the day of 0,10,20,30 and 40 of differentiation,the rate of the marker of photoreceptor cells,Recoverin,was 0.24±0.18%,0.72±0.55%,6.77±0.95%,16.49±1.99% and 24.14±3.04% respectively. Conclusions1.During the differentiation period,the ability of cell proliferation decreased.2.hESC could be induced into Retinal precursor cell and photoreceptor cell by adding cytokines combining the method of suspension and adherent culture.3.At day 20 of differentiation,the cells we acquired were mainly retinal precursor cells.And we chose the cells at the 20 th day of differentiation for the next research.Part ? The Efficacy and Safety of Retinal Precursor Cell Derived from Human Embryonic Stem Cell on RCS ratsObjectiveTo observe the survival,migration of RPC derived from hESC after transplanting into the subretinal space of RCS rats;to explore the effect of the RSC on the structure and function of retina of RCS rats,the safety was assessed also.MethodsAccording to the results of the part two,we chose the cells at day 20 of the differentiation.We chose the SSEA4-negative cells using FACS,then marked with CM-Dil before transplanted into the subretinal space of 3-week old RCS rats.At 4 week,8 week and 12 week after transplantation,we count the number of survived cell for transplanted group,sham-treated group and wild type group.At the same time,immunohistochemistry was performed at 4 week,8 week and 12 week after transplantation,and the thickness of outer nuclear layer was measured.Lastly,for transplanted group and sham-treated group,the examination of electroretinogram was performed at the day before the transplantation,4 week,8 week and 12 week after transplantation.RPCs derived from hESC(1×107 cells/100?l)were injected into the groin in six severe combined immune deficiency(SCID)mice for 12 weeks to observe the possible tumor formation and immunological rejection action;hESCs were injected into six SCID mice as control group.Animals were sacrificed and examined by a pathologist for microscopic pathological changes and evidence of tumor formation.Results1.Transplanted cells accumulated around the puncture points.Retina detached at the 4 week after transplanting;retina flatted slightly at the 8 week after transplanting.Transplanted cells could survive and migrate into outer nucleus layer.At the 4 week,8 week and 12 week after transplantation,the number of survived cells in one single horizon was 7.77±0.97,5.87±0.42,4.33±0.61,respectively.The difference was statistically significant(P<0.01).2.At the 4 week,8 week and 12 week after transplanted operation,the thickness of outer nucleus layer of transplanted group was 28.60±1.84 ?m,23.32±0.84 ?m and 19.82±1.18 ?m,respectively;the thickness of outer nucleus layer of sham-treated group was 7.12±1.01 ?m,6.70±0.52 ?m and 4.22±0.73 ?m,respectively.The difference of the thickness at the same time point was statistically significant(P<0.05).The thickness of outer nucleus layer of wild type group was 44.74±1.31 ?m,44.84±1.92 ?m,45.82±3.29 ?m,respectively at the same point with transplanted group.The difference between the cell-treated group and wild type group was statistically significant(P<0.05).3.Before the transplantation and at 12 week after transplantation,the differentiation of ERG b-wave amplitude was no statistically significant between transplanted group and sham-treated group(P>0.05).The differentiation of ERG b-wave amplitude was statistically significant between transplanted group and sham-treated group at the 4 week and 8 week after transplantation(P<0.05).4.No gross inflammatory reaction was observed in any of the animals.Teratoma was not observed in the experimental group.In contrast,teratoma were formed in two mice in the hESC group at 8 weeks after injection(33.3%).Conclusions1.RPC derived from hESC could survive and migrate in the subretinal space of RCS rats.The number of cells survived decreased with the time passed.2.RPC derived from hESC could delay the degeneration and atrophy of outer nucleus layer,and protect the retina structure of RCS rats.3.RPC derived from hESC improves the results of ERG of RCS rats when transplanted into the subretinal space of RCS rats.4.Preliminary safety assay shows that hESC-derived retinal precursor cells are not tumorigenic,and tumorigenicity of safety needs further study. |
PDF Full Text Request