Design and fabrication of high density borated polyethylene nanocomposites as a neutron shield

Background: polyethylene composites including boron can be used as an effective neutron shield. Our investigation focuses on manufacturing borated polyethylene nano-composite. The purpose of this study is to design a radiation shield for use in both neutron and gamma fields

Materials and Methods: borated polyethylene shields containing 2%, and 5% weight percentage of Boron nano-particles were constructed and their neutron attenuation was compared with pure polyethylene. Polycarbonate films were used to find the attenuation of Am-Be neutrons after passing the shields. Mechanical properties of the shields were finally compared

Results: mean [+/- SD] number of alpha tracks induced by neutrons passing through the shields, were found to be 1.0488×10[3]+/-128.98, 1.1972×10[3]+/-289.56and 1.5340×10[3]+/-206.52 for polyethylene with 5% by weight, polyethylene with 2% by weight boron nano-particles, and pure polyethylene, respectively. The neutron spectrum after each shield was also obtained by MCNP4C Monte Carlo simulations. On the other hand, borated polyethylene nano-composites showed higher tensile strength compared to that of pure polyethylene. Attenuation of neutrons measured in experiments and the result of MCNP simulation were in good agreement

Conclusion: a statistically significant difference was found between neutron attenuation by borated polyethylene nanocomposite made of 5% by weight boron and pure polyethylene. However, the difference between borated polyethylene nano-composite with 5% weight and 2% wt boron was not statistically significant

In vitro potential of human bone marrow and umbilical cord vein mesenchymal stem cells to differentiate into insulin producing cells

Mesenchymal stem cells [MSCs] derived from bone marrow are multi potent cells that have the capacity to trans-different!ate into a variety of cell types including insulin islet cells. However the efficiency is low. The aim of this study is to explore the potential of Marrow and Umbilical cord vein MSC to differentiate into functional islet like cells in vitro. BM-MSCs and UC-MSCs were obtained from healthy donors and were cultured. MSCs with high CD90, CD73, CD105, CD44 and very low CD34 and CD45 expression were differentiated into Islet-like cells, under defined conditions. Insulin and c-peptide positive cells were evaluated with immune-florescence and insulin release after glucose challenge was tested by ELISA. QRT-PCR was done to detect expression of insulin, Glut2, Nkx6.1 and Nkx2.2 at mRNA level. Our results showed that only BM-MSC can be differentiated to insulin secreting cells. About 15.8% +/- 2.6 and 13.5% +/- 5.5 of cells were positive for insulin and c-peptide, respectively. Our results revealed that expression of Insulin and Glut2 upregulated 20 fold changes at mRNA level. However they were not functional when treated by different concentration of glucose. Our results showed that only Human BM-MSCs, compared to umbilical cord vein MSCs, are able to differentiate into insulin producing cells in vitro

effect of systemic administration of dbcamp on neural stem cells migration in eae model of multiple sclerosis

Background and Purpose: Novel strategies of MS try to stimulate endogenous neural stem cells for demyelination repair. Increased levels of cAMP potentiate the repair mechanisms in CNS by activating PKA or independently. In the present study, we investigated the effect of dbcAMP on neural stem cells migration in experimental autoimmune encephalomyelitis [EAE] model of MS

Methods and Materials: Mice were immunized with 300 microg MOG peptide emulsified in complete Freund's adjuvant [CFA] and pertussis toxin [PT]. Control mice received CFA and PT. Groups of EAE- mice received i.p. injections of dbcAMP 10mg/kg from day 9-14 or 9-21. Animals were observed daily for neurological deficit. Nestin expression was used as a marker to detect neural stem cells. The number of Nestin+ cells in SVZ and olfactory bulb [OB] was evaluated using immunohistochemical staining. GraphPad Prism Version 5 was used for analyzing the data. For the clinical signs of EAE, the differences between the same days were compared by unpaired t-test. For the number of Nestin+ cells, the statistical differences between the groups were determined by one-way ANOVA and Tukey post-test

Results: EAE induction caused clinical signs and paralysis of tail and hind limbs. dbcAMP significantly reduced the incidence and severity of EAE in mice immunized with MOG. Maximum of scores reached 0.66 +/- 0.13 for dbcAMP treated mice [2.5 +/- 0.2 for EAE mice] on 21 dpi [day post inductin]. EAE induction did not change number of nestin+ cells in SVZ but it increased it in OB. With developing of scores on 21dpi, the number of cells decreased [5.66 +/- 1.20]. dbcAMP injection from 9-21 dpi increased these cells in SVZ. With developing of EAE scores on 21 dpi, the number of these cells in OB increased [19.5 +/- 2.04] and has significant differences with the control group. The treatment of EAE induced mice with dbcAMP from 9-21 dpi was assosiated with a significant elevation of Nestin+ cells in OB [40 +/- 2.73] [P<0.001].

Conclusion: Treatment with the dbcAMP and PKA activation effectively control the EAE signs via increasing the number of neural stem cells and inducing their migration from SVZ to OB and demyelinated lesions, and decrease the symptoms

[Large-scale expansion of human embryonic and induced pluripotent stem cells for cell therapy applications]

Successful isolation, derivation and culturing of human pluripotent stem cells, including human embryonic stem cells [hESCs] and human induced pluripotent stem [hiPSCs] cells in laboratory scale has opened new honzones for cell therapy applications such as tissue engineering and regenerative medicine. However, most of the cell therapy protocols using these unique cells require large number of fully defined and well-characterized cells with uniform properties. Hence, translating these valuable findings from laboratory scale research to clinical applications would involve development of robust dynamic and scalable culture systems being capable of producing large number of uniform and well-characterized cells under fully controlled conditions. Identification and meticulous investigation of most important parameters affecting on the performance of expansion, self renewal and differentiation of human pluripotent stem cell cultures such as cell line, culture medium, culture conditions and culture system are prerequisite steps for successful culture system development, which have attracted growing attention from researchers in recent years. This review will focus on recent research advances and achievements in culture system development for human pluripotent stem cells as well as current challenges in this filed

[Comparison of transplantation of human bone marrow mesenchymal stem cells and their derived neural like- progenitors to spinal cord injury rat model with injection]

The aim of the present study was to investigate transplantation of MSCs and their-derived Neural like progenitors [NLPs] into the spinal cord after injury and evaluate the survival, migration, differentiation properties of these cells in Rat spinal cord. NLPs were derived from MSCs [induced by bFGF [Fibroblast growth factor b], hEGF [Human Epidermal growth factor] and RA [Retinoic Acid]], and analyzed by flowcytometry and immuno fluorescence staining. MSCs and NLPs injected into model animals vein and collagen scaffold implanted into injured site. Behavioral testing was performed weekly for 5 weeks. Improvement of transplanted animals evaluated after 5 weeks. Unfortunately, Substantial changes were not observed among the rats after the transplantation. Immuno fluorescence staining analysis using human nuclei and BrdU antibodies confirmed survival and migration of hMSCs and NLPs into the injury site. Transplanted cells were found to adjacent segments located rostro-caudaly to the injury epicenter. Our findings indicate that hMSCs and NLPs couldn't facilitate recovery from spinal cord injury. However, these cells can express specific neuronal markers in injured site. There are many questions to be answered regarding this mechanism

[Isolation and characterization of human induced pluripotent stem cells-derived mesenchymal progenitors]

Isolating human induced pluripotent stem cells [hiPS]-derived mesenchymal progenitors as a new source of mesenchymal cells which can differentiate into different lineages like adipose and bone. After 7 days of hiPS1 culture on matrigle coated dishes, spindle like cells around colonies were removed by cell scraper. These cells that had mesenchymal like morphology was characterized after 4-6 passages. Mesenchymal cell surface markers CD73, CD90, CD105, CD29, CD44 and hematopoietic cell surface markers CD34 and CD45 were analyzed by flow cytometry and cells were differentiated to osteogenic and adipogenic lineages by defined medium. Flow cytometric analysis demonstrated that hi PS1 derived mesenchymal progenitors were%98.71 +/- 0.14 CD44+,%98.51 +/- 1.02 CD29+,%87.74 +/- 3.41 CD105+,%46.65 +/- 5.76 CD73+,%98.53 +/- 0.78 CD90+ and they did not express CD34 and CD45. These cells could be differentiated to osteogenic and adipogenic lineages. hiPS1 can make mesenchymal progenitors and these cells can be a suitable substitute for mesenchymal stem cells

Animal models of liver cirrhosis and fibrosis

Animal modeling is a crucial necessity in clinical studies of liver diseases. Authenticity of the data which are produced using this tool under different conditions and accuracy of the analyses and assessments which would be based on such data sets is completely dependent on the adoption of a standardized methodology for analyzes and assessment of these data sets. Cirrhosis and Fibrosis are among the most important diseases which are studied by animals modeling due to the fact that their final structure is usually similar among a verity of patients and also because they are the common end stage of most chronic liver diseases. Up to now, different approaches such as hepatotoxicity and surgical methods have been utilized to obtain cirrhotic or fibrotic models that either of which has its especial advantages and disadvantages. It is obvious that using models could indicate the production and treatment mechanisms of disease which we elucidate Fibrosis and cirrhosis here. Considering several animal models which were used for liver disease in the world, in this survey we try to explain why, what and how an animal must be choosen for modeling and how could be evaluated

Therapeutic potential of mouse bone marrow mesenchymal stem cells in carbon tetrachloride [CCI[4]]-lnduced liver fibrosis

To study the effect of allogenic bone marrow mesenchymal stem cells [BMMSCs] transplantation on carbon tetrachloride-induced liver fibrosis in mice. Fifty five female NMRI mice were divided in 5 groups, and to induce liver fibrosis CCL[4] intraperitonealy was injected 1 ml/Kg twice a week for 8 weeks 10[6] allogenic BMMSCs were infused in cell therapy group via tail vain at the end of 4[th] weeks. Liver samples were taken and evaluated with histopathologic and immunofluorescence techniques to determine the amount of fibrosis, cell homing and identity of the cells. Mice serum albumin level was measured as well. In the cell therapy group the amount of liver fibrosis and mortality rate decreased significantly [2.24 +/- 0.51% vs 3.48 +/- 0.6%, PO.05 and 27.3% vs 45.5%], respectively but there was no significant difference between their serum albumin level. These results were in compliance with low proportion of transplanted cells capable of producing albumin [0.23 +/- 0.08% of liver cells]. Because most transplanted cells were found in periportal area; they did not produce albumin. Conclusion: It seems that the major role of BMMSCs to reduce CCL[4]-induced liver fibrosis does not occur by their differentiation into hepatocyte but rather through other interaction pathways with injured liver tissue

Cell-based regenerative therapy as an alternative to liver transplantation for end-stage liver disease: experience from Iran

Several types of cells including mature hepatocytes, adult liver progenitor cells and human embryonic stem cells, fetal liver progenitor cells, bone marrow derived hematopoietic or mesenchymal stem cells, and um- bilical cord blood cells both in rodents and humans have been reported to be capable of self-replication, giving rise to daughter hepatocytes, both in vivo and in vitro. They have been shown to be able to repopulate liver in both animal models of liver injury and in patients with liver disease and to improve liver function. Human embryonic stem cell therapy seems to be a great promise for the treatment of liver cirrhosis, but there is no human clinical application due to ethical concerns or difficulties in harvesting or safely and efficiently expanding sufficient quantities. In contrast, adult bone marrow-derived hematopoietic or mesen- chymal stem cells, which can be easily and safely harvested, have been used in clinical trials to treat several chronic diseases including chronic liver disease. Cell therapy offers exciting promise for future treatment of cirrhosis and metabolic liver diseases, but significant technical hurdles remain that will only be overcome through years of intensive research. There is also serious concern about the long-term safety of stem cell therapy and the possibility of tumor development. Herein, we present our experience with cell therapy in treatment of chronic liver disease in Iran

[ effect of laminin and gelatin extracellular matrix on short-term cultivation of neonate mouse spermatogonial stem cells]

To compare the effect of laminin and gelatin on short-term culture of spermatogonial stem cells [SSCs] from neonatal mouse testes. Cell suspension containing SSCs were isolated from testes of 6 day-old mice and cultured in the presence of Glial-derived neuroterophic factor [GDNF], Epidermal Growth Factor [EGF] and Basic Fibroblastic Growth Factor [bFGF] on laminin-and gelatin-coated plates for 9 days. Number and area of colonies were measured in 5th, 7th and 9th days after culturing. At 9th day Immunostaining was used to detect expression of SSC markers, alpha 6-Integrin and beta 1-Integrin. moreover, the colonies were harvested and the percentage of alpha 6-Integrin and beta 1-Integrin positive cells was assessed by flowcytometery in both groups. Immunostaining analysis showed that our culture system contained SSC colonies as they were positive for alpha 6-Integrin and beta 1-Integrin. Additionally, the number of colonies those were formed on laminin were significantly higher in comparison with those of other group. but colony area was higher on gelatin. There was no significant difference in percentage of cells that expressed alpha 6-Integrin, beta 1-Integrin detected by flowcytometry in both groups. laminin as extracellular matrix cause to increase the number of neonate spermatogonial colonies and decrease the area of them [P

[Ultrastructural changes of sheep oocytes follow vitrification by different methods and in vitro maturation]

Our aim was determination of the sheep oocytes ultrastructural changes follow vitrification and in vitro maturation. Good quality isolated cumulus-oocyte complexes [COCs] were randomly divided into non-vitrified control, conventional straw, cryotop and solid surface vitrification groups. In the conventional and cryotop methods the vitrified COCs were plunged directly into liquid nitrogen [LN2], whereas in the solid surface group the vitrified COCs were cooled before plunging into LN2. Fresh and vitrified-warmed healthy COCs were matured in vitro and then their ultrastructural changes were evaluated. The results indicated that vitrification by cryotop and solid surface methods preserved the total arrangement of the ooplasm, whereas conventional straw vitrification disturbed the ooplasm organization. Additionally, the number of vacuoles in the ooplasm increased after vitrification, some of these vacuoles were filled partially or completely with lipids and some had filamentous scaffolding. Also, in the mature oocytes, the amount and the density of cortical granules decreased after conventional straw and solid surface vitrification. Cryotop group compared with other vitrification methods could preserve oocyte ultrastructure properly and create a condition the same as like as the control group

[In Vitro Production of Germ Cells from Stem Cells: Hypes and Hopes]

Several lines of evidence have reported that mouse ESCs can successfully differentiate into primordial germ cells [PGCs] as well as into mature male and female gametes. Human ESCs and adult stern cells [ASCs] can also differentiate into PGCs. Differentiation of ESCs into germ cells of various stages seems to he a spontaneous and quick process, probably due to the nature of ESCs themselves and the microenvironment Of the culture conditions that favor this process. Although the functionality of these LSC-delived gametes tenuous to he established. derivation of both male and female gametes from ESCs raises the possibility of using these gametes to gain a better understanding of basic reproductive biology and. in particular, in conjunction with nuclear transfer technology. to extend the potential for therapeutic cloning and the treatment or infertility

[ Effect of chick Embryonic Somites on Neural Rosette Formation in Mouse Embryonic Stem Cells]

The aim of the present study is to understand if EBs can generate neural rosette upon co-culture with chick embryo sornites. The mouse ES cells, line Royan Bi, were cultured in hanging drops to induce embryoid bodies [EBs] formation. Somites were isolated from the chick embryos and then embedded in alginate solution. Finally, alginate beads containing somites were co-cultured with EBs. RA was added to some EBs according to 2-12+12+ protocol Mean percentage of EBs containing early and late rosettes in somite, control and RA was 1456%, 2.6% and 0.0%, respectively and what is important to rosette formation in EBs was the preseice of neural inducing components as well as the time course of neural differentiation of EBs Chick embryonic somites can induce ES cells -derived EBs to generate rosttte structures with neuron formation capacity

Effect of extracellular matrix on differentiation of mouse embryonic stem cells into insulin-producing cells

Evaluation of extracellular matrics [ECMs] effect on differentiation of embryonic stem cells [ESCs] to pancreatic beta-cell. Mouse ESC line, Royan B1, was subjected to differentiation into beta-like cells in a three-step method: generation of embryoid bodies [EBs], spontaneous differentiation and induction by Nicotinamide onto different matrices including poly L-ornithine/laminin, gelatin, and two different dilution of matrigel [1:30, 1:100] and control group [no ECM]. At the final step, differentiated cells were analyzed for expression of some pancreas-specific genes using "semi-quantitative RT-PCR ", for detection of insulin and C-peptide presence in cells using "immunocytochemistry" and for the evaluation of the amount of secretd insulin in response to glucose Using "insulin secretion assay". The semi-quantitative RT-PCR analysis of differentiated cells on 1:30 matrigel coated-plates showed consistent higher expression of beta-cell specific markers including Insulin I, Insulin II, Slc2a2 in comparison to the other ECMs. The results of immunostainig for C-peptide showed no significant differences between the experimental groups and finally insulin secretion assay revealed that differentiated cells on 1:30 matrigel coated-plates secreted more insulin in response to glucose in comparison to the other ECMs. Our results suggest that type of ECM may influence ESC differentiation into insulin-secreting cells and 1:30 matrigel was more effective. However, the success rate of differentiation needs further investigations using other appropriate ECMs

Isolation of mesenchymal stem cells from dental pulp of exfoliated human deciduous teeth

The exfoliated human deciduous tooth [SHED] contain multipotent stem cells that identified to be a population of highly proliferative and clonogenic .These cells are capable of differentiating into a variety of cell types including neural cells, adipocytes, and odontoblasts. Normal exfoliated human deciduous incisors collected from six- to nine-years-old children. The pulp was separated from the crown and digested with collagenase .Single cell solutions were cultivated in alpha-MEM supplemented with ES-FCS. After two to three days, the cells reached confluency and were trypsinized and cultured for further passages. The passage-4 cells were analyzed with CD34, CD45, CD105, CD166, CD31, CD90 and CD146 markers that indicated these cells had a mesenchymal stem cell [MSC] identity. We examined the cells for Alkaline Phosphatase activity to investigate the mesenchymal [stromal] nature.Finally, the cells were differentiated into the osteoblastic and adipocytic lineages in different subcultures and analysed by RT-PCR and different staining protocols. Viable cells growing out of the explants showed elongated shapes in clusters. These cells showed alkaline phosphatase activity. Flow cytometry results revealed high expression of pluripotent stem cell markers .In some area of the osteoinductive cultures nodule-like structures were observed that showed red mineralizing area upon staining with Alizarin Red.In adipogenic cultures lipid vesicles appeared after five weeks of induction with Oil Red. This study show that pulp contains cells with high plasticity and proliferation capacity and can be easily isolated without any serious intervention

[ comparison of ultrastructural development of mouse embryonic stem cell-derived cardiomyocytes and normal invivo cardiomyocytes]

Stem cell biology has been the subject of much recent discussion. Embryonic stem [ES] cells, derived from the inner cell mass of the blastocyst stage of early mammalian embryos are expected to become a powerful tool in future regenerative medicine and developmental biology due to their capacity of selfrenewal and pluripotency. In the present study, the ultrastructural development of mouse ES cell derived cardiomyocytes was compared with invivo cardiomyocytes.. Cardiomyocytes were derived from mouse ES line [Royan B1] which developed spontaneously. The cultured cardiomyocytes were processed 3, 7, 14 and 21days after plating [day 7] for immuno histochemistry and transmission electron microscopy [TEM]. The in vivo cardiomyocytes were derived from16 days old fetuses and 2 and 8 days old pups. The beating cells expressed alpha-actinin. The maturation of the ultrastructure of cardiomyocytes depended on enhancement of development and expressed as myofibrillar bundle organization and exhibited intercalated discs, Z-disc, A, I, and H-bands, and M-line. While 7+21 days old cardiomyocytes showed all sarcomeric components such as A, I, and H-bands, Z-disc, and also M-line, T-tubule, sarcoplasmic reticulum and intercalated discs, early stage [7+3d, 7+7d and 7+14d] cardiomyocytes had few primary characteristics of subcellular structure. In fetal and 2-days old pups, the M-line was not visible. M-line was present in 8-days old pups frequently. Based on our data, mature cardiomyocytes can be produced from ES cells, and ES cell provide a good model for cardiomyocyte development. The cells can be used for cell therapy in future

[In vitro production and vitrification of bovine embryo]

It behooves the researchers to make attempts to conduct more studies; for, it deems crucial to utilize more up - to - date technologies in some special fields as cloning and producing trans genetic animals. These methods are supposed to proliferate the production of animals which are persistent against diseases, more milk production and pharmaceutical proteins. The aim of this study was in vitro production and verification of bovine embryo for next researches. After maturation and fertilization of oocyts, embryos Cultured in SOF medium at 39c temperature and%5 co2 in incubator for 8 day. Then 200 high quality blastocyst selected and vitrified [%40 ethylene glycol,%18 focal, 13 molar sacarose]. After thawing, embryos cullored for 24 hours and viability of them assessed by re-expanded embryos.%75 of oocyts fertilized and cleavaged,%20 of fertilized oocyts recived to blastocyst stage, percentage of post - thaw re - expanded blastocysts for 7 day and 8 day was%65 and%70 respectively. Even though post - thaw survival blastocyst rate on 7 day was higher than the 8 day, but difference between the two groups not significant. Rate of Fertilized oocyts, developed blastocysts and blastocysts survival after verification was as same as the other countries researchers

[Differentiation of human embryonic stem cells into insulin-secreting cells]

Type I diabetes mellitus is caused by autoimmune destruction of the insulin-producing beta-cells. A new potential method for curing the disease is transplantation of differentiated insulin- secreting cells from human embryonic stem cells. Human embryonic stem cell lines [Royan H1] were used to produce embryoid bodies. Differentiation carried out by growth factor-mediated selection of nestin positive cells. In final stage, these cells were expanded in the presence of bFGF, followed by addition of nicotinamide to promote differentiation of insulin- secreting cells. Cells were assayed by immunocytochemistry, RT-PCR, insulin secreting assay with Radio-immuno assay kit and Transmission Electron Microscopy. The cells were transplanted into immunosuppressed mice. Analysis of differentiation cells immunocytochemistry showed that these cells were insulin, glucagon, somatostatin and pancreatic polypeptide positive. RT-PCR reaction demonstrated the expression of pancreatic endocrine genes. Differentiation cells secreted insulin in response to glucose, but no significance difference in insulin concentration was observed with an increase in concentration of glucose. The implanted cells were vascularized and remained immunoreactive with insulin and glucagon. Transmission Electron microscopy of differentiate cells showed Golgi complexes, rough endoplasmic reticulum and a few granules but no true beta granules. The data showed that human embryonic stem cells can produce insulin secreting cells. However, more studies are needed to generate true beta cells

Culture and proliferation of human limbal stem cells in vitro

To evaluate the properties of cultured limbal stem cells for corneal surface reconstruction. Specimens of timbal explants were prepared from the Eye Bank of l.R. Iran. The explants were cultured and expanded on acellular amniotic membrane for 21 days. The cultured cells ''.ere evaluated for expression of Connexin 43 and keratinine K3 by immunocytochemistry and expression of K12 keratininc and P63 by RT-PCR. After 2 to 3 weeks, the limbal epithelial cells grew to form a sheet approximately 2x2 cm' in size on the amniotic membrane. On histological examination the epithelial sheet was composed of 4 to 5 cell layers at the margin of the sheet and from 1 to 4 cell layers in the area between the margin and the original explant tissue. Immunocytochemistry analysis showed Keratinine K3 and Connexin 43 were expressed in normal cornea. The markers were expressed weakly in cultured limbal cells. Also, RT-PC'R analysis revealed that P63 and K12 were expressed in cultured and normal limbal cells. The normal cornea expressed K3 and K12 but not P63 These data support the notion that expansion of limbal stem cells on amniotic membrane can be used of tranplantation to patients with limbal stem cell deficiency

[Early results of autologous cultivated limbal stem cell transplantaion in total limbal stem cell deficiency]

To report the early results of transplantation of autologous limbal stem cell cultivated on amniotic membrane in total limbal stem cell deficiency. Four eye of 4 patients with total unilateral stem cell deficiency secondary to severe chemical or thermal burn included. Stem cell deficiency was confirmed with impression cytology in all cases. Under topical anesthesia, a small limbal sector [1x1 mm] was removed from the sound eye and cultivated on amniotic membrane. Cell expansions were transplanted to the affected eye 2 weeks later. Patients were regularly followed up. At each follow-up visit, corneal eye examination with special attention recurrence or regression of vascularization, corneal pacification, and healing of epithelial defect was performed. Digital imaging was performed at each follow-up visit. Impression cytology was repeated in all cases after surgery. The patients were followed for 5-13 months. Decrease in corneal opacification and vascularization was obvious in 3 cases, in which the surface of the cornea was covered with corneal epithelium. Sectoral conjunctivalization was evident in these 3 cases, but their corneas were ready for corneal transplantation. The procedure failed in one case with total corneal conjunctivalization. Visual acuity improved in all cases. Autologous cultivated stem cell transplantation on amniotic membrane seems to be an effective way for stem cell transplantation. More definite judgment needs longer follow-up together with long-term results of corneal transplantation in these patients