Efficacy of rhNGF-loaded amniotic membrane transplantation for rabbit corneal epithelial and nerve regeneration.

AIM: To evaluate the efficacy of recombinant human nerve growth factor-loaded amniotic membrane (rhNGF-AM) on corneal epithelial and nerve regeneration in rabbit model. METHODS: Freshly prepared human amniotic membrane (AM) were immersed into PBS buffer containing 100 or 500 µg/mL rhNGF for 15, 30, and 60min at 4°C. The in vitro release kinetics of rhNGF was measured with ELISA. For in vivo evaluation, the AM were immersed with 500 µg/mL rhNGF for 30min. Fifty-seven rabbits were selected to establish corneal epithelial defect model. In addition to the 19 rabbits in control group, 38 rabbits received AM transplantation with or without rhNGF after the removal of central epithelium. Corneal epithelial defect area, sub-epithelial nerve fiber density, corneal sensitivity, rhNGF contents in resident AM and corneas were measured after the surgery. RESULTS: rhNGF was sustained release from the AM within 14d in vitro, with the positive correlation with initial immersion concentration. The immersion of AM in 500 µg/mL rhNGF for 30min achieved the most stable release within 14d. After transplantation in rabbit cornea, a high concentration of rhNGF in resident rhNGF-AM and cornea was maintained within 8d. Corneal epithelial healing, nerve fiber regeneration and the recovery of corneal sensitivity were significantly accelerated after the rhNGF-AM transplantation when compared to simple AM transplantation (all P<0.05). CONCLUSION: Simple immersion of AM achieves the sustained release of rhNGF, and promotes corneal epithelial wound healing and nerve regeneration, as well as the recovery of corneal sensitivity in rabbit.

Midterm outcomes of penetrating keratoplasty following allogeneic cultivated limbal epithelial transplantation in patients with bilateral limbal stem cell deficiency.

AIM: To evaluate the midterm outcomes of penetrating keratoplasty (PK) following allogeneic cultivated limbal epithelial transplantation (CLET) for bilateral total limbal stem cell deficiency (LSCD). METHODS: Ten patients (10 eyes) with bilateral LSCD were enrolled in this prospective noncomparative case series study. Each participant underwent PK approximately 6mo after a CLET. Topical tacrolimus, topical and systemic steroids, and oral ciclosporin were administered postoperatively. Best-corrected visual acuity (BCVA), intraocular pressure (IOP), ocular surface grading scores (OSS), corneal graft epithelial rehabilitation, persistent epithelial defect (PED), immunological rejection, and graft survival rate were assessed. RESULTS: The time interval between PK and allogeneic CLET was 6.90±1.29 (6-10)mo. BCVA improved from 2.46±0.32 logMAR preoperatively to 0.77±0.55 logMAR post-PK (P<0.001). Kaplan-Meier analysis of mean graft survival revealed graft survival rates of 100% at 12 and 24mo and 80.0% at 36mo. PEDs appeared in 5 eyes at different periods post-PK, and graft rejection occurred in 4 eyes. The total OSS decreased from 12.4±4.4 before allogeneic CLET to 1.4±1.51 after PK. CONCLUSION: A sequential therapy design of PK following allogeneic CLET can maintain a stable ocular surface with improved BCVA despite the relatively high graft rejection rate.

Effects of preservation time on proliferative potential of human limbal stem/progenitor cells.

AIM: To determine the proliferative potential and the maintenance of stem cell activity in stored human limbal tissues, and correlate this with the preservation time, cell viability and the expression of stem cell markers. METHODS: Thirty limbal rims were split into 4 parts and stored in corneal preservation medium at 4°C for 0, 1, 4, or 7 days. The limbal stem cell and mitotic markers P63, CK19, proliferating cell nuclear antigen (PCNA), and Ki67 were determined by immunohistochemical staining. The proliferative potential of limbal epithelial cells was assessed by cell viability, the ability of generating stratified epithelium, and colony forming assay. RESULTS: The stored tissues maintained limbal stratified structure to 7 days and exhibited comparable expression level of stem cell and mitotic markers. The proportion of viable cells decreased with the prolonged preservation time, while colony forming efficiency decreased from the 1(st) day and disappeared at the 4(th) day. When inoculated on amniotic membrane, the cells preserved for 1 day formed a stratified epithelium, while the cells from 4 days' preservation formed a discontinuous layer. CONCLUSION: The colony forming efficiency of limbal epithelial stem/progenitor cells decreased rapidly with the increasing preservation time, while the expression level of markers and capacity of forming epithelial monolayer on amniotic membrane decreased gradually. The limbal epithelial stem cells lost their function earlier than the lost expression level of stem cell markers. This may help us to better choose the appropriate preservation grafts for future limbal stem cell transplantation.

Comparison of the therapeutic effects of extracts from Spirulina platensis and amnion membrane on inflammation-associated corneal neovascularization.

AIM: To compare the therapeutic effects of polysaccharide extract from Spirulina platensis (PSP) and extract from amnion membrane (AME) on alkali burn-induced corneal neovascularization (CorNV). METHODS: PSP and AME were extracted from dry powder of Spirulina platensis and human aminion membrane respectively. Murine CorNV was induced by applying 1N sodiumhydroxide (NaOH) solution directly on the mice corneas. PSP and AME extracts were administered topically on the corneas 4 times daily for 7 days. The therapy effects of PSP and AME extracts were evaluated daily using slit-lamp. At the end of the therapy, corneas were harvested for H&E staining, masson trichrome staining, immunohistochemical study, and semi-quantification reverse transcriptive PCR (RT-PCR) was utilized for measurement of inflammation-related molecules. RESULTS: Topical application of PSP extract had significant therapeutic effects on CorNV that could be shown in various assays of the corneas. Compared with AME extract, PSP extract treatment was more effective in suppressing CorNV in terms of vessel length and levels of cluster of differentiation 31 (CD31) proteins or the angiogenesis related genes like vascular endothelial growth factor (VEGF), matrix metalloproteinase-2 (MMP2) and matrix metalloproteinase-9 (MMP9). PSP also inhibited inflammation more markedly by more effectively inhibiting mononuclear and polymorphonuclear cells infiltration into the corneal stroma and reducing levels of stromal cell-derived factor-1 (SDF1), tumor necrosis factor-alpha (TNFα) and macrophage inflammatory protein-3 (MIP3a). In additon, corneas of PSP group had a more regular and compact architecture of collagen with thinner corneal thickness than in the AME group. CONCLUSION: Polysaccharide extract from Spirulina platensis inhibited alkali burn-induced inflammation and CorNV more effectively than AME extract at the studied doses, thus may be used for the therapy of corneal diseases involving neovascularization and inflammation.

Differentiation of human bone marrow-derived mesenchymal stem cells into neural-like cells by co-culture with retinal pigmented epithelial cells.

AIM: To detect the differentiation effects of retinal cells or extracts on bone marrow-derived mesenchymal stem cells (BMSC). METHODS: Human fetal BMSC were previously labelled by carboxyfluorescein succinimidyl ester (CFSE), and co-cultured with retinal pigment epithelial (RPE) cells which were pre-treated with ultraviolet irradiation at a ratio of 1:1 to induce the differentiation of BMSC for up to 14 days. In some assays, a retinal extract of bovine retinal extract (BRE) was added to detect the potential effects of retinal component on the differentiation of BMSC. In addition, Neuron-specific enolase (NSE), Nestin and Glial fibrillary acidic protein (GFAP) immunostaining were performed to determine the characteristics of BMSC. RESULTS: The results indicated that by co-cultured with RPE cells, fetal BMSC were differentiated into neural-like cells expressing special neuronal markers Nestin, GFAP and NSE. And the expression of these markers was obviously increased by BRE. CONCLUSION: Retina derived cells and extracts can induce the differentiation of BMSC into neural-like cells.

[Progress of stem cells transplantation in the repair of damaged corneal surface].

Stem cells, such as limbal stem cells, oral mucosal cells, embryonic stem cells, mesenchymal stem cells, amniotic epithelial cells and skin stem cells, have shown to be able to repair many types of ocular surface damages. Based on the structural, functional and biological characteristics of corneal epithelium, the differentiation of embryonic and adult stem cells into corneal epithelial cells, the influences on the corneal wound healing and their respective clinical prospects and problems are reviewed.

Direct hepatic differentiation of mouse embryonic stem cells induced by valproic acid and cytokines.

AIM: To develop a protocol for direct hepatic lineage differentiation from early developmental progenitors to a population of mature hepatocytes. METHODS: Hepatic progenitor cells and then mature hepatocytes from mouse embryonic stem (ES) cells were obtained in a sequential manner, induced by valproic acid (VPA) and cytokines (hepatocyte growth factor, epidermal growth factor and insulin). Morphological changes of the differentiated cells were examined by phase-contrast microscopy and electron microscopy. Reverse transcription polymerase chain reaction and immunocytochemical analyses were used to evaluate the gene expression profiles of the VPA-induced hepatic progenitors and the hepatic progenitor-derived hepatocytes. Glycogen storage, cytochrome P450 activity, transplantation assay, differentiation of bile duct-like structures and tumorigenic analyses were performed for the functional identification of the differentiated cells. Furthermore, FACS and electron microscopy were used for the analyses of cell cycle profile and apoptosis in VPA-induced hepatic differentiated cells. RESULTS: Based on the combination of VPA and cytokines, mouse ES cells differentiated into a uniform and homogeneous cell population of hepatic progenitor cells and then matured into functional hepatocytes. The progenitor population shared several characteristics with ES cells and hepatic stem/progenitor cells, and represented a novel progenitor cell between ES and hepatic oval cells in embryonic development. The differentiated hepatocytes from progenitor cells shared typical characteristics with mature hepatocytes, including the patterns of gene expression, immunological markers, in vitro hepatocyte functions and in vivo capacity to restore acute-damaged liver function. In addition, the differentiation of hepatic progenitor cells from ES cells was accompanied by significant cell cycle arrest and selective survival of differentiating cells towards hepatic lineages. CONCLUSION: Hepatic cells of different developmental stages from early progenitors to matured hepatocytes can be acquired in the appropriate order based on sequential induction with VPA and cytokines.

[Expression of mouse telomerase reverse transcription in a mouse model of oxygen-induced retinopathy].

OBJECTIVE: To establish oxygen-induced retinal neovascularization in mice and to detect the expression of mTERT in mice. METHODS: It was an experimental study Establishment of oxygen-induced retinal neovascularization in mice. Thirty-two 7-day-old C57BL/6J mice were divided into oxygen-induced retinopathy group and control group without restriction of gender. In oxygen-induced retinopathy group, 16 mice were exposed to 75% +/- 2% oxygen for 5 days and then to room air; In control group, 16 mice were raised in room air. Observation of the retinal neovascularization. On the postnatal day 19, The mice's vena caudalis were perfused with 2% Evens blue solution. Eyeballs were enucleated and fixed in 4% paraformaldehyde for half an hour. Then the retina was separated and flat-mounted on the slide. The morphologic changes of retinal vessel were observed and captured under fluorescence microscope. Histological observation and vascular endothelial cells counting. The eyeballs were enucleated and then fixed. After paraffin imbedding, 4 microm serial slices, hematoxylin-eosin staining, select one section every 60 microm to count the endothelial cell nucleus that break through the inner limiting membrane. Expression of mTERT mRNA were confirmed by reverse-transcription polymerase chain reaction (RT-PCR). In the each group, the retina were all carefully dissected on the postnatal day 19. The total RNA was isolated and cDNA was synthesized before RT-PCR was performed. The PCR products were separated by 2% agarose gel electrophoresis and photographed. Expression of mTERT mRNA were confirmed by Real-time PCR The total RNA was isolated and cDNA was synthesized (The same procedure as RT-PCR). Fluorescent real-time quantitative polymerase chain reaction system (total 20 microl) was made. The Fluorescent signals were detected at 60 degrees C. The expression of mTERT were confirmed by immunohistochemistry. At P19, 4 microm cross sections were made in the hyperoxia-exposed and normal retinas. Sections were incubated with rabbit anti-Human/Mouse/Rat Telomerase 60 minutes at 37 degrees C. Anti-rabbit immunoglobulin G, depending on the primary antibody, was used as a secondary antibody for 30 min. Peroxidase activity was detected with the substrate diaminobenzidine. Permanent slides were covered with a 1.5 mm thick cover slip, examined using a light microscope and photographed. RESULTS: The central retina was nonperfused region at P12. The most of the central retina showed almost no perfusion and the radial vessels appeared tortuous and dilated at P14. Retinal neovascularization occurred at maximum between postnatal day 17 and postnatal day 19. Paraffin tissue slice with hematoxylin-eosin staining showed that in the control group the average counts of vascular endothelial cells which break through the inner limiting membrane were hardly seen, but in hyperoxia group were noticeably more than in the control group. Reverse-transcription polymerase chain reaction (RT-PCR) results: the mRNA of mTERT and bFGF in the retinopathy group were higher than in the control group (P < 0.05). Real-time PCR results: the expression of mTERT mRNA in the retinopathy group was noticeably higher than in the control group (F = 173.104, P < 0.05). Immunohistochemical staining showed that mTERT protein were positive in the retinal neovascularization of the hyperoxia group, but were negative in the retinal vessel of the control group. CONCLUSIONS: Telomerase reverse transcription and angiogenic correlation factors were up-regulated in a mouse model of oxygen-induced retinopathy, which may have therapeutic potential in the treatment with the neovascularization in retinopathy.

[TERT-siRNA inhibits oxygen-induced retinal neovascularization in mice].

OBJECTIVE: To investigate the inhibitory effect of small interfering RNA (siRNA) targeting TERT on murine retinal neovascularization and explore the feasibility of potential therapeutic approach in retinal vascular disease. METHODS: Two recombinant plasmids TERT siRNA (pSIREN-mTERT-1) and negative plasmid (pSIREN-mTERT-N) were constructed and 80 seven-day-old C57BL/6J mice were divided randomly into therapeutic group (A), negative plasmid group (B), oxygen-induced retinopathy group (C) and normal control group (D), 20 mice in each group. Group A, B and C were exposed to 75% +/- 2% oxygen for 5 days and then to room air, which induced mice retinal neovascularization. Groups A and B were injected two kinds of the above recombinant plasmid into the murine vitreous on the 12th day. The mice of group D were raised in normal oxygen circumstance. On the 19th day, 2% Evens blue angiography was used to observe the pattern of the retinal vascular. Expression of TERT mRNA were confirmed by reverse-transcription polymerase chain reaction (RT-PCR) and Real-time PCR. Histological observation and vascular endothelial cells counting were used to examine the effects of siRNA on the retinal neovascularization. RESULTS: Retinal flat after Evans blue angiography indicated that the vessels of group A formed a fine radial branching pattern, which was similar to normal mice. In group A, the retinal neovascularization reduced and the structure of retina were more regular than group B and C. At the same time the large vessels were distorted, neovascular clusters proliferated and fluorescence leaked in the middle and periphery area in group B and C. RT-PCR and Real-time PCR showed the expression of TERT mRNA was downregulated in group A compared with groups B and C (P < 0.05). Paraffin tissue slice with hematoxylin-eosin staining showed that the average counts of vascular endothelial cells which break through the inner limiting membrane in group A were less than groups B and C, the differences were significant (P < 0.05). CONCLUSION: Pathologic retinal neovascularization can be inhibited by specific TERT siRNA in vivo, which may be a novel efficient strategy against proliferative vasculopathies.

In vitro differentiation of embryonic stem cells into hepatocytes induced by fibroblast growth factors and bone morphological protein-4.

The feasibility of transforming embryonic endoderm into different cell types is tightly controlled by mesodermal and septum transversumal signalings during early embryonic development. Here, an induction protocol tracing embryonic liver development was designed, in which, three growth factors, acid fibroblast growth factor, basic fibroblast growth factor and bone morphological protein-4 that secreted from pre-cardiac mesoderm and septum transversum mesenchyme, respectively, were employed to investigate their specific potency of modulating the mature hepatocyte proportion during the differentiation process. Results showed that hepatic differentiation took place spontaneously at a low level, however, supplements of the three growth factors gave rise to a significant up-regulation of mature hepatocytes. Bone morphological protein-4 highlighted the differentiation ratio to 40-55%, showing the most effective promotion, and also exhibited a synergistic effect with the other two fibroblast factors, whereas no similar phenomenon was observed between the other two factors, which was reported for the first time. Our study not only provides a high-performance system of embryonic stem cells differentiating into hepatocytes, which would supply a sufficient hepatic population for related studies, but also make it clear of the inductive effects of three important growth factors, which could support for further investigation on the mechanisms of mesodermal and septumal derived signalings that regulate hepatic differentiation.

In vitro differentiation of hepatic progenitor cells from mouse embryonic stem cells induced by sodium butyrate.

Recently it was shown that embryonic stem (ES) cells could differentiate into hepatocytes both in vitro and in vivo, however, prospective hepatic progenitor cells have not yet been isolated and characterized from ES cells. Here we presented a novel 4-step procedure for the differentiation of mouse ES cells into hepatic progenitor cells and then hepatocytes. The differentiated hepatocytes were identified by morphological, biochemical, and functional analyses. The hepatic progenitor cells were isolated from the cultures after the withdrawal of sodium butyrate, which was characterized by scant cytoplasm, ovoid nuclei, the ability of rapid proliferation, expression of a series of hepatic progenitor cell markers, and the potential of differentiation into hepatocytes and bile duct-like cells under the proper conditions that favor hepatocyte and bile epithelial differentiation. The differentiation of hepatocytes from hepatic progenitor cells was characterized by a number of hepatic cell markers including albumin secretion, upregulated transcription of glucose-6-phosphatase and tyrosine aminotransferase, and functional phenotypes such as glycogen storage. The results from our experiments demonstrated that ES cells could differentiate into a novel bipotential hepatic progenitor cell and mature into hepatocytes with typical morphological, phenotypic and functional characteristics, which provides an useful model for the studies of key events during early liver development and a potential source of transplantable cells for cell-replacement therapies.

Cyclic adenosine 3',5'-monophosphate induces differentiation of mouse embryonic stem cells into cardiomyocytes.

Embryonic stem (ES) cells, derived from blastocyst-stage of early mammalian embryos, have the potential to differentiate into derivatives of all three embryonic germ layers. Here we reported the first evidence that murine pluripotent ES cells could be induced to differentiate into cardiomyocytes by cyclic adenosine 3',5'-monophosphate (cAMP) in vitro. Spontaneously beating of cardiac cell clusters began to be observed within the outgrowths of embryoid bodies (EBs) as early as 2 days after the onset of differentiation. By days 5-8 after induction, a maximum level of cardiomyocyte differentiation could be achieved. Incubation of EBs with cAMP at concentrations ranging from 0.01 mg/L to 1 mg/L resulted in a significant elevation in differentiation rate, reaching a maximum value of 44.0 +/- 1.3% at 0.03 mg/L of exposure. At 0.03 mg/L concentration point, an approximately 8.1-fold increase in cardiomyocyte differentiation was observed in comparison with 5.4 +/- 0.9% of untreated controls. The differentiation rate induced by cAMP was shown to be similar to that of RA/DMSO treated controls, indicating that cAMP has the same inducing effect as RA/DMSO. However, no significant co-inducing effects between cAMP and RA/DMSO were seen. Cardiomyocytes were evident as they expressed cardiac cell specific genes and protein markers including GATA4, Nkx2.5, beta-MHC, atrial natriuretic factor (ANF) and alpha-actin when analyzed by reverse transcription polymerase chain reaction (RT-PCR) and immunohistochemical staining. The results from the present study suggested a novel role of cAMP in cardiomyocyte differentiation and provided a new research model for the study of cardiac cell biology.

Generation of embryoid bodies from mouse embryonic stem cells cultured on STO feeder cells.

Embryoid bodies, which are similar to post-implantation egg-cylinder stage embryos, provide a model for the study of embryo development and stem cell differentiation. We describe here a novel method for generating embryoid bodies from murine embryonic stem (ES) cells cultured on the STO feeder layer. The ES cells grew into compact aggregates in the first 3 days of coculture, then became simple embryoid bodies (EBs) possessing primitive endoderm on the outer layer. They finally turned into cystic embryoid bodies after being transferred to Petri dishes for 1-3 days. Evaluation of the EBs in terms of morphology and differentiating potential indicates that they were typical in structure and could generate cells derived from the three germ layers. The results show that embryoid bodies can form not only in suspension culture but also directly from ES cells cultured on the STO feeder layer.

[Advances in the research of differentiation of embryonic stem cells into hepatocytes].

Orthotopic liver transplantation has proven to be effective in the treatment of a variety of life-threatening liver diseases, however, the limitations of donated organs available and long-term immunosuppression provided an impetus for developing alternative therapies. Cell replacement strategies have been one major effective approach for overcoming the obstacles of organ transplantation in recent years. The exogenous cells should be able to proliferate and differentiate into mature hepatic cells after grafting. Use of mature hepatocytes is also hampered by limited tissue source and inability to proliferate and maintain the function for a long term in vitro. Embryonic stem cells are immortal and pluripotent and may provide a novel cell source for potential cell therapy. This review summarizes the mechanisms of controlling early liver development and hepatic differentiation of visceral endoderm in embryoid bodies, and provides an overview of diverse differentiation systems in vitro and in vivo that were applied to hepatic research in recent years. Several studies have demonstrated that ES cell-derived hepatocytes can incorporate into liver tissue and function in vivo , but a few of them have shown complete restoration of liver function after transplantation into mice with liver diseases. Further studies should be made to exploit efficient methods and clinical applications of hepatocytes derived from ES cells in the future. In addition to clinical transplantation for treatment of liver diseases, ES cells can provide a valuable tool for drug discovery applications and study on of molecular basis of hepatic differentiation.