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Human pluripotent stem cell-derived retinal pigment epithelium (iRPE) is an attractive cell source for disease modeling and cell replacement therapy of retinal disorders with RPE defects. However, there are still challenges to develop appropriate culture conditions close to in vivo microenvironment to generate iRPE sheets, which mimic more faithfully the characteristics and functions of the human RPE cells. Here, we developed a simple, novel platform to construct authentic iRPE sheets using human amniotic membrane (hAM) as a natural scaffold. The decellularized hAM (dAM) provided a Bruch's membrane (BM)-like bioscaffold, supported the iRPE growth and enhanced the epithelial features, polarity distribution and functional features of iRPE cells. Importantly, RNA-seq analysis was performed to compare the transcriptomes of iRPE cells cultured on different substrates, which revealed the potential mechanism that dAM supported and promoted iRPE growth was the inhibition of epithelial-mesenchymal transition (EMT). The tissue-engineered iRPE sheets survived and kept monolayer when transplanted into the subretinal space of rabbits. All together, our results indicate that the dAM imitating the natural BM allows for engineering authentic human RPE sheets, which will provide valuable biomaterials for disease modeling, drug screening and cell replacement therapy of retinal degenerative diseases. STATEMENT OF SIGNIFICANCE: Engineered RPE sheets have a great advantage over RPE cell suspension for transplantation as they support RPE growth in an intact monolayer which RPE functions are dependent on. The substrates for RPE culture play a critical role to maintain the physiological functions of the RPE in stem cell therapies for patients with retinal degeneration. In this study, we constructed engineered iRPE sheets on the decellularized human amniotic membrane scaffolds, which contributed to enhancing epithelial features, polarity distribution and functional features of iRPE. dAM exhibited the ability of anti-epithelial mesenchymal transition to support iRPE growth. Furthermore, the results of transplantation in vivo demonstrated the feasibility of iRPE sheets in retina regenerative therapy. Engineering RPE sheets on dAM is a promising strategy to facilitate the development of iRPE replacement therapy and retinal disease modeling.

Background: An increasing number of HIV-negative cryptococcal meningitis (CM) patients have been reported with fatality approaching 30%.At present, HIV-negative CM patients are stratified according to clinical guidelines and clinical experience for individualized treatment, but the effect seems to be not ideal in clinical practice. Therefore, an accurate model that predict the prognosis for HIV-negative CM patients is needed to provide reference for precision treatment. Methods: This retrospective study involved 490 HIV-negative CM patients diagnosed between January 1, 1998, and March 31, 2022, by neurologists from 3 tertiary Chinese centres. Prognosis was evaluated at 10 weeks after the initiation of antifungal therapy. We used least absolute shrinkage and selection operator (LASSO) for feature filtering and developed a machine learning (ML) model to predict the prognosis in HIV-negative CM patients. Fifty-six patients from 2 other hospitals were analysed for external validation. An artificial intelligence (AI)-based detection model was also developed to automate the rapid counting of microscopic cryptococcal counts. Results:The final prediction model for HIV-negative CM patients comprised 8 variables: CSF cryptococcal count, CSF white blood cell (WBC), altered mental status, hearing impairment, CSF chloride levels, CSF opening pressure (OP), aspartate aminotransferase levels at admission and decreased rate of CSF cryptococcal count within 2 weeks after admission. The areas under the curve (AUCs) in the internal and external validation sets were 0.87 (95% CI 0.794-0.944) and 0.86 (95% CI 0.744-0.975), respectively. An AI model was trained to detect and count cryptococci, and the mean average precision (mAP) was 0.993. Additionally, an online and freely available platform for predicting prognosis and detecting and counting cryptococci in HIV-negative CM patients was established. Conclusions:A ML model for predicting prognosis in HIV-negative CM patients was built and validated, and the model might provide a reference for personalized treatment of HIV-negative CM patients. The change in the CSF cryptococcal count in the early phase of HIV-negative CM treatment can reflect the prognosis of the disease. In addition, utilizing AI to detect and count CSF cryptococci in HIV-negative CM patients can eliminate the interference of human factors in detecting cryptococci in CSF samples and reduce the workload of the examiner.

X-linked juvenile retinoschisis (XLRS), caused by the mutation of RS1 gene, is one of the most common causes of macular degeneration for male adolescents. The mutations and clinical manifestations of the disease are diverse. Neither the relationship between the genotypes and phenotypes, nor the radical treatment like gene therapy has been found by now. Retrospective studies have shown that carbonic anhydrase inhibitors can help reduce cysts. However, the specifically pharmacological mechanism remains unknown. Here, we culture induced pluripotent stem cells by drawing peripheral blood from a patient with XLRS, which are supposed to facilitate related researches.

Retinal degenerative diseases are the main causes of irreversible blindness without effective treatment. Pluripotent stem cells that have the potential to differentiate into all types of retinal cells, even mini-retinal tissues, hold huge promises for patients with these diseases and many opportunities in disease modeling and drug screening. However, the induction process from hPSCs to retinal cells is complicated and time-consuming. Here, we describe an optimized retinal induction protocol to generate retinal tissues with high reproducibility and efficiency, suitable for various human pluripotent stem cells. This protocol is performed without the addition of retinoic acid, which benefits the enrichment of cone photoreceptors. The advantage of this protocol is the quantification of EB size and plating density to significantly enhance the efficiency and repeatability of retinal induction. With this method, all major retinal cells sequentially appear and recapitulate the main steps of retinal development. It will facilitate downstream applications, such as disease modeling and cell therapy.

Background: Retinal degenerative disorders (RDs) are the main cause of blindness without curable treatment. Our previous studies have demonstrated that human-induced pluripotent stem cells can differentiate into retinal organoids with all subtypes of retina, which provides huge promise for treating these diseases. Before these methods can be realized, RD animal models are required to evaluate the safety and efficacy of stem cell therapy and to develop the surgical tools and procedures for cell transplantation in patients. This study involved the development of a monkey model of RD with controllable lesion sites, which can be rapidly prepared for the study of preclinical stem cell therapy among other applications. Methods: Sodium nitroprusside (SNP) in three doses was delivered into the monkey eye by subretinal injection (SI), and normal saline was applied as control. Structural and functional changes of the retinas were evaluated via multimodal imaging techniques and multifocal electroretinography (mfERG) before and after the treatment. Histological examination was performed to identify the target layer of the affected retina. The health status of monkeys was monitored during the experiment. Results: Well-defined lesions with various degrees of retinal degeneration were induced at the posterior pole of retina as early as 7 days after SNP SI. The damage of SNP was dose dependent. In general, 0.05 mM SNP caused mild structural changes in the retina, 0.1 mM SNP led to the loss of outer retinal layers, including the outer plexiform layer (OPL), outer nuclear layer (ONL), and retinal pigment epithelium (RPE), while 0.2 mM SNP impacted the entire layer of the retina and choroid. MfERG showed reduced amplitude in the damaged region. The structural and functional damages were not recovered at 7-month follow-up. Conclusion: A rapidly induced lesion site-controllable retinal degeneration monkey model was established by the subretinal administration of SNP, of which the optimal dose is 0.1 mM. This monkey model mimics the histological changes of advanced RDs and provides a valuable platform for preclinical assessment of stem cell therapy for RDs.

Background: Retinal degenerative disorders (RDs) are the main cause of blindness without curable treatment. Our previous studies have demonstrated that human induced pluripotent stem cells can differentiate into retinal organoids with all subtypes of retina, which provides huge promises for treating these diseases. Before it can be turned into reality, RD animal models are required to evaluate the safety and efficacy of stem cell therapy, and to develop the surgical tools and procedures for cell transplantation in patients. This study is to develop a monkey model of RD with controllable of lesion sites which can be rapidly prepared, for the studies of preclinical stem cell therapy among other applications.Methods: Sodium nitroprusside (SNP) in three doses was delivered into the monkey eye by subretinal injection (SI) and normal saline was applied as control. Structural and functional changes of the retinas were evaluated via multimodal imaging techniques and multifocal electroretinography (mfERG) before and after the treatment. Histological examination was performed to identify the target layer of the affected retina. The health status of monkeys was monitored during the experiment. Results: Well defined lesion with various degree of retinal degeneration was established at the posterior pole of retina as early as 7 days after SNP SI. The damage effect of SNP was dose-dependent. 0.05 mM SNP caused invisible structural changes in retina, similar to the control. 0.1 mM SNP led to the loss of outer retinal layer, including OPL, ONL and RPE, while 0.2 mM SNP impacted the entire layer of retina and choroid. MfERG showed reduced amplitude in the damaged region. The structural and functional damages were not recovered after 7-month follow-up. Conclusion: A simple, rapidly induced, lesion site-controllable, retinal degeneration model in monkey was established by the subretinal injection of 0.1 mM SNP. This monkey model closely mimics the histological changes of RDs, and provides a valuable platform for preclinical assessment of stem cell therapy.

CLCN2-related leukoencephalopathy (CC2L) is a rare disease due to autosomal recessive loss-of-function mutations in CLCN2 gene. We generated an induced pluripotent stem cell (iPSC) line (SKLOi001-A) from urine cells isolated from a CC2L patient carrying a homozygotic mutation: c.2257C>T (p.Arg753*) in CLCN2 gene via an integration-free methods. The established iPSC line kept the CLCN2 mutation and displayed a normal karyotype, expressed pluripotency markers, showed differentiation potential. This newly iPSC line could be served as a possible tool to unravel the mechanisms underlying CLCN2-associated diseases and screen drugs for the treatment.

Effective derivation of three-dimensional (3D) retinal tissue from human-induced pluripotent stem cells (hiPSCs) could provide models for drug screening and facilitate patient-specific retinal cell replacement therapy. However, some hiPSC lines cannot undergo 3D self-organization and show inadequate differentiation efficiency to meet clinical demand. In this study, we developed an optimized system for derivation of 3D retinal tissue. We found that the Wnt signaling pathway antagonist Dickkopf-related protein 1 (DKK-1) rescued the inability of differentiated retinal progenitors to self-organize. By evaluating DKK-1 expression and supplying DKK-1 if necessary, retinal organoids were differentiated from six hiPSC lines, which were reprogramed from three common initiating cell types. Retinal tissues derived from the optimized system were well organized and capable of surviving for further maturation. Thus, using this system, we generated retinal tissues from various hiPSC lines with high efficiency. This novel system has many potential applications in regenerative therapy and precision medicine.

Urine cells, a body trash, have been successfully reprogrammed into human induced pluripotent stem cells (U-hiPSCs) which hold a huge promise in regenerative medicine. However, it is unknown whether or to what extent U-hiPSCs can generate retinal cells so far. With a modified retinal differentiation protocol without addition of retinoic acid (RA), our study revealed that U-hiPSCs were able to differentiate towards retinal fates and form 3D retinal organoids containing laminated neural retina with all retinal cell types located in proper layer as in vivo. More importantly, U-hiPSCs generated highly mature photoreceptors with all subtypes, even red/green cone-rich photoreceptors. Our data indicated that a supplement of RA to culture medium was not necessary for maturation and specification of U-hiPSC-derived photoreceptors at least in the niche of retinal organoids. The success of retinal differentiation with U-hiPSCs provides many opportunities in cell therapy, disease modeling, and drug screening, especially in personalized medicine of retinal diseases since urine cells can be noninvasively collected from patients and their relatives.

RPE65-associated Leber congenital amaurosis (LCA) is one of highly heterogeneous, early onset, severe retinal dystrophy with at least 130 gene mutation sites identified. Their pathogenicity has not been directly clarified due to lack of diseased cells. Here, we generated human induced pluripotent stem cells (hiPSCs) from one putative LCA patient carrying two novel RPE65 mutations with c.200T>G (p.L67R) and c.430T>C (p.Y144H), named RPE65-hiPSCs, which were confirmed to contain the same mutations. The RPE65-hiPSCs presented typical morphological features with normal karyotype, expressed pluripotency markers, and developed teratoma in NOD-SCID mice. Moreover, the patient hiPSCs were able to differentiate towards retinal lineage fate and self-form retinal organoids with layered neural retina and RPE. All major retinal cell types including photoreceptors were also acquired overtime. Compared to healthy control, RPE cells from patient iPSCs had lower expression of RPE65, but similar phagocytic activity and VEGF secretion level. Our findings disclosed that these novel RPE65 gene mutations (c.200T>G and c.430T>C) had no influence on early stage of human retinal development, but down-regulated RPE65 expression in RPE cells. This study provided the valuable patient specific, disease targeted retinal organoids containing photoreceptor and RPE cells, which would facilitate the study of personalized pathogenic mechanisms of disease, drug screening, and cell replacement therapy.

This study was conducted to determine the dynamic Islet1 and Brn3 (POU4F) expression pattern in the human fetal retina and human-induced pluripotent stem cell- (hiPSC-) derived retinal organoid. Human fetal eyes from 8 to 27 fetal weeks (Fwks), human adult retina, hiPSC-derived retinal organoid from 7 to 31 differentiation weeks (Dwks), and rhesus adult retina were collected for cyrosectioning. Immunofluorescence analysis showed that Islet1 was expressed in retinal ganglion cells in the fetal retina, human adult retina, and retinal organoids. Unexpectedly, after Fwk 20, Brn3 expression gradually decreased in the fetal retina. In the midstage of development, Islet1 was detected in bipolar and developing horizontal cells. As the photoreceptor developed, the Islet1-positive cone precursors gradually became Islet1-negative/S-opsin-positive cones. This study highlights the distinguishing characteristics of Islet1 dynamic expression in human fetal retina development and proposes more concerns which should be taken regarding Brn3 as a cell-identifying marker in mature primate retina.

We have identified a discrete, focal telomere DNA expansion phenotype in the photoreceptor cell layer of normal, non-neoplastic human retinas. This phenotype is similar to that observed in a subset of human cancers, including a large fraction of tumors of the central nervous system, which maintain their telomeres via the non-telomerase-mediated alternative lengthening of telomeres (ALT) mechanism. We observed that these large, ultra-bright telomere DNA foci are restricted to the rod photoreceptors and are not observed in other cell types. Additionally, focus-positive rod cells are dispersed homogeneously throughout the posterior retinal photoreceptor cell layer and appear to be human-specific. We examined 108 normal human retinas obtained at autopsy from a wide range of ages. These large, ultra-bright telomere DNA foci were not observed in infants before 6 months of age; however, the prevalence of focus-positive rod cells dramatically increased throughout life. To investigate associations between this phenotype and retinal pathology, we assessed adult glaucoma (N = 29) and diabetic retinopathy (N = 38) cases. Focus-positive rod cells were prominent in these diseases. When compared to the normal group, after adjusting for age, logistic regression modeling revealed significantly increased odds of falling in the high category of focus-positive rod cells for glaucoma and diabetic retinopathy. In summary, we have identified a dramatic telomere alteration associated with aging and diseases affecting the retina.

Purpose: To assess the appropriate dose of sodium nitroprusside for establishing acute retinal photoreceptor degeneration models in rabbits. Methods: Sodium nitroprusside (SNP) was delivered intravitreously. Sixteen New Zealand White rabbits are divided into four groups randomly: 0.1 mM, 0.25 mM, 0.5 mM SNP intravitreal injection group (experimental groups), and normal saline intravitreal injection group (control group). Assessments included weight, anterior segment photography, fundus photography, Hematoxylin-eosin staining, immunofluorescence, multi-focal electroretinogram (mfERG) and pupillary direct light reflex were performed at baseline and day 28 after injection. The spectral domain optical coherence tomography (SD-OCT) and full field electroretinogram (fERG) were performed at baseline and day 1, 3, 7, 14, 21 and 28 after injection. Results: No complications and no significant different in weight were found among all groups. No obvious change was found by slit lamp and fundus photography after injection in all groups. In SD-OCT exams, a time-dependent and dose-dependent injury of photoreceptor was found in SNP injection groups (P

The derivation and maintenance of human pluripotent stem cells (hPSCs) in stable naive pluripotent states has a wide impact in human developmental biology. However, hPSCs are unstable in classical naive mouse embryonic stem cell (ESC) WNT and MEK/ERK signal inhibition (2i) culture. We show that a broad repertoire of conventional hESC and transgene-independent human induced pluripotent stem cell (hiPSC) lines could be reverted to stable human preimplantation inner cell mass (ICM)-like naive states with only WNT, MEK/ERK, and tankyrase inhibition (LIF-3i). LIF-3i-reverted hPSCs retained normal karyotypes and genomic imprints, and attained defining mouse ESC-like functional features, including high clonal self-renewal, independence from MEK/ERK signaling, dependence on JAK/STAT3 and BMP4 signaling, and naive-specific transcriptional and epigenetic configurations. Tankyrase inhibition promoted a stable acquisition of a human preimplantation ICM-like ground state via modulation of WNT signaling, and was most efficacious in efficiently reprogrammed conventional hiPSCs. Importantly, naive reversion of a broad repertoire of conventional hiPSCs reduced lineage-primed gene expression and significantly improved their multilineage differentiation capacities. Stable naive hPSCs with reduced genetic variability and improved functional pluripotency will have great utility in regenerative medicine and human disease modeling.

Numerous therapeutic procedures in modern medical research rely on the use of tissue engineering for the treatment of retinal diseases. However, the cell source and the transplantation method are still a limitation. Previously, it was reported that a self-organizing three-dimensional neural retina can be induced from human-induced pluripotent stem cells (hiPSCs). In this study, we disclose the generation of retinal ganglion cells (RGCs) from the neural retina and their seeding on a biodegradable poly (lactic-co-glycolic acid) (PLGA) scaffold to create an engineered RGC-scaffold biomaterial. Moreover, we explored the dendritic arbor, branching point, functional axon and action potential of the biomaterial. Finally, the cell-scaffold was transplanted into the intraocular environment of rabbits and rhesus monkeys. Statement of Significance As a part of the mammalian central nervous system (CNS), the retinal ganglion cell (RGC) shows little regenerative capacity. With the use of medical biomaterial for cells seeding and deliver, a new domain is now emerging that uses tissue engineering therapy for retinal disease. However, previous studies utilized RGCs from rodent model, which has limitations for human disease treatment. In the present study, we generated RGCs from hiPSCs-3D neural retina and then seeded these RGCs on PLGA scaffold to create an engineered RGC-scaffold biomaterial. Moreover, we assessed the transplantation method for biomaterial in vivo. Our study provides a technique to produce the engineered human RGC-scaffold biomaterial.

Retinal progenitor cells (RPC) are an excellent resource for retinal replacement therapy but usually unavailable. We attempted to induce bone marrow mesenchymal stromal cells (BMSC) into RPC.BMSC and embryonic day 13.5 (E13.5) RPC derived from wild-type or enhanced green fluorescence protein (EGFP) transgenic (Egfp(+/+)) mice were co-cultured in a transwell or re-aggregation system. Gene and protein expressions were investigated by reverse transcription-polymerase chain reaction (PCR) and immunofluorescence, respectively. Spontaneous cell fusion was evaluated by Chloromethylbenzamido derivative of 1,1'- dioctadecyl-3,3,3',3' - tetramethylindocarbocyanine perchlorate (CM-DiI) labeling together with EGFP tracing.BMSC from both wild-type and Egfp(+/+) mice displayed similar spindle shapes. The undifferentiated BMSC already expressed immature neural markers but did not express Nfl, Gfap or the retina-related genes Pax6, Math5 and Brn3b. When co-cultured with E13.5 RPC in the transwell system, BMSC displayed transient expression of early retinal development genes, including Pax6, Math5 and Brn3b at 3 days, as well as long-term expression of Nfl (up to 21 days). No expression of the late photoreceptor gene rhodopsin could be detected at any time. In re-aggregation co-culture, E13.5 RPC induced EGFP-positive BMSC to express not only the early retinal development genes but also the late gene rhodopsin. Furthermore, a small fraction of BMSC could be induced to express the synaptophysin protein. Re-aggregation co-culture of CM-DiI-labeled BMSC and EGFP-positive E13.5 RPC displayed minimal co-localization of the two fluorescence signals.E13.5 RPC are capable of inducing BMSC towards an RPC fate. The differentiation is independent of cell fusion. Cytokines and cell-cell interactions exert this induction effect, but they have different functions.

Purpose Retinal pigment epithelium (RPE) and neural retina could be generated concurrently through retinal organoid induction approaches using human induced pluripotent stem cells (hiPSCs), providing valuable sources for cell therapy of retinal degenerations. This study aims to enrich and expand hiPSC-RPE acquired with this platform and explore characteristics of serially passaged RPE cells. Methods RPE has been differentiated from hiPSCs with a published retinal organoid induction method. After detachment of neural retina on the 4th week, the remaining mixture was scraped from the dish and subjected to suspension culture for the formation of RPE spheroids. RPE sheets were isolated and digested for expansion. The cellular, molecular, and functional features of expanded RPE cells were evaluated by different assays. Results Under suspension culture, hiPSC-RPE spheroids with pigmentation self-formed were readily enriched by removing the non-retinal tissues. RPE sheets were further dissected and purified from the spheroids. The individualized RPE cells could be passaged every week for at least 5 times in serum medium, yielding large numbers of cells with high quality in a short period. In addition, when switched to a serum-free medium, the passaged RPE cells could mature in cellular, molecular, and physiological levels, including repigmentation, markers expression, and phagocytosis. Conclusions We developed a simple and novel RPE spheroids formation approach to enrich and expand hiPSC-RPE cells generated along with retinal neurons on a universal retinal organoid induction platform. This achievement will reduce the cost and time in producing retinal cells for basic and translational researches, in particular for retinal cell therapy.

Uric acid (UA) has been reported to be reduced in the serum of patients with multiple sclerosis (MS) and optic neuritis (ON). However, the relationship between UA and neuromyelitis optica (NMO) was unknown. NMO was claimed to be a distinct nosologic entity from MS. The aim of our study was to investigate the correlation between serum UA level and the clinical characteristics of NMO. The serum UA level was measured in 403 Chinese patients; 69 with NMO, 32 ON, 127 MS, 80 cerebral infarction (CI) patients, and 95 healthy controls (CTL). Serum UA level in NMO was significantly lower than that in CI (249.89 +/- 93.74 vs. 315.42 +/- 85.57 micromol/L, p = 0.004) and CTL (249.89 +/- 93.74 vs. 314.33 +/- 102.05 micromol/L, p0.0001). However, no difference was found between NMO and MS (p = 0.496) or NMO and ON (p = 0.858). When the analysis was performed in the female cohort separately, UA level was significantly lower in females than in males in all groups. It was also shown in our study that UA level in patients with NMO was not correlated with disease activity revealed by MRI, disease disability or duration of disease. Our results indicated a reduced serum UA level in patients with NMO.

Pluripotent stem cells can be induced from somatic cells by the transcription factors Oct3/4, Sox2, c-Myc and Klf4 when co-cultured with mouse embryonic fibroblast (MEF) feeder cells. To date, the role of the feeder cells in the reprogramming process remains unclear. In this study, using a comparative analysis, we demonstrated that MEF feeder cells did not accelerate reprogramming or increase the frequency of induced pluripotent stem (iPS) cell colonies. However, feeder conditions did improve the growth of primary iPS colonies and were necessary for passaging the primary colonies after reprogramming was achieved. We further developed a feeder-free culture system for supporting iPS growth and sustaining pluripotency by adding bFGF and activin A (bFA) to the medium. These data will facilitate the generation of human iPS cells without animal feeders for regenerative medicine.

The aim of this study was to investigate morphologic changes of retina and sclera in form-deprived myopic rabbits following intravitreal dopamine injection.Neonatal rabbits were monocularly deprived of form vision by suturing the right eyelids after natural eye opening. In the form deprivation (FD) group, the right eye received FD alone. In the dopamine-form deprivation (DA-FD) group, the deprived eye received an intravitreal injection of 20 microg of dopamine every 5 days for a total of 4 injections. In the saline-FD group, the deprived eye received saline injections to the same schedule as the DA-FD group. The untreated contralateral eyes were used as controls. After an 8-week treatment period, the effects of DA on sclera and retina anterior and posterior to the equator were evaluated by light and electron microscopy.Treated eyes in the FD and saline-FD groups developed form deprivation myopia. These eyes had markedly reduced scleral thickness and smaller diameter scleral collagen fibrils posterior to the equator. In addition, the normal gradient of fibril size from the outer to the inner layers of the posterior sclera was absent in the treated eyes of both the FD and saline-FD groups. In contrast, posterior scleral thickness was greater in DA-FD eyes than in contralateral controls. A distinct swelling of retinal pigment epithelium mitochondria was observed in the treated eye of the DA-FD group, but no obvious retinal abnormalities were found in the treated eyes of the other two groups.The sclera, especially posterior sclera, plays an important role in both the induction and inhibition of myopia. An additional finding was that changes in the sclera of rabbits with low myopia were similar to those of the sclera of other mammals with high myopia. The results of this study will contribute to the understanding of the mechanisms of myopia development and inhibition by intravitreal dopamine injection.

Microinjection of adult stem cells (ASCs) into blastocysts provides a classic model for studying ASC plasticity. To explore the molecular mechanisms that govern the reprogramming of ASCs, we evaluated the experimental model through microinjection of human epidermal stem cells (hEpiSCs) into mouse blastocysts. Mouse blastocysts underwent regular embryogenesis after microinjection of allogeneic cells, confirmed by morphological observation and embryo cell counting. hEpiSCs survive and integrate into mouse embryos, by monitoring the migration of injected cells at 2, 4, 12, 16 and 24 h. In this xenogeneic system, hEpiSCs could be reprogrammed within 24 h, as evidenced by the silencing of CK15 and Integrin beta 1 gene expression, without activation of Oct4 and Nanog. Microinjection of hEpiSCs into mouse blastocysts provides an efficient model for studying the molecular mechanisms of their plasticity. Moreover, the possibility of inducing pluripotent stem cells without transgenes or viruses can be entertained.

The serum uric acid (UA) levels were measured in 112 patients with multiple sclerosis (MS) and 794 patients with different types of other neurological diseases (OND) or healthy control group. Serum UA levels, along with relevant clinical parameters of MS and OND, were also investigated. MS patients had significantly lower UA levels than those with transient ischemia attack (344.6 ± 130.6 µmol/L, P = 0.000), cerebral hemorrhage (311.9 ± 104.7 µmol/L, P = 0.000), cerebral infarction (291.3 ± 101.6 µmol/L, P = 0.014) and the healthy control group (312.1 ± 92.8 µmol/L, P = 0.000). MS patients had significantly higher serum UA levels than those with cryptococcus meningitis or meningoencephalitis (178.9 ± 107.0 µmol/L, P = 0.000) and tuberculous meningitis or meningoencephalitis patients (175.7 ± 99.9 µmol/L, P = 0.000). There were no significant differences in UA levels between patients with MS and those with facial neuritis, viral meningitis or encephalitis, pulmonary tuberculosis, polymyositis or dermatomyositis, myasthenia gravis, subarachnoid hemorrhage, migraine, Guillain—Barre syndrome and myelitis. In addition, UA levels were independently correlated with gender and duration of MS, but neither with MRI activity, disability nor subtypes of the disease in MS patients. Our data suggest that UA has two biphasic functions: neuroprotective and injurious. Our studies may help physicians to deal with conditions having abnormal UA levels. Multiple Sclerosis 2008; 14: 188—196. http://msj.sagepub.com

Many forms of blindness result from the dysfunction or loss of retinal photoreceptors. Induced pluripotent stem cells (iPSCs) hold great potential for the modelling of these diseases or as potential therapeutic agents. However, to fulfill this promise, a remaining challenge is to induce human iPSC to recreate in vitro key structural and functional features of the native retina, in particular the presence of photoreceptors with outer-segment discs and light sensitivity. Here we report that hiPSC can, in a highly autonomous manner, recapitulate spatiotemporally each of the main steps of retinal development observed in vivo and form three-dimensional retinal cups that contain all major retinal cell types arranged in their proper layers. Moreover, the photoreceptors in our hiPSC-derived retinal tissue achieve advanced maturation, showing the beginning of outer-segment disc formation and photosensitivity. This success brings us one step closer to the anticipated use of hiPSC for disease modelling and open possibilities for future therapies.

Purpose We sought to determine if human induced pluripotent stem cells (iPSCs) derived from blood could produce optic vesicle-like structures (OVs) with the capacity to stratify and express markers of intercellular communication. Methods Activated T-lymphocytes from a routine peripheral blood sample were reprogrammed by retroviral transduction to iPSCs. The T-lymphocyte-derived iPSCs (TiPSCs) were characterized for pluripotency and differentiated to OVs using our previously published protocol. TiPSC-OVs were then manually isolated, pooled, and cultured en masse to more mature stages of retinogenesis. Throughout this stepwise differentiation process, changes in anterior neural, retinal, and synaptic marker expression were monitored by PCR, immunocytochemistry, and/or flow cytometry. Results TiPSCs generated abundant OVs, which contained a near homogeneous population of proliferating neuroretinal progenitor cells (NRPCs). These NRPCs differentiated into multiple neuroretinal cell types, similar to OV cultures from human embryonic stem cells and fibroblast-derived iPSCs. In addition, portions of some TiPSC-OVs maintained their distinctive neuroepithelial appearance and spontaneously formed primitive laminae, reminiscent of the developing retina. Retinal progeny from TiPSC-OV cultures expressed numerous genes and proteins critical for synaptogenesis and gap junction formation, concomitant with the emergence of glia and the upregulation of thrombospondins in culture. Conclusions We demonstrate for the first time that human blood-derived iPSCs can generate retinal cell types, providing a highly convenient donor cell source for iPSC-based retinal studies. We also show that cultured TiPSC-OVs have the capacity to self-assemble into rudimentary neuroretinal structures and express markers indicative of chemical and electrical synapses.

Parstatin is a 41-mer peptide formed by proteolytic cleavage on activation of the PAR1 receptor. The authors recently showed that parstatin is a potent inhibitor of angiogenesis. The purpose of the present study was to evaluate the therapeutic effect of parstatin on ocular neovascularization.Choroidal neovascularization was generated in mice using laser-induced rupture of Bruch's membrane and was assessed after 14 days after perfusion of FITC-dextran. Oxygen-induced retinal neovascularization was established in neonatal mice by exposing them to 75% O(2) at postnatal day (P)7 for 5 days and then placing them in room air for 5 days. Evaluation was performed on P17 after staining with anti-mouse PECAM-1. The effect of parstatin was tested after intravitreal administration. The effects of subconjunctival-injected parstatin on corneal neovascularization and inflammation in rats were assessed 7 days after chemical burn-induced corneal neovascularization. Retinal leukostasis in mice was assessed after perfusion with FITC-conjugated concanavalin A.Parstatin potently inhibited choroidal neovascularization with an IC(50) of approximately 3 μg and a maximum inhibition of 59% at 10 μg. Parstatin suppressed retinal neovascularization with maximum inhibition of 60% at 3 μg. Ten-microgram and 30-μg doses appeared to be toxic to the neonatal retina. Subconjunctival parstatin inhibited corneal neovascularization, with 200 μg the most effective dose (59% inhibition). In addition, parstatin significantly inhibited corneal inflammation and VEGF-induced retinal leukostasis. In all models tested, scrambled parstatin was without any significant effect.Parstatin is a potent antiangiogenic agent of ocular neovascularization and may have clinical potential in the treatment of angiogenesis-related ocular disorders.

Bilirubin is the end product of heme catabolism by heme oxygenases. Although bilirubin has long been considered as a toxic waste product, it is now recognized as an endogenous antioxidant. It has been reported that bilirubin is an effective treatment in both acute and chronic experimental autoimmune encephalomyelitis (EAE) disease models. However, the relationship between bilirubin and multiple sclerosis (MS) has not been fully explored. The serum bilirubin concentrations were measured in 340 individuals comprising 88 healthy subjects, 133 patients with MS and 119 patients with cerebral infarction. Serum total bilirubin (Tbil), direct bilirubin (Dbil) and indirect bilirubin (Ibil) concentrations were significantly lower in patients with MS than in either patients with cerebral infarction or healthy controls (p0.001). The correlation identified between bilirubin and MS was still highly significant when the effect of gender was eliminated. Among patients with MS, Tbil, Dbil and Ibil concentrations were lower in patients with MS with longer duration (2 years), less disabling disease (Expanded Disability Status Scale score3), and inactive MRI appearance, although the differences did not reach statistical significance. Our results suggest that there are reduced serum bilirubin concentrations in patients with MS.

Purpose Primary open-angle glaucoma (POAG) is a leading cause of irreversible blindness worldwide, and its pathogenesis is still unknown. The purpose of this study was to determine molecular changes in membrane proteins in trabecular meshwork (TM) cells from POAG patients compared to those of age-matched normal controls. Methods Two-dimensional (2-D) gel electrophoresis profiles of membrane extracts from normal and glaucomatous TM cells were compared. The desired spots were identified after trypsin digestion and mass spectrometric analysis. Based on the results, a calcium-dependant membrane-binding protein, copine1, was further approached for a possible role in glaucomatous TM cells. The intracellular calcium concentration ([Ca2+]i) of TM cells was increased by incubating with calcium ionophore, A23187. Relative quantification real-time polymerase chain reaction (PCR) and western blot analysis measured copine1 expression and localization both in untreated and A23187-treated TM cells. Results Real-time PCR and western blot analysis confirmed that copine1 mRNA and protein expression were upregulated in glaucomatous TM cells when compared to normal ones. The cell distribution studies further showed that copine1 existed both in the membrane and cytoplasm fractions of glaucomatous TM cells but existed exclusively in cytoplasm fractions of their normal counterparts. More importantly, an influx of Ca2+ markedly promoted the translocation of copine1 from the cytoplasm to membranes in glaucomatous TM cells. Conclusions Copine1 is upregulated in plasma membranes of TM cells in individuals with POAG, which may be partly explained by its Ca2+-dependent translocation from the cytoplasm to the membranes. Investigation of the role and functions of copine1 in TM cells should offer new insight into the abnormal intracellular Ca2+-signaling pathway in glaucomatous TM and help to clarify the molecular mechanism of POAG.

Retinoblastoma (RB) is the most common malignant tumor of the retina in human children. Although it has been hypothesized for a long time that RB derives from multipotent retinal stem cells (RSCs) or retinoblasts, the direct evidence that the presence of tumorigenic RSCs in RB tumors is still lacking. Some studies indicate that malignant tumors contain tumor stem cells similar to their normal tissue stem cell counterparts. With in vitro culture and differentiation method we demonstrate that tumorigenic retinal stem-like cells (RSLCs) indeed exist in RB lesions and that RB tumor-derived cultures encompass undifferentiated cells capable of extensive proliferation as clonal nonadherent neurospheres and can differentiate into different retinal cells in vitro. Interestingly, cultured cells expressed retinal development related genes including nestin, CD133, pax6, chx10 and Rx, and overexpressed Bmi-1, a gene required for self-renewal and proliferation of stem cells. Significantly, when these cultured cells were intraocularly transplanted into SCID mice, they gave rise to new tumors with histomorphological features and immunophenotypes similar to their parental primary RBs. The results show that RBs contain tumorigenic RSLCs that contribute to tumorigenesis. This study provides a new insight to investigate the histogenesis of RBs and establishes a model for other RB research.

To find the better method of eosinophilic granulocytes staining by comparison between HE staining and Giemsa stainingTwenty conjunctivitis patients(40 eyes) were studied. The infected palpabral conjunctiva of both eyes were scroped and stained separately with Giemsa staining and HE staining.Under microscope, eosinophilic granulocytes were more clear by Giemsa staining than HE staining.In showing eosinophilic granulocytes, Giemsa staining was better than HE staining.

To establish model of retinoblastoma subcutaneously in NOD-SCID mice and study rules of formation and distribution of retinoblastoma metastasis.Retinoblastoma cells SO-RB50 were inoculated subcutaneously in NOD-SCID mice. Animal acts and tumor formation, growth and metastasis in NOD-SCID mice were observed. Primary and metastatic tumors were studied pathohistologically by HE and immunohistochemical staining.The latent periods of tumor growth were 12-19 days and the taken rate of tumor was 100%. 32 days later, 5 NOD-SCID mice were found with tumors that had metastasized to areas mainly located in the abdominal cavity and the side of the kidney; the metastatic time of tumors in the mice also differed. The tumor cells of the primary nodules and the metastasis were similar with human retinoblastoma cells and positive in immunohistochemical staining of NSE.The subcutaneous model of retinoblastoma in NOD-SCID mice showed a high taken rate and a short latent period of tumor, which had a high metastatic rate and was the best model in research of behaviors of retinoblastoma at present.

Background and purpose The purpose of this study was to describe and evaluate neuroimaging findings of patients with neurosyphilis. Methods The neuroimaging studies of 14 patients with documented neurosyphilis were reviewed. Diagnosis was established in 14 patients with cerebrospinal fluid for a Treponema Pallidum Particle Agglutination (TPPA) test. All patients had reactive TPPA and Unheated Serum Regain test (USR) in their sera. Imaging studies included plain, contrast-enhanced CT of the brain, plain and gadolinium-enhanced MR, and MR angiography. Results In the 14 HIV-negative patients with neurosyphilis, CT and MR showed the presence of cerebral infarction in six cases, arteritis in four cases, nonspecific white matter lesion in three cases, acute syphilitic meningitis in one case and normal neuroimaging finding in one case. In addition, 4 in 14 patients had general paresis, and MRI showed high signal intensity on T2 -weighted images involving frontotemporal lobes, hippocampus and periventricular area. Treatment with penicillin significantly diminished the size of these high signal intensity on T2-weighted images with general paresis. Conclusion These results suggest that MR and CT images have some characteristic manifestations in patients of neurosyphilis. Because early diagnosis and treatment of neurosyphilis are crucial to avoid persistent brain damage, the neuroimaging findings are valuable adjunct to clinical diagnosis and to provide useful information to follow-up after therapy.

The pluripotent embryonic stem cells can differentiate into various kinds of normal tissues. There is no previous report on the differentiation of embryonic stem cell in the intraocular environment. In this paper, the authors tried to investigate the intraocular growth character of mice embryonic stem cells in nude mice.Murine embryonic stem cells were cultured and maintained in an undifferentiated state in vitro. They were transplanted into the right eyes of 20 nude mice by microinjection under operating microscope. Animal eye observation, light microscope and immunohistochemical examinations were implemented.Two to three days after transplantation, small pieces of gray-white material could be viewed in the vitreous cavity. Between the 15th and 20th day, the gray-white mass grew into the anterior chamber in 4 nude mice eyes. Then, the mass at the anterior chamber extended extraocularly. On the 30th day, a remarkable proptosis was observed in two of the four nude mice. In 6 to 45 days, the mice were executed for morphological examination which showed the following typical structures: (1) Undifferentiated cells with prominent nucleolius. (2) Flexner-Wintersteiner-like rosettes. (3) Medulloepithelioma-like structure: the cells were arranged in sheets, cords, tubes, and cysts. (4) Large, spindle-or astrocyte-like cells. (5) Cartilage-like structure. Immunohistochemically, most of the cells were highly positive in NSE staining and a few cells were moderately positive in GFAP staining.Both animal eye findings and morphologic examinations certificated that the transplanted embryonic stem cells could grow in the eyes of nude mice and differentiate into intraocular medulloepithelioma.

To clone the variable region gene of light chain of monoclonal antibody against human retinoblastoma and to analyze the characterization of its nucleotide sequence as well as amino acid sequence.Total RNA was extracted from 3C6 hybridoma cells secreting specific monoclonal antibody (McAb) against human retinoblastoma (RB), then transcripted reversely into cDNA with olig-dT primers. The variable region of the light chain (VL) gene fragments was amplified using polymerase chain reaction(PCR) and further cloned into pGEM -T Easy vector. Then, 3C6 VL cDNA was sequenced by Sanger's method. Homologous analysis was done by NCBI BLAST.The complete nucleotide sequence of 3C6 VL cDNA consisted of 321 bp encoding 107 amino acid residues, containing four workframe regions (FRs) and three complementarity-determining regions(CDRs) as well as the typical structure of two cys residues. The sequence is most homological to a member of the Vk9 gene family, and its chain utilizes the Jkl gene segment.The light chain variable region gene of the McAb against human RB was amplified successfully, which belongs to the Vk9 gene family and utilizes Vk-Jkl gene rearrangement. This study lays a good basis for constructing a recombinant antibody and for making a new targeted therapeutic agents against retinoblastoma.