Min Luo

Effects of let-7b and TLX on the proliferation and differentiation of retinal progenitor cells in vitro

MicroRNAs manifest significant functions in brain neural stem cell (NSC) self-renewal and differentiation through the post-transcriptional regulation of neurogenesis genes. Let-7b is expressed in the mammalian brain and regulates NSC proliferation and differentiation by targeting the nuclear receptor TLX, which is an essential regulator of NSC self-renewal. Whether let-7b and TLX act as important regulators in retinal progenitor cell (RPC) proliferation and differentiation remains unknown. Here, our data show that let-7b and TLX play important roles in controlling RPC fate determination in vitro. Let-7b suppresses TLX expression to negatively regulate RPC proliferation and accelerate the neuronal and glial differentiation of RPCs. The overexpression of let-7b downregulates TLX levels in RPCs, leading to reduced RPC proliferation and increased neuronal and glial differentiation, whereas antisense knockdown of let-7b produces robust TLX expression,enhanced RPC proliferation and decreased differentiation. Moreover, the inhibition of endogenous TLX by small interfering RNA suppresses RPC proliferation and promotes RPC differentiation. Furthermore, overexpression of TLX rescues let-7b-induced proliferation deficiency and weakens the RPC differentiation enhancement caused by let-7b alone. These results suggest that let-7b, by forming a negative feedback loop with TLX, provides a novel model to regulate the proliferation and differentiation of retinal progenitors in vitro.

Bone marrow mesenchymal stem cells stimulate proliferation and neuronal differentiation of retinal progenitor cells.

During retina development, retinal progenitor cell (RPC) proliferation and differentiation are regulated by complex inter- and intracellular interactions. Bone marrow mesenchymal stem cells (BMSCs) are reported to express a variety of cytokines and neurotrophic factors, which have powerful trophic and protective functions for neural tissue-derived cells. Here, we show that the expanded RPC cultures treated with BMSC-derived conditioned medium (CM) which was substantially enriched for bFGF and CNTF, expressed clearly increased levels of nuclear receptor TLX, an essential regulator of neural stem cell (NSC) self-renewal, as well as betacellulin (BTC), an EGF-like protein described as supporting NSC expansion. The BMSC CM- or bFGF-treated RPCs also displayed an obviously enhanced proliferation capability, while BMSC CM-derived bFGF knocked down by anti-bFGF, the effect of BMSC CM on enhancing RPC proliferation was partly reversed. Under differentiation conditions, treatment with BMSC CM or CNTF markedly favoured RPC differentiation towards retinal neurons, including Brn3a-positive retinal ganglion cells (RGCs) and rhodopsin-positive photoreceptors, and clearly diminished retinal glial cell differentiation. These findings demonstrate that BMSCs supported RPC proliferation and neuronal differentiation which may be partly mediated by BMSC CM-derived bFGF and CNTF, reveal potential limitations of RPC culture systems, and suggest a means for optimizing RPC cell fate determination in vitro.

Reciprocal Actions of microRNA-9 and TLX in the Proliferation and Differentiation of Retinal Progenitor Cells

Recent research has demonstrated critical roles of a number of microRNAs (miRNAs) in stem cell proliferation and differentiation. miRNA-9 (miR-9) is a brain-enriched miRNA. Whether miR-9 has a role in retinal progenitor cell (RPC) proliferation and differentiation remains unknown. In this study, we show that miR-9 plays an important role in RPC fate determination. The expression of miR-9 was inversely correlated with that of the nuclear receptor TLX, which is an essential regulator of neural stem cell self-renewal. Overexpression of miR-9 downregulated the TLX levels in RPCs, leading to reduced RPC proliferation and increased neuronal and glial differentiation, and the effect of miR-9 overexpression on RPC proliferation and differentiation was inhibited by the TLX overexpression; knockdown of miR-9 resulted in increased TLX expression as well as enhanced proliferation of RPCs. Furthermore, inhibition of endogenous TLX by small interfering RNA suppressed RPC proliferation and promoted RPCs to differentiate into retinal neuronal and glial cells. These results suggest that miR-9 and TLX form a feedback regulatory loop to coordinate the proliferation and differentiation of retinal progenitors.

An in vitro comparison study: the effects of fetal bovine serum concentration on retinal progenitor cell multipotentiality.

Retinal progenitor cells (RPCs) are an excellent resource for retinal replacement therapy, because they show enormous potential to differentiate into retinal-specific cell types. While the differentiating influence of serum has long been appreciated, the effects of serum concentration on RPC differentiation into specified retinal neural cells have not been investigated. Using cultured murine RPCs, this study compared the effects of different levels of fetal bovine serum (FBS) (1%, 5%, 10% and 20%) on RPC differentiation in vitro. RPC multipotentiality was assessed by using quantitative polymerase chain reaction (qPCR) to determine the relative expression levels of 10 genes involved in retinal development. In addition, analyses of cell morphology and retinal development-related protein expression were performed using microscopy and immunocytochemistry. The data revealed that 1% FBS-induced cultures preferentially generated rhodopsin- and PKC-α-positive cells. Calbindin and AP2α expression levels were greater in 5% FBS-induced cultures. Brn3a was expressed at similar levels in 1%, 5% and 10% FBS treatment conditions but diminished in 20% FBS conditions. Twenty percent FBS induced more glial fibrillary acid protein (GFAP)-immunoreactive cells corresponding to glia populations. These findings suggest that the concentration of FBS plays an important role in RPC differentiation in vitro. Treatment with low levels of FBS favors differentiation of rhodopsin-positive photoreceptors, interneurons and retinal ganglion cells (RGCs), while high FBS concentrations preferentially induce differentiation of glia cells. These results are expected to facilitate research in the treatment of neurodegenerative retinal diseases.

An in vitro comparison of two different subpopulations of retinal progenitor cells for self-renewal and multipotentiality

Retinal progenitor cells (RPCs) show enormous potential for the treatment of retinal degenerative diseases. It is well known that in vitro cultures of RPCs comprise suspension spheres and adherent cells, but the differences between the two cell populations are not fully understood. In this study, cultured RPCs were sorted into suspension and adherent cells. Analyses of cell morphology, cell growth and retinal progenitor-related expression markers were performed using quantitative polymerase chain reaction (qPCR) and immunocytochemistry to identify the proliferative and multipotent capacity of the cells in vitro. The data showed that both the suspension and adherent cells were maintained in an undifferentiated state, although the former exhibited a greater proliferative potential than the latter. Immunocytochemistry analysis indicated that the two subsets of RPCs were able to differentiate into different retinal cells in the presence of fetal bovine serum (FBS); the adherent cells were more likely to differentiate toward the β3-tubulin-, AP2α- and Map2-positive neuronal lineage, while the suspension cells were more effective at differentiating into rod photoreceptors, which was consistent with the qPCR results. These findings suggest that adherent RPCs may be a potential candidate for retinal interneuron or ganglion cell substitution therapies, whereas suspension RPCs may be preferred for photoreceptor cell replacement.

Ocular Complications of Human Immunodeficiency Virus Infection in Eastern China

PURPOSE To investigate ocular complications in patients with HIV/AIDS in eastern China during the time of highly active antiretroviral therapy (HAART). DESIGN Prospective study. METHODS This study was carried out from August 1, 2009 to July 31, 2010. Recruited HIV/AIDS patients underwent a series of surveys and ophthalmologic and laboratory examinations (including CD4 level) at enrollment. RESULTS In this study, all 787 HIV/AIDS patients (1574 eyes) had a history of HAART. Of these patients, 28.72% (95% CI = 0.26-0.32) had a history of systemic disease and 26.30% (95% CI = 0.23-0.29) had ocular complications. Of these ocular complications, cytomegalovirus retinitis (CMVR) had the highest prevalence (10.6%, 83/787) and ocular microangiopathy had the second-highest prevalence (9.4%, 74/787). Among the patients with CMVR, 16.9% (14/83) suffered from immune recovery uveitis (IRU). Furthermore, 3.4% (27/787) of the recruited AIDS patients had neuro-ophthalmologic disorders. The mean logMAR visual acuity of the group with ocular complications was 0.47 ± 0.64, which was significantly different from the asymptomatic group (0.17 ± 0.39, P < .001). The median CD4 T-cell count of the group with ocular complications is 43 cells/μL, which was significantly different from the asymptomatic group (116.5 cells/μL, P < .001). CONCLUSIONS The study shows a high rate of treatable ocular complications among patients with HIV/AIDS in eastern China. HIV/AIDS treatment programs in China must be prepared to identify ocular complications and refer patients to the correct treatment facilities.

Clinical study of Acrysof IQ aspheric intraocular lenses.

Purpose:  To determine whether implantation of an aspheric intraocular lens (SN 60 WF Alcon) results in reduced spherical aberration and improved contrast sensitivity after cataract surgery.

Visual performance of Acrysof ReSTOR compared with a monofocal intraocular lens following implantation in cataract surgery

The aim of this study was to compare the visual performance of Acrysof ReSTOR and Acrysof Natural intraocular lenses (IOLs) following cataract surgery. A randomized prospective study was performed in which 64 eyes (51 patients) were divided randomly into two groups. Monofocal IOLs (Acrysof Natural) were implanted into 34 eyes (27 patients) and multifocal IOLs (Acrysof ReSTOR) were implanted into 30 eyes (24 patients) using phacoemulsification surgery. The corrected distance visual acuity, near visual acuity, pseudoaccommodation, contrast sensitivity (CS) and wavefront analysis were measured at 1 week, 1 month and 3 months after surgery. The distance vision of the monofocal and ReSTOR patients improved equally with glasses (P<0.05). A greater improvement in near vision without glasses was observed in the ReSTOR-implanted patients (P<0.01). The CS values of the multifocal IOL group were significantly lower than those of the monofocal IOL group for all spatial frequencies tested (P<0.05). The spherical aberration was significantly higher in the multifocal IOL group compared with the monofocal IOL group (P<0.05). We observed no differences in coma between the two groups. The difference in the amplitude of pseudoaccommodation between the two groups was statistically significant (−3.14±0.91 D in the ReSTOR group vs. −1.03±0.33 D in the Natural group, P<0.01). The improvement in near vision was significantly more evident in the ReSTOR patients. Compared with the monofocal IOL lens, the multifocal lens is able to increase the amplitude of pseudoaccommodation. However, increased spherical aberration may contribute to lower CS values in the multifocal IOL group.

Clinicopathological characteristics and surgical outcomes of divided nevus of the eyelids: a decade's experience on 73 cases.

Currently, there is a lack of comprehensive data regarding clinicopathologic characteristics and surgical treatment of divided nevus of the eyelids. Here clinicopathologic features and reconstructive procedures performed on 73 patients were described. We showed that the lesion was randomly located on both eyes with a predominant incidence in females. Affected regions included the following: eyelid only (n = 40, 54.8%); medial canthus involvement (n = 4, 5.5%); medial canthus, caruncle, and bulbar conjunctiva involvement (n = 5, 6.8%); lateral canthus involvement (n = 18, 24.7%); full eyelids (n = 6, 8.2%); and cheek extension (n = 9, 12.3%). Lesions presented as a pigmented macule (n = 18, 24.7%) or mass (n = 55, 75.3%). Ptosis was present in 26 (35.6%) cases leading to visual field defects (n = 9, 12.3%). The puncta were covered in 13 (17.8%) cases. Overall, surgical results were satisfactory. Pathologically, lesions were either intradermal (n = 40, 54.8%), junctional (n = 15, 20.5%), or compound (n = 18, 24.7%). Because of the lack of systemic data regarding the rare entity of divided nevus of the eyelids, this study improves our understanding of this lesion, and it should be treated with individualized reconstructive procedures. Incorporation of pediatric patient population in the future will further extend our knowledge in dealing with this disorder.

Progress of stem/progenitor cell-based therapy for retinal degeneration

Retinal degeneration (RD), such as age-related macular degeneration (AMD) and retinitis pigmentosa, is one of the leading causes of blindness. Presently, no satisfactory therapeutic options are available for these diseases principally because the retina and retinal pigmented epithelium (RPE) do not regenerate, although wet AMD can be prevented from further progression by anti-vascular endothelial growth factor therapy. Nevertheless, stem/progenitor cell approaches exhibit enormous potential for RD treatment using strategies mainly aimed at the rescue and replacement of photoreceptors and RPE. The sources of stem/progenitor cells are classified into two broad categories in this review, which are (1) ocular-derived progenitor cells, such as retinal progenitor cells, and (2) non-ocular-derived stem cells, including embryonic stem cells, induced pluripotent stem cells, and mesenchymal stromal cells. Here, we discuss in detail the progress in the study of four predominant stem/progenitor cell types used in animal models of RD. A short overview of clinical trials involving the stem/progenitor cells is also presented. Currently, stem/progenitor cell therapies for RD still have some drawbacks such as inhibited proliferation and/or differentiation in vitro (with the exception of the RPE) and limited long-term survival and function of grafts in vivo. Despite these challenges, stem/progenitor cells represent the most promising strategy for RD treatment in the near future.