ng Ji

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.

Electrospun SF/PLCL nanofibrous membrane: a potential scaffold for retinal progenitor cell proliferation and differentiation

Biocompatible polymer scaffolds are promising as potential carriers for the delivery of retinal progenitor cells (RPCs) in cell replacement therapy for the repair of damaged or diseased retinas. The primary goal of the present study was to investigate the effects of blended electrospun nanofibrous membranes of silk fibroin (SF) and poly(L-lactic acid-co-ε-caprolactone) (PLCL), a novel scaffold, on the biological behaviour of RPCs in vitro. To assess the cell-scaffold interaction, RPCs were cultured on SF/PLCL scaffolds for indicated durations. Our data revealed that all the SF/PLCL scaffolds were thoroughly cytocompatible, and the SF:PLCL (1:1) scaffolds yielded the best RPC growth. The in vitro proliferation assays showed that RPCs proliferated more quickly on the SF:PLCL (1:1) than on the other scaffolds and the control. Quantitative polymerase chain reaction (qPCR) and immunocytochemistry analyses demonstrated that RPCs grown on the SF:PLCL (1:1) scaffolds preferentially differentiated toward retinal neurons, including, most interestingly, photoreceptors. In summary, we demonstrated that the SF:PLCL (1:1) scaffolds can not only markedly promote RPC proliferation with cytocompatibility for RPC growth but also robustly enhance RPCs’ differentiation toward specific retinal neurons of interest in vitro, suggesting that SF:PLCL (1:1) scaffolds may have potential applications in retinal cell replacement therapy in the future.

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.

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.

Effects of RPE‑conditioned medium on the differentiation of hADSCs into RPE cells, and their proliferation and migration

Age-related macular degeneration (AMD) is associated with the dysfunction and death of the retinal pigment epithelium (RPE). Recently, there has been increasing interest in stem cell-derived RPE cells for cell replacement therapies, such as those for AMD. The present study investigated whether RPE-conditioned medium (RPECM) could promote the differentiation of human adipose tissue-derived mesenchymal stromal cells (hADSCs) into RPE cells, and enhance the proliferation and migration of these cells. Reverse-transcription quantitative polymerase chain reaction analysis demonstrated that RPECM induced hADSCs to differentiate into cells expressing RPE markers, including retinoid isomerohydrolase (RPE65), cytokeratin (CK8) and Bestrophin, which were identified to be significantly upregulated by ~10-fold, 3.5-fold and 2.4-fold, respectively, compared with the control group [hADSCs cultured in ADSC-conditioned medium (ADSCCM)]. The immunocytochemistry and western blot analysis results demonstrated that the protein levels of RPE65, CK8 and Bestrophin were significantly increased in RPECM-treated hADSCs. In addition, Cell Counting Kit-8 analysis demonstrated that RPECM promoted the proliferation of induced cells. RPECM also increased the expression level of the cell proliferative marker Ki-67. Furthermore, to evaluate the migration potential, cell migration assays were performed. These assays demonstrated that following RPECM treatment hADSCs migrated more quickly compared with the control group. The results of the present study suggest that RPECM induces hADSCs to differentiate into RPE cells with higher proliferative and migratory potentials, which may aid in applications for hADSCs in RPE regenerative therapy.

Decellularized matrix of adipose-derived mesenchymal stromal cells enhanced retinal progenitor cell proliferation via the Akt/Erk pathway and neuronal differentiation

BACKGROUND AIMS Retinal progenitor cells (RPCs) are a promising cell therapy treatment for retinal degenerative diseases. However, problems with limited proliferation ability and differentiation preference toward glia rather than neurons restrict the clinical application of these RPCs. The extracellular matrix (ECM) has been recognized to provide an appropriate microenvironment to support stem cell adhesion and direct cell behaviors, such as self-renewal and differentiation. METHODS In this study, decellularized matrix of adipose-derived mesenchymal stromal cells (DMA) was manufactured using a chemical agent method (0.5% ammonium hydroxide Triton + 20 mmol/L NH4OH) in combination with a biological agent method (DNase solution), and the resulting DMA were evaluated by scanning electron microscopy (SEM) and immunocytochemistry. The effect of DMA on RPC proliferation and differentiation was evaluated by quantitative polymerase chain reaction, Western blot and immunocytochemistry analysis. RESULTS DMA was successfully fabricated, as demonstrated by SEM and immunocytochemistry. Compared with tissue culture plates, DMA may effectively enhance the proliferation of RPCs by activating Akt and Erk phosphorylation; when the two pathways were blocked, the promoting effect was reversed. Moreover, DMA promoted the differentiation of RPCs toward retinal neurons, especially rhodopsin- and recoverin-positive photoreceptors, which is the most interesting class of cells for retinal degeneration treatment. CONCLUSIONS These results indicate that DMA has important roles in governing RPC proliferation and differentiation and may contribute to the application of RPCs in treating retinal degenerative diseases.

Poly(1,3-propylene sebacate) and Poly(sebacoyl diglyceride): A Pair of Potential Polymers for the Proliferation and Differentiation of Retinal Progenitor Cells

Using suitable polymers as a carrier for growing and delivering retinal progenitor cells (RPCs) is a promising therapeutic strategy in retinal cell-replacement therapy. Herein recently developed polymer, poly(sebacoyl diglyceride) (PSeD), is selected and its nonhydroxylized counterpart poly(1,3-propylene sebacate) (PPS) is designed to evaluate their potentials for RPC growth and future RPC application. The structures and mechanical properties of the polymers are characterized. The cytocompatibility and effects of these polymers on RPC proliferation, differentiation, and migration are systematically investigated in vitro. Our data show that PPS and PSeD display excellent cytocompatibility with low expression of inflammation and apoptosis factors, which benefit RPC growth. In proliferation assays reveal that RPCs expands well on the polymers, but PPS performs the best for RPC expansion, indicating that PPS can remarkably promote RPC proliferation. In differentiation conditions, RPCs grown on PSeD are more likely to differentiate toward retinal neurons, including photoreceptors, the most interesting type of cells for retinal cell-replacement therapy. Additionally, our results demonstrate that RPCs grown on PSeD display an outstanding ability to migrate. In conclusion, PPS can markedly promote RPC proliferation, whereas PSeD can enhance RPC differentiation toward retinal neurons, suggesting that PSeD and PPS have potential applications in future retinal cell-replacement therapies.