Junzhao Chen

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.

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.

Electrospun nanofibrous SF/P(LLA-CL) membrane: a potential substratum for endothelial keratoplasty

Background Cornea transplant technology has progressed markedly in recent decades, allowing surgeons to replace diseased corneal endothelium by a thin lamellar structure. A thin, transparent, biocompatible, tissue-engineered substratum with corneal endothelial cells for endothelial keratoplasty is currently of interest. Electrospinning a nanofibrous structure can simulate the extracellular matrix and have beneficial effects for cell culture. Silk fibroin (SF) has good biocompatibility but poor mechanical properties, while poly(l-lactic acid-co-ε-caprolactone) (P(LLA-CL)) has good mechanical properties but poor biocompatibility. Blending SF with P(LLA-CL) can maintain the advantages of both these materials and overcome their disadvantages. Blended electrospun nanofibrous membranes may be suitable for regeneration of the corneal endothelium. The aim of this study was to produce a tissue-engineered construct suitable for endothelial keratoplasty. Methods Five scaffolds containing different SF:P(LLA-CL) blended ratios (100:0, 75:25, 50:50, 25:75, 0:100) were manufactured. A human corneal endothelial (B4G12) cell line was cultured on the membranes. Light transmission, speed of cell adherence, cell viability (live-dead test), cell proliferation (Ki-67, BrdU staining), and cell monolayer formation were detected on membranes with the different blended ratios, and expression of some functional genes was also detected by real-time polymerase chain reaction. Results Different blended ratios of scaffolds had different light transmittance properties. The 25:75 blended ratio membrane had the best transmittance among these scaffolds. All electrospun nanofibrous membranes showed improved speed of cell adherence when compared with the control group, especially when the P(LLA-CL) ratio increased. The 25:75 blended ratio membranes also had the highest cell proliferation. B4G12 cells could form a monolayer on all scaffolds, and most functional genes were also stably expressed on all scaffolds. Only two genes showed changes in expression. Conclusion All blended ratios of SF:P(LLA-CL) scaffolds were evaluated and showed good biocompatibility for cell adherence and monolayer formation. Among them, the 25:75 blended ratio SF:P(LLA-CL) scaffold had the best transmittance and the highest cell proliferation. These attributes further the potential application of the SF:P(LLA-CL) scaffold for corneal endothelial transplantation.

Electrospun silk fibroin/poly(lactide-co-ε-caprolactone) nanofibrous scaffolds for bone regeneration

Background Tissue engineering has become a promising therapeutic approach for bone regeneration. Nanofibrous scaffolds have attracted great interest mainly due to their structural similarity to natural extracellular matrix (ECM). Poly(lactide-co-ε-caprolactone) (PLCL) has been successfully used in bone regeneration, but PLCL polymers are inert and lack natural cell recognition sites, and the surface of PLCL scaffold is hydrophobic. Silk fibroin (SF) is a kind of natural polymer with inherent bioactivity, and supports mesenchymal stem cell attachment, osteogenesis, and ECM deposition. Therefore, we fabricated hybrid nanofibrous scaffolds by adding different weight ratios of SF to PLCL in order to find a scaffold with improved properties for bone regeneration. Methods Hybrid nanofibrous scaffolds were fabricated by blending different weight ratios of SF with PLCL. Human adipose-derived stem cells (hADSCs) were seeded on SF/PLCL nanofibrous scaffolds of various ratios for a systematic evaluation of cell adhesion, proliferation, cytotoxicity, and osteogenic differentiation; the efficacy of the composite of hADSCs and scaffolds in repairing critical-sized calvarial defects in rats was investigated. Results The SF/PLCL (50/50) scaffold exhibited favorable tensile strength, surface roughness, and hydrophilicity, which facilitated cell adhesion and proliferation. Moreover, the SF/PLCL (50/50) scaffold promoted the osteogenic differentiation of hADSCs by elevating the expression levels of osteogenic marker genes such as BSP, Ocn, Col1A1, and OPN and enhanced ECM mineralization. In vivo assays showed that SF/PLCL (50/50) scaffold improved the repair of the critical-sized calvarial defect in rats, resulting in increased bone volume, higher trabecular number, enhanced bone mineral density, and increased new bone areas, compared with the pure PLCL scaffold. Conclusion The SF/PLCL (50/50) nanofibrous scaffold facilitated hADSC proliferation and osteogenic differentiation in vitro and further promoted new bone formation in vivo, suggesting that the SF/PLCL (50/50) nanofibrous scaffold holds great potential in bone tissue regeneration.

Targeted transplantation of human umbilical cord blood endothelial progenitor cells with immunomagnetic nanoparticles to repair corneal endothelium defect.

Corneal endothelial dysfunction involves progressive corneal edema and loss of visual acuity, which result in the need for corneal transplantation. The global shortage of donor corneas limits the development of the surgery. Reconstruction of a bioengineered corneal endothelium might resolve this problem. Various scaffolds have been used, but poor biocompatibility and degradation limit their applications. In this study, a novel method of targeted cellular transplantation without permanent residence of cell carriers in the host was proposed. Human umbilical cord blood endothelial progenitor cells (UCB EPCs) were labeled with CD34 immunomagnetic nanoparticles. The efficiency of the magnet attraction was evaluated in vitro with a simple device simulating the anterior chamber. The UCB EPCs labeled with nanoparticles were transplanted into the anterior chamber of rabbits with magnet attraction. The results indicated that labeling the nanoparticles did not affect the proliferation of the UCB EPCs. The in vitro study indicated that the magnet could directionally attract UCB EPCs labeled with nanoparticles. The in vivo study indicated that the corneas in rabbits transplanted with UCB EPCs labeled with nanoparticles and magnet attraction became relatively transparent with little edema. These results showed that UCB EPCs labeled with CD34 immunomagnetic nanoparticles could be attracted directionally by a magnet and could repair corneal endothelial defects, providing a promising cell therapy for corneal endothelial dysfunction.

Adenosine Triphosphate-Induced Rabbit Corneal Endothelial Cell Proliferation in vitro via the P2Y2-PI3K/Akt Signaling Axis

Purpose: To investigate the effect of the ATP-P2Y2-PI3K/Akt signaling axis on promoting rabbit corneal endothelial cell (RCEC) proliferation in vitro. Methods: Five concentrations of adenosine triphosphate (ATP; 1, 10, 25, 50 and 100 μM) were added to RCECs, and the cell proliferation was detected using Cell Counting Kit-8 (CCK8) and Ki67 immunohistochemical staining. Other P2Y2 receptor agonists and antagonists were added to the cells, and the proliferation effect was evaluated using CCK8 to determine the involvement of the P2Y2 receptor. Changes in the expression of phosphorylated Akt in RCECs treated with different concentrations of extracellular ATP and the duration of extracellular ATP on Akt phosphorylation were investigated using Western blotting. The pharmacological profiles with or without the PI3K/Akt pathway inhibitors were also determined using Western blotting. Results: We found that 10 μM ATP strongly promoted RCEC proliferation in vitro. Additionally, 25 μM ATP had a proliferation effect, whereas other concentrations (1, 50 and 100 μM) had no effect compared with the control group. Selective P2Y2 receptor agonists (UTP, ATPγS and Ap4A) showed the same promotion effect, while P2Y2 antagonists and PI3K/Akt inhibitors inhibited the effect of ATP. Moreover, phosphorylated Akt could be induced by the addition of extracellular ATP at all five concentrations and lasted for 1 h. This phosphorylation was prevented by PI3K/Akt inhibitors and a P2Y2 antagonist. Conclusions: These findings showed that 10 μM ATP markedly promoted RCEC proliferation via the P2Y2-PI3K/Akt signaling axis.

Dynamic changes of tear fluid after cosmetic transcutaneous lower blepharoplasty measured by optical coherence tomography.

PURPOSE To investigate the dynamic changes of tear fluid after cosmetic transcutaneous lower blepharoplasty. DESIGN Prospective, observational case series study. METHODS A total of 60 eyes of 30 patients with transcutaneous lower blepharoplasty were involved in the study. All subjects completed the Ocular Surface Disease Index questionnaire and underwent tear film break-up time measurements and the Schirmer test before surgery and 1 week, 1 month, and 3 months after surgery. Tear meniscus parameters measured by optical coherence tomography were recorded. Lower lid margin reflex distance was also measured. At each time point after surgery, these parameters were compared with the baseline parameters. The incidence of dry eye and chemosis after surgery was assessed. RESULTS The Ocular Surface Disease Index score increased significantly 1 week after surgery (P < .01). The Schirmer test values were significantly decreased 1 week postoperatively (P < .01), and they returned to baseline by 3 months. There was no significant change in tear film break-up time before and after surgery. The tear meniscus height, depth, and cross section area values were significantly increased at 1 week postoperatively, decreased at 1 month (P < .01), and returned to baseline at 3 months (P > .05). The cornea-lower eyelid angle significantly decreased at 1 week postoperatively (P < .01), increased at 1 month (P < .01), and returned to baseline by 3 months (P > .05). The lower lid margin reflex distance was increased 1 week after surgery (P < .01), and gradually recovered but had not returned to the baseline 3 months after surgery (P < .01). The increased tear meniscus height had a positive correlation with lower lid margin reflex distance. The incidence of dry eye and chemosis at 1 week postoperatively was 16.7% and 15%, respectively. CONCLUSIONS Cosmetic transcutaneous lower blepharoplasty affects ocular surface and tear fluid, which leads to dryness, tearful eyes, and chemosis. However, this influence is temporary after surgery, and the symptoms resolve within 3 months. A positive correlation between tear meniscus height and lower lid margin reflex distance was observed. Assessment of the tear meniscus with optical coherence tomography provided a quick, noninvasive, and quantitative method to investigate the dynamic changes of the tear fluid after blepharoplasty.

Rabbit Model of Corneal Endothelial Injury Established Using the Nd: YAG Laser

Purpose: To develop a new rabbit model of corneal endothelial injury using a neodymium-doped yttrium aluminum garnet (Nd:YAG) laser. Methods: The corneal endothelia of 2 groups of New Zealand white rabbits were treated with an Nd:YAG laser in a uniformly scattered fashion. Rabbits in group A underwent laser burns on the whole corneal endothelium, including the limbus area, whereas rabbits in group B were subjected to laser burns in the central 9-mm diameter zone of the endothelium. Slit-lamp biomicroscopy, optical coherence tomography, applanation tonometry, confocal microscopy, scanning electron microscopy, and histological examinations were performed during 4 weeks of follow-up. Results: In both groups, dotted or focal corneal endothelium defects were directly observed. The stroma was intact. Right after laser application, a series of clinical manifestations appeared, including subepithelial and stromal edema, increased central corneal thickness, and corneal opacity. Laser burn had more notable effects in group A than in group B. In both groups, we observed no damage to the intraocular structures, and intraocular pressure was normal after laser treatment. Conclusions: Nd:YAG laser treatment in a 9-mm diameter zone of the endothelium can effectively induce bullous keratopathy in a rabbit, whereas treatment for the entire corneal endothelium maintains bullous keratopathy for a longer period. The procedure is simple and reproducible, and it retains normal intraocular structures. This study provided a promising model for future research into endothelial cell damage and for the development of new therapies.

Electrospun collagen/poly(L-lactic acid‑co‑ε‑caprolactone) scaffolds for conjunctival tissue engineering

Conjunctival injuries are general but intractable ocular surface diseases, the sequelae of which are particularly challenging to treat. A promising therapy for conjunctival injuries is to employ biodegradable scaffolds to deliver conjunctival epithelial cells for repairing damaged or diseased conjunctiva. In the present study, an ultrathin porous nanofibrous scaffold was fabricated by using collagen and poly(L-lactic acid-co-ε-caprolactone) (PLCL) and displayed a thickness of 20 µm, with a high porosity and an average fiber diameter of 248.83±26.44 nm. Conjunctival epithelial cells seeded on the scaffolds proliferated well and had a high cell viability. Reverse-transcription quantitative PCR showed the expression of conjunctival epithelial cell-specific genes; in addition, there was no significant difference in the inflammatory gene expression between cells grown on collagen/PLCL scaffolds and tricalcium phosphate scaffolds. After co-culture for 2 weeks in vitro, epithelial cell stratification was observed using hematoxylin and eosin staining, exhibiting three to four epithelial-cell layers. In conclusion, these results suggested that collagen/PLCL scaffolds have potential application for repairing conjunctival epithelial coloboma.