Mesenchymal Stem Cells and Exosomes: A Novel Therapeutic Approach for Corneal Diseases.

The cornea, with its delicate structure, is vulnerable to damage from physical, chemical, and genetic factors. Corneal transplantation, including penetrating and lamellar keratoplasties, can restore the functions of the cornea in cases of severe damage. However, the process of corneal transplantation presents considerable obstacles, including a shortage of available donors, the risk of severe graft rejection, and potentially life-threatening complications. Over the past few decades, mesenchymal stem cell (MSC) therapy has become a novel alternative approach to corneal regeneration. Numerous studies have demonstrated the potential of MSCs to differentiate into different corneal cell types, such as keratocytes, epithelial cells, and endothelial cells. MSCs are considered a suitable candidate for corneal regeneration because of their promising therapeutic perspective and beneficial properties. MSCs compromise unique immunomodulation, anti-angiogenesis, and anti-inflammatory properties and secrete various growth factors, thus promoting corneal reconstruction. These effects in corneal engineering are mediated by MSCs differentiating into different lineages and paracrine action via exosomes. Early studies have proven the roles of MSC-derived exosomes in corneal regeneration by reducing inflammation, inhibiting neovascularization, and angiogenesis, and by promoting cell proliferation. This review highlights the contribution of MSCs and MSC-derived exosomes, their current usage status to overcome corneal disease, and their potential to restore different corneal layers as novel therapeutic agents. It also discusses feasible future possibilities, applications, challenges, and opportunities for future research in this field.

Current Advances in Regenerative Strategies for Dry Eye Diseases: A Comprehensive Review.

Dry eye disease (DED) is an emerging health issue affecting millions of individuals annually. Ocular surface disorders, such as DED, are characterized by inflammation triggered by various factors. This condition can lead to tear deficiencies, resulting in the desiccation of the ocular surface, corneal ulceration/perforation, increased susceptibility to infections, and a higher risk of severe visual impairment and blindness. Currently, the clinical management of DED primarily relies on supportive and palliative measures, including the frequent and lifelong use of different lubricating agents. While some advancements like punctal plugs, non-steroidal anti-inflammatory drugs, and salivary gland autografts have been attempted, they have shown limited effectiveness. Recently, there have been promising developments in the treatment of DED, including biomaterials such as nano-systems, hydrogels, and contact lenses for drug delivery, cell-based therapies, biological approaches, and tissue-based regenerative therapy. This article specifically explores the different strategies reported so far for treating DED. The aim is to discuss their potential as long-term cures for DED while also considering the factors that limit their feasibility and effectiveness. These advancements offer hope for more effective and sustainable treatment options in the future.

Induction of Corneal Endothelial-like Cells from Mesenchymal Stem Cells of the Umbilical Cord.

Because of the limited differentiation capacity of human corneal endothelial cells (CECs), stem cells have emerged as a potential remedy for corneal endothelial dysfunction (CED). This study aimed to demonstrate the differentiation of human umbilical cord-derived mesenchymal stem cells (UC-MSCs) into CECs and to investigate the efficacy of MSC-induced CEC injection into the anterior chamber in a rabbit model of CED. Human UC-MSCs were differentiated into CECs using medium containing glycogen synthase kinase 3ß inhibitor and two types of Rho-associated protein kinase inhibitors. In the MSC-induced CECs, CEC-specific proteins were identified through immunohistochemistry and changes in CEC-specific gene expressions over time were confirmed through quantitative RT-PCR. When MSC-induced CECs were injected into a rabbit model of CED, corneal opacity and neovascularization were improved compared with the non-transplanted control or MSC injection group. We also confirmed that MSC-induced CECs were well engrafted as evidenced by human mitochondrial DNA in the central cornea of an animal model. Therefore, we demonstrated the differentiation of UC-MSCs into CECs in vitro and demonstrated the clinical efficacy of MSC-induced CEC injection, providing in vivo evidence that MSC-induced CECs have potential as a treatment option for CED.

XLRS Rat with Rs1-/Y Exon-1-Del Shows Failure of Early Postnatal Outer Retina Development.

We generated a Long Evans transgenic rat with targeted deletion of the whole Rs1 exon-1 and evaluated the pathological retinal phenotype of this Rs1-/Y rat model of X-linked retinoschisis (XLRS). The Rs1-/Y rat exhibited very early onset and rapidly progressive photoreceptor degeneration. The outer limiting membrane (OLM) was disrupted and discontinuous by post-natal day (P15) and allowed photoreceptor nuclei to dislocate from the outer nuclear layers (ONL) into the sub-retinal side of the OLM. Dark-adapted electroretinogram (ERG) a-wave and b-wave amplitudes were considerably reduced to only 20-25% of WT by P17. Microglia and Müller glial showed cell marker activation by P7. Intravitreal application of AAV8-RS1 at P5-6 induced RS1 expression by P15 and rescued the inner nuclear layer (INL) and outer plexiform layer (OPL) cavity formation otherwise present at P15, and the outer-retinal structure was less disrupted. This Rs1-/Y exon-1-del rat model displays substantially faster rod cell loss compared to the exon-1-del Rs1-KO mouse. Most unexpected was the rapid appearance of schisis cavities between P7 and P15, and then cavities rapidly disappeared by P21/P30. The rat model provides clues on the molecular and cellular mechanisms underlying XLRS pathology in this model and points to a substantial and early changes to normal retinal development.

Therapeutic Potency of Induced Pluripotent Stem-Cell-Derived Corneal Endothelial-like Cells for Corneal Endothelial Dysfunction.

Corneal endothelial cells (CECs) do not proliferate or recover after illness or injury, resulting in decreased cell density and loss of pump/barrier function. Considering the shortage of donor cornea, it is vital to establish robust methods to generate CECs from induced pluripotent stem cells (iPSCs). We investigated the efficacy and safety of transplantation of iPSC-derived CECs into a corneal endothelial dysfunction (CED) rabbit model. iPSCs were generated from human fibroblasts. We characterized iPSCs by demonstrating the gene expression of the PSC markers OCT4, SOX2, TRA-1-60, and NANOG, teratoma formation, and differentiation into three germ layers. Differentiation of iPSCs into CECs was induced via neural crest cell (NCC) induction. CEC markers were detected using immunofluorescence and gene expression was analyzed using quantitative real-time PCR (qRT-PCR). After culturing iPSC-derived NCCs, we found the expression of zona occludens-1 (ZO-1) and Na+/K+ ATPase and a hexagonal morphology. ATP1A1, COL8A1, and AQP1 mRNA expression was higher in iPSC-derived CECs than in iPSCs and NCCs. We performed an injection of iPSC-derived CECs into the anterior chamber of a CED rabbit model and found improved levels of corneal transparency. We also found increased numbers of ZO-1- and ATP1A1-positive cells in rabbit corneas in the iPSC-derived CEC transplantation group. Usage of the coating material vitronectin (VTN) and fasudil resulted in good levels of CEC marker expression, demonstrated with Western blotting and immunocytochemistry. Combination of the VTN coating material and fasudil, instead of FNC mixture and Y27632, afforded the best results in terms of CEC differentiation's in vitro and in vivo efficacy. Successful transplantation of CEC-like cells into a CED animal model confirms the therapeutic efficacy of these cells, demonstrated by the restoration of corneal clarity. Our results suggest that iPSC-derived CECs can be a promising cellular resource for the treatment of CED.

Aqueous humor induces lymphatic regression on the ocular surface.

PURPOSE: This study is to investigate the potential effect of aqueous humor on already formed lymphatic vessels of the ocular surface including the conjunctiva and the cornea. METHODS: Aqueous humor harvested from fresh bovine or murine eyeballs were used in the study. It was injected into the subconjunctival space of Prox-1-GFP (green fluorescent protein) transgenic mice. Pre-existing conjunctival lymphatics were observed in vivo using our advanced live imaging system. Additionally, ex vivo tissue cultures were performed in aqueous humor with normal conjunctival tissues or inflamed corneas with newly formed lymphatic vessels. Time lapse images were taken by an advanced live cell imaging system with an incubator. Moreover, human primary microdermal lymphatic endothelial cell culture system was employed to evaluate the effect of aqueous humor on lymphatic tube regression in vitro. RESULTS: Aqueous humor induced lymphatic regression in both normal conjunctiva and inflamed corneas. It also led to the regression of formed lymphatic tubes by the lymphatic endothelial cells in vitro. CONCLUSIONS: This study provides the first direct and real time live imaging evidence showing that aqueous humor induces lymphatic regression. Further investigation promises for divulging new mechanisms and therapeutic strategies to treat lymphatic diseases that occur both inside and outside the eye.

miR-15a/16 inhibits TGF-beta3/VEGF signaling and increases retinal endothelial cell barrier proteins.

Hyperglycemia is a significant risk factor for diabetic retinopathy and induces multiple biochemical changes, including inflammation and endothelial dysfunction in the retina. Alterations in microRNA expression have been implicated in the pathological responses of diabetic retinopathy and the manipulation of microRNA may provide powerful strategy for therapeutics. Among the predicted targets of miR-15a and -16 are TGF-beta3, SMAD2/3, and VEGF, all of which are known to play a role in vascular endothelial functions. The purpose of this study was to investigate the hypothesis that miR-15a/16 inhibits TGF-beta3/VEGF signaling to maintain retinal endothelial cell barrier protein levels. Human primary retinal endothelial cells (REC) were maintained in normal (5mM) glucose or transferred to high glucose medium (25mM) for 3days. REC were transfected with miRNA mimics (hsa-miR-15a-5p and -16-5p). Retinal lysates from miR-15a-transgenic mice were also analyzed. We demonstrated that overexpression of miR-15a/16 resulted in decreased TGF-beta3 signaling and VEGF levels in cultured REC grown in high glucose conditions. In addition, the levels of tight junction proteins, zonula occludens-1 (ZO-1) and occludin, were elevated in REC following overexpression of miR-15a and -16. Overexpression of miR-15a and -16 played a role in reducing cellular permeability through inhibition of VEGF signaling in REC cultured under high glucose conditions. Using miR-15a-transgenic mice, we demonstrated the regulatory role of miR-15a on TGF-beta3 signaling and tight junction proteins in vivo. Our outcomes suggest that miR-15a/16 maintain the retinal endothelial cell barrier by reducing TGFbeta3/VEGF signaling and increasing levels of key tight junction proteins.

miR-146a suppresses STAT3/VEGF pathways and reduces apoptosis through IL-6 signaling in primary human retinal microvascular endothelial cells in high glucose conditions.

microRNA (miRNA) play critical roles in the pathological processes of diabetic retinopathy, including inflammatory responses, insulin signaling, and angiogenesis. In addition to their regulatory functions on gene expression, miRNA is considered as a potential therapeutic target, as well as a diagnostic marker for many diseases. Our understanding on the pathological mechanisms underlying diabetic retinopathy is still incomplete and additional investigations are required to develop novel therapeutic strategies. The aim of this study was to investigate our hypothesis that miR-146a plays a role in suppressing pro-inflammatory pathways, involving STAT3 and VEGF, through regulating IL-6 signaling to reduce apoptosis of human retinal endothelial cells (REC) in high glucose conditions. Human REC were cultured in normal (5mM) glucose or high glucose medium (25mM) for 3days. We performed transfections on REC with miRNA mimics (hsa-miR-146a-5p). Overexpression of miR-146a reduced IL-6 levels, STAT3 phosphorylation, and VEGF levels in REC cultured in high glucose. Cellular apoptosis was decreased in REC overexpressing miR-146a, as demonstrated by the inhibition of DNA fragmentation. More importantly, we demonstrated that the regulatory role of miR-146a on STAT3/VEGF and apoptosis was mediated by IL-6 receptor signaling in REC. Overall, we report that miR-146a suppressed IL-6 signaling, leading to reduced levels of STAT3 and VEGF in REC in high glucose conditions, leading to decreased apoptosis. The outcome suggests that miR-146a is a potential molecular target for inhibiting inflammation and apoptosis in the diabetic retina through the suppression of the IL-6-mediated STAT3/VEGF pathway.

miR-15a/16 reduces retinal leukostasis through decreased pro-inflammatory signaling.

BACKGROUND: Hyperglycemia is a significant risk factor for diabetic retinopathy and induces increased inflammatory responses and retinal leukostasis, as well as vascular damage. Although there is an increasing amount of evidence that miRNA may be involved in the regulation in the pathology of diabetic retinopathy, the mechanisms by which miRNA mediate cellular responses to control onset and progression of diabetic retinopathy are still unclear. The purpose of our study was to investigate the hypothesis that miR-15a/16 inhibit pro-inflammatory signaling to reduce retinal leukostasis. METHODS: We generated conditional knockout mice in which miR-15a/16 are eliminated in vascular endothelial cells. For the in vitro work, human retinal endothelial cells (REC) were cultured in normal (5 mM) glucose or transferred to high glucose medium (25 mM) for 3 days. Transfection was performed on REC in high glucose with miRNA mimic (hsa-miR-15a-5p, hsa-miR-16-5p). Statistical analyses were done using unpaired Student t test with two-tailed p value. p < 0.05 was considered significant. Data are presented as mean ± SEM. RESULTS: We demonstrated that high glucose conditions decreased expression of miR-15a/16 in cultured REC. Overexpression of miR-15a/16 with the mimic significantly decreased pro-inflammatory signaling of IL-1ß, TNFα, and NF-κB in REC. In vivo data demonstrated that the loss of miR-15a/16 in vascular cells led to increased retinal leukostasis and CD45 levels, together with upregulated levels of IL-1ß, TNFα, and NF-κB. CONCLUSIONS: The data indicate that miR-15a/16 play significant roles in reducing retinal leukostasis, potentially through inhibition of inflammatory cellular signaling. Therefore, we suggest that miR-15a/16 offer a novel potential target for the inhibition of inflammatory mediators in diabetic retinopathy.

miR-146a Attenuates Inflammatory Pathways Mediated by TLR4/NF-κB and TNFα to Protect Primary Human Retinal Microvascular Endothelial Cells Grown in High Glucose.

Pathological mechanisms underlying diabetic retinopathy are still not completely understood. Increased understanding of potential cellular pathways responsive to hyperglycemia is essential to develop novel therapeutic strategies for diabetic retinopathy. A growing body of evidence shows that microRNA (miRNA) play important roles in pathological mechanisms involved in diabetic retinopathy, as well as possessing potential as novel therapeutic targets. The hypothesis of this study was that miR-146a plays a key role in attenuating hyperglycemia-induced inflammatory pathways through reduced TLR4/NF-κB and TNFα signaling in primary human retinal microvascular endothelial cells (REC). We cultured human REC in normal (5 mM) glucose or transferred to high glucose medium (25 mM) for 3 days. Transfection was performed on REC with miRNA mimic (hsa-miR-146a-5p). Our results demonstrate that miR-146a expression was decreased in human REC cultured in high glucose. Overexpression of miR-146a using mimics reduced the levels of TLR4/NF-κB and TNFα in REC cultured in high glucose. Both MyD88-dependent and -independent signaling were decreased by miR-146a overexpression in REC in high glucose conditions. The results suggest that miR-146a is a potential therapeutic target for reducing inflammation in REC through inhibition of TLR4/NF-κB and TNFα. Our study will contribute to understanding of diabetic retinal pathology, as well as providing important clues to develop therapeutics for clinical applications.

miR-15b/16 protects primary human retinal microvascular endothelial cells against hyperglycemia-induced increases in tumor necrosis factor alpha and suppressor of cytokine signaling 3.

BACKGROUND: Mechanisms underlying the pathology of diabetic retinopathy are still not completely understood. Increased understanding of potential cellular pathways responsive to hyperglycemia is essential to develop novel therapeutic strategies for diabetic retinopathy. Emerging evidence shows the impact of microRNA (miR) as a potential novel therapeutic target. The purpose of our study was to test the hypothesis that miR-15b and miR-16 are altered by hyperglycemia in retinal endothelial cells (REC), and that miR-15b/16 play key roles in regulating insulin signaling through a reduction in TNFα- and suppressor of cytokine signaling 3 (SOCS3)-mediated insulin resistance pathways. METHODS: Human REC were maintained in normal (5 mM) glucose or transferred to high-glucose medium (25 mM) for 3 days. REC were transfected with miRNA mimics (hsa-miR-15b-5p and hsa-miR-16-5p) 48 h before cell harvest. A final concentration of 30 nM was used when transfected separately (miR-15b and miR-16) and 15 nM was used in combination (miR-15b + miR-16). A negative control group was treated with an equal concentration of a mimic negative control. The levels of miRNA overexpression were verified using quantitative reverse transcription-polymerase chain reaction and real-time PCR. Western blot analyses were performed to study the levels of phosphorylated Akt (Serine 473), Akt, SOCS3, insulin receptor, phosphorylated insulin receptor (tyrosine 1150/1151), and insulin receptor phosphorylated on Tyr960. In addition, ELISA was used to examine cleaved caspase 3 and TNFα. Analyses were done using unpaired Student t test. Data are presented as mean ± S.E.M. RESULTS: We demonstrated that the expression of miR-15b and miR-16 was reduced in human REC cultured in hyperglycemia. Overexpression of miR-15b and/or miR-16 reduced TNFα and SOCS3 levels, while increasing insulin-like growth factor binding protein-3 (IGFBP-3) levels and the phosphorylation of insulin receptor (IR)(Tyr1150/1151) in REC cultured in hyperglycemia. These, in turn, led to an increase of Akt phosphorylation and decreased cleavage of caspase 3. CONCLUSIONS: miR-15b and miR-16 play a role in the inhibition of insulin resistance via reduced TNFα and SOCS3 signaling and increased IGFBP-3 levels, resulting in REC protection from hyperglycemia-induced apoptosis. This outcome suggests that both miR-15b and miR-16 are potential therapeutic targets for therapeutics for the diabetic retina.

High throughput characterization of adult stem cells engineered for delivery of therapeutic factors for neuroprotective strategies.

Mesenchymal stem cells (MSCs) derived from bone marrow are a powerful cellular resource and have been used in numerous studies as potential candidates to develop strategies for treating a variety of diseases. The purpose of this study was to develop and characterize MSCs as cellular vehicles engineered for delivery of therapeutic factors as part of a neuroprotective strategy for rescuing the damaged or diseased nervous system. In this study we used mouse MSCs that were genetically modified using lentiviral vectors, which encoded brain-derived neurotrophic factor (BDNF) or glial cell-derived neurotrophic factor (GDNF), together with green fluorescent protein (GFP). Before proceeding with in vivo transplant studies it was important to characterize the engineered cells to determine whether or not the genetic modification altered aspects of normal cell behavior. Different culture substrates were examined for their ability to support cell adhesion, proliferation, survival, and cell migration of the four subpopulations of engineered MSCs. High content screening (HCS) was conducted and image analysis performed. Substrates examined included: poly-L-lysine, fibronectin, collagen type I, laminin, entactin-collagen IV-laminin (ECL). Ki67 immunolabeling was used to investigate cell proliferation and Propidium Iodide staining was used to investigate cell viability. Time-lapse imaging was conducted using a transmitted light/environmental chamber system on the high content screening system. Our results demonstrated that the different subpopulations of the genetically modified MSCs displayed similar behaviors that were in general comparable to that of the original, non-modified MSCs. The influence of different culture substrates on cell growth and cell migration was not dramatically different between groups comparing the different MSC subtypes, as well as culture substrates. This study provides an experimental strategy to rapidly characterize engineered stem cells and their behaviors before their application in long-term in vivo transplant studies for nervous system rescue and repair.

Etanercept restores normal insulin signal transduction in ß2-adrenergic receptor knockout mice.

BACKGROUND: Inhibition of TNFα protects the retina against diabetic-like changes in rodent models. The mechanism by which TNFα induces deleterious retinal changes is not known. Previously, we have shown that TNFα can inhibit normal insulin signal transduction, leading to increased apoptosis in both retinal endothelial cells (REC) and Müller cells. Additionally, ß2-adrenergic receptor knockout mice (ß2KO) have increased TNFα levels and decreased insulin receptor activity. In this study, we hypothesized that inhibition of TNFα in ß2KO mice would increase normal insulin signaling, leading to improved retinal function. METHODS: C57BL6 or ß2KO mice were left untreated or treated with etanercept (0.3 mg/kg subcutaneously, 3× a week) for 2 months. Electroretinogram analyses were done before treatment was initiated and after two months of treatment with etanercept on all mice. Western blot or ELISA analyses were done on whole retinal lysates from all four groups of mice for TNFα, suppressor of cytokine signaling 3 (SOCS3), insulin receptor, and apoptotic proteins. RESULTS: Etanercept significantly reduced TNFα levels in ß2KO mice, leading to increased insulin receptor phosphorylation on tyrosine 1150/1151. SOCS3 levels were increased in ß2KO mice, which were reduced after etanercept treatment. Pro-apoptotic proteins were reduced in etanercept-treated ß2KO mice. Etanercept improved ERG amplitudes in ß2KO mice. CONCLUSIONS: Inhibition of TNFα by etanercept protects the retina likely through reduced TNFα-mediated insulin resistance, leading to reduced apoptosis.

Pioglitazone normalizes insulin signaling in the diabetic rat retina through reduction in tumor necrosis factor α and suppressor of cytokine signaling 3.

Dysfunctional insulin signaling is a key component of type 2 diabetes. Little is understood of the effects of systemic diabetes on retinal insulin signaling. A number of agents are used to treat patients with type 2 diabetes to normalize glucose levels and improve insulin signaling; however, little has been done to investigate the effects of these agents on retinal insulin signal transduction. We hypothesized that pioglitazone, a peroxisome proliferator-activated receptor γ (PPARγ) agonist, would normalize retinal insulin signal transduction through reduced tumor necrosis factor α (TNFα) and suppressor of cytokine signaling 3 (SOCS3) activities in whole retina and retinal endothelial cells (REC) and Müller cells. To test this hypothesis, we used the BBZDR/Wor type 2 diabetic rat model, as well as REC and Müller cells cultured in normoglycemia and hyperglycemic conditions, to investigate the effects of pioglitazone on TNFα, SOCS3, and downstream insulin signal transduction proteins. We also evaluated pioglitazone's effects on retinal function using electroretinogram and markers of apoptosis. Data demonstrate that 2 months of pioglitazone significantly increased electroretinogram amplitudes in type 2 diabetic obese rats, which was associated with improved insulin receptor activation. These changes occurred in both REC and Müller cells treated with pioglitazone, suggesting that these two cell types are key to insulin resistance in the retina. Taken together, these data provide evidence of impaired insulin signaling in type 2 diabetes rats, which was improved by increasing PPARγ activity. Further investigations of PPARγ actions in the retina may provide improved treatment options.

ß1-adrenergic receptor stimulation by agonist Compound 49b restores insulin receptor signal transduction in vivo.

PURPOSE: Determine whether Compound 49b treatment ameliorates retinal changes due to the lack of ß2-adrenergic receptor signaling. METHODS: Using retinas from 3-month-old ß2-adrenergic receptor-deficient mice, we treated mice with our novel ß1-/ß2-adrenergic receptor agonist, Compound 49b, to assess the effects of adrenergic agonists acting only on ß1-adrenergic receptors due to the absence of ß2-adrenergic receptors. Western blotting or enzyme-linked immunosorbent assay (ELISA) analyses were performed for ß1- and ß2-adrenergic receptors, as well as key insulin resistance proteins, including TNF-α, SOCS3, IRS-1(Ser307), and IR(Tyr960). Analyses were also performed on key anti- and proapoptotic proteins: Akt, Bcl-xL, Bax, and caspase 3. Electroretinogram analyses were conducted to assess functional changes, while histological assessment was conducted for changes in retinal thickness. RESULTS: A 2-month treatment of ß2-adrenergic receptor-deficient mice with daily eye drops of 1 mM Compound 49b, a novel ß1- and ß2-adrenergic receptor agonist, reversed the changes in insulin resistance markers (TNF-α and SOCS3) observed in untreated ß2-adrenergic receptor-deficient mice, and concomitantly increased morphological integrity (retinal thickness) and functional responses (electroretinogram amplitude). These results suggest that stimulating ß1-adrenergic receptors on retinal endothelial cells or Müller cells can compensate for the loss of ß2-adrenergic receptor signaling on Müller cells, restore insulin signal transduction, reduce retinal apoptosis, and enhance retinal function. CONCLUSIONS: Since our previous studies with ß1-adrenergic receptor knockout mice confirmed that the reverse also occurs (ß2-adrenergic receptor stimulation can compensate for the loss of ß1-adrenergic receptor activity), it appears that increased activity in either of these pathways alone is sufficient to block insulin resistance-based retinal cell apoptosis.

Multipotent adult hippocampal progenitor cells maintained as neurospheres favor differentiation toward glial lineages.

Adult hippocampal progenitor cells (AHPCs) are generally maintained as a dispersed monolayer population of multipotent neural progenitors. To better understand cell-cell interactions among neural progenitors and their influences on cellular characteristics, we generated free-floating cellular aggregates, or neurospheres, from the adherent monolayer population of AHPCs. Results from in vitro analyses demonstrated that both populations of AHPCs were highly proliferative under maintenance conditions, but AHPCs formed in neurospheres favored differentiation along a glial lineage and displayed greater migrational activity than the traditionally cultured AHPCs. To study the plasticity of AHPCs from both populations in vivo, we transplanted green fluorescent protein (GFP)-expressing AHPCs via intraocular injection into the developing rat eyes. Both AHPC populations were capable of surviving and integrating into developing host central nervous system, but considerably more GFP-positive cells were observed in the retinas transplanted with neurosphere AHPCs, compared to adherent AHPCs. These results suggest that the culture configuration during maintenance for neural progenitor cells (NPCs) influences cell fate and motility in vitro as well as in vivo. Our findings have implication for understanding different cellular characteristics of NPCs according to distinct intercellular architectures and for developing cell-based therapeutic strategies using lineage-committed NPCs.

Characterization of structure and function of the mouse retina using pattern electroretinography, pupil light reflex, and optical coherence tomography.

OBJECTIVE: To perform in vivo analysis of retinal functional and structural parameters in healthy mouse eyes. ANIMAL STUDIED: Adult C57BL/6 male mice (n = 37). PROCEDURES: Retinal function was evaluated using pattern electroretinography (pERG) and the chromatic pupil light reflex (cPLR). Structural properties of the retina and nerve fiber layer (NFL) were evaluated using spectral-domain optical coherence tomography (SD-OCT). RESULTS: The average pERG amplitudes were found to be 11.2 ± 0.7 µV (P50-N95, mean ± SEM), with an implicit time for P50-N95 interval of 90.4 ± 5.4 ms. Total retinal thickness was 229.5 ± 1.7 µm (mean ± SEM) in the area centralis region. The thickness of the retinal nerve fiber layer (mean ± SEM) using a circular peripapillary retinal scan centered on the optic nerve was 46.7 ± 0.9 µm (temporal), 46.1 ± 0.9 µm (superior), 45.8 ± 0.9 µm (nasal), and 48.4 ± 1 µm (inferior). The baseline pupil diameter was 2.1 ± 0.05 mm in darkness, and 1.1 ± 0.05 and 0.56 ± 0.03 mm after stimulation with red (630 nm, luminance 200 kcd/m(2)) or blue (480 nm, luminance 200 kcd/m(2)) light illumination, respectively. CONCLUSIONS: Pattern electroretinography, cPLR and SD-OCT analysis are reproducible techniques, which can provide important information about retinal and optic nerve function and structure in mice.

Integrins contribute to initial morphological development and process outgrowth in rat adult hippocampal progenitor cells.

Adult rat hippocampal progenitor cells (AHPCs) are self-renewing, multipotent neural progenitor cells (NPCs) that can differentiate into neurons, oligodendrocytes, and astrocytes. AHPCs contact a variety of molecular cues within their surrounding microenvironment via integrins. We hypothesize that integrin receptors are important for NPCs. In this study, we have examined the distribution of integrins in neuronal-like, oligodendrocyte-like, and astrocyte-like AHPCs when grown on substrates that support integrin-mediated adhesion (laminin, fibronectin), and those that do not (poly-L: -ornithine, PLO) using immunocytochemistry as well as characterized the phenotypic differentiation of AHPCs plated on laminin and fibronectin. Focal adhesions were prominent in AHPCs plated on purified substrates, but were also found in AHPCs plated on PLO. The focal adhesions observed in AHPCs plated on PLO substrates may be formed by self-adhesion to the endogenously produced laminin or fibronectin. We have demonstrated that integrins contribute to the initial morphological differentiation of AHPCs, as inhibition of fibronectin binding with the competitive inhibitor echistatin significantly decreased the number of processes and microspikes present in treated cells, and also decreased overall cell area. Finally, we have characterized the genetic profile of a subset of integrins and integrin-related genes in the AHPCs using reverse transcriptase polymerase chain reaction. These results demonstrate an important role of integrins, in vitro, for the initial morphological differentiation of AHPCs.

Differentiation and behavior of human neural progenitors on micropatterned substrates and in the developing retina.

In this study we investigated the differentiation of human neural progenitor cells (hNPCs) in vitro to evaluate their differentiation potential and in vivo to explore their viability and behavior following transplantation. Progenitors were maintained as neurospheres in media containing basic fibroblast growth factor and epidermal growth factor. Micropatterned polystyrene substrates were fabricated and coated with ECL (entactin, collagen, and laminin) to provide physical and chemical guidance during the differentiation of the hNPCs. The hNPCs growing on the micropatterned substrates showed no differences in proliferation or differentiation potential compared with those hNPCs growing on the nonpatterned substrates. However, hNPCs cultured on the micropatterned substrates were aligned in the direction of the micropattern compared with those cells growing on the nonpatterned substrates. Furthermore, hNPC migration was directed in alignment with the micropatterned substrates. Transplantation of the hNPCs into the developing retina was used to evaluate their behavior in vivo. Cells displayed extensive survival, differentiation, and morphological integration following xenotransplant into the retina, even in the absence of immunosuppression. Taken together, our results show that these multipotent hNPCs are a neurogenic progenitor population that can be maintained in culture for extended periods. Although the micropatterned substrates have no major effect on the proliferation or differentiation of the hNPCs, they clearly promoted alignment and directed neurite outgrowth along the pattern as well as directing migration of the cells. These approaches may provide important strategies to guide the growth and differentiation of NPCs in vitro and in vivo.