Developing a model for aqueous deficient dry eye secondary to periglandular cicatrizing conjunctivitis.

PURPOSE: The current study used various techniques to develop a rabbit animal model of lacrimal gland damage caused by scarring conjunctivitis in the periglandular area. METHODS: Left eyes of New Zealand white rabbits were injected with 0.1 ml of 1M NaOH subconjunctivally around superior and inferior lacrimal gland orifices (Group 1, n = 4), touched with 1M NaOH for 100 s to the superior and inferior fornices with conjunctival denuding (Group 2; n = 4), and electrocauterization to the ductal opening area (Group 3; n = 4). The ocular surface staining, Schirmer I, lacrimal gland, and conjunctival changes were observed at baseline,1, 4, 8, and 12 weeks. The degree of glandular inflammation, conjunctival fibrosis (Masson Trichrome), and goblet cell density (PAS) were also assessed. RESULTS: At 12 weeks, the lacrimal glands of group 1 rabbits with periglandular injection showed severe inflammation with mean four foci/10HPF and a significant mean reduction in the Schirmer values by 7.6 mm (P = 0.007). Lacrimal glands had diffuse acinar atrophy, loss of myoepithelial cells, and ductular dilatation. The overlying conjunctiva showed fibrosis, goblet cell loss, and corneal vascularization in the inferotemporal quadrant. No lacrimal gland or ocular surface changes were observed in groups 2 and 3 at 12 weeks, except for localized subconjunctival fibrosis. CONCLUSION: Periglandular injection of 0.1 ml of 1M NaOH induced extensive lacrimal gland damage with reduced secretion and scarring in the subconjunctival plane compared to direct cauterization or direct NaOH contact to the ductal orifices of the rabbit lacrimal gland.

Revisiting rabbit models for keratoconus: A long-term study on collagenase-induced disease progression.

Keratoconus (KC) is a degenerative disorder resulting from the degradation of the stromal collagen fibril network in the cornea, leading to its thinning and conical deformation. Various studies have established animal models of KC by using the collagenase type II enzyme to gain a better understanding of the pathogenesis, however, long-term monitoring or follow-up of the models have not been reported so far. This study evaluates the long-term stability of collagenase type II-induced KC in a rabbit model. Six New Zealand rabbits were divided into 4 study groups with 3 eyes per group. The groups were control (group 1), 0.5% proparacaine + 5 min collagenase treatment on day 0 and day 30 (group 2), 0.5% proparacaine + 10 min collagenase treatment on day 0 (group 3) and, mechanical debridement + 2 min collagenase treatment on day 0 (group 4). Inflammation was observed in group 4 till week 10. Significant decrease in the central corneal thickness was observed in group 3 by week 4 (p < 0.001) however, the thickness was regained in the subsequent follow-ups in all the groups. Keratography results showed no changes in Km values but an increased astigmatic power in all groups. Scanning electron microscopy images revealed thinner collagen fibrils arranged in a mesh-like pattern above the uniform layer of the collagen lamellae in the central part of the treated corneas. Similarly, histological staining revealed loosely packed stromal fibrils in the anterior portion of the cornea which corroborates with the immunofluorescent staining results. This study revealed the remodeling of the corneal structure by eight weeks of collagenase treatment. Consequently, the possibility of creating a rabbit keratoconus model induced by collagenase may warrant further consideration.

Development and validation of a reliable rabbit model of limbal stem cell deficiency by mechanical debridement using an ophthalmic burr.

A simple and reproducible method is necessary to generate reliable animal models of limbal stem cell deficiency (LSCD) for assessing the safety and efficacy of new therapeutic modalities. This study aimed to develop and validate a rabbit model of LSCD through mechanical injury. The corneal and limbal epithelium of New Zealand White rabbits (n = 18) were mechanically debrided using an ophthalmic burr (Algerbrush II) with a 1.0-mm rotating head after 360° conjunctival peritomy. The debrided eyes were serially evaluated for changes in corneal opacity, neo-vascularization, epithelial defect and corneal thickness using clinical photography, slit lamp imaging, fluorescein staining, and anterior segment optical coherence tomography scanning (AS-OCT). Following this, an assessment of histopathology and phenotypic marker expression of the excised corneas was conducted. The experimental eyes were grouped as mild (n = 4), moderate (n = 10), and severe (n = 4) based on the grade of LSCD. The moderate group exhibited abnormal epithelium, cellular infiltration in the stroma, and vascularization in the central, peripheral, and limbal regions of the cornea. The severe group demonstrated central epithelial edema, peripheral epithelial thinning with sparse goblet cell population, extensive cellular infiltration in the stroma, and dense vascularization in the limbal region of the cornea. A significant decrease in the expression of K12 and p63 (p < 0.0001) was observed, indicating the loss of corneal epithelium and limbal epithelial stem cells in the LSCD cornea. This study demonstrates that the Alger brush-induced mechanical debridement model provides a reliable model of LSCD with comprehensive clinic-pathological features and that is well suited for evaluating novel therapeutic and regenerative approaches.

Human cornea-derived extracellular matrix hydrogel for prevention of post-traumatic corneal scarring: A translational approach.

Corneal scarring and opacification are a significant cause of blindness affecting millions worldwide. The current standard of care for corneal blindness is corneal transplantation, which suffers from several drawbacks. One alternative approach that has shown promise is the use of xenogeneic corneal extracellular matrix (ECM), but its clinical applicability is challenging due to safety concerns. This study reports the innovative use of human cornea-derived ECM to prevent post-traumatic corneal scarring. About 30 - 40% of corneas donated to the eye banks do not meet the standards defined for clinical use and are generally discarded, although they are completely screened for their safety. In this study, human cornea-derived decellularized ECM hydrogel was prepared from the non-transplantation grade human cadaveric corneas obtained from an accredited eye-bank. The prepared hydrogel was screened for its efficacy against corneal opacification following an injury in an animal model. Our in vivo study revealed that, the control collagen-treated group developed corneal opacification, while the prophylactic application of human cornea-derived hydrogel effectively prevented corneal scarring and opacification. The human hydrogel-treated corneas were indistinguishable from healthy corneas and comparable to those treated with the xenogeneic bovine corneal hydrogel. We also demonstrated that the application of the hydrogel retained the biological milieu including cell behavior, protein components, optical properties, curvature, and nerve regeneration by remodeling the corneal wound after injury. The hydrogel application is also sutureless, resulting in faster corneal healing. We envision that this human cornea-derived ECM-based hydrogel has potential clinical application in preventing scarring from corneal wounding. STATEMENT OF SIGNIFICANCE: There are significant challenges surrounding corneal regeneration after injury due to extensive scarring. Although there is substantial research on corneal regeneration, much of it uses synthetic materials with chemical cross-linking methods or xenogeneic tissue-based material devices which have to undergo exhaustive safety analysis before clinical trials. Herein, we demonstrate the potential application of a human corneal extracellular matrix hydrogel without any additional materials for scarless corneal tissue regeneration, and a method to reduce the wasting of donated allogenic corneal tissue from eye banks. We found no difference in efficacy between the usage of human tissues compared to xenogeneic sources. This may help ease clinical translation and can be used topically without sutures as an outpatient procedure.

A review of rabbit models of meibomian gland dysfunction and scope for translational research.

Dry eye disease (DED) is an emerging global health concern with meibomian gland dysfunction (MGD) being the most common subtype of DED. Despite being quite prevalent, the pathophysiological mechanisms governing MGD are poorly understood. Animal models for MGD can be a valuable resource to advance our understanding of this entity and explore novel diagnostic and therapeutic modalities. Although a lot of literature on rodent MGD models exists, a comprehensive review on rabbit animal models is lacking. Rabbits offer a great advantage over other animals as models for studying both DED and MGD. Rabbits have a widely exposed ocular surface and meibomian gland anatomy comparable with humans, which makes performing dry eye diagnostic tests possible using clinically validated imaging platforms. The existing MGD models in rabbits can broadly be classified as pharmacologically induced and surgically induced models. Most models show keratinization of the meibomian gland orifice with plugging as the final common pathway for developing MGD. Thus, understanding the advantages and disadvantages of each rabbit MGD model can help researchers choose the appropriate experimental plan based on the objective of the study. In this review, we discuss the comparative anatomy of the meibomian glands in humans and rabbits, various rabbit models of MGD, translational applications, unmet needs, and future directions in developing MGD models in rabbits.

Anti-SARS-CoV-2 potential of Cissampelos pareira L. identified by connectivity map-based analysis and in vitro studies.

BACKGROUND: Viral infections have a history of abrupt and severe eruptions through the years in the form of pandemics. And yet, definitive therapies or preventive measures are not present. Herbal medicines have been a source of various antiviral compounds such as Oseltamivir, extracted using shikimic acid from star anise (Illicium verum) and Acyclovir from Carissa edulis are FDA (Food and Drug Administration) approved antiviral drugs. In this study, we dissect the anti-coronavirus infection activity of Cissampelos pareira L (Cipa) extract using an integrative approach. METHODS: We analysed the signature similarities between predicted antiviral agents and Cipa using the connectivity map ( https://clue.io/ ). Next, we tested the anti-SARS-COV-2 activity of Cipa in vitro. Molecular docking analyses of constituents of with key targets of SARS-CoV2 protein viz. spike protein, RNA­dependent RNA­polymerase (RdRp) and 3C­like proteinase. was also performed. A three-way comparative analysis of Cipa transcriptome, COVID-19 BALF transcriptome and CMAP signatures of small compounds was also performed. RESULTS: Several predicted antivirals showed a high positive connectivity score with Cipa such as apcidin, emetine, homoharringtonine etc. We also observed 98% inhibition of SARS-COV-2 replication in infected Vero cell cultures with the whole extract. Some of its prominent pure constituents e.g. pareirarine, cissamine, magnoflorine exhibited 40-80% inhibition. Comparison of genes between BALF and Cipa showed an enrichment of biological processes like transcription regulation and response to lipids, to be downregulated in Cipa while being upregulated in COVID-19. CMAP also showed that Triciribine, torin-1 and VU-0365114-2 had positive connectivity with BALF 1 and 2, and negative connectivity with Cipa. Amongst all the tested compounds, Magnoflorine and Salutaridine exhibited the most potent and consistent strong in silico binding profiles with SARS-CoV2 therapeutic targets.

Antimicrobial silver nanoparticle-photodeposited fabrics for SARS-CoV-2 destruction.

Surfaces containing antiviral nanoparticles could play a crucial role in minimizing the virus spread further, specifically for COVID-19. Here in, we have developed a facile and durable antiviral and antimicrobial fabric containing photodeposited silver nanoparticles. Scanning and transmission electron microscopy, UV-VIS spectroscopy, and XPS are used to characterize the silver nanoparticles deposited cloth. It is evident that Ag0/Ag+ redox couple is formed during fabrication, which acts as an active agent. Antiviral testing results show that silver nanoparticles deposited fabric exhibits 97% viral reduction specific to SARS-CoV-2. Besides its excellent antiviral property, the modified fabric also offers antimicrobial efficiency when tested with the airborne human pathogenic bacteria Escherichia coli and fungi Aspergillus Niger. The direct photodeposition provides Ag-O-C interaction leads to firmly grafted nanoparticles on fabric allow the modified fabric to sustain the laundry durability test. The straightforward strategy to prepare an efficient antimicrobial cloth can attract rapid large-scale industrial production.

Gramicidin S and Melittin - Potential anti-viral therapeutic peptides to treat SARS-CoV-2 infection

The COVID19 pandemic has resulted in multipronged approaches for treatment of the disease. Since de novo discovery of drugs is time consuming, repurposing of molecules is now considered as one of the alternative strategies to treat COVID19. Antibacterial peptides are being recognized as attractive candidates for repurposing to treat viral infections. In this study, we describe the anti-SARS-CoV-2 activity of gramicidin S and melittin peptides obtained from Bacillus brevis and bee venom respectively. Our in vitro antiviral assay results showed significant decrease in the viral load compared to the untreated group with no/very less cytotoxicity. The EC50 values for gramicidin S and melittin are calculated as 1.571g and 0.656g respectively. Both the peptides treated to the SARS-CoV-2 infected Vero cells showed viral clearance from 12 hours onwards with a maximal clearance after 24 hours post infection. Based on proteome analysis it was found that more than 250 proteins were found to be differentially regulated in the gramicidin S and melittin treated SARS-CoV-2 infected Vero cells against control SARS-CoV-2 infected Vero cells after 24 and 48 hours post infection. The identified proteins were found to be associated in the metabolic and mRNA processing of the Vero cells post-treatment and infection. Both these peptides could be attractive candidates for repurposing to treat SARS-CoV-2 infection.

Adhatoda Vasica attenuates inflammatory and hypoxic responses in preclinical mouse models: potential for repurposing in COVID-19-like conditions.

BACKGROUND: COVID-19 pneumonia has been associated with severe acute hypoxia, sepsis-like states, thrombosis and chronic sequelae including persisting hypoxia and fibrosis. The molecular hypoxia response pathway has been associated with such pathologies and our recent observations on anti-hypoxic and anti-inflammatory effects of whole aqueous extract of Adhatoda Vasica (AV) prompted us to explore its effects on relevant preclinical mouse models. METHODS: In this study, we tested the effect of whole aqueous extract of AV, in murine models of bleomycin induced pulmonary fibrosis, Cecum Ligation and Puncture (CLP) induced sepsis, and siRNA induced hypoxia-thrombosis phenotype. The effect on lung of AV treated naïve mice was also studied at transcriptome level. We also determined if the extract may have any effect on SARS-CoV2 replication. RESULTS: Oral administration AV extract attenuates increased airway inflammation, levels of transforming growth factor-ß1 (TGF-ß1), IL-6, HIF-1α and improves the overall survival rates of mice in the models of pulmonary fibrosis and sepsis and rescues the siRNA induced inflammation and associated blood coagulation phenotypes in mice. We observed downregulation of hypoxia, inflammation, TGF-ß1, and angiogenesis genes and upregulation of adaptive immunity-related genes in the lung transcriptome. AV treatment also reduced the viral load in Vero cells infected with SARS-CoV2. CONCLUSION: Our results provide a scientific rationale for this ayurvedic herbal medicine in ameliorating the hypoxia-hyperinflammation features and highlights the repurposing potential of AV in COVID-19-like conditions.

Long term observation of ocular surface alkali burn in rabbit models: Quantitative analysis of corneal haze, vascularity and self-recovery.

Limbal Stem Cell Deficiency (LSCD), caused due to corneal injury, primarily by chemical/alkali burns, leads to compromised vision. Recently, several animal models of corneal alkali burn injury have become available. The majority of the studies with these animal models start interventions soon after the injury. However, in the clinical setting, there is a considerable delay before the intervention is initiated. Detailed knowledge of the molecular, histopathological, and clinical parameters associated with the progression of the injury leading to LSCD is highly desirable. In this context, we set out to investigate clinical, histopathological parameters of ocular surface alkali burn over a long period of time, post-injury. Limbal stem cell-deficient animal models of rabbits were created by alkali burn using sodium hydroxide, which was then assessed for their progression towards LSCD by grading the alkali burn, corneal haze, and vascularization. Additionally, cells present on the corneal surface after the burn was investigated by histology and immunophenotyping. Grading of rabbit eyes post-alkali burn had shown complete conjunctivalization in 80% (n = 12/15) of the rabbits with the alkali burn grade score of 3.88 ± 0.29 in three months and remained stable at four months (4.12 ± 0.24). However, ocular surface showed self-healing in 20% (n = 3/15) of the rabbits with a score of 1.67 ± 0.34 in four months irrespective of similar alkali injury. These self-healing corneas exhibited decreased opacity score from 2.51 ± 0.39 to 0.66 ± 0.22 (p = 0.002) and regressed vascularity from 1.66 ± 0.41 to 0.66 ± 0.33 in one to nine months, respectively. Restoration of the corneal phenotype (CK3+) was observed in central and mid-peripheral regions of the self-healing corneas, and histology revealed the localization of inflammatory cells to the peripheral cornea when compared to conjunctivalized and scarred LSCD eyes. Our study shows the essentiality to consider the time required for surgical intervention after the corneal alkali injury in rabbit models as evident from their tendency to self-heal and restore corneal phenotype without therapy. Such information on the possibility of self-healing should be useful in further studies as well as determining interventional timings and strategy during clinical presentation of corneal alkali burns.

Dry Swab Method of sample collection for SARS-CoV2 testing can be used for culturing virus

Back groundEarlier studies suggested the use of dry swab method for SARS-CoV-2 detection as it does not need VTM and subsequent RNA extraction step making the process cheaper, safer and faster. In this study we explore whether the virus in the dry swab is viable and can be cultured and propagated. MethodSwabs were spiked with SARS-CoV-2 and stored in three different conditions: a) as dry swab (SD, eluted in 1 mL DMEM), b) in 1 mL of Viral Transport Medium (SVTM), and c) in 1 mL of Tris-EDTA buffer (STE). The sample groups were stored either at room temperature (RT, 25{degrees}C{+/-}1{degrees}C) or at 4{degrees}C for 1, 4, 8, 12, 24, 48 and 72 hours before being used as viral inoculums for the propagation studies in Vero cells. ResultsThe RT-qPCR data suggests that SD incubated both at RT and 4{degrees}C harbors viral particles that are viable and culturable at par with SVTM and STE. ConclusionThe dry swab method, in addition to its advantages in detection of the virus, also renders viable viral particles that can be cultured and propagated.

Prevention of Corneal Myofibroblastic Differentiation In Vitro Using a Biomimetic ECM Hydrogel for Corneal Tissue Regeneration.

Corneal scarring is one of the major causes of blindness, affecting millions worldwide. Despite recent advancements in surgical strategies, there is an unmet need for a clinically feasible material and methods to prevent scarring following corneal injury. In this study, we report the potential utility of a hydrogel derived from cadaveric animal corneas, using a decellularized corneal matrix hydrogel (abbreviated as dCMH), which is prepared by a simple method. This hydrogel is easily injectable, biocompatible, and has the ability to maintain good shape-retention properties at 37 °C, which make it suitable for in vivo applications. Furthermore, our gene expression studies and immunofluorescence studies indicate that dCMH maintains the morphology and function of keratocytes in vitro and prevents their transdifferentiation to myofibroblasts. From the above results, it is evident that dCMH maintains the keratocytes with the ability to regenerate the corneal defect without scar. We thus suggest a simple yet effective approach for corneal tissue decellularization and that dCMH can be a promising material for prophylaxis against blinding scar formation in an injured cornea.

HspB5 protects mouse neural stem/progenitor cells from paraquat toxicity.

INTRODUCTION: HspB5 (αB-crystallin) is known to be involved in a variety of cellular functions, including, protection of cells from oxidative damage and inhibiting apoptosis. Neural stem/progenitor cells (NSPCs) have significant therapeutic value, especially in the NSC/NPC transplantation therapy. However, the viability of the transplanted NSPCs remains low because of various factors, including oxidative stress. OBJECTIVE: The current investigation explored the possible role of HspB5 in the protection of mouse NSPCs (mNSPCs) against paraquat-induced toxicity. METHODS: The recombinant human HspB5 was expressed in E.coli and was purified using gel filtration and Ion-exchange chromatography. The biophysical characterization of HspB5 was carried out using DLS, CD, and Analytical Ultracentrifugation (SV); the chaperone activity of HspB5 was determined by alcohol dehydrogenase aggregation assay. We have subjected the mNSPCs to paraquat-induced oxidative stress and monitored the protective ability of HspB5 by MTT assay and Hoechst-PI staining. Furthermore, increase in the expression of the anti-apoptotic protein, procaspase-3 was monitored using western blotting. RESULTS: The recombinant HspB5 was purified to its homogeneity and was characterized using various biophysical techniques. The externally added FITC-labeled HspB5 was found to be localized within the cytoplasm of mNSPCs. Our Immunocytochemistry results showed that the externally added FITC-labeled HspB5 not only entered the cells but also conferred cytoprotection against paraquat-induced toxicity. The protective events were monitored by a decrease in the PI-positive cells and an increase in the procaspase-3 expression through Immunocytochemistry and Western blotting respectively. CONCLUSION: Our results clearly demonstrate that exogenously added recombinant human HspB5 enters the mNSPCs and confers protection against paraquat toxicity.

Design and synthesis of substituted dihydropyrimidinone derivatives as cytotoxic and tubulin polymerization inhibitors.

An operationally simple Biginelli protocol was employed for the synthesis of new C6-carbon based aryl α-haloacrylamide-linked dihydropyrimidinone derivatives. The synthesized compounds were appraised for their in vitro antiproliferative potential against a selected panel of human cancer cell lines especially MCF-7 (human breast cancer), MDA-MB-231 (human breast cancer), HCT-116 (human colon cancer), HCT-15 (human colorectal adenocarcinoma), HT-29 (human colon adenocarcinoma) and DU145 (human prostate cancer) along with normal lung fibroblasts (HFL-1). Preferably, compounds containing α-haloacrylamide (10a-g) functionality were found to exhibit most significant cytotoxicity (IC50 value 0.54 ±â€¯0.12 to 8.35 ±â€¯0.82 µM) against the listed cancer cell lines, particularly towards breast cancer cell lines MCF-7 and MDA-MB-231 (IC50 value 0.54 ±â€¯0.12 to 3.70 ±â€¯0.24 µM). In the seam of synthesized compounds, compound 10f exhibited potent antiproliferative activity against breast cancer cell lines namely MCF-7 (IC50 value 0.54 ±â€¯0.12 µM) and MDA-MB-231 (IC50 value 1.18 ±â€¯0.32 µM). Further to understand the underlying apoptosis mechanisms, different staining techniques such as AO/EB, DCFDA, and DAPI staining were performed. To know the extent of apoptosis and loss of mitochondrial membrane potential in MCF-7 cell lines, annexin V-FITC/PI and JC-1 were performed. Cell cycle analysis revealed that compound 10f arrested the cells at G2/M phase in a dose-dependent manner. The compound 10f also found to exhibit significant inhibition of tubulin polymerization (IC50 of 6.91 ±â€¯0.43 µM) with microtubule destabilizing properties. Molecular docking studies also revealed that compound 10f efficiently interacted with critical catalytically active residues Ser178, Val238, and Val318 of the α/ß-tubulin by a hydrogen bond.

Inflammation, vascularization and goblet cell differences in LSCD: Validating animal models of corneal alkali burns.

Limbal stem cell deficiency (LSCD) is one of the serious cause of visual impairment and blindness with loss of corneal clarity and vascularization. Factors such as ocular burns (acids, lime, thermal), genetic disorders or infections results in the loss of limbal stem cells leading to LSCD. Reliable animal models of LSCD are useful for understanding the pathophysiology and developing novel therapeutic approaches. The purpose of the present study was to validate small and large animal models of LSCD by immunohistochemcal, clinical and histopathological comparison with human. The animal models of LSCD were created by topical administration of sodium hydroxide on the ocular surface of C57BL/6 mice (m, n = 12) and New Zealand white rabbits (r, n = 12) as per the standard existing protocol. Human corneal specimens (h, n = 12) were obtained from tissue bank who had chemical burn-induced LSCD. All samples were either paraffin embedded or frozen in cryogenic medium and the sections were processed for Hematoxylin-Eosin and Periodic Acid-Schiff staining to analyse the morphology and histopathological features of the corneal surface such as vascularization, inflammation, presence of goblet cells, epithelial hyperplasia and keratinization. Immunofluorescence was performed to distinguish between corneal (CK3+), conjunctival (CK19+) and epidermal (CK10+) epithelial phenotype. Histological analysis of corneal specimens from the three groups showed the presence of goblet cells (h:83%, m:50%, r:50%, p = 0.014), epithelial hypertrophy (h:92%, m:50%, r:66.6%, p = 0.04), epithelial hyperplasia (h:50%, m:17%, r:17%, p = 0.18), intra epithelial edema (h:42%, m:33%, r:100%, p = 0.02), stromal inflammation (h:100%, m:67%, r:67%, p = 0.01) and stromal vascularization (h:100%, m:50%, r:67%), in varying proportions. Immunostaining showed presence of total LSCD (CK19 + and/or CK10+, CK3-) in 92% of human and 50% of animal specimens. While partial LSCD (CK19 + and/or CK10+, CK3+) was seen in 8% of human and 50% of animal specimens. Our study shows the significant differences in the extent of vascularization, inflammation, epithelial thickness and goblet cell formation in mice and rabbit models of LSCD when compared to post-chemical burn LSCD in human corneas. In both mice and rabbit models complete LSCD developed in only 50% of cases and this important fact needs to be considered when working with animal models of LSCD.

Gene Delivery Approaches for Mesenchymal Stem Cell Therapy: Strategies to Increase Efficiency and Specificity.

A significant number of clinical trials have been undertaken to explore the use of mesenchymal stem cells (MSCs) for the treatment of several diseases such as Crohn's disease, diabetes, bone defects, myocardial infarction, stroke etc., Due to their efficiency in homing to the tissue injury sites, their differentiation potential, the capability to secrete a large amount of trophic factors and their immunomodulatory effects, MSCs are becoming increasingly popular and expected to be one of the promising therapeutic approaches. However, challenges associated with the isolation of pure MSC populations, their culture and expansion, specific phenotypic characterization, multi-potential differentiation and challenges of efficient transplantation limit their usage. The current strategies of cell-based therapies emphasize introducing beneficial genes, which will improve the therapeutic ability of MSCs and have better homing efficiency. The continuous improvement in gene transfer technologies has broad implications in stem cell biology. Although viral vectors are efficient vehicles for gene delivery, construction of viral vectors with desired genes, their safety and immunogenicity limit their use in clinical applications. We review current gene delivery approaches, including viral and plasmid vectors, for transfecting MSC with beneficial genes. The review also discusses the use of a few emerging technologies that could be used to improve the transfer/induction of desirable genes for cell therapy.

Transfection of arginine decarboxylase gene increases the neuronal differentiation of neural progenitor cells.

Growing evidence suggests that the clinical use of neural progenitor cells (NPCs) is hampered by heterogeneity, poor neuronal yield and low survival rate. Recently, we reported that retrovirus-delivered human arginine decarboxylase (hADC) genes improve cell survival against oxidative insult in murine NPCs in vitro. This study investigates whether the induced expression of hADC gene in mNPCs induces any significant change in the cell fate commitment. The evaluation of induced hADC gene function was assessed by knockdown of hADC gene using specific siRNA. The hADC gene delivery triggered higher expression of N-CAM, cell adhesion molecule and MAP-2, neuronal marker. However, the hADC gene knockdown showed downregulation of N-CAM and MAP-2 expression suggesting that hADC gene delivery favors cell fate commitment of mNPCs towards neuronal lineage. Neurite outgrowth was significantly longer in the hADC infected cells. The neurotrophic signal, BDNF aided in the neuronal commitment, differentiation, and maturation of hADC-mNPCs through PI3K and ERK1/2 activation. The induction of neuron-like differentiation is believed to be regulated by the expression of GSK-3ß and Wnt/ß-catenin signaling pathways. Our findings suggest that hADC gene delivery favors cell fate commitment of mNPCs towards neuronal lineage, bring new advances in the field of neurogenesis and cell therapy.

Foil assisted replica molding for fabrication of microfluidic devices and their application in vitro.

We present a simple, rapid, benchtop, Foil Assisted Rapid Molding (FARM) method for the fabrication of microfluidic devices. This novel technique involves the use of aluminium foil, pen and an X-Y plotter to create semi-circular or plano-concave, shallow microchannels. It is an easy do-it-yourself (DIY) technique for creating a microfluidic device in three simple steps: (1) create a channel design using the CAD software, (2) plot the patterns on aluminium foil and (3) use the reverse of the engraved foil as a mold to create microfluidic devices. In this report, we present a detailed study of the proposed method by varying a range of parameters such as foil thickness, tip material, and tip sizes and by investigating their effect on the creation of channels with varying geometry. Furthermore, we demonstrated the cytocompatibility of these devices in vitro.

Modulation of stem cell differentiation by the influence of nanobiomaterials/carriers.

Stem cells, either neural [NSCs] or mesenchymal [MSCs], possess tremendous untapped potential for cell therapy. Unlike the NSCs, MSCs are multi-potent and they have high self-renewal capability and broad tissue distribution. Since they do not produce significant immune rejection on post-transplantation; they are better suited for cell-based therapies. However, several critical issues need to be addressed to maximize stem cell-derived therapeutic effects. The key factor affecting the therapeutic application of stem cells is exposure to hostile conditions in vivo such as oxidative stress, which results in considerably low survival rate of these cells at transplanted sites, thereby reducing the therapeutic efficiency. Such limitation has led scientists to design clinically relevant, innovative and multifaceted solutions including the use of nanobiomaterials. Use of cytocompatible nanobiomaterials holds great promise and has gained attention of researchers, worldwide. Various nanobiomaterials are being explored to increase the survival efficiency and direct differentiation of stem cells to generate tissue-specific cells for biomedical research and futuristic therapies. These materials have superior cytocompatability, mechanical, electrical, optical, catalytic and magnetic properties. Non-invasive visualization of the biological system has been developed using magnetic nanoparticles and magnetic resonance imaging [MRI] approaches. Apart from viral vectors, non-viral carriers such as DNA nano carriers, single stranded RNA nanoparticles, liposomes and carbon nanotubes/wires are being exploited for gene delivery into stem cells. This article reviews potential application of various biocompatible nanomaterials in stem cell research and development.

Agmatine improves cognitive dysfunction and prevents cell death in a streptozotocin-induced Alzheimer rat model.

PURPOSE: Alzheimer's disease (AD) results in memory impairment and neuronal cell death in the brain. Previous studies demonstrated that intracerebroventricular administration of streptozotocin (STZ) induces pathological and behavioral alterations similar to those observed in AD. Agmatine (Agm) has been shown to exert neuroprotective effects in central nervous system disorders. In this study, we investigated whether Agm treatment could attenuate apoptosis and improve cognitive decline in a STZ-induced Alzheimer rat model. MATERIALS AND METHODS: We studied the effect of Agm on AD pathology using a STZ-induced Alzheimer rat model. For each experiment, rats were given anesthesia (chloral hydrate 300 mg/kg, ip), followed by a single injection of STZ (1.5 mg/kg) bilaterally into each lateral ventricle (5 µL/ventricle). Rats were injected with Agm (100 mg/kg) daily up to two weeks from the surgery day. RESULTS: Agm suppressed the accumulation of amyloid beta and enhanced insulin signal transduction in STZ-induced Alzheimer rats [experimetal control (EC) group]. Upon evaluation of cognitive function by Morris water maze testing, significant improvement of learning and memory dysfunction in the STZ-Agm group was observed compared with the EC group. Western blot results revealed significant attenuation of the protein expressions of cleaved caspase-3 and Bax, as well as increases in the protein expressions of Bcl2, PI3K, Nrf2, and γ-glutamyl cysteine synthetase, in the STZ-Agm group. CONCLUSION: Our results showed that Agm is involved in the activation of antioxidant signaling pathways and activation of insulin signal transduction. Accordingly, Agm may be a promising therapeutic agent for improving cognitive decline and attenuating apoptosis in AD.