Bone marrow mesenchymal stem cell-derived extracellular vesicles promote corneal epithelial repair and suppress apoptosis via modulation of Caspase-3 in vitro.

Corneal injuries are the major cause of blindness and visual impairment. Available treatments are limited by their efficacy and side effects. Mesenchymal stem cell-derived extracellular vesicles are presumed as functional equivalents and potential candidates for cell-free therapy. This study reports isolation and characterization of extracellular vesicles from human bone marrow mesenchymal stem cells and evaluates their role in mediating epithelial repair and apoptosis in cultured corneal epithelial cells through scratch assay, PCR, immunofluorescence, and flow cytometry in vitro. The isolated extracellular vesicles were spherical, < 150 nm in diameter, and characterized as CD9+, CD63+, CD81+, TSG101+, and Calnexin-. Further, these vesicles promoted corneal epithelial repair by enhancing proliferation and suppressed apoptosis by regulating the expression of BAD, P53, BCL-2, and cleaved CASPASE-3. Thus, our results suggest that BM-MSC-EVs might have the potential to be used for the treatment of injury-induced corneal epithelial defects. Clinical translation of this work would require further investigations.

Mesenchymal stem cell-based adjunctive therapy for Pseudomonas aeruginosa-induced keratitis: A proof-of-concept in-vitro study.

PURPOSE: Pseudomonas aeruginosa-induced keratitis is one of the most severe and challenging forms of corneal infection, owing to its associated intense inflammatory reactions leading to corneal necrosis and dense corneal scar with loss of vision. Since mesenchymal stem cells (MSCs) are reported to possess antimicrobial and immunomodulatory properties, they can be tested as an adjuvant treatment along with the antibiotics which are the current standard of care. This study aims to investigate the anti-bacterial and immunomodulatory roles of human bone marrow MSC-derived conditioned medium (MSC-CM) in P. aeruginosa-infected human corneal epithelial cells (HCECs) in vitro. METHODS: The effect of MSC-CM on the growth of clinical isolates of P. aeruginosa was evaluated by colony-forming unit assay. The expression of inflammatory cytokines (IL-6 and TNF-α) and an antimicrobial peptide (Lipocalin 2) in lipopolysaccharide-treated MSCs and HCECs was analyzed through ELISA. Corneal epithelial repair following infection with P. aeruginosa was studied through scratch assay. RESULTS: Compared to control (P. aeruginosa (5*105) incubated in DMEM (1 ml) at 37 °C for 16 h), MSC-CM significantly: i) inhibits the growth of P. aeruginosa (159*109 vs. 104*109 CFU/ml), ii) accelerates corneal epithelial repair following infection with P. aeruginosa (9% vs. 24% closure of the wounded area after 12 h of infection), and iii) downregulates the lipopolysaccharide-induced expression of IL-6, TNF-α and Lipocalin 2 in HCECs. A combination of MSC-CM with an antibiotic, Ciprofloxacin moderately regulated the expression of IL-6, TNF-α, and Lipocalin 2. CONCLUSION: MSC-CM holds promise as an adjunctive therapeutic approach for P. aeruginosa-induced corneal epithelial damage.

Sunset Yellow protects against oxidative damage and exhibits chemoprevention in chemically induced skin cancer model.

Skin cancer and other skin-related inflammatory pathologies are rising due to heightened exposure to environmental pollutants and carcinogens. In this context, natural products and repurposed compounds hold promise as novel therapeutic and preventive agents. Strengthening the skin's antioxidant defense mechanisms is pivotal in neutralizing reactive oxygen species (ROS) and mitigating oxidative stress. Sunset Yellow (SY) exhibits immunomodulatory characteristics, evidenced by its capacity to partially inhibit the secretion of proinflammatory cytokines, regulate immune cell populations, and modulate the activation of lymphocytes. This study aimed to investigate the antioxidant and anti-genotoxic properties of SY using in-silico, in vitro, and physiochemical test systems, and to further explore its potential role in 7,12-dimethylbenz(a) anthracene (DMBA)/ 12-o-tetradecanoylphorbol-13-acetate (TPA)-induced two-stage skin carcinogenesis. In vitro experiments showed that pre-treatment of SY significantly enhanced the cell viability of HaCaT cells when exposed to tertiary-Butyl Hydrogen Peroxide (tBHP). This increase was accompanied by reduced ROS levels, restoration of mitochondrial membrane potential, and notable reduction in DNA damage in (SY + tBHP) treated cells. Mechanistic investigations using DPPH chemical antioxidant activity test and potentiometric titrations confirmed SY's antioxidant properties, with a standard reduction potential ( E o ) of 0.211 V. Remarkably, evaluating the effect of topical application of SY in DMBA/TPA-induced two-step skin carcinogenesis model revealed dose-dependent decreases in tumor latency, incidence, yield, and burden over 21-weeks. Furthermore, computational analysis and experimental validations identified GSK3ß, KEAP1 and EGFR as putative molecular targets of SY. Collectively, our findings reveal that SY enhances cellular antioxidant defenses, exhibits anti-genotoxic effects, and functions as a promising chemopreventive agent.

Autoregulation of Pax6 in neuronal cells is mediated by Pax6(5a), Pax6(ΔPD), SPARC, and p53.

BACKGROUND: Pax6, a multifunctional protein and a transcriptional regulator is critical for optimal functioning of neuronal cells. It is known that alternatively spliced Pax6 isoforms and co-expressed interacting proteins mediate cell/tissue specific autoregulation of Pax6, however, underlying mechanism(s) are poorly understood. METHODS AND RESULTS: We used Neuro-2a cells to explore the mechanism of autoregulation of Pax6 in neuronal cells whereas NIH/3T3 cells were used as control. We first studied the transcript expression of the three Pax6 isoforms: Pax6, Pax6(5a), and Pax6(ΔPD); and the two co-expressed Pax6-interacting partners: SPARC and p53 in normal and overexpressed conditions, through the semi-quantitative RT-PCR. Further, we used the luciferase reporter assay to study the binding and transactivation of the three Pax6 isoforms: Pax6, Pax6(5a), and Pax6(ΔPD) to their respective promoters: P0, P1, and Pα; followed by that of the two co-expressed Pax6-interacting partners: SPARC and p53 to the Pax6-P1 promoter. Expression and distribution of Pax6, Pax6(5a) and Pax6(ΔPD), their binding to Pax6-promoters (P0, P1, and Pα) and transactivation were modulated in transfected Neuro-2a cells. CONCLUSION: Our results suggest that autoregulation of Pax6 in neuronal cells is driven by a promoter dependent mechanism which is mediated by spliced variants [Pax6(5a) and Pax6(ΔPD)] and interacting proteins (SPARC and p53) of Pax6.

Human Umbilical Cord-Derived Mesenchymal Stem Cells Promote Corneal Epithelial Repair In Vitro.

Corneal injuries are among the leading causes of blindness and vision impairment. Trauma, infectious keratitis, thermal and chemical (acids and alkali burn) injuries may lead to irreversible corneal scarring, neovascularization, conjunctivalization, and limbal stem cell deficiency. Bilateral blindness constitutes 12% of total global blindness and corneal transplantation remains a stand-alone treatment modality for the majority of end-stage corneal diseases. However, global shortage of donor corneas, the potential risk of graft rejection, and severe side effects arising from long-term use of immunosuppressive medications, demands alternative therapeutic approaches. Umbilical cord-derived mesenchymal stem cells can be isolated in large numbers using a relatively less invasive procedure. However, their role in injury induced corneal repair is largely unexplored. Here, we isolated, cultured and characterized mesenchymal stem cells from human umbilical cord, and studied the expression of mesenchymal (CD73, CD90, CD105, and CD34), ocular surface and epithelial (PAX6, WNT7A, and CK-8/18) lineage markers through immunofluorescence. The cultured human limbal and corneal epithelial cells were used as controls. Scratch assay was used to study the corneal epithelial repair potential of umbilical cord-derived mesenchymal stem cells, in vitro. The in vitro cultured umbilical cord-derived mesenchymal stem cells were plastic adherent, showed trilineage differentiation and expressed: mesenchymal markers CD90, CD105, CD73; epithelial marker CK-8/18, and ocular lineage developmental markers PAX6 and WNT-7A. Our findings suggest that umbilical cord-derived mesenchymal stem cells promote repair of the injured corneal epithelium by stimulating the proliferation of corneal epithelial cells, in vitro. They may serve as a potential non-ocular source of stem cells for treating injury induced bilateral corneal diseases.

Limbal Epithelial and Mesenchymal Stem Cell Therapy for Corneal Regeneration.

Corneal pathologies are a major cause of blindness and visual impairment, especially in the developing world. However, not only is there a global shortage of donor corneal tissue, but a significant proportion of these blinding pathologies also carry an unfavourable long-term prognosis for conventional corneal transplantation. In the last few decades, there has been a spurt of research on developing alternate approaches to address corneal blindness, including stem cell therapy. After the discovery of epithelial stem cells at the limbus, successful cell-based approaches to treat severe ocular surface disease were developed and have subsequently become widely practised across the world. More recently, mesenchymal stem cells were identified near the epithelial stem cells at the limbus, providing a unique opportunity to develop regenerative therapies for both corneal epithelial and stromal pathologies. This review firstly emphasises on qualifying limbal stem cells as either epithelial or mesenchymal and then summarises all the existing knowledge on both cell types and their individual roles in corneal regeneration.  The review describes the history, indications, techniques, and outcomes of the different methods of limbal epithelial stem cell transplantation and elaborates on the potential applications of limbal mesenchymal stem cell therapy.

Therapeutic efficacy of different routes of mesenchymal stem cell administration in corneal injury.

PURPOSE: Corneal injuries are associated with significant impairment in vision. Mesenchymal stem cells (MSCs) have been shown to limit inflammation and promote tissue repair at the ocular surface. Here, we evaluate the efficacies of different modes of MSC delivery (topical, subconjunctival, intraperitoneal [IP] and intravenous [IV]) to promote tissue repair and restore corneal transparency in a murine model of corneal injury. METHODS: MSCs were purified from the bone marrow of C57BL/6  mice and expanded using plastic adherence in vitro. Corneal injury was created using an Algerbrush, and 0.5 × 106 MSCs/mouse were administered via topical, subconjunctival, IP or IV routes. Qdot-labeled MSCs were employed to determine the effect of route of administration on corneal and conjunctival MSC frequencies. Corneal opacity scores were calculated using ImageJ. Expression of inflammatory cytokines was quantified by qPCR, and infiltration of CD45+ cells was evaluated by flow cytometry. RESULTS: Subconjunctival or IV administration results in increased frequencies of MSCs in ocular surface tissues following corneal injury, relative to topical or intraperitoneal delivery. Subconjunctival or IV administration reduces: (i) corneal opacity, (ii) tissue fibrosis as quantified by α-Sma expression, (iii) the expression of inflammatory cytokines (Il-1ß and Tnf-α) and (iv) CD45+ inflammatory cell infiltration relative to untreated injured control animals. Administration via subconjunctival or IV routes was observed to accelerate corneal repair by restoring tissue architecture and epithelial integrity. CONCLUSIONS: Our data suggest that subconjunctival or IV delivery of MSCs has superior therapeutic efficacy compared to topical or IP delivery following corneal injury.

Mesenchymal Stromal Cells Modulate Corneal Alloimmunity via Secretion of Hepatocyte Growth Factor.

Mesenchymal stromal cells (MSCs) are multipotent stem cells that participate in tissue repair and possess considerable immunomodulatory potential. MSCs have been shown to promote allograft survival, yet the mechanisms behind this phenomenon have not been fully defined. Here, we investigate the capacity of MSCs to suppress the allogeneic immune response by secreting the pleiotropic molecule hepatocyte growth factor (HGF). Using an in vivo mouse model of corneal transplantation, we report that MSCs promote graft survival in an HGF-dependent manner. Moreover, our data indicate that topically administered recombinant HGF (a) suppresses antigen-presenting cell maturation in draining lymphoid tissue, (b) limits T-helper type-1 cell generation, (c) decreases inflammatory cell infiltration into grafted tissue, and (d) is itself sufficient to promote transplant survival. These findings have potential translational implications for the development of HGF-based therapeutics. Stem Cells Translational Medicine 2019;8:1030-1040.

Optimizing the role of limbal explant size and source in determining the outcomes of limbal transplantation: An in vitro study.

PURPOSE: Simple limbal epithelial transplantation (SLET) and cultivated limbal epithelial transplantation (CLET) are proven clinical techniques for treating limbal stem cell deficiency (LSCD). However, the ideal size and number of the limbal explants required for transplantation has not been clearly elucidated. This in vitro study aimed to determine the optimal limbal explant size required for complete corneal epithelialization by characterizing the cell expansion. METHODS: Limbal explants obtained from both live and cadaveric biopsies were cultured on the denuded amniotic membrane. Explant size and the explant cell outgrowth (expansion) were measured using ImageJ software with respect to days. Cultures were characterized by assessing the rate of proliferation of cells with 5-bromo-2'-deoxyuridine (BrdU) assay along with the expression of different stem cell markers (ABCG2, p63α), corneal epithelial (CK3+12) and adherens junction molecules (E-Cadherin) by immunofluorescence. RESULTS: Explants from live biopsies had 80% growth potential in vitro whereas 40% of the cadaveric tissue failed to grow. Minimum explant sizes of 0.3 mm2 for live and ≥0.5 mm2 for cadaveric tissue had a mean expansion areas of 182.39±17.06 mm2 and 217.59±16.91 mm2 respectively suggesting adequate growth potential of the explants. Mean total percentage of proliferative cells was 31.80±3.81 in live and 33.49±4.25 in cadaveric tissue expansion. The expression was noted to be similar in cells cultured from cadaveric compared to cells cultured from live limbal tissue with respect to ABCG2, p63α, CK(3+12) and E-cadherin. CONCLUSION: Our findings show that a minimal amount of 0.3 mm2 live tissue would be sufficient for ample limbal cell expansion in vitro. Cadaveric explants <0.5 mm2 had poor growth potential. However, larger explants (≥ 0.5 mm2) had growth rate and proliferative potential similar to the live tissue. These findings could prove to be critical for clinical success especially while attempting cadaveric limbal transplantation. This study provides a novel clinical strategy for enhancing efficacy of the limbal transplantation surgery and opens the probability of even using the cadaveric tissue by considering the size of explant.

Level of PAX5 in differential diagnosis of non-Hodgkin's lymphoma.

BACKGROUND & OBJECTIVES: The PAX5, a paired box transcription factor and B-cell activator protein (BSAP), activates B-cell commitment genes and represses non-B-cell lineage genes. About 14 transcript variants of PAX5 have been observed in human. Any alteration in its expression pattern leads to lymphogenesis or associated diseases and carcinogenesis in non-lymphoid tissues. Its mechanisms of function in pathophysiology of non-Hodgkin's lymphoma (NHL) are unclear. This study was intended to explore influence of PAX5 in cascade of NHL pathogenesis and diagnosis. METHODS: Samples of 65 patients were evaluated by immunohistochemical staining for cellular localization of PAX5, CD19, CD3, cABL, p53, Ras and Raf and by TUNEL assay, RNA-isolation and reverse transcriptase (RT)-PCR, w0 estern blot analysis, and lactate dehydrogenase (LDH) specific staining. RESULTS: B-cell type NHL patients were positive for PAX5, p53, Ras, CD19, Raf and CD3. All of them showed TUNEL-positive cells. The differential expression pattern of PAX5, CD19, p53, CD3, Zap700 , HIF 1α, Ras, Raf and MAPK (mitogen-activated protein kinase) at the levels of transcripts and proteins was observed. The LDH assay showed modulation of LDH4 and LDH5 isoforms in the lymph nodes of NHL patients. INTERPRETATION & CONCLUSIONS: The histological observations suggested that the patients represent diverse cases of NHL like mature B-cell type, mature T-cell type and high grade diffuse B-cell type NHL. The findings indicate that patients with NHL may also be analyzed for status of PAX5, CD19 and ZAP70, and their transcriptional and post-translational variants for the differential diagnosis of NHL and therapy.

Pax6 influences expression patterns of genes involved in neuro- degeneration.

BACKGROUND: Pax6, a highly conserved multifunctional transcription factor, has been critical for neurogenesis and neuronal plasticity. It is presumed that if level of Pax6 approaches either low or null, critical genes responsible for maintaining functional status of neurons or glia would be modulated. PURPOSE: Therefore, it has been intended to explore possibility of either direct or indirect influence of Pax6 in neurodegeneration. METHODS: The cell lines having origin of murine embryonic fibroblast (Pax6-non expressing, NIH3T3-cell line), murine neuroblastoma (Pax6-expressing brain-derived, Neuro-2a-cell line), and human glioblastoma-astrocytoma (U87MG) were cultured and maintained in a CO2 incubator at 37°C and 5% CO2 in DMEM containing 10% fetal bovine serum. The knockdown of endogenous Pax6 in Neuro-2a cells was achieved through siRNA based gene knock-down approach. The efficiency and validation of knock-down was done by real time PCR. The knock-down of Pax6 was successfully achieved. RESULTS: The levels of expression of transcripts of some of the proposed putative markers of neurodegeneration like Pax6, S100ß, GFAP, BDNF, NGN2, p73α, p73δ, LDH, SOD, and Catalase were analyzed in Pax6 knockdown condition for analysis of role of Pax6 in neurodegeneration. Since the Pax6 has been proposed to bind to promoter sequences of catalase, and catalase suppresses TGFß, relative lower levels of catalase in Neuro-2a and U-87MG as compared to NIH-3T3 indicates a possible progressive dominant negative impact of Pax6. However, presence of SOD and LDH indicates alternative protective mechanism. CONCLUSION: Presence of BDNF and TGFß indicates association between them in glioblastoma-astrocytoma. Therefore, Pax6 seems to be involved directly with p53 and TGFß mediated pathways and indirectly with redox-sensitive pathway regulation. The neurodegenerative markers S100ß, GFAP, BDNF, NGN2, p73α, p73δ, observed downregulated in Pax6 knockdown condition suggest Pax6-mediated regulation of these markers. Observations enlighten Pax6-mediated influences on cascades of genes involved in growth, differentiation and maturation of neurons and glia.

Science and Art of Cell-Based Ocular Surface Regeneration.

The potential cause of blindness worldwide includes diseases of the cornea, ocular surface (limbal stem cell deficiency, allergic conjunctivitis, dry eye diseases), and retinal diseases. The presence of stem cells (limbal stem cells) in the basal region of the limbus makes it an important tool for the ocular regeneration and also in maintaining the transparency of eye by replacing the corneal epithelium continuously. Various surgical modalities have been developed like cultured limbal epithelial transplantation, cultured oral mucosal epithelial transplantation, simple limbal epithelial transplantation, etc., utilizing the cell-based regenerative properties to treat limbal disorder. Cell-based therapies for ocular repair and regeneration comprise a major hope by therapies involving the mesenchymal stem cells, embryonic stem cells, and limbal stem cells for the restoration of vision in individuals whose ocular tissue has been irreversibly damaged by disease or trauma. This review explores critical needs in human disease mainly the ocular problem where cell-based therapeutics is exceptionally well suited and also the use of animal models, various artificial scaffolds, as well as advancement in clinical technique to challenge the current demand to overcome corneal blindness.