Cellular reparative mechanisms of mesenchymal stem cells for retinal diseases

The use of multipotent mesenchymal stem cells (MSCs) has been reported as promising for the treatment of numerous degenerative disorders including the eye. In retinal degenerative diseases, MSCs exhibit the potential to regenerate into retinal neurons and retinal pigmented epithelial cells in both in vitro and in vivo studies. Delivery of MSCs was found to improve retinal morphology and function and delay retinal degeneration. In this review, we revisit the therapeutic role of MSCs in the diseased eye. Furthermore, we reveal the possible cellular mechanisms and identify the associated signaling pathways of MSCs in reversing the pathological conditions of various ocular disorders such as age-related macular degeneration (AMD), retinitis pigmentosa, diabetic retinopathy, and glaucoma. Current stem cell treatment can be dispensed as an independent cell treatment format or with the combination of other approaches. Hence, the improvement of the treatment strategy is largely subjected by our understanding of MSCs mechanism of action

Human mesenchymal stem cells expressing erythropoietin enhance survivability of retinal neurons against oxidative stress : an in-vitro study

Retinal degeneration is a prominent feature in ocular disorders. In exploring possible treatments, Mesenchymal Stem Cells (MSCs) have been recognized to yield therapeutic role for retinal degenerative diseases. Studies have also displayed that erythropoietin (EPO) administration into degenerative retina models confers significant neuroprotective actions in limiting pathological cell death. In this study, we aimed to use MSCs to deliver EPO and to evaluate the ability of EPO to rescue retinal neurons from dying upon reactive oxidative stress induction. We derived human MSCs from Wharton's jelly (hWJMSCs) of the umbilical cord and cells were transduced with lentivirus particles encoding EPO and a reporter gene of green fluorescent protein (GFP). The supernatants of both transduced and non-transduced cells were collected and used as a pre-conditioning medium for Y79 retinoblastoma cells (retinal neuron cell line) following exposure to glutamate induction. Retinal cells exposed to glutamate showed reduced mitochondrial depolarization and enhanced improvement in cell viability when incubated with pre-conditioned media of transduced cells. Our results established a proof-of-concept that MSCs could be used as a candidate for the delivery of EPO therapeutic gene in the treatment of retinal degenerations

Photoreceptor therapy: generation of neurosphere-like cells from human mesenchymal stem cells expressing erythropoietin

The loss of photoreceptors is a major concern implicated in age-macular degeneration (AMD), a type of neurodegenerative disorder. Failure to prescribe a suitable treatment due to the lack of understanding of the molecular pathogenesis, and limited capacity to compensate irreparably damaged photoreceptors in the retina have greatly contributed to the progression of visual dysfunction. Our previous study has shown that Mesenchymal Stem Cells (MSCs) expressing erythropoietin (EPO) could commit into photoreceptor cell lineage. However, the efficiency of cell differentiation is limited. The present study aims to explore the capacity of these MSCs to form neurospheres. The cells were transduced with lentiviral particles encoding for human EPO and green fluorescent protein (GFP) genes, culture-expanded and sorted before subjected for differentiation induction into neural precursor cells. Our results showed that MSC-EPO developed into larger neurosphere and expressed relatively higher expression of nestin compared with MSCs alone when cultured under neural induction medium. These preliminary findings suggested that MSC-EPO have greater neurogenic potential than MSCs alone. Further study is needed to evaluate the possibilities of neurosphere to delete differentiate into functional photoreceptor cells. We believe that the success of neurosphere expansion may potentially be useful in scaling up the manufacturing of photoreceptors in a shorter time and at an efficient cost for retinal cell replacement therapy

Overcoming the challenge of transduction of human T-cells with chimeric antigen receptor (CAR) specific for ERBB2 antigen

Breast cancer is one of the most common malignancies among woman. Decades of scientific study have linked the overexpression of ERBB2 antigen to aggressive tumors. To target aggressive breast cancer, chimeric antigen receptor (CAR) technology can be utilized. For this, human T-cells are transduced with a gene sequence encoding a CAR that is specific for tumor-associated antigens (TAAs). These genetically-engineered CAR transduced T-cells (CAR-T cells) are able to target the tumor antigen without the need for major histocompatibility complex (MHC) recognition, rendering it a potentially universal immunotherapeutic option. However, efficient transduction of therapeutic gene into human T-cells and further cell expansion are challenging. In this study, we reported a successful optimization of a transduction protocol using spinoculation on CD3+ T-cells with different concentrations of lentiviral plasmid encoding the CAR gene. CD3+T-cells were isolated from the peripheral blood mononuclear cells (PBMCs). The constructed CAR gene was inserted into a lentiviral plasmid containing the green fluorescent protein (GFP) tag and lentiviral particles were produced. These lentiviral particles were used to transduce activated T-cells by spinoculation. T-cells were activated using Dynabead-conjugated CD3/CD28 human T-cell activator and interleukin-2 (IL-2) before transduction. CD3+ T-cells were selected and GFP expression, which indicated transduction, was observed. Future studies will focus on in vitro and in vivo models to determine the efficiency of CAR-T cells in specifically targeting ERBB2-expressing cells

Overcoming the challenge of transduction of human T-cells with chimeric antigen receptor (CAR) specific for ERBB2 antigen

Breast cancer is one of the most common malignancies among woman. Decades of scientific study have linked the overexpression of ERBB2 antigen to aggressive tumors. To target aggressive breast cancer, chimeric antigen receptor (CAR) technology can be utilized. For this, human T-cells are transduced with a gene sequence encoding a CAR that is specific for tumor-associated antigens (TAAs). These genetically-engineered CAR transduced T-cells (CAR-T cells) are able to target the tumor antigen without the need for major histocompatibility complex (MHC) recognition, rendering it a potentially universal immunotherapeutic option. However, efficient transduction of therapeutic gene into human T-cells and further cell expansion are challenging. In this study, we reported a successful optimization of a transduction protocol using spinoculation on CD3+ T-cells with different concentrations of lentiviral plasmid encoding the CAR gene. CD3+T-cells were isolated from the peripheral blood mononuclear cells (PBMCs). The constructed CAR gene was inserted into a lentiviral plasmid containing the green fluorescent protein (GFP) tag and lentiviral particles were produced. These lentiviral particles were used to transduce activated T-cells by spinoculation. T-cells were activated using Dynabead-conjugated CD3/CD28 human T-cell activator and interleukin-2 (IL-2) before transduction. CD3+ T-cells were selected and GFP expression, which indicated transduction, was observed. Future studies will focus on in vitro and in vivo models to determine the efficiency of CAR-T cells in specifically targeting ERBB2-expressing cells

Characterisation of erythropoietin gene-modified human mesenchymal stem cells and anti-apoptotic effect of glutamate excitotoxicity in a retinal neuron cell line

Retinal degeneration is a prominent feature in ocular disorders. In exploring possible treatments, Mesenchymal Stem Cells (MSCs) have been recognised to yield therapeutic role for retinal degenerative diseases. Studies have also shown that erythropoietin (EPO) administration into degenerative retina models confers significant neuroprotective actions in limiting pathological cell death. For this reason, introducing anti-apoptotic proteins, such as erythropoietin (EPO), may exhibit a superior effect in enhancing beneficial activity of MSCs and hence, the treatment in retinal degenerative disorders. The objective of this study was to characterise EPO gene-modified human MSCs and evaluate its anti-apoptotic effect of glutamate excitotoxicity in a retinal neuron cell line. MSCs derived from the human Wharton’s jelly of umbilical cord were cultured, expanded, and characterised for immunophenotypical expression of MSC surface markers and multipotency differentiation potentials. Following that, MSCs were genetically modified to carry EPO through lentiviral transduction. The cells were transduced with lentivirus particles encoding EPO and green fluorescent protein (GFP), as a reporter gene. The cultured MSCs displayed plastic adherence properties and formed spindle-shaped cells that resembled a fibroblast. MSC immunophenotyping revealed high expression of CD90, CD73 (SH3), CD105 (SH2), CD29, and HLA-ABC but lack of expression for CD34, CD14, CD45, CD80, and CD86. Furthermore, MSCs were capable to undergo bilineage mesenchymal differentiation into adipocytes and osteocytes. EPO-expressing MSCs (MSC-EPO) also demonstrated a greater capacity to promote cell differentiation into nestin-expressing neurospheres when compared to non-transduced cells. The supernatants of the transduced and non-transduced cells were collected and used as a pre-conditioning medium for Y79 retinoblastoma cells (retinal neuron cell line), following exposure to glutamate treatment to induce apoptosis. Cellular recovery of human retinoblastoma (Y79) subjected to glutamate at a toxic dose was assessed following incubation with supernatants harvested from EPO transduced MSCs. Retinal cells exposed to glutamate showed enhanced improvement in cell viability and reduced mitochondrial depolarization when incubated with the preconditioned medium collected from EPO-transduced cells. The outcome of this study established a proof-of-concept that MSCs could be used as a candidate for the delivery of EPO therapeutic gene in the treatment of retinal degenerations and that generated MSC-EPO can further differentiate into neural lineage that may serve as an alternative for cell replacement therapy for degenerating retinal neurons

Empowering Mesenchymal Stem Cells for Ocular Degenerative Disorders

Multipotent mesenchymal stem cells (MSCs) have been employed in numerous pre-clinical and clinical settings for various diseases. MSCs have been used in treating degenerative disorders pertaining to the eye, for example, age-related macular degeneration, glaucoma, retinitis pigmentosa, diabetic retinopathy, and optic neuritis. Despite the known therapeutic role and mechanisms of MSCs, low cell precision towards the targeted area and cell survivability at tissue needing repair often resulted in a disparity in therapeutic outcomes. In this review, we will discuss the current and feasible strategy options to enhance treatment outcomes with MSC therapy. We will review the application of various types of biomaterials and advances in nanotechnology, which have been employed on MSCs to augment cellular function and differentiation for improving treatment of visual functions. In addition, several modes of gene delivery into MSCs and the types of associated therapeutic genes that are important for modulation of ocular tissue function and repair will be highlighted

Image_1_Human Mesenchymal Stem Cells Expressing Erythropoietin Enhance Survivability of Retinal Neurons Against Oxidative Stress: An In Vitro Study.TIF

<p>Retinal degeneration is a prominent feature in ocular disorders. In exploring possible treatments, Mesenchymal Stem Cells (MSCs) have been recognized to yield therapeutic role for retinal degenerative diseases. Studies have also displayed that erythropoietin (EPO) administration into degenerative retina models confers significant neuroprotective actions in limiting pathological cell death. In this study, we aimed to use MSCs to deliver EPO and to evaluate the ability of EPO to rescue retinal neurons from dying upon reactive oxidative stress induction. We derived human MSCs from Wharton’s jelly (hWJMSCs) of the umbilical cord and cells were transduced with lentivirus particles encoding EPO and a reporter gene of green fluorescent protein (GFP). The supernatants of both transduced and non-transduced cells were collected and used as a pre-conditioning medium for Y79 retinoblastoma cells (retinal neuron cell line) following exposure to glutamate induction. Retinal cells exposed to glutamate showed reduced mitochondrial depolarization and enhanced improvement in cell viability when incubated with pre-conditioned media of transduced cells. Our results established a proof-of-concept that MSCs could be used as a candidate for the delivery of EPO therapeutic gene in the treatment of retinal degenerations.</p

Genetically-modified human mesenchymal stem cells to express erythropoietin enhances differentiation into retinal photoreceptors: an in-vitro study

Dysfunctional or death of retinal photoreceptors is an irreversible phenomenon that is closely associated with a broad range of retinal degenerative diseases, such as retinitis pigmentosa and age-related macular degeneration (AMD), resulting in successive loss of visual function and blindness. In search for viable treatment for retinal degenerative diseases, mesenchymal stem cells (MSCs) has demonstrated promising therapeutic capabilities to repair and replace damaged photoreceptor cells in both in vitro and in vivo conditions. Nevertheless, the dearth of MSC differentiation capacity into photoreceptors has limited its use in cell replacement therapy. Erythropoietin (EPO) has vital role in early neural retinal cell differentiation and demonstrated rescue potential on dying photoreceptor cells. Hence, we aimed to evaluate the differentiation capacity of MSCs into photoreceptor cells in the presence of human EPO protein. We derived the MSC from human Wharton's jelly of umbilical cord and transduced the cells with lentivirus particles encoding EPO and green fluorescent protein (GFP) as reporter gene. The transduced cells were selectively cultured and induced to differentiate into photoreceptors by exposing to photoreceptor differentiation cocktail. Our preliminary results showed that transduced cells exposed to induction medium had an enhanced differentiation capacity when compared to non-transduced cells. Our results demonstrated a novel strategy to increase the yield of in vitro photoreceptor differentiation and may be potentially useful in improving the efficiency of stem cell transplantation for ocular disorders