Ben Mead

Intravitreally Transplanted Dental Pulp Stem Cells Promote Neuroprotection and Axon Regeneration of Retinal Ganglion Cells After Optic Nerve Injury

PURPOSE To investigate the potential therapeutic benefit of intravitreally implanted dental pulp stem cells (DPSCs) on axotomized adult rat retinal ganglion cells (RGCs) using in vitro and in vivo neural injury models. METHODS Conditioned media collected from cultured rat DPSCs and bone marrow-derived mesenchymal stem cells (BMSCs) were assayed for nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), and neurotrophin-3 (NT-3) secretion using ELISA. DPSCs or BMSCs were cocultured with retinal cells, with or without Fc-TrK inhibitors, in a Transwell system, and the number of surviving βIII-tubulin⁺ retinal cells and length/number of βIII-tubulin⁺ neurites were quantified. For the in vivo study, DPSCs or BMSCs were transplanted into the vitreous body of the eye after a surgically induced optic nerve crush injury. At 7, 14, and 21 days postlesion (dpl), optical coherence tomography (OCT) was used to measure the retinal nerve fiber layer thickness as a measure of axonal atrophy. At 21 dpl, numbers of Brn-3a⁺ RGCs in parasagittal retinal sections and growth-associated protein-43⁺ axons in longitudinal optic nerve sections were quantified as measures of RGC survival and axon regeneration, respectively. RESULTS Both DPSCs and BMSCs secreted NGF, BDNF, and NT-3, with DPSCs secreting significantly higher titers of NGF and BDNF than BMSCs. DPSCs, and to a lesser extent BMSCs, promoted statistically significant survival and neuritogenesis/axogenesis of βIII-tubulin⁺ retinal cells in vitro and in vivo where the effects were abolished after TrK receptor blockade. CONCLUSIONS Intravitreal transplants of DPSCs promoted significant neurotrophin-mediated RGC survival and axon regeneration after optic nerve injury.

Paracrine-Mediated Neuroprotection and Neuritogenesis of Axotomised Retinal Ganglion Cells by Human Dental Pulp Stem Cells: Comparison with Human Bone Marrow and Adipose-Derived Mesenchymal Stem Cells

We have investigated and compared the neurotrophic activity of human dental pulp stem cells (hDPSC), human bone marrow-derived mesenchymal stem cells (hBMSC) and human adipose-derived stem cells (hAMSC) on axotomised adult rat retinal ganglion cells (RGC) in vitro in order to evaluate their therapeutic potential for neurodegenerative conditions of RGC. Using the transwell system, RGC survival and length/number of neurites were quantified in coculture with stem cells in the presence or absence of specific Fc-receptor inhibitors to determine the role of NGF, BDNF, NT-3, VEGF, GDNF, PDGF-AA and PDGF-AB/BB in stem cell-mediated RGC neuroprotection and neuritogenesis. Conditioned media, collected from cultured hDPSC/hBMSC/hAMSC, were assayed for the secreted growth factors detailed above using ELISA. PCR array determined the hDPSC, hBMSC and hAMSC expression of genes encoding 84 growth factors and receptors. The results demonstrated that hDPSC promoted significantly more neuroprotection and neuritogenesis of axotomised RGC than either hBMSC or hAMSC, an effect that was neutralized after the addition of specific Fc-receptor inhibitors. hDPSC secreted greater levels of various growth factors including NGF, BDNF and VEGF compared with hBMSC/hAMSC. The PCR array confirmed these findings and identified VGF as a novel potentially therapeutic hDPSC-derived neurotrophic factor (NTF) with significant RGC neuroprotective properties after coculture with axotomised RGC. In conclusion, hDPSC promoted significant multi-factorial paracrine-mediated RGC survival and neurite outgrowth and may be considered a potent and advantageous cell therapy for retinal nerve repair.

Stem cell treatment of degenerative eye disease

Stem cell therapies are being explored extensively as treatments for degenerative eye disease, either for replacing lost neurons, restoring neural circuits or, based on more recent evidence, as paracrine-mediated therapies in which stem cell-derived trophic factors protect compromised endogenous retinal neurons from death and induce the growth of new connections. Retinal progenitor phenotypes induced from embryonic stem cells/induced pluripotent stem cells (ESCs/iPSCs) and endogenous retinal stem cells may replace lost photoreceptors and retinal pigment epithelial (RPE) cells and restore vision in the diseased eye, whereas treatment of injured retinal ganglion cells (RGCs) has so far been reliant on mesenchymal stem cells (MSC). Here, we review the properties of non-retinal-derived adult stem cells, in particular neural stem cells (NSCs), MSC derived from bone marrow (BMSC), adipose tissues (ADSC) and dental pulp (DPSC), together with ESC/iPSC and discuss and compare their potential advantages as therapies designed to provide trophic support, repair and replacement of retinal neurons, RPE and glia in degenerative retinal diseases. We conclude that ESCs/iPSCs have the potential to replace lost retinal cells, whereas MSC may be a useful source of paracrine factors that protect RGC and stimulate regeneration of their axons in the optic nerve in degenerate eye disease. NSC may have potential as both a source of replacement cells and also as mediators of paracrine treatment.

Mesenchymal stromal cell-mediated neuroprotection and functional preservation of retinal ganglion cells in a rodent model of glaucoma.

BACKGROUND AIMS Glaucoma is a leading cause of irreversible blindness involving loss of retinal ganglion cells (RGC). Mesenchymal stromal cells (MSC) have shown promise as a paracrine-mediated therapy for compromised neurons. It is, however, unknown whether dental pulp stem cells (DPSC) are effective as a cellular therapy in glaucoma and how their hypothesized influence compares with other more widely researched MSC sources. The present study aimed to compare the efficacy of adipose-derived stem cells, bone marrow-derived MSC (BMSC) and DPSC in preventing the loss of RGC and visual function when transplanted into the vitreous of glaucomatous rodent eyes. METHODS Thirty-five days after raised intraocular pressure (IOP) and intravitreal stem cell transplantation, Brn3a(+) RGC numbers, retinal nerve fibre layer thickness (RNFL) and RGC function were evaluated by immunohistochemistry, optical coherence tomography and electroretinography, respectively. RESULTS Control glaucomatous eyes that were sham-treated with heat-killed DPSC had a significant loss of RGC numbers, RNFL thickness and function compared with intact eyes. BMSC and, to a greater extent, DPSC provided significant protection from RGC loss and RNFL thinning and preserved RGC function. DISCUSSION The study supports the use of DPSC as a neuroprotective cellular therapy in retinal degenerative disease such as glaucoma.

Comparative evaluation of methods for estimating retinal ganglion cell loss in retinal sections and wholemounts.

To investigate the reliability of different methods of quantifying retinal ganglion cells (RGCs) in rat retinal sections and wholemounts from eyes with either intact optic nerves or those axotomised after optic nerve crush (ONC). Adult rats received a unilateral ONC and after 21 days the numbers of Brn3a+, βIII-tubulin+ and Islet-1+ RGCs were quantified in either retinal radial sections or wholemounts in which FluoroGold (FG) was injected 48 h before harvesting. Phenotypic antibody markers were used to distinguish RGCs from astrocytes, macrophages/microglia and amacrine cells. In wholemounted retinae, counts of FG+ and Brn3a+ RGCs were of similar magnitude in eyes with intact optic nerves and were similarly reduced after ONC. Larger differences in RGC number were detected between intact and ONC groups when images were taken closer to the optic nerve head. In radial sections, Brn3a did not stain astrocytes, macrophages/microglia or amacrine cells, whereas βIII-tubulin and Islet-1 did localize to amacrine cells as well as RGCs. The numbers of βIII-tubulin+ RGCs was greater than Brn3a+ RGCs, both in retinae from eyes with intact optic nerves and eyes 21 days after ONC. Islet-1 staining also overestimated the number of RGCs compared to Brn3a, but only after ONC. Estimates of RGC loss were similar in Brn3a-stained radial retinal sections compared to both Brn3a-stained wholemounts and retinal wholemounts in which RGCs were backfilled with FG, with sections having the added advantage of reducing experimental animal usage.

Concise Review: Dental Pulp Stem Cells: A Novel Cell Therapy for Retinal and Central Nervous System Repair

Dental pulp stem cells (DPSC) are neural crest‐derived ecto‐mesenchymal stem cells that can relatively easily and non‐invasively be isolated from the dental pulp of extracted postnatal and adult teeth. Accumulating evidence suggests that DPSC have great promise as a cellular therapy for central nervous system (CNS) and retinal injury and disease. The mode of action by which DPSC confer therapeutic benefit may comprise multiple pathways, in particular, paracrine‐mediated processes which involve a wide array of secreted trophic factors and is increasingly regarded as the principal predominant mechanism. In this concise review, we present the current evidence for the use of DPSC to repair CNS damage, including recent findings on retinal ganglion cell neuroprotection and regeneration in optic nerve injury and glaucoma. Stem Cells 2017;35:61–67

Dental pulp stem cells: a novel cell therapy for retinal and central nervous system repair

Dental pulp stem cells (DPSC) are neural crest‐derived ecto‐mesenchymal stem cells that can relatively easily and non‐invasively be isolated from the dental pulp of extracted postnatal and adult teeth. Accumulating evidence suggests that DPSC have great promise as a cellular therapy for central nervous system (CNS) and retinal injury and disease. The mode of action by which DPSC confer therapeutic benefit may comprise multiple pathways, in particular, paracrine‐mediated processes which involve a wide array of secreted trophic factors and is increasingly regarded as the principal predominant mechanism. In this concise review, we present the current evidence for the use of DPSC to repair CNS damage, including recent findings on retinal ganglion cell neuroprotection and regeneration in optic nerve injury and glaucoma. Stem Cells 2017;35:61–67

Decorin Reduces Intraocular Pressure and Retinal Ganglion Cell Loss in Rodents Through Fibrolysis of the Scarred Trabecular Meshwork

PURPOSE To investigate whether Decorin, a matrikine that regulates extracellular matrix (ECM) deposition, can reverse established trabecular meshwork (TM) fibrosis, lower IOP, and reduce progressive retinal ganglion cell (RGC) death in a novel rodent model of TM fibrosis. METHODS Adult rats had intracameral (IC) injections of human recombinant (hr) TGF-β over 30 days (30 d; to induce TM fibrosis, raise IOP, and initiate RGC death by 17 d) or PBS (controls) and visually evoked potentials (VEP) were measured at 30 d to evaluate resultant visual pathway dysfunction. In some animals TGF-β injections were stopped at 17 d when TM fibrosis and IOP were consistently raised and either hrDecorin or PBS IC injections were administered between 21 d and 30 d. Intraocular pressure was measured biweekly and eyes were processed for immunohistochemical analysis of ECM deposition to assess TM fibrosis and levels of matrix metalloproteinases (MMP) and tissue inhibitors of matrix metalloproteinases (TIMP) to assess fibrolysis. The effect of hrDecorin treatment on RGC survival was also assessed. RESULTS Transforming growth factor-β injections caused sustained increases in ECM deposition in the TM and raised IOP by 17 d, responses that were associated with 42% RGC loss and a significant decrease in VEP amplitude measured at 30 d. Decorin treatment from 17 d reduced TGF-β-induced TM fibrosis, increased levels of MMP2 and MMP9 and lowered TIMP2 levels, and lowered IOP, preventing progressive RGC loss. CONCLUSIONS Human recombinant Decorin reversed established TM fibrosis and lowered IOP, thereby rescuing RGC from progressive death. These data provide evidence for the candidacy of hrDecorin as a treatment for open-angle glaucoma.

Dental pulp stem cells, a paracrine-mediated therapy for the retina

The functional loss that occurs after retinal/optic nerve injury is permanent and can arise through trauma or neurodegenerative conditions such as glaucoma. Neurotrophic factors (NTFs) promote survival of injured retinal ganglion cells (RGCs) and regeneration of their axons, suggesting their clinical utility to prevent further damage and restore lost function. Delivery of optimal concentrations of NTFs to RGCs is difficult to achieve by injection but single implants of stem cells which naturally secrete multiple NTFs for sustained periods better addresses this problem. This review discusses a relatively new source of adult stem cells, the dental pulp stem cells, and compares their efficacy and feasibility with other stem cells, such as the well-studied bone marrow-derived mesenchymal stem cells (BMSCs), in the context of cellular therapy for the retina.

Mesenchymal stem cell therapy for retinal ganglion cell neuroprotection and axon regeneration.

Retinal ganglion cells (RGCs) are responsible for propagating signals derived from visual stimuli in the eye to the brain, along their axons within the optic nerve to the superior colliculus, lateral geniculate nucleus and visual cortex of the brain. Damage to the optic nerve either through trauma, such as head injury, or degenerative disease, such as glaucoma causes irreversible loss of function through degeneration of non-regenerating RGC axons and death of irreplaceable RGCs, ultimately leading to blindness (Berry et al., 2008). The degeneration of RGCs and their axons is due to the loss of the necessary source of retrogradely transported neurotrophic factors (NTFs) being hindered by axonal injury. NTFs are survival factors for neurons and play a pivotal part in axon regeneration. Stem cells particularly mesenchymal stem cells (MSCs) have been shown to possess a natural intrinsic capacity for paracrine support, releasing multiple signalling molecules including NTFs. By transplanting MSCs into the vitreous, they are positioned adjacent to the injured retina to provide paracrine-mediated therapy for the retinal neuronal cells (Johnson et al., 2010a; Mead et al., 2013). Additionally, MSCs may be pre-differentiated into supportive glial-like cells, such as Schwann cells, which could further increase their potential for paracrine support of injured neurons (Martens et al., 2013). Thus, MSCs have received considerable attention as a new cellular therapy for both traumatic and degenerative eye disease, acting as an alternative source of NTFs, protecting injured RGCs and promoting regeneration of their axons (Figure 1).