Caroline Hicks

Human Retinal Progenitor Cell Transplantation Preserves Vision

Background: Human retinal progenitor cells (hRPCs) are expandable in vitro and represent a possible therapy for retinal degenerative diseases. Results: In a rat model of retinal degeneration, transplantation of hRPCs preserved photoreceptors and visual function. Conclusion: Subretinal injection of hRPCs rescues photoreceptors without causing adverse effects. Significance: This study provides proof of concept for hRPC transplantation and paves the way for further studies and human trials. Cell transplantation is a potential therapeutic strategy for retinal degenerative diseases involving the loss of photoreceptors. However, it faces challenges to clinical translation due to safety concerns and a limited supply of cells. Human retinal progenitor cells (hRPCs) from fetal neural retina are expandable in vitro and maintain an undifferentiated state. This study aimed to investigate the therapeutic potential of hRPCs transplanted into a Royal College of Surgeons (RCS) rat model of retinal degeneration. At 12 weeks, optokinetic response showed that hRPC-grafted eyes had significantly superior visual acuity compared with vehicle-treated eyes. Histological evaluation of outer nuclear layer (ONL) characteristics such as ONL thickness, spread distance, and cell count demonstrated a significantly greater preservation of the ONL in hRPC-treated eyes compared with both vehicle-treated and control eyes. The transplanted hRPCs arrested visual decline over time in the RCS rat and rescued retinal morphology, demonstrating their potential as a therapy for retinal diseases. We suggest that the preservation of visual acuity was likely achieved through host photoreceptor rescue. We found that hRPC transplantation into the subretinal space of RCS rats was well tolerated, with no adverse effects such as tumor formation noted at 12 weeks after treatment.

In Vivo and In Vitro Characterization of the Angiogenic Effect of CTX0E03 Human Neural Stem Cells

CTX0E03 is a human neural stem cell line previously reported to reduce sensory motor deficits in a middle cerebral artery occlusion (MCAo) model of stroke. The objective of this study was to investigate if CTX0E03 treatment promotes angiogenesis. As stroke leads to damage of the vasculature in the brain, angiogenesis may contribute to the functional recovery. To test this hypothesis, the angiogenic activity of CTX0E03 was assessed both in vitro and in vivo. In vitro, CTX0E03 expression of trophic and proangiogenic factors was determined by real-time RT-PCR, Western blot, and ELISA, and its angiogenic activity was investigated in well-established angiogenesis assays. In vivo, angiogenesis was investigated in naive mice and MCAo rat brain and was evaluated by immunohistochemistry (IHC) using Von Willebrand factor (VWF), a marker of blood vessel formation, and BrdU/CD31 double labeling in naive mice only. In vitro results showed that CTX0E03-conditioned medium and coculture significantly increased total tubule formation compared with controls (p = 0.002 and p = 0.0008, respectively). Furthermore, CTX0E03 cells were found to be in direct association with the tubules by ICC. In vivo CTX0E03-treated brains demonstrated a significant increase in areas occupied by VWF-positive microvessels compared with vehicle-treated naive mice (two-way ANOVA, Interaction p < 0.05, Treatment p < 0.0001, Time p < 0.0) and MCAo rat (p = 0.001 unpaired t test, Welchs correction). CTX0E03-treated naive mouse brains showed an increase in BrdU/CD31 colabeling. In conclusion, in vitro CTX0E03 cells express proangiogenic factors and may promote angiogenesis by both release of paracrine factors and direct physical interaction. Furthermore, in vivo CTX0E03-treated rodent brains exhibited a significant increase in microvessels at the site of implantation compared with vehicle-injected groups. Taken together these data suggest that CTX0E03 cell therapy may provide significant benefit to stroke patients through upregulation of angiogenesis in the ischemic brain.

Clinical-Grade Human Neural Stem Cells Promote Reparative Neovascularization in Mouse Models of Hindlimb Ischemia

Objective—CTX0E03 (CTX) is a clinical-grade human neural stem cell (hNSC) line that promotes angiogenesis and neurogenesis in a preclinical model of stroke and is now under clinical development for stroke disability. We evaluated the therapeutic activity of intramuscular CTX hNSC implantation in murine models of hindlimb ischemia for potential translation to clinical studies in critical limb ischemia. Approach and Results—Immunodeficient (CD-1 Foxnu/nu) mice acutely treated with hNSCs had overall significantly increased rates and magnitude of recovery of surface blood flow (laser Doppler), limb muscle perfusion (fluorescent microspheres, P<0.001), and capillary and small arteriole densities in the ischemic limb (fluorescence immunohistochemistry, both P<0.001) when compared with the vehicle-treated group. Hemodynamic and anatomic improvements were dose related and optimal at a minimum dose of 3×105 cells. Dose-dependent improvements in blood flow and increased vessel densities by hNSC administration early after ischemia were confirmed in immunocompetent CD-1 and streptozotocin-induced diabetic mice, together with marked reductions in the incidence of necrotic toes (P<0.05). Delayed administration of hNSCs, 7 days after occlusion, produced restorative effects when comparable with acute treatment of 35 days after hindlimb ischemia. Histological studies in hindlimb ischemia immunocompetent mice for the first 7 days after treatment revealed short-term hNSC survival, transient elevation of early host muscle inflammatory, and angiogenic responses and acceleration of myogenesis. Conclusions—hNSC therapy represents a promising treatment option for critical limb ischemia.

Implantation of c-mycERTAM Immortalized Human Mesencephalic-Derived Clonal Cell Lines Ameliorates Behavior Dysfunction in a Rat Model of Parkinson’s Disease

Human neural stem cells offer the hope that a cell therapy treatment for Parkinsons disease (PD) could be made widely available. In this study, we describe two clonal human neural cell lines, derived from two different 10-week-old fetal mesencephalic tissues and immortalized with the c-mycER(TAM) transgene. Under the growth control of 4-hydroxytamoxifen, both cell lines display stable long-term growth in culture with a normal karyotype. In vitro, these nestin-positive cells are able to differentiate into tyrosine hydroxylase (TH)-positive neurons and are multipotential. Implantation of the undifferentiated cells into the 6-OHDA substantia nigral lesioned rat model displayed sustained improvements in a number of behavioral tests compared with noncell-implanted, vehicle-injected controls over the course of 6 months. Histological analysis of the brains showed survival of the implanted cells but no evidence of differentiation into TH-positive neurons. An average increase of approximately 26% in host TH immunoreactivity in the lesioned dorsal striatum was observed in the cell-treated groups compared to controls, with no difference in loss of TH cell bodies in the lesioned substantia nigra. Further analysis of the cell lines identified a number of expressed trophic factors, providing a plausible explanation for the effects observed in vivo. The exact mechanisms by which the implanted human neural cell lines provide behavioral improvements in the PD model are not completely understood; however, these findings provide evidence that cell therapy can be a potent treatment for PD acting through a mechanism independent of dopaminergic neuronal cell replacement.

The Development of Stem Cell-derived Exosomes as a Cell-free Regenerative Medicine

A successful strategy in regenerative medicine over the last decade has been the translation of stem cell therapy to repair diseased or damaged tissue in a wide range of indications, despite limited evidence attributing any therapeutic benefit to cell survival or differentiation. Recent findings, however, have demonstrated that the conditioned media from stem cell cultures can produce similar efficacious effects compared to those observed for cells. This has led to the stem cell paracrine hypothesis, proposing that secreted factors released from the stem cells contribute significantly to their beneficial effects. It has been well documented that stem cells have the ability to release a range of growth factors, cytokines and chemokines relevant to their function; however, these factors are released at levels too low to account for the reported therapeutic effects. Further purification of the conditioned media has since identified that not only are small molecules released by the stem cells, but so too are a large quantity of membrane-bound vesicles, including exosomes, in a functionally relevant manner. In this review, we present our current understanding and explore the evidence supporting the development of stem cell-derived exosomes as a cell-free regenerative medicine.

Differentiation of a Human Neural Stem Cell Line on Three Dimensional Cultures, Analysis of MicroRNA and Putative Target Genes

Neural stem cells (NSCs) are capable of self-renewal and differentiation into neurons, astrocytes and oligodendrocytes under specific local microenvironments. In here, we present a set of methods used for three dimensional (3D) differentiation and miRNA analysis of a clonal human neural stem cell (hNSC) line, currently in clinical trials for stroke disability (NCT01151124 and NCT02117635, Clinicaltrials.gov). HNSCs were derived from an ethical approved first trimester human fetal cortex and conditionally immortalized using retroviral integration of a single copy of the c-mycERTAMconstruct. We describe how to measure axon process outgrowth of hNSCs differentiated on 3D scaffolds and how to quantify associated changes in miRNA expression using PCR array. Furthermore we exemplify computational analysis with the aim of selecting miRNA putative targets. SOX5 and NR4A3 were identified as suitable miRNA putative target of selected significantly down-regulated miRNAs in differentiated hNSC. MiRNA target validation was performed on SOX5 and NR4A3 3’UTRs by dual reporter plasmid transfection and dual luciferase assay.