In vivo study to assess dosage of allogeneic pig retinal progenitor cells: Long-term survival, engraftment, differentiation and safety.

Despite notable efforts and significant therapeutical advances, age-related macular degeneration remains the single most common reason for vision loss. Retinal progenitor cells (RPCs) are considered promising candidates for cellular treatments that repair and restore vision. In this allogenic study, the phenotypic profile of pig and human RPCs derived using similar manufacturing processes is compared. The long-term (12-week) survival of green fluorescent protein-pig retinal progenitor cells GFP-pRPC after subretinal transplantation into normal miniature pig (mini-pig) retina is investigated. Human eyes are both anatomically and physiologically mimicked by pig eyes, so the pig is an ideal model to show an equivalent way of delivering cells, immunological response and dosage. The phenotypic equivalency of porcine and clinically intended human RPCs was established. Thirty-nine mini-pigs are used in this study, and vehicle-injected eyes and non-injected eyes serve as controls. Six groups are given different dosages of pRPCs, and the cells are found to survive well in all groups. At 12 weeks, strong evidence of integration is indicated by the location of the grafted cells within the neuro-retina, extension of processes to the plexiform layers and expression of key retinal markers such as recoverin, rhodopsin and synaptophysin. No immunosuppression is used, and no immune response is found in any of the groups. No pRPC-related histopathology findings are reported in the major organs investigated. An initial dose of 250 k cells in 100 µl of buffer is established as an appropriate initial dose for future human clinical trials.

Preclinical imaging methods for assessing the safety and efficacy of regenerative medicine therapies.

Regenerative medicine therapies hold enormous potential for a variety of currently incurable conditions with high unmet clinical need. Most progress in this field to date has been achieved with cell-based regenerative medicine therapies, with over a thousand clinical trials performed up to 2015. However, lack of adequate safety and efficacy data is currently limiting wider uptake of these therapies. To facilitate clinical translation, non-invasive in vivo imaging technologies that enable careful evaluation and characterisation of the administered cells and their effects on host tissues are critically required to evaluate their safety and efficacy in relevant preclinical models. This article reviews the most common imaging technologies available and how they can be applied to regenerative medicine research. We cover details of how each technology works, which cell labels are most appropriate for different applications, and the value of multi-modal imaging approaches to gain a comprehensive understanding of the responses to cell therapy in vivo.

Efficacy and Safety of Human Retinal Progenitor Cells.

PURPOSE: We assessed the long-term efficacy and safety of human retinal progenitor cells (hRPC) using established rodent models. METHODS: Efficacy of hRPC was tested initially in Royal College of Surgeons (RCS) dystrophic rats immunosuppressed with cyclosporine/dexamethasone. Due to adverse effects of dexamethasone, this drug was omitted from a subsequent dose-ranging study, where different hRPC doses were tested for their ability to preserve visual function (measured by optokinetic head tracking) and retinal structure in RCS rats at 3 to 6 months after grafting. Safety of hRPC was assessed by subretinal transplantation into wild type (WT) rats and NIH-III nude mice, with analysis at 3 to 6 and 9 months after grafting, respectively. RESULTS: The optimal dose of hRPC for preserving visual function/retinal structure in dystrophic rats was 50,000 to 100,000 cells. Human retinal progenitor cells integrated/survived in dystrophic and WT rat retina up to 6 months after grafting and expressed nestin, vimentin, GFAP, and ßIII tubulin. Vision and retinal structure remained normal in WT rats injected with hRPC and there was no evidence of tumors. A comparison between dexamethasone-treated and untreated dystrophic rats at 3 months after grafting revealed an unexpected reduction in the baseline visual acuity of dexamethasone-treated animals. CONCLUSIONS: Human retinal progenitor cells appear safe and efficacious in the preclinical models used here. TRANSLATIONAL RELEVANCE: Human retinal progenitor cells could be deployed during early stages of retinal degeneration or in regions of intact retina, without adverse effects on visual function. The ability of dexamethasone to reduce baseline visual acuity in RCS dystrophic rats has important implications for the interpretation of preclinical and clinical cell transplant studies.

The Effect of Transient Local Anti-inflammatory Treatment on the Survival of Pig Retinal Progenitor Cell Allotransplants.

PURPOSE: The development of photoreceptor replacement therapy for retinal degenerative disorders requires the identification of the optimal cell source and immunosuppressive regimen in a large animal model. Allotransplants are not acutely rejected in swine subretinal space, although it is not known if survival can be improved with immunosuppression. Here we investigated the survival and integration of expanded pig retinal progenitor cells (pRPCs) in normal recipients with and without transient anti-inflammatory suppression. METHODS: pRPCs were derived from the neural retina of E60 GFP transgenic pigs, expanded for six passages, characterized, and transplanted into the subretinal space of 12 pigs. Six recipients received a single intravitreal injection of rapamycin and dexamethasone. RESULTS: pRPCs expressed the photoreceptor development genes Sox2, Pax6, Lhx2, Crx, Nrl, and Recoverin in vitro. Transplanted cells were identified in 9 out of 12 recipients 4 weeks after the injection. pRPCs integrated primarily into the photoreceptor inner segment layer and outer nuclear layer with single cells present in the inner nuclear layer. Donor cells remained recoverin-positive and acquired rhodopsin. We did not observe any signs of graft proliferation. The immunosuppression did not affect the survival or distribution of grafts. No macrophage infiltration or loss of retinal structure was observed in either group. CONCLUSIONS: Local immunosuppression with rapamycin and dexamethasone does not improve the outcome of pRPC allotransplantation into the subretinal space. TRANSLATIONAL RELEVANCE: Survival and integration of pRPC together with the lack of graft proliferation suggests that allogeneic RPC transplantation without transient immunosuppression is a favorable approach for photoreceptor cell replacement.

Concise review: workshop review: understanding and assessing the risks of stem cell-based therapies.

The field of stem cell therapeutics is moving ever closer to widespread application in the clinic. However, despite the undoubted potential held by these therapies, the balance between risk and benefit remains difficult to predict. As in any new field, a lack of previous application in man and gaps in the underlying science mean that regulators and investigators continue to look for a balance between minimizing potential risk and ensuring therapies are not needlessly kept from patients. Here, we attempt to identify the important safety issues, assessing the current advances in scientific knowledge and how they may translate to clinical therapeutic strategies in the identification and management of these risks. We also investigate the tools and techniques currently available to researchers during preclinical and clinical development of stem cell products, their utility and limitations, and how these tools may be strategically used in the development of these therapies. We conclude that ensuring safety through cutting-edge science and robust assays, coupled with regular and open discussions between regulators and academic/industrial investigators, is likely to prove the most fruitful route to ensuring the safest possible development of new products.

Human retinal progenitor cell transplantation preserves vision.

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.

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 Fox(nu/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 3×10(5) 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.

Assessing the safety of stem cell therapeutics.

Unprecedented developments in stem cell research herald a new era of hope and expectation for novel therapies. However, they also present a major challenge for regulators since safety assessment criteria, designed for conventional agents, are largely inappropriate for cell-based therapies. This article aims to set out the safety issues pertaining to novel stem cell-derived treatments, to identify knowledge gaps that require further research, and to suggest a roadmap for developing safety assessment criteria. It is essential that regulators, pharmaceutical providers, and safety scientists work together to frame new safety guidelines, based on "acceptable risk," so that patients are adequately protected but the safety "bar" is not set so high that exciting new treatments are lost.

The neural stem cell line CTX0E03 promotes behavioral recovery and endogenous neurogenesis after experimental stroke in a dose-dependent fashion.

BACKGROUND: This study investigated behavioral recovery in rats following implanting increasing doses of CTX0E03 cells into the putamen ipsilateral to the stroke damage. Postmortem histological analysis investigated possible mechanisms of behavioral recovery. METHODS: . At 4 weeks after middle cerebral artery occlusion (MCAO), rats were treated with 4500, 45,000, or 450,000 CTX0E03 cells or vehicle implanted into the putamen with testing on a battery of tasks preocclusion and postocclusion. Histological examination of brains included assessment of lesion volumes, implant cell survival and differentiation, changes to host brain matrix, angiogenesis, and neurogenesis using immunohistochemical methods. RESULTS: . Statistically significant dose-related recovery in sensorimotor function deficits (bilateral asymmetry test [BAT; P < .0002] in the mid- and high-dose groups and rotameter test after amphetamine exposure [P < .05] in the high-dose group) was found in the CTX0E03 cell implanted groups compared to the vehicle group. In-life functional improvements correlated with cell dose, though did not correlate with survival of CTX0E03 cells measured at postmortem. Surviving CTX0E03 cells differentiated into oligodendroglial and endothelial phenotypes. MCAO-induced reduction of neurogenesis in the subventricular zone (SVZ) was partially restored to that observed in sham operated controls. No adverse CTX0E03 cell-related effects were observed during in-life observations or on tissue histology. CONCLUSIONS: . This study found that the implantation of CTX0E03 human neural stem cells in rats after MCAO stroke promoted significant behavioral recovery depending on cell dose. The authors propose a paracrine trophic mechanism, which is triggered early after CTX0E03 cell implantation, and which in turn targets restoration of neurogenesis in the SVZ of MCAO rats.

Implantation of c-mycER TAM 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 Parkinson's 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.

Development of a human neural stem cell line for use in recovery from disability after stroke.

A clonal human neural stem cell line (ReN001) has been developed for clinical use in the treatment of stable disability after stroke. This cell line has been conditionally immortalized using the fusion transgene c-mycERTAM to allow controlled expansion when cultured in the presence of 4-hydroxytamoxifen. The cell line has been banked and fully characterized to assure there is genetic stability and no phenotypic drift with extended passages. In vivo studies determined the ability of the cell line to survive after implantation into damaged brain and its efficacy in the reduction of chronic behavioural dysfunction after implantation into a rodent model of stroke damage. A further study was conducted in this model and a dose-dependent effect was observed on behavioural recovery. No safety or toxicology issues were identified in in vivo studies with this cell line, which made REN001 a strong candidate for one of the first cell-based IND applications to be submitted to the Food and Drug Administration in the United States for consideration for the treatment of stroke in humans.

Differential development of neuronal physiological responsiveness in two human neural stem cell lines.

BACKGROUND: Neural stem cells (NSCs) are powerful research tools for the design and discovery of new approaches to neurodegenerative disease. Overexpression of the myc family transcription factors in human primary cells from developing cortex and mesencephalon has produced two stable multipotential NSC lines (ReNcell VM and CX) that can be continuously expanded in monolayer culture. RESULTS: In the undifferentiated state, both ReNcell VM and CX are nestin positive and have resting membrane potentials of around -60 mV but do not display any voltage-activated conductances. As initially hypothesized, using standard methods (stdD) for differentiation, both cell lines can form neurons, astrocytes and oligodendrocytes according to immunohistological characteristics. However it became clear that this was not true for electrophysiological features which designate neurons, such as the firing of action potentials. We have thus developed a new differentiation protocol, designated 'pre-aggregation differentiation' (preD) which appears to favor development of electrophysiologically functional neurons and to lead to an increase in dopaminergic neurons in the ReNcell VM line. In contrast, the protocol used had little effect on the differentiation of ReNcell CX in which dopaminergic differentiation was not observed. Moreover, after a week of differentiation with the preD protocol, 100% of ReNcell VM featured TTX-sensitive Na+-channels and fired action potentials, compared to 25% after stdD. Currents via other voltage-gated channels did not appear to depend on the differentiation protocol. ReNcell CX did not display the same electrophysiological properties as the VM line, generating voltage-dependant K+ currents but no Na+ currents or action potentials under either stdD or preD differentiation. CONCLUSION: These data demonstrate that overexpression of myc in NSCs can be used to generate electrophysiologically active neurons in culture. Development of a functional neuronal phenotype may be dependent on parameters of isolation and differentiation of the cell lines, indicating that not all human NSCs are functionally equivalent.

Making stem cell lines suitable for transplantation.

Human stem cells, progenitor cells, and cell lines have been derived from embryonic, fetal, and adult sources in the search for graft tissue suitable for the treatment of CNS disorders. An increasing number of experimental studies have shown that grafts from several sources survive, differentiate into distinct cell types, and exert positive functional effects in experimental animal models, but little attention has been given to developing cells under conditions of good manufacturing practice (GMP) that can be scaled up for mass treatment. The capacity for continued division of stem cells in culture offers the opportunity to expand their production to meet the widespread clinical demands posed by neurodegenerative diseases. However, maintaining stem cell division in culture long term, while ensuring differentiation after transplantation, requires genetic and/ or oncogenetic manipulations, which may affect the genetic stability and in vivo survival of cells. This review outlines the stages, selection criteria, problems, and ultimately the successes arising in the development of conditionally immortal clinical grade stem cell lines, which divide in vitro, differentiate in vivo, and exert positive functional effects. These processes are specifically exemplified by the murine MHP36 cell line, conditionally immortalized by a temperature-sensitive mutant of the SV40 large T antigen, and cell lines transfected with the c-myc protein fused with a mutated estrogen receptor (c-mycERTAM), regulated by a tamoxifen metabolite, but the issues raised are common to all routes for the development of effective clinical grade cells.

A conditionally immortal clonal stem cell line from human cortical neuroepithelium for the treatment of ischemic stroke.

Transplantation of neural stem cells into the brain is a novel approach to the treatment of chronic stroke disability. For clinical application, safety and efficacy of defined, stable cell lines produced under GMP conditions are required. To this end, a human neural stem cell line, CTX0E03, was derived from human somatic stem cells following genetic modification with a conditional immortalizing gene, c-mycER(TAM). This transgene generates a fusion protein that stimulates cell proliferation in the presence of a synthetic drug 4-hydroxy-tamoxifen (4-OHT). The cell line is clonal, expands rapidly in culture (doubling time 50-60 h) and has a normal human karyotype (46 XY). In the absence of growth factors and 4-OHT, the cells undergo growth arrest and differentiate into neurons and astrocytes. Transplantation of CTX0E03 in a rat model of stroke (MCAo) caused statistically significant improvements in both sensorimotor function and gross motor asymmetry at 6-12 weeks post-grafting. In addition, cell migration and long-term survival in vivo were not associated with significant cell proliferation. These data indicate that CTX0E03 has the appropriate biological and manufacturing characteristics necessary for development as a therapeutic cell line.

Effects of implantation site of dead stem cells in rats with stroke damage.

Searching for valid control grafts, we assessed the performance of rats subjected to middle cerebral artery occlusion (MCAO) and grafted with freeze-thawed dead stem cells into sites previously used for active grafts (ipsilateral and contralateral striatum and ventricle) on bilateral asymmetry and water maze tests. We expected to find that sham grafted groups had impairments equivalent to those of MCAO-only controls, relative to intact controls. This proved to be the case for contralateral and intraventricular grafts, and for asymmetry in rats with ipsilateral grafts. However, spatial learning was substantially impaired and lesion volume was increased by 55% with ipsilateral dead cell grafts. Exacerbation of stroke effects indicates potential hazards in the use of dead cells for sham grafts.

Transplantation of neural stem cells in a rat model of stroke: assessment of short-term graft survival and acute host immunological response.

The use of progenitors and stem cells for neural grafting is promising, as these not only have the potential to be maintained in vitro until use, but may also prove less likely to evoke an immunogenic response in the host, when compared to primary (fetal) grafts. We investigated whether the short-term survival of a grafted conditionally immortalised murine neuroepithelial stem cell line (MHP36) (2 weeks post-implantation, 4 weeks post-ischaemia) is influenced by: (i) immunosuppression (cyclosporin A (CSA) vs. no CSA), (ii) the local (intact vs. lesioned hemisphere), or (iii) global (lesioned vs. sham) brain environment. MHP36 cells were transplanted ipsi- and contralateral to the lesion in rats with middle cerebral artery occlusion (MCAo) or sham controls. Animals were either administered CSA or received no immunosuppressive treatment. A proliferation assay of lymphocytes dissociated from cervical lymph nodes, grading of the survival of the grafted cells, and histological evaluation of the immune response revealed no significant difference between animals treated with or without CSA. There was no difference in survival or immunological response to cells grafted ipsi- or contralateral to the lesion. Although a local upregulation of immunological markers (MHC class I, MHC class II, CD45, CD11b) was detected around the injection site and the ischaemic lesion, these were not specifically upregulated in response to transplanted cells. These results provide evidence for the low immunogenic properties of MHP36 cells during the initial period following implantation, known to be associated with an acute host immune response and ensuing graft rejection.

Effects of implantation site of stem cell grafts on behavioral recovery from stroke damage.

BACKGROUND AND PURPOSE: Findings that MHP36 stem cells grafted into intact parenchyma contralateral to the lesion induced by middle cerebral artery occlusion promoted recovery from stroke deficits led us to investigate whether implantation site of stem cells affects the functional efficacy of MHP36 grafts. METHODS: MHP36 cells (200 000/8 microL) were implanted in the left (n=8) or right (n=9) parenchyma or infused into the right ventricle (intraventricular; n=7) 2 to 3 weeks after stroke induced by 60 minutes of intraluminal right middle cerebral artery occlusion. Additionally, intact (n=11) and stroke (n=7) control groups were tested for 14 weeks in bilateral asymmetry, rotation bias, and spatial learning tasks before histological investigation of cell distribution and differentiation. RESULTS: Rats with left and right parenchymal grafts showed reduced bilateral asymmetry but no improvement in spatial learning. Conversely, spatial learning improved in rats with intraventricular grafts, but marked asymmetry persisted. No grafted group showed reduced amphetamine-induced rotation bias or reduced lesion volume relative to stroke controls. In all grafted groups, cells occupied both sides of the brain. A third of cells grafted in the striatum crossed the midline to occupy homologous regions in intact and lesioned hemispheres and differentiated into site-appropriate phenotypes. CONCLUSIONS: After stroke, both the intact and lesioned hemispheres attract grafted stem cells, suggesting repair processes that utilize cells both for local repair and to augment plastic changes in contralateral motor pathways. However, differential effects of parenchymal and intraventricular grafts suggest that different mechanisms are implicated in recovery from cognitive and sensorimotor deficits induced by stroke.

The distribution of 5-HT(6) receptors in rat brain: an autoradiographic binding study using the radiolabelled 5-HT(6) receptor antagonist [(125)I]SB-258585.

We used the highly selective 5-HT(6) receptor radioligand [(125)I]SB-258585 (4-iodo-N-[4-methoxy-3-(4-methylpiperazin-1-yl)phenyl]benzene-sulfonamide) to perform autoradiographic binding studies on the rat brain. High levels of specific binding occurred in the corpus striatum, nucleus accumbens, Islands of Calleja and the olfactory tubercle. A high level of binding also appeared in the choroid plexus. Moderate levels occurred in several regions of the hippocampal formation and in certain regions of the cerebral cortex, thalamus, hypothalamus, and substantia nigra; and very low levels in the globus pallidus, cerebellum, other mesencephalic regions, and the rhombencephalon. Displacement of total binding with 10 microM unlabelled SB-214111 (4-bromo-N-[4-methoxy-3-(4-methylpiperazin-1-yl)phenyl]benzene-sulfonamide), another selective 5-HT(6) receptor antagonist, or 10 microM unlabelled methiothepin, reduced binding to barely discernible levels. Some animals received unilateral injections of 6-hydroxydopamine (6-OHDA) into the median forebrain bundle to lesion the nigro-striatal pathway before autoradiographic examination. Effectiveness of the 6-OHDA lesions in the substantia nigra and striatum was confirmed with tyrosine hydroxylase immunohistochemistry. Such lesions resulted in no significant changes in [(125)I]SB-SB258585 binding in any brain region examined, suggesting that 5-HT(6) receptors in the striatum are not located on dendritic, somatic or terminal elements of dopaminergic neurones. Thus, the striatal binding sites seen in this study may be on intrinsic GABAergic or cholinergic neurones, or on terminals of projection neurones from the thalamus or cerebral cortex. The 5-HT(6) receptor ligand binding seen here in the striatum, accumbens, olfactory tubercle, Islands of Calleja, cerebral cortex and hippocampus are in concordance with previous immunohistochemical studies, and suggest a possible involvement of 5-HT(6) receptors in locomotor control, cognition, memory, and control of affect. The high levels of binding observed in the choroid plexus in this study have not been reported before. This finding suggests that 5-HT(6) receptors could play a role in the control of cerebrospinal fluid dynamics.