Intracerebral implantation of human neural stem cells and motor recovery after stroke: multicentre prospective single-arm study (PISCES-2).

BACKGROUND: Human neural stem cell implantation may offer improved recovery from stroke. We investigated the feasibility of intracerebral implantation of the allogeneic human neural stem cell line CTX0E03 in the subacute-chronic recovery phase of stroke and potential measures of therapeutic response in a multicentre study. METHODS: We undertook a prospective, multicentre, single-arm, open-label study in adults aged >40 years with significant upper limb motor deficits 2-13 months after ischaemic stroke. 20 million cells were implanted by stereotaxic injection to the putamen ipsilateral to the cerebral infarct. The primary outcome was improvement by 2 or more points on the Action Research Arm Test (ARAT) subtest 2 at 3 months after implantation. FINDINGS: Twenty-three patients underwent cell implantation at eight UK hospitals a median of 7 months after stroke. One of 23 participants improved by the prespecified ARAT subtest level at 3 months, and three participants at 6 and 12 months. Improvement in ARAT was seen only in those with residual upper limb movement at baseline. Transient procedural adverse effects were seen, but no cell-related adverse events occurred up to 12 months of follow-up. Two deaths were unrelated to trial procedures. INTERPRETATION: Administration of human neural stem cells by intracerebral implantation is feasible in a multicentre study. Improvements in upper limb function occurred at 3, 6 and 12 months, but not in those with absent upper limb movement at baseline, suggesting a possible target population for future controlled trials. FUNDING: ReNeuron, Innovate UK (application no 32074-222145). TRIAL REGISTRATION NUMBER: EudraCT Number: 2012-003482-18.

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.

c-MycERTAM transgene silencing in a genetically modified human neural stem cell line implanted into MCAo rodent brain.

BACKGROUND: The human neural stem cell line CTX0E03 was developed for the cell based treatment of chronic stroke disability. Derived from fetal cortical brain tissue, CTX0E03 is a clonal cell line that contains a single copy of the c-mycERTAM transgene delivered by retroviral infection. Under the conditional regulation by 4-hydroxytamoxifen (4-OHT), c-mycERTAM enabled large-scale stable banking of the CTX0E03 cells. In this study, we investigated the fate of this transgene following growth arrest (EGF, bFGF and 4-OHT withdrawal) in vitro and following intracerebral implantation into a mid-cerebral artery occluded (MCAo) rat brain. In vitro, 4-weeks after removing growth factors and 4-OHT from the culture medium, c-mycERTAM transgene transcription is reduced by ~75%. Furthermore, immunocytochemistry and western blotting demonstrated a concurrent decrease in the c-MycERTAM protein. To examine the transcription of the transgene in vivo, CTX0E03 cells (450,000) were implanted 4-weeks post MCAo lesion and analysed for human cell survival and c-mycERTAM transcription by qPCR and qRT-PCR, respectively. RESULTS: The results show that CTX0E03 cells were present in all grafted animal brains ranging from 6.3% to 39.8% of the total cells injected. Prior to implantation, the CTX0E03 cell suspension contained 215.7 (SEM = 13.2) copies of the c-mycERTAM transcript per cell. After implantation the c-mycERTAM transcript copy number per CTX0E03 cell had reduced to 6.9 (SEM = 3.4) at 1-week and 7.7 (SEM = 2.5) at 4-weeks. Bisulfite genomic DNA sequencing of the in vivo samples confirmed c-mycERTAM silencing occurred through methylation of the transgene promoter sequence. CONCLUSION: In conclusion the results confirm that CTX0E03 cells downregulated c-mycERTAM transgene expression both in vitro following EGF, bFGF and 4-OHT withdrawal and in vivo following implantation in MCAo rat brain. The silencing of the c-mycERTAM transgene in vivo provides an additional safety feature of CTX0E03 cells for potential clinical application.

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.

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.

Human Neural Stem Cell Therapy for Chronic Ischemic Stroke: Charting Progress from Laboratory to Patients.

Chronic disability after stroke represents a major unmet neurologic need. ReNeuron's development of a human neural stem cell (hNSC) therapy for chronic disability after stroke is progressing through early clinical studies. A Phase I trial has recently been published, showing no safety concerns and some promising signs of efficacy. A single-arm Phase II multicenter trial in patients with stable upper-limb paresis has recently completed recruitment. The hNSCs administrated are from a manufactured, conditionally immortalized hNSC line (ReNeuron's CTX0E03 or CTX), generated with c-mycERTAM technology. This technology has enabled CTX to be manufactured at large scale under cGMP conditions, ensuring sufficient supply to meets the demands of research, clinical development, and, eventually, the market. CTX has key pro-angiogenic, pro-neurogenic, and immunomodulatory characteristics that are mechanistically important in functional recovery poststroke. This review covers the progress of CTX cell therapy from its laboratory origins to the clinic, concluding with a look into the late stage clinical future.

Stem cell transplantation after middle cerebral artery occlusion.

Stem cell lines have been and are being developed to treat damage in the central nervous system after stroke. Stem cells are able to migrate to areas of damage and to differentiate into neurons and glia. Grafts of murine stem cells have been shown to promote recovery from behavioral dysfunction after stroke. We have developed protocols to optimize behavioral testing, animal recovery, and stem cell delivery after middle cerebral artery occlusion. In this chapter we discuss study protocols aimed at integrating in vitro preparation of cells, small animal surgery, behavioral testing batteries, and histological analysis.

Turning Regenerative Medicine Breakthrough Ideas and Innovations into Commercial Products.

The TERMIS-Europe (EU) Industry committee intended to address the two main critical issues in the clinical/commercial translation of Advanced Therapeutic Medicine Products (ATMP): (1) entrepreneurial exploitation of breakthrough ideas and innovations, and (2) regulatory market approval. Since January 2012, more than 12,000 publications related to regenerative medicine and tissue engineering have been accepted for publications, reflecting the intense academic research activity in this field. The TERMIS-EU 2014 Industry Symposium provided a reflection on the management of innovation and technological breakthroughs in biotechnology first proposed to contextualize the key development milestones and constraints of allocation of financial resources, in the development life-cycle of radical innovation projects. This was illustrated with the biofuels story, sharing similarities with regenerative medicine. The transition was then ensured by an overview of the key identified challenges facing the commercialization of cell therapy products as ATMP examples. Real cases and testimonies were then provided by a palette of medical technologies and regenerative medicine companies from their commercial development of cell and gene therapy products. Although the commercial development of ATMP is still at the proof-of-concept stage due to technology risks, changing policies, changing markets, and management changes, the sector is highly dynamic with a number of explored therapeutic approaches, developed by using a large diversity of business models, both proposed by the experience, pitfalls, and successes of regenerative medicine pioneers, and adapted to the constraint resource allocation and environment in radical innovation projects.

Three-dimensional structure and survival of newly formed blood vessels after focal cerebral ischemia.

Penumbra tissue becomes highly angiogenic after ischaemic stroke in man, and the re-establishment of a functional vasculature might be beneficial to patients. Unilateral ischaemia was induced in male Sprague-Dawley rats by permanent occlusion of the distal left middle cerebral artery (MCAO). Animals with stroke were kept alive for 1, 7, 14, 21 or 28 days after which time they were terminally anaesthetized. Vascular casts of infarcted areas, analyzed by scanning electron microscopy demonstrated that radially arranged neocortical arterioles and venules lost their regular patterns within one day of occlusion, and soon afterwards started to form a very dense network of anastomosing microvessels. At 1 week, vascular budding was visible at many sites. The smallest microvessels (4-10 microm) formed connections with the surrounding proliferating vessels similar to those in the normal brain. Survival of microvascular endothelial cells (ECs) was studied by double labeling of tissue sections using immunohistochemistry and antibodies to caspase-3, and TUNEL staining for apoptotic cells. ECs demonstrated intensive staining for caspase-3 and also staining by TUNEL, particularly near the infarct border, 14 days post-MCAO. These data support the hypothesis that growing blood vessels in ischaemic tissue form new connections, the pattern of which is similar to that in normal rat brain, but different to those formed in growing tumours. This normal growth pattern might be essential in future therapies involving induction of vascularization and neuroprotection to enhance long-term survival of the penumbra.

Human neural progenitor cell engraftment increases neurogenesis and microglial recruitment in the brain of rats with stroke.

MAIN OBJECTIVES: Stem cell transplantation is to date one of the most promising therapies for chronic ischemic stroke. The human conditionally immortalised neural stem cell line, CTX0E03, has demonstrable efficacy in a rodent model of stroke and is currently in clinical trials. Nonetheless, the mechanisms by which it promotes brain repair are not fully characterised. This study investigated the cellular events occurring after CTX0E03 transplantation in the brains of rats that underwent ischemic stroke. METHODS: We focused on the endogenous proliferative activity of the host brain in response to cell transplantation and determined the identity of the proliferating cells using markers for young neurons (doublecortin, Dcx) and microglia (CD11b). So as to determine the chronology of events occurring post-transplantation, we analysed the engrafted brains one week and four weeks post-transplantation. RESULTS: We observed a significantly greater endogenous proliferation in the striatum of ischemic brains receiving a CTX0E03 graft compared to vehicle-treated ischemic brains. A significant proportion of these proliferative cells were found to be Dcx+ striatal neuroblasts. Further, we describe an enhanced immune response after CTX0E03 engraftment, as shown by a significant increase of proliferating CD11b+ microglial cells. CONCLUSIONS: Our study demonstrates that few Dcx+ neuroblasts are proliferative in normal conditions, and that this population of proliferative neuroblasts is increased in response to stroke. We further show that CTX0E03 transplantation after stroke leads to the maintenance of this proliferative activity. Interestingly, the preservation of neuronal proliferative activity upon CTX0E03 transplantation is preceded and accompanied by a high rate of proliferating microglia. Our study suggests that microglia might mediate in part the effect of CTX0E03 transplantation on neuronal proliferation in ischemic stroke conditions.

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.

TrkAd5: A novel therapeutic agent for treatment of inflammatory pain and asthma.

Elevated levels of nerve growth factor have been linked to the onset and persistence of many pain-related disorders and asthma. Described here are the design, expression, refolding, and purification of a monomeric (nonstrand-swapped) form of the binding domain of the nerve growth factor receptor, designated TrkAd5. We have shown that TrkAd5 produced recombinantly binds nerve growth factor with picomolar affinity. TrkAd5 has been characterized using a variety of biophysical and biochemical assays and is shown here to be stable in both plasma and urine. The palliative effects of TrkAd5 are demonstrated in animal models of inflammatory pain and allergic asthma. We conclude that TrkAd5 will prove effective in ameliorating both acute and chronic conditions where nerve growth factor acts as a mediator and suggest a role for its application in vivo as a novel therapeutic.

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.