Michael McGrogan

Clonal Human (hNT) Neuron Grafts for Stroke Therapy : Neuropathology in a Patient 27 Months after Implantation

Although grafted cells may be promising therapy for stroke, survival of implanted neural cells in the brains of stroke patients has never been documented. Human NT2N (hNT) neurons derived from the NTera2 (NT2) teratocarcinoma cell line were shown to remain postmitotic, retain a neuronal phenotype, survive >1 year in host rodent brains and ameliorate motor and cognitive impairments in animal models of ischemic stroke. Here we report the first postmortem brain findings of a phase I clinical stroke trial patient implanted with human hNT neurons adjacent to a lacunar infarct 27 months after surgery. Neurofilament immunoreactive neurons were identified in the graft site, fluorescent in situ hybridization revealed polyploidy in groups of cells at this site just like polyploid hNT neurons in vitro, and there was no evidence of a neoplasm. These findings indicate that implanted hNT neurons survive for >2 years in the human brain without deleterious effects.

Clinical Outcomes of Transplanted Modified Bone Marrow–Derived Mesenchymal Stem Cells in Stroke: A Phase 1/2a Study

Background and Purpose— Preclinical data suggest that cell-based therapies have the potential to improve stroke outcomes. Methods— Eighteen patients with stable, chronic stroke were enrolled in a 2-year, open-label, single-arm study to evaluate the safety and clinical outcomes of surgical transplantation of modified bone marrow–derived mesenchymal stem cells (SB623). Results— All patients in the safety population (N=18) experienced at least 1 treatment-emergent adverse event. Six patients experienced 6 serious treatment-emergent adverse events; 2 were probably or definitely related to surgical procedure; none were related to cell treatment. All serious treatment-emergent adverse events resolved without sequelae. There were no dose-limiting toxicities or deaths. Sixteen patients completed 12 months of follow-up at the time of this analysis. Significant improvement from baseline (mean) was reported for: (1) European Stroke Scale: mean increase 6.88 (95% confidence interval, 3.5–10.3; P<0.001), (2) National Institutes of Health Stroke Scale: mean decrease 2.00 (95% confidence interval, −2.7 to −1.3; P<0.001), (3) Fugl-Meyer total score: mean increase 19.20 (95% confidence interval, 11.4–27.0; P<0.001), and (4) Fugl-Meyer motor function total score: mean increase 11.40 (95% confidence interval, 4.6–18.2; P<0.001). No changes were observed in modified Rankin Scale. The area of magnetic resonance T2 fluid-attenuated inversion recovery signal change in the ipsilateral cortex 1 week after implantation significantly correlated with clinical improvement at 12 months (P<0.001 for European Stroke Scale). Conclusions— In this interim report, SB623 cells were safe and associated with improvement in clinical outcome end points at 12 months. Clinical Trial Registration— URL: https://www.clinicaltrials.gov. Unique identifier: NCT01287936.

Notch-Induced Rat and Human Bone Marrow Stromal Cell Grafts Reduce Ischemic Cell Loss and Ameliorate Behavioral Deficits in Chronic Stroke Animals

Gene transfection with Notch 1 intracellular domain and subsequent growth factor treatment stimulate neuron-like differentiation of bone marrow stromal cells (BMSCs). Here, we examined the potential of transplanting Notch-induced BMSCs to exert therapeutic effects in a rat model of chronic ischemic stroke. In experiment 1, Notch-induced rat BMSCs were intrastriatally transplanted in rats at 1 month after being subjected to transient occlusion of middle cerebral artery (MCAo). Compared to post-stroke/pretransplantation level, significant improvements in locomotor and neurological function were detected in stroke rats that received 100 k and 200 k BMSCs, but not in those that received 40 k BMSCs. Histological results revealed 9%-15% graft survival, which dose-dependently correlated with behavioral recovery. At 5 weeks post-transplantation, some grafted BMSCs were positive for the glial marker GFAP (about 5%), but only a few cells (2-5 cells per brain) were positive for the neuronal marker NeuN. However, at 12 weeks post-transplantation, where the number of GFAP-positive BMSCs was maintained (5%), there was a dramatic increase in NeuN-positive BMSCs (23%). In experiment 2, Notch-induced human BMSCs were intrastriatally transplanted in rats at 1 month following the same MCAo model. Improvements in both locomotor and neurological function were observed from day 7 to day 28 post-transplantation, with the high dose (180 k) displaying significantly better behavioral recovery than the low dose (90 k) or vehicle. There were no observable adverse behavioral effects during this study period that also involved chronic immunosuppression of all animals. Histological analyses revealed a modest 5%-7% graft survival, with few (<1%) cells expressing an intermediate MAP2 neuronal marker, but not glial or oligodendroglial markers. In addition, striatal peri-infarct cell loss was significantly reduced in transplanted stroke animals compared to vehicle-treated stroke animals. The present study demonstrates the potential of Notch-induced BMSC cell therapy for patients presenting with fixed ischemic stroke.

Human Mesenchymal Stromal Cells and Their Derivative, SB623 Cells, Rescue Neural Cells via Trophic Support Following In Vitro Ischemia

Cell transplantation is a promising treatment strategy for many neurological disorders, including stroke, which can target multiple therapeutic mechanisms in a sustained fashion. We investigated the ability of human mesenchymal stromal cells (MSCs) and MSC-derived SB623 cells to rescue neural cells via trophic support following an in vitro stroke model. Following oxygen glucose deprivation, cortical neurons or hippocampal slices were cocultured with either MSCs or SB623 cells separated by a semiporous membrane (prohibits cell–cell contact) or with MSC- or SB623 cell-conditioned medium. MSCs, SB623 cells, MSC-conditioned media, and SB623 cell-conditioned media all significantly reduced neural cell damage/death compared to untreated conditions, and the rescue effect of the conditioned media was dose dependent. We identified 11 neurotrophic factors secreted by MSCs and/or SB623 cells. This study emphasizes the importance of trophic support provided by marrow-derived cells, which likely contributes to the efficacy of cell therapy for brain injury.

Evaluation of surgical techniques for neuronal cell transplantation used in patients with stroke.

Transplantation of cultured neuronal cells was performed in two human clinical trials after safety and efficacy was demonstrated in animal models of stroke. The studies tested the utility of human neuronal cellular transplantation into and around the small stroke volume. We developed a stereotactic surgical technique for cell delivery and evaluated that method in 26 patients with basal ganglia region motor stroke. Human neuronal cells (hNT cells; LBS neurons) were delivered frozen then thawed and formulated on the morning of surgery. Patients in a first trial received 2 or 6 million cells in three or nine implants, and in a second trial, 5 or 10 million in 25 implants. A novel cell delivery cannula was designed, manufactured, tested, and used in surgery. Immediate postoperative CT scans and later serial MR scans were used to evaluate the surgical site. Tests on the cell implantation cannula showed that the cells were not damaged and remained viable after injection. All patients underwent uncomplicated surgeries. Cells could be implanted within a 2-h period, maintaining viability of the preparation. Serial evaluations (maximum 5 years) showed no cell-related adverse serologic or imaging-defined effects. One patient had burr hole drainage of an asymptomatic chronic subdural hematoma. Human neuronal cells can be produced in culture and implanted stereotactically into the brains of patients with stroke. Surgical cell delivery did not lead to new neurological deficits, and imaging studies showed no adverse effects. The cannula used allowed precise injection of the clinical cell dose within a time period that maintained cell viability.

Glial Cell Line-Derived Neurotrophic Factor―Secreting Genetically Modified Human Bone Marrow-Derived Mesenchymal Stem Cells Promote Recovery in a Rat Model of Parkinson's Disease

Parkinsons disease (PD) is a neurodegenerative disease characterized by progressive degeneration of nigrostriatal dopaminergic (DA) neurons. The therapeutic potential of glial cell line‐derived neurotrophic factor (GDNF), the most potent neurotrophic factor for DA neurons, has been demonstrated in many experimental models of PD. However, chronic delivery of GDNF to DA neurons in the brain remains an unmet challenge. Here, we report the effects of GDNF‐releasing Notch‐induced human bone marrow‐derived mesenchymal stem cells (MSC) grafted into striatum of the 6‐hydroxydopamine (6‐OHDA) progressively lesioned rat model of PD. Human MSC, obtained from bone marrow aspirates of young, healthy adult volunteers, were transiently transfected with the intracellular domain of the Notch1 gene (NICD) to generate SB623 cells. SB623 cells expressing GDNF and/or humanized Renilla green fluorescent protein (hrGFP) following lentiviral transduction or nontransduced cells were stereotaxically placed into rat striatum 1 week after a unilateral partial 6‐OHDA striatal lesion. At 4 weeks, rats that had received GDNF‐transduced SB623 cells had significantly decreased amphetamine‐induced rotation compared with control rats, although this effect was not observed in rats that received GFP‐transduced or nontransduced SB623 cells. At 5 weeks, rejuvenated tyrosine hydroxylase‐immunoreactive (TH‐IR) fibers that appeared to be host DA axons were observed in and around grafts. This effect was more prominent in rats that received GDNF‐secreting cells and was not observed in controls. These observations suggest that human bone‐marrow derived MSC, genetically modified to secrete GDNF, hold potential as an allogeneic or autologous stem cell therapy for PD.

Extracellular matrix produced by bone marrow stromal cells and by their derivative, SB623 cells, supports neural cell growth

Several studies have shown the benefits of transplanting bone marrow‐derived multipotent mesenchymal stromal cells (MSC) into neurodegenerative lesions of the central nervous system, despite a low engraftment rate and the poor persistence of grafts. It is known that the extracellular matrix (ECM) modulates neuritogenesis and glial growth, but little is known about effects of MSC‐derived ECM on neural cells. In this study, we demonstrate in vitro that the ECM produced by MSC can support neural cell attachment and growth. We also compare the neurosupportive properties of MSC to the MSC derivative, SB623 cells, which is being developed as a cell therapy for stroke. Embryonic rat brain cortical cells cultured for 3 weeks on human MSC‐ and SB623 cell‐derived ECM exhibit about a 1.5 and 3 times higher metabolic activity, respectively, compared with the cultures grown on poly‐D‐lysine (PDL), although the initial neural cell adhesion to cell‐derived ECM and PDL is similar. The MSC‐ and SB623 cell‐derived ECM protects neural cells from nutrient and growth factor deprivation. Under the conditions used, only neurons grow on PDL. In contrast, both MSC‐ and SB623 cell‐derived ECMs support the growth of neurons, astrocytes, and oligodendrocytes, as demonstrated by immunostaining. Morphologically, neurons on cell‐derived ECM form more complex and extended neurite networks than those cultured on PDL. Together, these data indicate that the beneficial effect of MSC and SB623 cells in neurotransplantation could be explained in part by the neurosupportive properties of the ECM produced by these cells.

Reversal of dopaminergic degeneration in a parkinsonian rat following micrografting of human bone marrow-derived neural progenitors.

Parkinsons disease (PD) is a common neurodegenerative disease characterized by the selective loss of dopaminergic (DA) neurons in the midbrain. Various types of stem cells that have potential to differentiate into DA neurons are being investigated as cellular therapies for PD. Stem cells also secrete growth factors and therefore also may have therapeutic effects in promoting the health of diseased DA neurons in the PD brain. To address this possibility in an experimental model of PD, bone marrow-derived neuroprogenitor-like cells were generated from bone marrow procured from healthy human adult volunteers and their potential to elicit recovery of damaged DA axons was studied in a partial lesion rat model of PD. Following collection of bone marrow, mesenchymal stem cells (MSC) were isolated and then genetically modified to create SB623 cells by transient transfection with the intracellular domain of the Notch1 gene (NICD), a modification that upregulates expression of certain neuroprogenitor markers. Ten deposits of 0.5 μl of SB623 cell suspension adjusted from 6,000 to 21,000 cells/μl in PBS or PBS alone were stereotaxically placed in the striatum 1 week after the nigrostriatal projection had been partially lesioned in adult F344 rats by injection of 6-hydroxydopamine (6-OHDA) into the striatum. At 3 weeks, a small number of grafted SB623 cells survived in the lesioned striatum as visualized by expression of the human specific nuclear matrix protein (hNuMA). In rats that received SB623 cells, but not in control rats, dense tyrosine hydroxylase immunoreactive (TH-ir) fibers were observed around the grafts. These fibers appeared to be rejuvenated host DA axons because no TH-ir in soma of surviving SB623 cells or coexpression of TH and hNuMA-ir were observed. In addition, dense serotonin immunoreactive (5-HT-ir) fibers were observed around grafted SB623 cells and these fibers also appeared to be of the host origin. Also, in some SB623 grafted rats that were sacrificed within 2 h of dl-amphetamine injection, hot spots of c-Fos-positive nuclei that coincided with rejuvenated dense TH fibers around the grafted SB623 cells were observed, suggesting increased availability of DA in these locations. Our observations suggest that NICD-transfected MSC hold potential as a readily available autologous or allogenic cellular therapy for ameliorating the degeneration of DA and 5-HT neurons in PD patients.

Dopaminergic phenotype of hNT cells in vitro.

We investigated the catecholaminergic nature of cultured hNT neurons previously treated either for 4 or 5 weeks with retinoic acid (RA). There were significantly more tyrosine hydroxylase (TH)-positive neurons (60%) in cultures treated for 4 weeks with RA compared to 5 week-treated cultures (</=15%). Furthermore, numerous TH-positive hNT cells were also immunoreactive to dopamine transporter (DAT), dopamine receptor (D2) and aldehyde dehydrogenase (AHD-2), an enzyme exclusively expressed by dopaminergic (DA) ventral mesencephalic (VM) precursors. Thus this cell line has all the necessary cellular machinery to produce functional DA neurons and therefore is a good alternative tissue source to fetal VM.

Stem Cell Recruitment of Newly Formed Host Cells via a Successful Seduction? Filling the Gap between Neurogenic Niche and Injured Brain Site

Here, we report that a unique mechanism of action exerted by stem cells in the repair of the traumatically injured brain involves their ability to harness a biobridge between neurogenic niche and injured brain site. This biobridge, visualized immunohistochemically and laser captured, corresponded to an area between the neurogenic subventricular zone and the injured cortex. That the biobridge expressed high levels of extracellular matrix metalloproteinases characterized initially by a stream of transplanted stem cells, but subsequently contained only few to non-detectable grafts and overgrown by newly formed host cells, implicates a novel property of stem cells. The transplanted stem cells manifest themselves as pathways for trafficking the migration of host neurogenic cells, but once this biobridge is formed between the neurogenic site and the injured brain site, the grafted cells disappear and relinquish their task to the host neurogenic cells. Our findings reveal that long-distance migration of host cells from the neurogenic niche to the injured brain site can be achieved through transplanted stem cells serving as biobridges for initiation of endogenous repair mechanisms. This is the first report of a stem cell-paved “biobridge”. Indeed, to date the two major schools of discipline in stem cell repair mechanism primarily support the concept of “cell replacement” and bystander effects of “trophic factor secretion”. The present novel observations of a stem cell seducing a host cell to engage in brain repair advances basic science concepts on stem cell biology and extracellular matrix, as well as provokes translational research on propagating this stem cell-paved biobridge beyond cell replacement and trophic factor secretion for the treatment of traumatic brain injury and other neurological disorders.