Transplantation of human central nervous system stem cells - neuroprotection in retinal degeneration.

Stem cells derived from the human brain and grown as neurospheres (HuCNS-SC) have been shown to be effective in treating central neurodegenerative conditions in a variety of animal models. Human safety data in neurodegenerative disorders are currently being accrued. In the present study, we explored the efficacy of HuCNS-SC in a rodent model of retinal degeneration, the Royal College of Surgeons (RCS) rat, and extended our previous cell transplantation studies to include an in-depth examination of donor cell behavior and phenotype post-transplantation. As a first step, we have shown that HuCNS-SC protect host photoreceptors and preserve visual function after transplantation into the subretinal space of postnatal day 21 RCS rats. Moreover, cone photoreceptor density remained relatively constant over several months, consistent with the sustained visual acuity and luminance sensitivity functional outcomes. The novel findings of this study include the characterization and quantification of donor cell radial migration from the injection site and within the subretinal space as well as the demonstration that donor cells maintain an immature phenotype throughout the 7 months of the experiment and undergo very limited proliferation with no evidence of uncontrolled growth or tumor-like formation. Given the efficacy findings and lack of adverse events in the RCS rat in combination with the results from ongoing clinical investigations, HuCNS-SC appear to be a well-suited candidate for cell therapy in retinal degenerative conditions.

Transplanted stem cell-secreted vascular endothelial growth factor effects poststroke recovery, inflammation, and vascular repair.

Cell transplantation offers a novel therapeutic strategy for stroke; however, how transplanted cells function in vivo is poorly understood. We show for the first time that after subacute transplantation into the ischemic brain of human central nervous system stem cells grown as neurospheres (hCNS-SCns), the stem cell-secreted factor, human vascular endothelial growth factor (hVEGF), is necessary for cell-induced functional recovery. We correlate this functional recovery to hVEGF-induced effects on the host brain including multiple facets of vascular repair and its unexpected suppression of the inflammatory response. We found that transplanted hCNS-SCns affected multiple parameters in the brain with different kinetics: early improvement in blood-brain barrier integrity and suppression of inflammation was followed by a delayed spatiotemporal regulated increase in neovascularization. These events coincided with a bimodal pattern of functional recovery, with, an early recovery independent of neovascularization, and a delayed hVEGF-dependent recovery coincident with neovascularization. Therefore, cell transplantation therapy offers an exciting multimodal strategy for brain repair in stroke and potentially other disorders with a vascular or inflammatory component.

Subretinal Transplantation of Human Central Nervous System Stem Cells Stimulates Controlled Proliferation of Endogenous Retinal Pigment Epithelium.

PURPOSE: The loss of retinal pigment epithelial (RPE) cells is a feature common to age-related macular degeneration (AMD) and retinitis pigmentosa (RP) and multiple early phase clinical trials are underway testing the safety of RPE cell replacement for these diseases. We examined whether transplantation of human neural stem cells into the subretinal space could enhance the endogenous proliferative capacity of the host RPE cell to regenerate. METHODS: Human central nervous system stem cells (HuCNS-SC) were isolated from enzymatically treated brain tissue using flow cytometry. Pigmented dystrophic Royal College of Surgeons (RCS) and S334ter-4 rats treated with oral bromodeoxyuridine (BrdU) received a unilateral subretinal injection of 1.0 × 105 HuCNS-SC cells at either postnatal day 21 or 60. Animals were sacrificed at 90, 120, and 150 days of age. Eyes were fixed processed for cryostat sectioning. Sections were immunostained with Stem101, Ku80, RPE65, OTX1/2, BrdU, and CRALBP antibodies and analyzed via confocal microscopy. RESULTS: RCS rats that received transplantation of HuCNS-SC had significantly more (approximately 3-fold) Ki67-positive or BrdU-labelled host RPE cells adjacent to the HuCNS-SC graft than controls. Significantly increased host RPE cell proliferation as a result of HuCNS-SC transplantation also was confirmed in S334ter-line 4 transgenic rats with higher proliferation observed in animals with longer posttransplantation periods. CONCLUSIONS: These results suggest that controlled proliferation of endogenous RPE by HuCNS-SC may provide another mechanism by which RPE cell diseases could be treated. TRANSLATIONAL RELEVANCE: Engaging the capacity for endogenous RPE cell regeneration in atrophic diseases may be a novel therapeutic strategy for degenerative diseases of the RPE and retina.

Long-Term Safety, Immunologic Response, and Imaging Outcomes following Neural Stem Cell Transplantation for Pelizaeus-Merzbacher Disease.

Four boys with Pelizaeus-Merzbacher disease, an X-linked leukodystrophy, underwent transplantation with human allogeneic central nervous system stem cells (HuCNS-SC). Subsequently, all subjects were followed for an additional 4 years in this separate follow-up study to evaluate safety, neurologic function, magnetic resonance imaging (MRI) data, and immunologic response. The neurosurgical procedure, immunosuppression, and HuCNS-SC transplantation were well tolerated and all four subjects were alive at the conclusion of the study period. At year 2, all subjects exhibited diffusion MRI changes at the implantation sites as well as in more distant brain regions. There were persistent, increased signal changes in the three patients who were studied up to year 5. Two of four subjects developed donor-specific HLA alloantibodies, demonstrating that neural stem cells can elicit an immune response when injected into the CNS, and suggesting the importance of monitoring immunologic parameters and identifying markers of engraftment in future studies.

Central nervous system therapy for lysosomal storage disorders.

Most lysosomal storage disorders are characterized by progressive central nervous system impairment, with or without systemic involvement. Affected individuals have an array of symptoms related to brain dysfunction, the most devastating of which is neurodegeneration following a period of normal development. The blood-brain barrier has represented a significant impediment to developing therapeutic approaches to treat brain disease, but novel approaches-including enzyme replacement, small-molecule, gene, and cell-based therapies-have given children afflicted by these conditions and those who care for them hope for the future.

Cellular therapy for childhood neurodegenerative disease. Part I: rationale and preclinical studies.

Successful cellular replacement in the diseased human central nervous system (CNS) faces numerous hurdles. In this first installment of a 2-part review, the authors report on the preclinical challenges involved in preparing for a major Phase I trial investigating the safety of human neural stem cell transplantation in a lysosomal storage disorder. Specifically, they discuss choice of the ideal disease for treatment, best donor cell type and source for implantation, the in vitro and in vivo methods used to estimate safety and efficacy, the challenges to noninvasive tracking of cells after transplantation, and the unique issues related to the immunology of CNS cellular transplantation.

Cellular therapy for childhood neurodegenerative disease. Part II: clinical trial design and implementation.

Cellular replacement therapy attempts to improve functioning of the diseased human central nervous system (CNS). In this second installment of a 2-part review, the authors discuss the major challenges to the translation of in vitro and animal studies of neural stem cell (NSC) therapy in the clinical setting. This analysis details the problems unique to the design of clinical trials using human NSCs, outlines patient selection practices, describes surgical techniques for cellular transplantation, and reviews the regulatory issues and ethical concerns in trials involving neurologically impaired children.

Congenital glioblastoma multiforme: case report and review of the literature.

Congenital glioblastoma multiforme is a rare primary brain tumor that has a unique biology distinct from pediatric and adult variants. In this report, we present a case of congenital glioblastoma with complicated management course. A literature review of previously reported cases is included to illustrate the epidemiology and natural history of this disease. A 9-month-old male infant developed acute lethargy, hemiparesis and unilaterally dilated pupil. Imaging studies revealed a large hemispheric tumor, resulting in significant midline shift suggestive of impending herniation. Emergent tumor cystic fluid drainage was performed at initial presentation. A frontotemporoparietal craniotomy was performed on the following day to attempt a gross total resection. Adjuvant chemotherapy consisting of oral temozolomide was administered. The patient eventually succumbed 4 months later due to aggressive tumor progression. Congenital glioblastoma should be included in the differential diagnosis of infants with large intracranial tumors. Although surgical intervention may increase survival, the overall outcome remains poor despite maximal multimodal treatment.

Emerging Safety of Intramedullary Transplantation of Human Neural Stem Cells in Chronic Cervical and Thoracic Spinal Cord Injury.

BACKGROUND: Human central nervous system stem cells (HuCNS-SC) are multipotent adult stem cells with successful engraftment, migration, and region-appropriate differentiation after spinal cord injury (SCI). OBJECTIVE: To present data on the surgical safety profile and feasibility of multiple intramedullary perilesional injections of HuCNS-SC after SCI. METHODS: Intramedullary free-hand (manual) transplantation of HuCNS-SC cells was performed in subjects with thoracic (n = 12) and cervical (n = 17) complete and sensory incomplete chronic traumatic SCI. RESULTS: Intramedullary stem cell transplantation needle times in the thoracic cohort (20 M HuCNS-SC) were 19:30 min and total injection time was 42:15 min. The cervical cohort I (n = 6), demonstrated that escalating doses of HuCNS-SC up to 40 M range were well tolerated. In cohort II (40 M, n = 11), the intramedullary stem cell transplantation needle times and total injection time was 26:05 ± 1:08 and 58:14 ± 4:06 min, respectively. In the first year after injection, there were 4 serious adverse events in 4 of the 12 thoracic subjects and 15 serious adverse events in 9 of the 17 cervical patients. No safety concerns were considered related to the cells or the manual intramedullary injection. Cervical magnetic resonance images demonstrated mild increased T2 signal change in 8 of 17 transplanted subjects without motor decrements or emerging neuropathic pain. All T2 signal change resolved by 6 to 12 mo post-transplant. CONCLUSION: A total cell dose of 20 M cells via 4 and up to 40 M cells via 8 perilesional intramedullary injections after thoracic and cervical SCI respectively proved safe and feasible using a manual injection technique.

Glutaric acidemia type I: a neurosurgical perspective. Report of two cases.

Glutaric acidemia type I (GA-I) is a rare, autosomal recessive metabolic disorder that leads to severe dystonia, basal ganglia degeneration, and bilaterally enlarged anterior middle cranial fossae. The current management of this disease includes early diagnosis with newborn screening, prevention of catabolism, carnitine supplementation, and a strict dietary protein restriction. Neurosurgical evaluation and intervention may be necessary in patients with structural lesions associated with this disease. In this report, the authors present two pediatric patients with GA-I and discuss the neurosurgical aspects of this rare medical disorder.

Profile of daily life in children with brain tumors: an assessment of health-related quality of life.

PURPOSE: The survival of children with CNS tumors approaches 70%, yet health-related quality of life (HRQOL) has not been investigated rigorously in this population. We aimed to show that universal assessment of HRQOL could be obtained easily by using the PedsQL 4.0 and to provide a composite profile of their daily lives. PATIENTS AND METHODS: The PedsQL was administered to all patients seen in the neuro-oncology clinic at Lucile Packard Children's Hospital (Palo Alto, CA) from December 2001, to September 2002. Patients were compared with healthy controls by using two-sided t tests to evaluate statistically significant differences. RESULTS: One hundred thirty-four patients (73 male; mean age +/- standard deviation, 11.8 +/- 5.4 years; 55 had low-grade glioma, 32 had medulloblastoma/primitive neuroectodermal tumor/embryonal tumor, 17 had malignant astrocytoma, nine had germ-cell tumor, and 21 had other types of tumors) were assessed, each in less than 20 minutes. Scores on both child and parent-proxy reports for the total HRQOL, psychosocial, physical, emotional, social, and school-functioning scales were all significantly lower than controls (P < .01). Patients with low-grade glioma were reported to have the highest total HRQOL. Children receiving radiation therapy (XRT) but no chemotherapy had significantly lower total, psychosocial, emotional, and social functioning than those receiving other treatments, including XRT plus chemotherapy. CONCLUSION: The PedsQL can be used to assess HRQOL rapidly and easily in children with CNS tumors, who have significantly worse HRQOL than healthy children. Children receiving XRT fare worse overall; chemotherapy added to XRT does not seem to worsen HRQOL. Assessment of HRQOL should be included as an outcome in future clinical trials.

Identification of phenotypic neural stem cells in a pediatric astroblastoma.

OBJECT: The goal of this study was to illustrate the findings of a significant subpopulation of cells within a pediatric astroblastoma that have the specific cell surface phenotype found on known human neural stem cells. METHODS: Cells with a cell surface marker profile characteristic of human neural stem cells were isolated using fluorescence-activated cell sorting from a mostly nonmitotic astroblastoma removed from the brain of an 11-year-old girl. An unusually high proportion (24%) of the cells were CD133 positive and CD24, CD34, and CD45 negative (CD133(+)CD24(-)CD34(-)CD45(-) cells), the phenotypic antigenic pattern associated with neural stem cells; very few CD133-positive cells were not also CD24, CD34, and CD45 negative. Some cells (12%) were CD34 positive, indicating the presence within the tumor of hematopoietic stem cells. Cells formed cytospheres that resembled neurospheres when seeded into stem cell media and coexpressed beta-tubulin and glial fibrillary acidic protein (GFAP) but did not express the oligodendrocyte marker O4. Cell proliferation was demonstrated by incorporation of bromodeoxyuridine. The cells lost their capacity for self-renewal in vitro after four to six passages, although they continued to coexpress beta-tubulin and GFAP. The cells did not differentiate into neurons or astrocytes when placed in differentiation medium. CONCLUSIONS: Although this astroblastoma contained a high proportion of phenotypic neural stemlike cells, the cells had limited proliferative capacity and multipotency. Their role in astroblastoma formation and growth is unknown.

Progress toward automated metabolic profiling of human serum: comparison of CPMG and gradient-filtered NMR analytical methods.

The investigation of drug delivery and metabolism requires the analysis of molecules in complicated biological matrices such as human serum. In NMR-based metabonomic analysis, T(2) relaxation editing with a CPMG filter is commonly used to suppress background signals from proteins and other endogenous components. Radio frequency pulse imperfections and incomplete irradiation across the spectral bandwidth can cause phase and baseline distortions in CPMG spectra. These distortions are exacerbated by water suppression techniques. Baseline correction methods included in commercially available data processing software packages may be incapable of producing artifact-free spectra. To increase the analytical precision of metabolic profiling, one NMR spectroscopist may be responsible for manually phasing and baseline correcting hundreds of spectra individually to remove operator-dependent variations, significantly reducing throughput. For metabonomic analysis of human serum, it was observed that the application of a pulsed field gradient filter produced (1)H NMR spectra well suited to automatic phasing routines. Superior baseline characteristics, an increased tolerance to radio frequency pulse imperfections, and improved water suppression were achieved. A concomitant reduction in signal intensity compared with the CPMG method was easily recovered by increasing the number of scans. Principal component analysis (PCA) of spectra, acquired under a variety of experimental conditions, revealed the improved reproducibility and robustness of (1)H NMR pulsed field gradient-filtered metabonomic analyses of serum compared to the CPMG method.

Radiation therapy for intracranial germ cell tumors.

PURPOSE: To review the combined experiences of University of California, San Francisco, and Stanford University Medical Center in the treatment of intracranial germ cell tumors (GCT) and to assess the impact of craniospinal radiation (CSI) on patterns of relapse, progression-free survival (PFS), and overall survival (OS). PATIENTS AND METHODS: Ninety-three patients received radiation for newly diagnosed intracranial GCTs, including 49 germinomas, 16 nongerminomatous GCTs (NGGCT), and 28 with no biopsy. Median follow-up for surviving patients was 4.5 years (range 0.25-34). Tests for variables correlating with OS and PFS were conducted using Cox proportional hazards model. RESULTS: Five-year PFS and OS rates were 60% +/- 15% and 68% +/- 14% for patients with NGGCT and 88% +/- 5% and 93% +/- 4% for those with germinoma. Of 6 patients with localized NGGCT who did not receive CSI, 1 experienced an isolated spinal recurrence but was salvaged. Of 41 patients with localized germinoma, 6 who received CSI and 35 who did not, no isolated spinal cord relapses occurred. Twenty-one patients with localized germinoma received neither CSI nor whole brain radiation. Of these, none of 18 with ventricular radiation relapsed. One of 3 patients with primary tumor radiation relapsed intracranially but had only received 11 Gy at initial treatment. On multivariate analysis, germinoma histology but not CSI correlated with improved PFS and OS. CONCLUSION: CSI is not indicated in the treatment of localized germinomas. For patients with localized germinomas treated with radiation alone, we recommend ventricular irradiation followed by primary tumor boost to a total of 45-50 Gy.

Downregulation of cerebrospinal fluid production in patients with chronic hydrocephalus.

OBJECT: The goal of this study was to determine the effect of hydrocephalus on cerebrospinal fluid (CSF) production rates in patients with acute and chronic hydrocephalus. METHODS: The authors studied CSF production both in patients presenting with acute and chronic hydrocephalus, and patients with Parkinson disease (PD) of a similar mean age, whose CSF production was known to be normal. A modification of the Masserman method was used to measure CSF production through a ventricular catheter. The CSF production rates (means +/- standard deviations) in the three groups were then compared. The patients with PD had a mean CSF production rate of 0.42 +/- 0.13 ml/minute; this value lies within the normal range measured using this technique. Patients with acute hydrocephalus had a similar CSF production rate of 0.4 +/- 0.13 ml/minute, whereas patients with chronic hydrocephalus had a significantly decreased mean CSF production rate of 0.25 +/- 0.08 ml/minute. CONCLUSIONS: The authors postulate that chronic increased intracranial pressure causes downregulation of CSF production.

Clinical translation of human neural stem cells.

Human neural stem cell transplants have potential as therapeutic candidates to treat a vast number of disorders of the central nervous system (CNS). StemCells, Inc. has purified human neural stem cells and developed culture conditions for expansion and banking that preserve their unique biological properties. The biological activity of these human central nervous system stem cells (HuCNS-SC®) has been analyzed extensively in vitro and in vivo. When formulated for transplantation, the expanded and cryopreserved banked cells maintain their stem cell phenotype, self-renew and generate mature oligodendrocytes, neurons and astrocytes, cells normally found in the CNS. In this overview, the rationale and supporting data for pursuing neuroprotective strategies and clinical translation in the three components of the CNS (brain, spinal cord and eye) are described. A phase I trial for a rare myelin disorder and phase I/II trial for spinal cord injury are providing intriguing data relevant to the biological properties of neural stem cells, and the early clinical outcomes compel further development.

Phagocytosis of photoreceptor outer segments by transplanted human neural stem cells as a neuroprotective mechanism in retinal degeneration.

PURPOSE: Transplantation of human central nervous system stem cells (HuCNS-SC) into the subretinal space of Royal College of Surgeons (RCS) rats preserves photoreceptors and visual function. To explore possible mechanism(s) of action underlying this neuroprotective effect, we performed a detailed morphologic and ultrastructure analysis of HuCNS-SC transplanted retinas. METHODS: The HuCNS-SC were transplanted into the subretinal space of RCS rats. Histologic examination of the transplanted retinas was performed by light and electron microscopy. Areas of the retina adjacent to HuCNS-SC graft (treated regions) were analyzed and compared to control sections obtained from the same retina, but distant from the transplant site (untreated regions). RESULTS: The HuCNS-SC were detected as a layer of STEM 121 immunopositive cells in the subretinal space. In treated regions, preserved photoreceptor nuclei, as well as inner and outer segments were identified readily. In contrast, classic signs of degeneration were observed in the untreated regions. Interestingly, detailed ultrastructure analysis revealed a striking preservation of the photoreceptor-bipolar-horizontal cell synaptic contacts in the outer plexiform layer (OPL) of treated areas, in stark contrast with untreated areas. Finally, the presence of phagosomes and vesicles exhibiting the lamellar structure of outer segments also was detected within the cytosol of HuCNS-SC, indicating that these cells have phagocytic capacity in vivo. CONCLUSIONS: This study reveals the novel finding that preservation of specialized synaptic contacts between photoreceptors and second order neurons, as well as phagocytosis of photoreceptor outer segments, are potential mechanism(s) of HuCNS-SC transplantation, mediating functional rescue in retinal degeneration.

Central nervous system stem cell transplantation for children with neuronal ceroid lipofuscinosis.

OBJECT: Infantile and late-infantile neuronal ceroid lipofuscinoses (NCLs) are invariably fatal lysosomal storage diseases associated with defects in lysosomal enzyme palmitoyl-protein thioesterase 1 (PPT-1) or tripeptidyl peptidase 1 (TPP1) activity. Previous preclinical studies have demonstrated that human CNS stem cells (HuCNS-SCs) produce both PPT-1 and TPP1 and result in donor cell engraftment and reduced accumulation of storage material in the brain when tested in an NCL mouse model. METHODS: HuCNS-SC transplantation was tested in an open-label dose-escalation Phase I clinical trial as a potential treatment for infantile and late-infantile NCL. Study design included direct neurosurgical transplantation of allogeneic HuCNS-SCs into the cerebral hemispheres and lateral ventricles accompanied by 12 months of immunosuppression. RESULTS: Six children with either the infantile or late-infantile forms of NCL underwent low- (3 patients) and high- (3 patients) dose transplantation of HuCNS-SCs followed by immunosuppression. The surgery, immunosuppression, and cell transplantation were well tolerated. Adverse events following transplantation were consistent with the underlying disease, and none were directly attributed to the donor cells. Observations regarding efficacy of the intervention were limited by the enrollment criteria requiring that patients be in advanced stages of disease. CONCLUSIONS: This study represents the first-in-human clinical trial involving transplantation of a purified population of human neural stem cells for a neurodegenerative disorder. The feasibility of this approach and absence of transplantation-related serious adverse events support further exploration of HuCNS-SC transplantation as a potential treatment for select subtypes of NCL, and possibly for other neurodegenerative disorders.

Neural stem cell engraftment and myelination in the human brain.

Pelizaeus-Merzbacher disease (PMD) is a rare leukodystrophy caused by mutation of the proteolipid protein 1 gene. Defective oligodendrocytes in PMD fail to myelinate axons, causing global neurological dysfunction. Human central nervous system stem cells (HuCNS-SCs) can develop into oligodendrocytes and confer structurally normal myelin when transplanted into a hypomyelinating mouse model. A 1-year, open-label phase-1 study was undertaken to evaluate safety and to detect evidence of myelin formation after HuCNS-SC transplantation. Allogeneic HuCNS-SCs were surgically implanted into the frontal lobe white matter in four male subjects with an early-onset severe form of PMD. Immunosuppression was administered for 9 months. Serial neurological evaluations, developmental assessments, and cranial magnetic resonance imaging (MRI) and MR spectroscopy, including high-angular resolution diffusion tensor imaging (DTI), were performed at baseline and after transplantation. The neurosurgical procedure, immunosuppression regimen, and HuCNS-SC transplantation were well tolerated. Modest gains in neurological function were observed in three of the four subjects. No clinical or radiological adverse effects were directly attributed to the donor cells. Reduced T1 and T2 relaxation times were observed in the regions of transplantation 9 months after the procedure in the three subjects. Normalized DTI showed increasing fractional anisotropy and reduced radial diffusivity, consistent with myelination, in the region of transplantation compared to control white matter regions remote to the transplant sites. These phase 1 findings indicate a favorable safety profile for HuCNS-SCs in subjects with PMD. The MRI results suggest durable cell engraftment and donor-derived myelin in the transplanted host white matter.

Resolution of syringomyelia after release of tethered cord.

BACKGROUND: Syringomyelia is an abnormal cystic dilatation of the spinal cord caused by excessive accumulation of CSF. Patients can develop various neurologic deficits secondary to untreated syringomyelia, some of which can be permanent despite surgical intervention. CASE DESCRIPTION: The authors present a patient with syringomyelia, aortic coarctation, and tethered cord syndrome. Serial radiographic imaging demonstrated initial significant reduction of the thoracic syrinx after coarctation repair and release of tethered cord. However, subsequent follow-up imaging revealed partial recurrence. CONCLUSION: This case provides evidence of a possible cause-effect relationship between syringomyelia and tethered cord. It demonstrates the indication of surveillance imaging of the entire spine to ensure that all potential etiologies of syringomyelia are identified and treated. Furthermore, it illustrates the complex dynamic nature of syrinx physiology and reinforces the importance of serial follow-up studies after surgical intervention.