Human foetal brain tissue as quality control when developing stem cells towards cell replacement therapy for neurological diseases.

Human foetal brain tissue has been used in experimental and clinical trials to develop cell replacement therapy in neurodegenerative disorders such as Parkinson's disease and Huntington's disease. These pioneering clinical studies have shown proof of principle that cell replacement therapy can be effective and is worthwhile to develop as a therapeutic strategy for repairing the damaged brain. However, because of the limited availability of foetal brain material, and difficulties in producing standardized and quality-tested cell preparations from this source, there have been extensive efforts in investigating the potential use of alternative cell sources for generating a large number of transplantable, authentic neural progenitors and neurons. In this review, we highlight the value of using human foetal tissue as a reference material for quality control of acquired cell fate of in vitro generated neurons before and after transplantation.

Increased cell suspension concentration augments the survival rate of grafted tyrosine hydroxylase immunoreactive neurons.

The poor survival rate (5-20%) of grafted embryonic dopamine (DA) neurons is one of the primary factors preventing cell replacement from becoming a viable treatment for Parkinson's disease. Previous studies have demonstrated that graft volume impacts grafted DA neuron survival, indicating that transplant parameters influence survival rates. However, the effects of mesencephalic cell concentration on grafted DA neuron survival have not been investigated. The current study compares the survival rates of DA neurons in grafts of varying concentrations. Mesencephalic cell suspensions derived from E14 Fisher 344 rat pups were concentrated to 25,000, 50,000, 100,000 and 200,000 cells/microl and transplanted into two 0.5 microl sites in the 6-OHDA-denervated rat striatum. Animals were sacrificed 10 days and 6 weeks post-transplantation for histochemical analysis of striatal grafts. The absolute number of DA neurons per graft increased proportionally to the total number of cells transplanted. However, our results show that the 200,000 cells/microl group exhibited significantly higher survival rates (5.48+/-0.83%) compared to the 25,000 cells/microl (2.81+/-0.39%) and 50,000 cells/microl (3.36+/-0.51%) groups (p=0.02 and 0.03, respectively). Soma size of grafted DA neurons in the 200,000 cells/microl group was significantly larger than that of the 25,000 cells/microl (p<0.0001) and 50,000 cells/microl groups (p=0.004). In conclusion, increasing the concentration of mesencephalic cells prior to transplantation, augments the survival and functionality of grafted DA neurons. These data have the potential to identify optimal transplantation parameters that can be applied to procedures utilizing stem cells, neural progenitors, and primary mesencephalic cells.

Optimizing the transplant dose of a human neuronal cell line graft to treat SCI pain in the rat.

Neuropathic pain is a prevalent and difficult problem in the setting of spinal cord injury (SCI). The use of cellular transplant therapy to treat this pain has been successful with the use of a human neuronal cell line, hNT2.17 [M.J. Eaton, S.Q. Wolfe, M.A. Martinez, M. Hernandez, C. Furst, J. Huang, B.R. Frydel, O. Gomez-Marin, Subarachnoid transplant of a human neuronal cell line attenuates chronic allodynia and hyperalgesia after excitotoxic SCI in the rat, J. Pain 8 (2007) 33-50]. Intrathecal transplant of these cells potently reverses behavioral hypersensitivity after excitotoxic spinal cord injury in the rat model. This study focuses on delineating the optimal dose of these cell grafts in the same model. Two weeks after intraspinal injection of quisqualic acid (QUIS) with subsequent behavioral hypersensitivity, terminally differentiated hNT2.17 cells were transplanted into 300 g Wistar-Furth rats in a logarithmic variation of doses: 10(6), 10(5) and 10(3) cells. Behavioral hypersensitivity testing was performed weekly for 6 weeks following transplant. The dose of 10(6) cells (or approximately 3 million/kg) potently and permanently reversed both cutaneous allodynia (CA) and thermal hyperalgesia (TH). Reduced transplant doses of the hNT2.17 cell line did not permanently reverse behavioral hypersensitivity, suggesting that there is an optimal dose that can be used as a clinical tool to treat SCI-associated neuropathic pain.

Human adult olfactory neuroepithelial derived progenitors retain telomerase activity and lack apoptotic activity.

Olfactory epithelium (OE) contains a population of progenitors responsible for its life-long regenerative capacity. Procedures for the isolation of these progenitors have been established [F.J. Roisen, K.M. Klueber, C.L. Lu, L.M. Hatcher, A. Dozier, C.B. Shields, Adult human olfactory stem cells, Brain Res., 890 (2001) 11-12.] and over 40 patient-specific cell lines from adult postmortem OE and endoscopic biopsy from patients undergoing nasal sinus surgery have been obtained. As these cells emerged in primary cultures, they formed neurospheres (NSFCs). The purpose of the present study was to further characterize these adult human olfactory-derived progenitors. Subcultures of the NSFCs have been passaged nearly 200 times, with a mitotic cycle of 18-20 h. Telomerase activity remains in stem cells; therefore, ELISA was employed to determine the telomerase activity of different lines and passages. Since progenitors undergo low levels of apoptosis, the levels of apoptosis were also examined in these populations. The levels of telomerase and apoptotic activity in 12 NSFC lines remained relatively constant irrespective of donor age, culture duration, or sex. To further study the apoptotic characteristics of the NSFCs, nine different caspases (cysteine proteases) known to be critical in apoptosis were evaluated using gene-microarrays comparing cells from a single line at passages 14, 88, and 183. No increases were found in caspase activity in all passages studied. ELISA confirmed the absence of caspase activity over the entire range of passages. This study further suggests that NSFCs can be obtained and used from patients, irrespective of age, sex, or time in culture without altered viability expanding the potential utility of these cells for autologous transplantation and possible diagnostic testing.

Towards a definition of recovery of function.

In this review we consider recovery of function after spinal cord injury, and, in particular, recovery improved following intraspinal cellular transplants. Some recovery occurs spontaneously and this can be especially dramatic in neonates, supporting the notion that developing and adult spinal cord respond differently to injury. Recovery can be improved in both neonates and adults by appropriate cellular transplants into the injury site. We describe several functional tests used in animals with spinal lesions and transplants. We compare the effects of transplants of fetal tissue and genetically modified fibroblasts into neonatal and adult injury sites on recovery of motor and sensorimotor function. Fetal tissue transplants support greater recovery and elicit more regeneration in neonates than in adults. Transplants of fibroblasts modified to produce neurotrophic factors however support both recovery and axonal growth even in adults. The contribution of the transplant to recovery is shown by the loss of function that follows a second lesion just rostral to the original lesion/transplant site. The effect of the re-lesion indicates that the recovery is mediated by the presence of the transplant but the way in which transplants act to promote recovery may include a number of mechanisms, including regeneration and sprouting, neuroprotection, and modifications of organization of spared CNS structures.

[Results and outlooks of using cell technologies in the treatment of neurological diseases]. / Itogi i perspektivy ispol'zovaniia kletochnykh tekhnologii v lechenii nevrologicheskikh zabolevanii.

An attempt was undertaken in the last decade of the 20th century to use a principally new approach to the treatment of neurological diseases--cell therapy. Main efforts were focused on developing a method related with replacement of neurons dying in neurodegenerative pathology, primarily, in Parkinson disease (PD). Outlined below are the key elements of the technology:--ensuring, in experiment, of a prolonged therapeutic effect in transplantation, to the affected part, first of embryonic neurons of the animal of the same species (allografting) and then of homologous embryonic neurons of man (heterografting);--obtaining, standardization and preparation (for transplantation) of embryonic nervous tissue of man; transplantation of embryonic nervous tissue of man to the brain of patient and evaluation, in situ, of the functional activity of its neurons; and evaluation of the therapeutic effect of grafting. Cell suspension of meseencephalon of 6-9 week human fetus containing around 10% of differentiating dopaminergic neurons was used for grafting in PD. Embryonic dopaminergic neurons, administered stereotactically into the striatum of patient, established synaptic links with neurons of the recipient, which was accompanied by the onset of synthesis and reverse uptake of dopamine (DA) as well as by the onset of spontaneous and stimulated release of DA. Neurografting ensured a temporary improvement of the condition in a part of PD patients but did not cure them. Moreover, such positive therapeutic effect was registered only in patients with the akineticorigid but not trembling variation of the disease. Hence, although there was a certain progress in clinical neurografting, the approach cannot be now recommended for introduction in neurology and neurosurgery. The limited therapeutic effect of the treatment method is primarily explained by a low rate of survival of transplanted dopaminergic neurons and, consequently, by the persisting DA deficit in patient's body. Therefore, the outlooks for perfecting the cell technology are related with increasing the survival rate of implanted dopaminergic neurons and with stimulating the innervation of target neurons in patient's striatum as well as with using the neural (glia) and non-neural (fibroblasts, myoblasts) cells with modified gene and stem cells. Finally, despite a certain progress of advancing the cell technology in neurology the approach still needs more research, which would enable further clinical trials.

Stereotactic MR imaging for planning neural transplantation: a reliable technique at 3 Tesla?

The purpose of this study was to assess the accuracy of high field (3 Tesla) MR in target localization for stem cell transplantation. Three patients with Huntington's disease were imaged with a stereotactic frame in place for both MRI and CT. Quality assurance procedures and manual shimming were performed before each MRI study to minimize image distortion. The images were fused using multi-modality rigid body image registration software. Image fusion demonstrated the MR images to be in agreement with CT to within 1.5 mm, as assessed by measuring the coordinates of markers on the frame and on the shape and size of the lateral ventricles. Target coordinates for transplantation were selected from the MR images. Postoperative imaging confirmed accurate graft placement.

Fetal porcine ventral mesencephalon graft. Determination of the optimal gestational age for implantation in parkinsonian patients.

Human fetal ventral mesencephalon tissue has been used as dopaminergic striatal implants in Parkinsonian patients, so far with variable effects. Fetuses from animals that breed in large litters, e.g., pigs, have been considered as alternative donors of dopaminergic tissue. The optimal gestational age of the porcine fetal donors has not been studied systematically. We collected ventral mesencephalic (VM) tissue from fetal pigs, embryonal ages E21, E28, E42, and E70, and examined the viability of the fetal VM cells after dissociation, the expression of tyrosine hydroxylase (TH) in culture, the presence of catecholamines, and the cellular survival and outgrowth up to 10 months after intrastriatal implantation in rats. The highest viability was found in suspensions prepared from E28 fetuses. The highest number of TH-positive cells was found in cell cultures prepared from E28 VM tissue. Explants with a gestational age of 28 and 42 days contained the largest amount of dopamine. Only E28-derived grafts showed TH-cell survival after implantation in rat striatum. Our results show that a gestational age of 28 days must be considered to be the optimal age for dopaminergic tissue derived from pig fetuses for therapeutic use as intrastriatal grafts in Parkinsonian patients.

Ethical issues in neurografting of human embryonic cells.

During the last decade neurotransplantation has developed into a technique with the possible potential to repair damaged or degenerating human brain. Effective neurotransplantation has so far been based on the use of fetal brain tissue derived from aborted embryos or fetuses. The ethical issues related to this new therapeutic approach therefore not only concern the possible adverse side effects for a neural graft-receiving patient, but also the relationship between the requirements for fetal tissue and the decision-making process for induced abortion. Although for decades human embryos and fetuses have been the subject of biomedical studies, and, in principle, their use has therefore not been seen as ethically objectionable, the above points made it necessary to reconsider the moral issues. The present paper points out several of these issues, both from the donor and acceptor (patient) point of view. The conclusion is that under a series of restrictions intended to prevent the use of grafts from encouraging induced abortions and to maintain high standards of respect for life and human dignity, neurotransplantation using embryonic or fetal brain tissue parts cannot be rejected on moral grounds.

Intracerebral transplantation of fetal ventral mesencephalon for patients with advanced Parkinson's disease. Methodology and 6-month to 1-year follow-up in 3 patients.

In order to launch a new transplantation program for Parkinson's disease (PD), we evaluated the safety and efficacy of fetal ventral mesencephalic grafts in 3 patients with advanced PD. Inclusion criteria and clinical evaluation followed strictly the Core Assessment Program for Intracerebral Transplantation. The transplantation procedure was based on the technique previously described by the groups in Lund (Sweden) and Créteil (France). The putamen contralateral to the site of predominant symptoms was unilaterally grafted in all patients. Magnetic resonance (MR)-based stereotactic guidance with multiplanar correlation was used to define 3 implantation trajectories in the precommissural, commissural, and postcommissural putamen. Fetal ventral mesencephalon was prepared from 6- to 8-week-old human embryos obtained from same-day abortions. Under general anesthesia, 8 deposits of 3 microliters of the fetal tissue were placed 1 mm apart along each implantation trajectory using a customized microsyringe and needle attached to the stereotactic frame. The patients recovered uneventfully from the neurosurgical procedure. Early postoperative MR clearly showed the implantation trajectories reaching the putamen in all patients. The follow-up period was of 12, 9 and 6 months, for each of the 3 patients, respectively. Clinical changes appeared between 3 and 6 months after transplantation and consisted of an increase in the 'on' periods and in quantitative bilateral improvement in the motor timed tests. There was an improvement of the Unified Parkinson's Disease Rating Scale score and an improvement of rigidity. Tremor was unchanged, and there was a slight and transient increase in dyskinesias. Neuropsychological follow-up revealed slight frontal alterations in 2 patients. Positron emission tomography demonstrated an increase of 18F-fluorodopa uptake in the grafted site. Adverse events include a reversible Cushing syndrome secondary to immunosuppression in 1 patient and a transient episode of confusion in another. The results of this study, designed as a prerequisite for a wider transplantation program, are in accordance with those previously reported by others and show that, using standardized neurosurgical techniques and methods of evaluation, transplantation is a reproducible and safe therapeutic approach which provides clinical benefits to patients with advanced PD.