Thomas B. Freeman

Adult Bone Marrow Stromal Cells Differentiate into Neural Cells in Vitro

Bone marrow stromal cells (BMSC) normally give rise to bone, cartilage, and mesenchymal cells. Recently, bone marrow cells have been shown to have the capacity to differentiate into myocytes, hepatocytes, and glial cells. We now demonstrate that human and mouse BMSC can be induced to differentiate into neural cells under experimental cell culture conditions. BMSC cultured in the presence of EGF or BDNF expressed the protein and mRNA for nestin, a marker of neural precursors. These cultures also expressed glial fibrillary acidic protein (GFAP) and neuron-specific nuclear protein (NeuN). When labeled human or mouse BMSC were cultured with rat fetal mesencephalic or striatal cells, a small proportion of BMSC-derived cells differentiated into neuron-like cells expressing NeuN and glial cells expressing GFAP.

A Double-blind Controlled Trial of Bilateral Fetal Nigral Transplantation in Parkinson's Disease

Thirty‐four patients with advanced Parkinsons disease participated in a prospective 24‐month double‐blind, placebo‐controlled trial of fetal nigral transplantation. Patients were randomized to receive bilateral transplantation with one or four donors per side or a placebo procedure. The primary end point was change between baseline and final visits in motor component of the Unified Parkinsons Disease Rating Scale in the practically defined off state. There was no significant overall treatment effect (p = 0.244). Patients in the placebo and one‐donor groups deteriorated by 9.4 ± 4.25 and 3.5 ± 4.23 points, respectively, whereas those in the four‐donor group improved by 0.72 ± 4.05 points. Pairwise comparisons were not significant, although the four‐donor versus placebo groups yielded a p value of 0.096. Stratification based on disease severity showed a treatment effect in milder patients (p = 0.006). Striatal fluorodopa uptake was significantly increased after transplantation in both groups and robust survival of dopamine neurons was observed at postmortem examination. Fifty‐six percent of transplanted patients developed dyskinesia that persisted after overnight withdrawal of dopaminergic medication (“off”‐medication dyskinesia). Fetal nigral transplantation currently cannot be recommended as a therapy for PD based on these results.Ann Neurol 2003;54:403–414

Lewy body–like pathology in long-term embryonic nigral transplants in Parkinson's disease

Fourteen years after transplantation into the striatum of an individual with Parkinsons disease, grafted nigral neurons were found to have Lewy body–like inclusions that stained positively for α-synuclein and ubiquitin and to have reduced immunostaining for dopamine transporter. These pathological changes suggest that Parkinsons disease is an ongoing process that can affect grafted cells in the striatum in a manner similar to host dopamine neurons in the substantia nigra. These findings have implications for cell-based therapies and for understanding the cause of Parkinsons disease.

Neuropathological Evidence of Graft Survival and Striatal Reinnervation after the Transplantation of Fetal Mesencephalic Tissue in a Patient with Parkinson's Disease

BACKGROUND Trials are under way to determine whether fetal nigral grafts can improve motor function in patients with Parkinsons disease. Some studies use fluorodopa uptake on positron-emission tomography (PET) as a marker of graft viability, but fluorodopa uptake does not distinguish between host and grafted neurons. There has been no direct evidence that grafts of fetal tissue can survive and innervate the striatum. METHODS We studied a 59-year-old man with advanced Parkinsons disease who received bilateral grafts of fetal ventral mesencephalic tissue in the postcommissural putamen. The tissue came from seven embryos between 6 1/2 and 9 weeks after conception. The patient died 18 months later from a massive pulmonary embolism. The brain was studied with the use of tyrosine hydroxylase immunohistochemical methods. RESULTS After transplantation, the patient had sustained improvement in motor function and a progressive increase in fluorodopa uptake in the putamen on PET scanning. On examination of the brain, each of the large grafts appeared to be viable. Each was integrated into the host striatum and contained dense clusters of dopaminergic neurons. Processes from these neurons had grown out of the grafts and provided extensive dopaminergic reinnervation to the striatum in a patch-matrix pattern. Ungrafted regions of the putamen showed sparse dopaminergic innervation. We could not identify any sprouting of host dopaminergic processes. CONCLUSIONS Grafts of fetal mesencephalic tissue can survive for a long period in the human brain and restore dopaminergic innervation to the striatum in patients with Parkinsons disease. In the patient we studied, clinical improvement and enhanced fluorodopa with uptake on PET scanning were associated the survival of the grafts and dopaminergic reinnervation of the striatum.

Oligodendrocyte dysfunction in schizophrenia and bipolar disorder

BACKGROUND Results of array studies have suggested abnormalities in expression of lipid and myelin-related genes in schizophrenia. Here, we investigated oligodendrocyte-specific and myelination-associated gene expression in schizophrenia and bipolar affective disorder. METHODS We used samples from the Stanley brain collection, consisting of 15 schizophrenia, 15 bipolar affective disorder, and 15 control brains. Indexing-based differential display PCR was done to screen for differences in gene expression in schizophrenia patients versus controls. Results were cross-validated with quantitative PCR, which was also used to investigate expression profiles of 16 other oligodendrocyte and myelin genes in schizophrenia and bipolar disorder. These genes were further investigated with an ongoing microarray analysis. FINDINGS Results of differential display and quantitative PCR analysis showed a reduction of key oligodendrocyte-related and myelin-related genes in schizophrenia and bipolar patients; expression changes for both disorders showed a high degree of overlap. Microarray results of the same genes investigated by quantitative PCR correlated well overall. INTERPRETATION Schizophrenia and bipolar brains showed downregulation of key oligodendrocyte and myelination genes, including transcription factors that regulate these genes, compared with control brains. These results lend support to and extend observations from other microarray investigations. Our study also showed similar expression changes to the schizophrenia group in bipolar brains, which thus lends support to the notion that the disorders share common causative and pathophysiological pathways.

Mitochondrial dysfunction in schizophrenia: evidence for compromised brain metabolism and oxidative stress.

The etiology and pathophysiology of schizophrenia remain unknown. A parallel transcriptomics, proteomics and metabolomics approach was employed on human brain tissue to explore the molecular disease signatures. Almost half the altered proteins identified by proteomics were associated with mitochondrial function and oxidative stress responses. This was mirrored by transcriptional and metabolite perturbations. Cluster analysis of transcriptional alterations showed that genes related to energy metabolism and oxidative stress differentiated almost 90% of schizophrenia patients from controls, while confounding drug effects could be ruled out. We propose that oxidative stress and the ensuing cellular adaptations are linked to the schizophrenia disease process and hope that this new disease concept may advance the approach to treatment, diagnosis and disease prevention of schizophrenia and related syndromes.

Functional fetal nigral grafts in a patient with Parkinson's disease: Chemoanatomic, ultrastructural, and metabolic studies

A patient with Parkinsons disease received bilateral fetal human nigral implants from six donors aged 6.5 to 9 weeks post‐conception. Eighteen months following a post‐operative clinical course characterized by marked improvement in clinical function, this patient died from events unrelated to the grafting procedure. Post‐mortem histological analyses revealed the presence of viable grafts in all 12 implant sites, each containing a heterogeneous population of neurons and glia. Approximately 210,146 implanted tyrosine hydroxylase‐immunoreactive (TH‐ir) neurons were found. A greater number of TH‐ir grafted neurons were observed in the right (128,162) than the left (81,905) putamen. Grafted TH‐ir neurons were organized in an organotypic fashion. These cells provided extensive TH‐ir and dopamine transporter‐ir innervation to the host striatum which occurred in a patch‐matrix fashion. Quantitative evaluations revealed that fetal nigral grafts reinnervated 53% and 28% of the post‐commissural putamen on the right and left side, respectively. Grafts on the left side innervated a lesser area of the striatum, but optical density measurements were similar on both sides. There was no evidence that the implants induced sprouting of host TH‐ir systems. Electron microscopic analyses revealed axo‐dendritic and occasional axo‐axonic synapses between graft and host. In contrast, axo‐somatic synapses were not observed. In situ hybridization for TH mRNA revealed intensely hybridized grafted neurons which far exceeded TH mRNA expression within residual host nigral cells. In addition, γ‐amino butyric acid (GABA)‐ergic neurons were observed within the graft that formed a dense local neuropil which was confined to the implant site. Serotonergic neurons were not observed within the graft. Cytochrome oxidase activity was increased bilaterally within the grafted post‐commissural putamen, suggesting increased metabolic activity. In this regard, a doubling of cytochrome oxidase activity was observed within the grafted post‐commissural putamen bilaterally relative to the non‐grafted anterior putamen. The grafts were hypovascular relative to the surrounding striatum and host substantia nigra. Blood vessels within the graft stained intensely for GLUT‐1, suggesting that this marker of blood‐brain barrier function is present within human nigral allografts. Taken together, these data indicate that fetal nigral neurons can survive transplantation, functionally reinnervate the host putamen, establish synaptic contacts with host neurons, and sustain many of the morphological and functional characteristics of normal nigral neurons following grafting into a patient with PD.

Bilateral human fetal striatal transplantation in Huntington’s disease

BackgroundTransplanted striatal cells have been demonstrated to survive, grow, establish afferent and efferent connections, and improve behavioral signs in animal models of Huntington’s disease (HD). ObjectiveTo evaluate feasibility and safety and to provide preliminary information regarding the efficacy of bilateral human fetal striatal transplantation in HD. MethodsSeven symptomatic patients with genetically confirmed HD underwent bilateral stereotactic transplantation of two to eight fetal striata per side in two staged procedures. Tissue was dissected from the lateral half of the lateral ventricular eminence of donors 8 to 9 weeks postconception. Subjects received cyclosporine for 6 months. ResultsThree subjects developed subdural hemorrhages (SDHs) and two required surgical drainage. One subject died 18 months after surgery from probable cardiac arrhythmia secondary to severe atherosclerotic cardiac disease. Autopsy demonstrated clearly demarcated grafts of typical developing striatal morphology, with host-derived dopaminergic fibers extending into the grafts and no evidence of immune rejection. Other adverse events were generally mild and transient. Mean Unified HD Rating Scale (UHDRS) motor scores were 32.9 ± 6.2 at baseline and 29.7 ± 7.5 12 months after surgery (p = 0.24). Post-hoc analysis, excluding one subject who experienced cognitive and motor deterioration after the development of symptomatic bilateral SDHs, found that UHDRS motor scores were 33.8 ± 6.2 at baseline and 27.5 ± 5.2 at 12 months (p = 0.03). ConclusionsTransplantation of human fetal striatal cells is feasible and survival of transplanted cells was demonstrated. Patients with moderately advanced HD are at risk for SDH after transplantation surgery.

Use of placebo surgery in controlled trials of a cellular-based therapy for Parkinson's disease.

Surgical procedures are frequently introduced into general practice on the basis of uncontrolled studies that are less rigorous than those required for the approval of medical interventions.1 The s...

Transplanted dopaminergic neurons develop PD pathologic changes: A second case report

This report describes pathological changes within the grafted neurons of another patient with Parkinsons disease (PD) who died 14 years posttransplantation. Although numerous healthy appearing grafted neurons were present at this long‐term time point, some displayed Lewy bodies as evidenced by alpha‐synuclein, ubiquitin, and thioflavin‐S staining. Additionally, there was a general loss of dopamine transporter‐immunoreactivity in grafted neurons. Some grafted cell displayed a loss of tyrosine hydroxylase. These data support the emerging concept that PD‐like pathology is seen in young grafted neurons when they survive long term.