Karim Mukhida

Dopamine neurons implanted into people with Parkinson's disease survive without pathology for 14 years

Postmortem analysis of five subjects with Parkinsons disease 9–14 years after transplantation of fetal midbrain cell suspensions revealed surviving grafts that included dopamine and serotonin neurons without pathology. These findings are important for the understanding of the etiopathogenesis of midbrain dopamine neuron degeneration and future use of cell replacement therapies.

GDNF therapy for Parkinson’s disease

With an increase in the aging population, the incidence of Parkinson’s disease (PD), a disabling neurodegenerative disorder mainly affecting motor function, will inevitably present a challenge to an already overburdened healthcare system. Current medical and surgical therapies offer symptomatic relief but do not provide a cure. Experimental studies suggest that GDNF has the ability to protect degenerating dopamine neurons in PD as well as promote regeneration of the nigrostriatal dopamine system. However, clinical trials of GDNF infusion to date remain inconclusive. This review will examine the experimental and clinical evidence of GDNF use in PD with particular focus on its potential as an effective therapy in the treatment of PD.

Spinal GABAergic transplants attenuate mechanical allodynia in a rat model of neuropathic pain.

Injury to the spinal cord or peripheral nerves can lead to the development of allodynia due to the loss of inhibitory tone involved in spinal sensory function. The potential of intraspinal transplants of GABAergic cells to restore inhibitory tone and thus decrease pain behaviors in a rat model of neuropathic pain was investigated. Allodynia of the left hind paw was induced in rats by unilateral L5– 6 spinal nerve root ligation. Mechanical sensitivity was assessed using von Frey filaments. Postinjury, transgenic fetal green fluorescent protein mouse GABAergic cells or human neural precursor cells (HNPCs) expanded in suspension bioreactors and differentiated into a GABAergic phenotype were transplanted into the spinal cord. Control rats received undifferentiated HNPCs or cell suspension medium only. Animals that received either fetal mouse GABAergic cell or differentiated GABAergic HNPC intraspinal transplants demonstrated a significant increase in paw withdrawal thresholds at 1 week post‐transplantation that was sustained for 6 weeks. Transplanted fetal mouse GABAergic cells demonstrated immunoreactivity for glutamic acid decarboxylase and GABA that colocalized with green fluorescent protein. Intraspinally transplanted differentiated GABAergic HNPCs demonstrated immunoreactivity for GABA and β‐III tubulin. In contrast, intraspinal transplantation of undifferentiated HNPCs, which predominantly differentiated into astrocytes, or cell suspension medium did not affect any behavioral recovery. Intraspinally transplanted GABAergic cells can reduce allodynia in a rat model of neuropathic pain. In addition, HNPCs expanded in a standardized fashion in suspension bioreactors and differentiated into a GABAergic phenotype may be an alternative to fetal cells for cell‐based therapies to treat chronic pain syndromes.

Erythropoietin and GDNF enhance ventral mesencephalic fiber outgrowth and capillary proliferation following neural transplantation in a rodent model of Parkinson's disease.

Low dopaminergic cell survival and suboptimal fiber reinnervation are likely major contributing factors for the limited benefits of neural transplantation in Parkinsons disease (PD) patients. Glial cell lined‐derived neurotrophic factor (GDNF) has been shown to enhance dopaminergic cell survival and fiber outgrowth of the graft site as well as promote behavioral recovery in rodent models of PD, while erythropoietin (EPO) can produce dopaminergic neuroprotective effects against 6‐hydroxydopamine (6‐OHDA) exposure on cultured neurons and 1‐methyl‐4‐phenyl‐1,2,3,6‐tetrahydropyridine‐treated mice. The aim of this study was to determine if fetal ventral mesencephalic (FVM) tissue exposed to hibernation media containing a combination of GDNF and EPO could enhance dopaminergic graft survival, striatal reinnervation and functional recovery in a 6‐OHDA rodent model of PD. FVM tissue was dissected from 14‐day‐old rat fetuses and placed for 6 days in hibernation media alone, and in hibernation media that received either a daily administration of GDNF, EPO or a combination of GDNF and EPO. Following hibernation, FVM cells were transplanted as a single cell suspension into the striatum of unilateral 6‐OHDA‐lesioned rats. Rotational behavioral assessment revealed animals that received FVM tissue exposed to GDNF, EPO or the combination of both drugs had accelerated functional recovery. Immunohistochemical and stereological assessment revealed a significant increase in graft fiber density and angiogenesis into the graft when compared with control. These findings suggest that the hibernation of FVM tissue in a combination of GDNF and EPO can enhance graft efficacy and may have important implications for tissue preparation protocols for clinical neural transplantation in PD.

Expansion of Human Neural Precursor Cells in Large-Scale Bioreactors for the Treatment of Neurodegenerative Disorders

The transplantation of in vitro expanded human neural precursor cells (hNPCs) represents a potential new treatment alternative for individuals suffering from incurable neurodegenerative disorders such as Parkinsons disease (PD) and Huntingtons disease (HD). However, in order for cell restorative therapy to have widespread therapeutic significance, it will be necessary to generate unlimited quantities of clinical grade hNPCs in a standardized method. We report here that we have developed a serum‐free medium and scale‐up protocols that allow for the generation of clinical quantities of human telencephalon‐derived hNPCs in 500‐mL computer‐controlled suspension bioreactors. The average hNPC aggregate diameter in the bioreactors was maintained below a target value of 500 μm by controlling the liquid shear field. The human cells, which were inoculated at 105 cells/mL, exhibited a doubling time of 84 h, underwent a 36‐fold expansion over the course of 18 days, and maintained an average viability of over 90%. The bioreactor‐derived hNPCs retained their nestin expression following expansion and were able to differentiate into glial and neuronal phenotypes under defined conditions. It has also been demonstrated that these hNPCs differentiated to a GABAergic phenotype that has recently been shown to be able to restore functional behavior in rat models of HD and neuropathic pain (Mukhida, K. et al. Stem Cells 2007; DOI 10.1634/stemcells.2007–0326). This study demonstrates that clinical quantities of hNPCs can be successfully and reproducibly generated under standardized conditions in computer‐controlled suspension bioreactors.

Bioreactor Expansion of Human Neural Precursor Cells in Serum-Free Media Retains Neurogenic Potential

Human neural precursor cells (hNPCs), harvested from somatic tissue and grown in vitro, may serve as a source of cells for cell replacement strategies aimed at treating neurodegenerative disorders such as Parkinsons disease (PD), Huntingtons disease (HD), and intractable spinal cord pain. A crucial element in a robust clinical production method for hNPCs is a serum‐free growth medium that can support the rapid expansion of cells while retaining their multipotency. Here, we report the development of a cell growth medium (PPRF‐h2) for the expansion of hNPCs, achieving an overall cell‐fold expansion of 1013 over a period of 140 days in stationary culture which is significantly greater than other literature results. More importantly, hNPC expansion could be scaled‐up from stationary culture to suspension bioreactors using this medium. Serial subculturing of the cells in suspension bioreactors resulted in an overall cell‐fold expansion of 7.8 × 1013 after 140 days. These expanded cells maintained their multipotency including the capacity to generate large numbers of neurons (about 60%). In view of our previous studies regarding successful transplantation of the bioreactor‐expanded hNPCs in animal models of neurological disorders, these results have demonstrated that PPRF‐h2 (containing dehydroepiandrosterone, basic fibroblast growth factor and human leukemia inhibitory factor) can successfully facilitate the production of large quantities of hNPCs with potential to be used in the treatment of neurodegenerative disorders. Biotechnol. Bioeng. 2010. 105: 823–833.

Survival, differentiation, and migration of bioreactor- expanded human neural precursor cells in a model of Parkinson disease in rats

OBJECT Fetal tissue transplantation for Parkinson disease (PD) has demonstrated promising results in experimental and clinical studies. However, the widespread clinical application of this therapeutic approach is limited by a lack of fetal tissue. Human neural precursor cells (HNPCs) are attractive candidates for transplantation because of their long-term proliferation activity. Furthermore, these cells can be reproducibly expanded in a standardized fashion in suspension bioreactors. In this study the authors sought to determine whether the survival, differentiation, and migration of HNPCs after transplantation depended on the region of precursor cell origin, intracerebral site of transplantation, and duration of their expansion. METHODS Human neural precursor cells were isolated from the telencephalon, brainstem, ventral mesencephalon, and spinal cord of human fetuses 8-10 weeks of gestational age, and their differentiation potential characterized in vitro. After expansion in suspension bioreactors, the HNPCs were transplanted into the striatum and substantia nigra of parkinsonian rats. Histological analyses were performed 7 weeks posttransplantation. RESULTS The HNPCs isolated from various regions of the neuraxis demonstrated diverse propensities to differentiate into astrocytes and neurons and could all successfully expand under standardized conditions in suspension bioreactors. At 7 weeks posttransplantation, survival and migration were significantly greater for HNPCs obtained from the more rostral brain regions. The HNPCs differentiated predominantly into astrocytes after transplantation into the striatum or substantia nigra regions, and thus no behavioral improvement was observed. CONCLUSIONS Understanding the regional differences in HNPC properties is prerequisite to their application for PD cell restoration strategies.

A multitarget basal ganglia dopaminergic and GABAergic transplantation strategy enhances behavioural recovery in parkinsonian rats

The current transplantation paradigm for Parkinsons disease that places foetal dopaminergic cells in the striatum neither normalizes neuronal activity in basal ganglia structures such as the substantia nigra (SN) and subthalamic nucleus (STN) nor leads to complete functional recovery. It was hypothesized that restoration of parkinsonian deficits requires inhibition of the pathological overactivity of the STN and SN in addition to restoration of dopaminergic activity in the striatum. To achieve inhibition, a multitargeted basal ganglia transplantation strategy using GABAergic cells derived from either foetal striatal primordia (FSP) cells or human neural precursor cells (hNPCs) expanded in suspension bioreactors was investigated. In hemiparkinsonian rats, transplantation of foetal rat dopaminergic cells in the striatum in conjunction with GABAergic grafts in the STN and/or SN promoted significant improvement in forelimb akinesia and motor function compared to transplantation of intrastriatal dopaminergic grafts alone or in conjunction with undifferentiated hNPCs. In culture, FSP cells exhibited neuronal electrophysiological properties. However, recordings from GABAergic hNPCs revealed limited ionic conductances and an inability to fire action potentials. Despite this, they were almost as efficacious as FSP cells in inducing functional recovery following transplantation, suggesting that such recovery may have been mediated by secretion of GABA rather than by functional integration into the host. Thus, restoration of dopaminergic activity to the striatum in concert with inhibition of the STN and SN by GABAergic grafts may be beneficial for improving clinical outcomes in patients with Parkinsons disease and potential clinical application of this strategy may be enhanced by the use of differentiated hNPCs.

A review of bioreactor protocols for human neural precursor cell expansion in preparation for clinical trials.

Tissue-specific human neural precursor cells (hNPCs) can be isolated from various regions of the developing or adult central nervous system and may serve as a viable source of cells in cell replacement therapies for the treatment of neurodegenerative disorders. However, in order for cell replacement strategies to become a routine therapeutic option for the treatment of neurodegenerative disorders, hNPCs should be generated under standardized and controlled conditions. Studies over the last two decades have focused on developing cell growth media and cell handling protocols for expansion and differentiation of hNPCs in culture. Key studies have reported the development of serum-free growth media and large-scale computer-controlled suspension bioreactors that can support high cell proliferation rates (doubling times < 3 days), multipotentiality, and potential neurogenic differentiation (more than 60% neurons). Moreover, bioengineering studies have focused on controlling culture conditions in suspension bioreactors including inoculation, hydrodynamics of culture, oxygen and nutrients transfer to the cells, monitoring in situ physiological parameters using process control techniques, and expansion for extended periods of time. In addition, in vitro and in vivo characterization of hNPCs have been performed, providing information on stem/progenitor cell characteristics, cell surface analysis, and appropriate type of cells to use in transplantation studies.

Pituitary apoplexy following cardiac surgery.

First described in case reports by Bailey in 1898,1 Bleibtreu in 1905,2 and Dingley and Lond in 1932,3 and finally described as a clinical syndrome by Brougham and colleagues in 1950,4 pituitary apoplexy refers to the potentially life-threatening acute hemorrhage or infarction of a pituitary tumour. Its presentation, related to meningeal irritation and compression of the optic chiasm and parasellar structures, is variable5-7 but typically includes the sudden onset of headache, nausea, vomiting, ophthalmoplegia, altered mental status, and hormonal dysfunction.8 Pituitary apoplexy has an incidence of 0.6-16.6% in pituitary adenomas6,9-12 and may occur spontaneously but is also associated with trauma, anticoagulation, radiation therapy, mechanical ventilation, and surgery.13 Pituitary apoplexy after cardiac surgery is an exceptionally rare perioperative complication, and all but one reported case have been managed surgically. Additionally, while spontaneous remission of endocrinopathies following pituitary apoplexy of hormonallyactive tumours is well-described,14-17 resolution of nonfunctioning tumours following apoplexy is rare.18,19 A case is presented in which no surgical management of pituitary apoplexy was required as the apoplectic event led to the spontaneous resolution of a previously undiagnosed nonfunctioning pituitary macroadenoma following cardiac surgery.