Neural progenitor NT2N cell lines from teratocarcinoma for transplantation therapy in stroke

https://doi.org/10.1016/j.pneurobio.2008.04.005Get rights and content

Abstract

This review article discusses recent progress on the use of teratocarcinoma-derived Ntera2/D1 neuron-like cells (NT2N cells, also called hNT cells) as graft source for cell transplantation in stroke. Laboratory evidence has demonstrated the therapeutic potential of NT2N cells in stroke therapy. Phase I and II clinical trials have shown the cells’ feasibility, safety and tolerability profiles in stroke patients. Despite these novel features of NT2N cells, the transplantation regimen remains to be optimized. Moreover, determining the mechanisms underlying the grafts’ beneficial effects, specifically demonstrating functional synaptic connections between host brain and NT2N cell grafts, warrants further examination. The major limiting factor for initiating a large clinical trial is the cells’ highly potent proliferative property due to their cancerous origin, thereby raising the concern that these cells may revert to a neoplastic state over time after transplantation. To this end, we explored a proof-of-concept “retroviral” strategy to further establish the post-mitotic status of NT2N cells by transfecting these cells with the transcription factor Nurr1, in addition to the standard treatment with retinoic acid and mitotic inhibitors. This new cell line NT2N.Nurr1 displays an expedited neuronal commitment and secretes a high level of the neurotrophic factor glial cell line-derived neurotrophic factor (GDNF), and when transplanted into the rodent stroke brain expressed neuronal phenotype and reduced behavioral impairments which are comparable, if not more robust, than those produced by NT2N cells. Such highly potent neuronal lineage commitment and neurotrophic factor secretory function of NT2.Nurr1 cells make them an appealing graft source for transplantation therapy.

Introduction

The “non-regenerative central nervous system” dogma is arguably a historical past. Accumulating scientific evidence has demonstrated that diseased or aging brain cells can potentially be rescued and have their functions restored. Although endogenous cell repair mechanisms alone may not fully reverse the disease or aging process, augmenting the host restorative potential with exogenous cell therapy may improve the neuroprotective or neuroregenerative outcome (Borlongan et al., 2000a, Borlongan et al., 2006, Kondziolka et al., 2002, Lo et al., 2005, Fisher and Henninger, 2007, Bliss et al., 2007, Chopp et al., 2007, Borlongan and Hess, 2008). Indeed, neural transplantation has shown therapeutic benefits in animal models of neurological disorders, with limited clinical trials underway in Parkinson's disease (Lindvall and Hagell, 2001, Kordower et al., 1996), Huntington's disease (Freeman et al., 2000) and stroke (Kondziolka et al., 2005, Kondziolka et al., 2006). The use of neuroteratocarcinoma cells for transplantation therapy in stroke is one such approach that has transitioned from the laboratory into the clinic (Kondziolka et al., 2000, Kondziolka et al., 2005, Nelson et al., 2002).

The target patient population for cell transplantation has been chronically ill patients, thus it is not surprising that stroke patients have been a focus of attention for this therapy. Stroke is the third leading cause of death and affects over a half-million people each year in the United States alone, with about 3 million stroke survivors suffering from significant cognitive and functional disabilities (Kondziolka et al., 2002, Lo et al., 2005, Fisher and Henninger, 2007, Bliss et al., 2007, Chopp et al., 2007, Borlongan and Hess, 2008). Rehabilitation therapy has helped some stroke survivors recover, but many patients, however, still experience permanent loss of independent function. The cost for rehabilitation and lost wages is estimated at $30 billion each year and thus represents a significant financial impact on society (Kondziolka et al., 2002, Lo et al., 2005, Fisher and Henninger, 2007, Bliss et al., 2007, Chopp et al., 2007, Borlongan and Hess, 2008). Current stroke treatments are typically limited to supportive care and secondary stroke prevention, resulting in only limited improvement in cognitive and motor function. Although more than 300 experimental stroke treatments have reached clinical trials, to date only intravenous tissue plasminogen activator (tPA) administration has been effective in ameliorating the neurological deficits arising from acute stroke (The National Institute of Neurological Disorders and Stroke rt-PA Stroke Study Group, 1995). However, tPA treatment remains problematic because of its extremely limited window of efficacy, which should be administered within 3 h of stroke onset, thereby only benefiting less than 3% of ischemic stroke patients (Kondziolka et al., 2002, Lo et al., 2005, Fisher and Henninger, 2007, Bliss et al., 2007, Chopp et al., 2007, Borlongan and Hess, 2008). These statistics project a significant unmet clinical need that warrants investigations of novel therapies, such as neural transplantation, for treating stroke (Fig. 1).

The laboratory studies on NT2/NT2N transplantation focus on two rodent stroke models, namely the proximal middle cerebral artery occlusion (MCAO) model which produces primarily striatal infarcts with portions of the motor cortex also damaged, and the distal middle cerebral artery ligation (MCAl) which causes highly localized cortical damage without affecting the striatum. The recapitulation of the human condition by animal stroke model remains a challenge in laboratory research. Human stroke is heterogenous in nature. At best, the MCAO is deemed as a good model for subcortical infarcts in humans, while the MCAl more closely resembles cortical infarcts seen in the clinic. However, these pathological correlates of the stroke model in humans may not completely mimic the behavioral deficits seen in stroke patients, in that while motor abnormalities are detected in both stroke models, cognitive deficits, especially the complex tasks shown to be impaired in the clinic, remain to be fully characterized in stroke animals. Accordingly, much laboratory work is required to improve existing stroke animal models, and warrants the need to develop new ones (e.g., femtosecond laser-induced “heterogenous” ischemia; Nishimura et al., 2006) in order to closely mimic the human condition that will allow better evaluation of experimental therapeutics for stroke.

In this review paper, we discuss mostly preclinical and clinical transplantation studies on stroke, but also highlight relevant reports from other CNS disorders (e.g., Parkinson's disease, Huntington's disease) in order to provide a general perspective on the status of NT2/NT2N cells and similar neural progenitor cells for treating brain disorders (Table 1).

Section snippets

Cell transplantation and brain disorders

Cell transplantation for brain disorders has evolved primarily as a strategy to replace dead or dying cells as a result of aging or disease-related injury. An equally logical rationale in the pursuit of cell transplantation is to deliver exogenous proteins into the central nervous system (CNS), since cell death is associated with alterations in CNS protein levels. Accordingly, finding a transplantable and transfectable cell type that has the potential to become neurons and to replace injured

Turning teratocarcinomas into neurons

Teratocarcinoma-derived cells have been suggested as a neural progenitor cell line. The process of generating neurons from teratocarcinomas involves treatment with retinoic acid (RA) and mitotic inhibitors. In particular, an embryonal carcinoma cell line (NT2 cells) derived from a human teratocarcinoma can be induced to differentiate into post-mitotic neuron-like cells referred to as NT2N neurons (Trojanowski et al., 1997, Kleppner et al., 1995, Miyazono et al., 1995, Miyazono et al., 1996).

Mitotic NT2 cell grafts into rodent brains

Survival, proliferation, and differentiation of parent NT2 cells are affected by the host microenvironment as revealed by transplantation studies that entail grafting of these cells into different regions of the normal, i.e., non-injured brains of sub-acute combined immunodeficient (SCID) mice and nude mice (Kleppner et al., 1995, Miyazono et al., 1995, Miyazono et al., 1996). The anatomical site into which the NT2 cells were implanted significantly influenced the neoplastic state of NT2 cells.

Cell dose and survival rate of graft influence therapeutic efficacy

Intracerebral transplantation of NT2N cells in the rat brain at 1 month after an experimental stroke ameliorated ischemia-induced behavioral dysfunctions as early as 1 month post-transplantation (Borlongan et al., 1998a, Borlongan et al., 1998b, Borlongan et al., 1998c). The reversal of motor abnormalities in stroke rats produced by NT2N cell grafts was significantly greater than the behavioral recovery seen with transplantation of fetal striatal cells. The pre-transplantation viability and

Clinical trials of NT2N cell grafts in stroke patients

The Food and Drug Administration approved Phase I clinical trials of transplantation of NT2N neurons to evaluate this therapy in the treatment of patients with stable stroke. NT2N cells were transplanted into patients with basal ganglia stroke and fixed motor deficits, including 12 patients aged 44–75 years with an infarct of 6 months to 6 years who were stable for at least 2 months (Kondziolka et al., 2000). Serial evaluations at 12–18 months showed no adverse cell-related serologic or

Genetic engineering of NT2N cells

Laboratory studies and the two clinical trials to date have shown maintenance of post-mitotic status and absence of tumorgenecity in the transplanted NT2N cells in stroke animal models and patients. However, the possibility that NT2N cells may revert to their neoplastic state has been raised because whereas the NT2N differentiation process to achieve their neuronal phenotype commitment involves exogenous signals (RA and mitotic inhibitors), their “genotype” is one that likely retains a

NT2N.Nurr1: a new NT2N sister cell line

Nurr1 is a transcription factor that belongs to the orphan nuclear receptor superfamily of transcription factors that is highly expressed in midbrain dopaminergic neurons and has been implicated in the development and maintenance of the dopaminergic system (Jankovic et al., 2005). Moreover, Nurr1-overexpressing embryonic stem (ES) cells could be differentiated into dopaminergic neurons with high proportion (Chung et al., 2002). In our recent study, Nurr1 gene was transduced into NT2 cells with

Overall conclusions

In view of accumulating evidence and limited clinical trials providing support for stem/progenitor cell therapy for stroke, this review paper offers experimental and clinical guidance on the use of human neuroteratocarcinoma NT2N cell transplantation that may be extrapolated to other stem and progenitor cells now in the pipeline for clinical trials in stroke. Our call is to continuously evaluate in the laboratory the safety, tolerability, toxicity and efficacy of these stem/progenitor cells.

References (122)

  • P. Gortz et al.

    Neuronal network properties of human teratocarcinoma cell line-derived neurons

    Brain Res.

    (2004)
  • H. Hida et al.

    Dopamine-denervation enhances the trophic activity in striatum: evaluation by morphological and electrophysiological development in PC12D cells

    Neurosci. Res.

    (1997)
  • W.D. Hill et al.

    The NF-kappaB inhibitor diethyldithiocarbamate (DDTC) increases brain cell death in a transient middle cerebral artery occlusion model of ischemia

    Brain Res. Bull.

    (2001)
  • J. Jankovic et al.

    The role of Nurr1 in the development of dopaminergic neurons and Parkinson's disease

    Prog. Neurobiol.

    (2005)
  • P. Kofler et al.

    Liposome-mediated gene transfer into established CNS cell lines, primary glial cells, and in vivo

    Cell Transplant.

    (1998)
  • D. Kondziolka et al.

    Neural transplantation for stroke

    J. Clin. Neurosci.

    (2002)
  • V.M. Lee et al.

    Neurobiology of human neurons (NT2N) grafted into mouse spinal cord: implications for improving therapy of spinal cord injury

    Prog. Brain Res.

    (2000)
  • Y. Li et al.

    Cell proliferation and differentiation from ependymal, subependymal and choroid plexus cells in response to stroke in rats

    J. Neurol. Sci.

    (2002)
  • M. Lu et al.

    Global test statistics for treatment effect of stroke and traumatic brain injury in rats with administration of bone marrow stromal cells

    J. Neurosci. Methods

    (2003)
  • P. Lu et al.

    Neural stem cell constitutively secrete neurotrophic factors and promote extensive host axonal growth after spinal cord injury

    Exp. Neurol.

    (2003)
  • T. Matsuoka et al.

    The GABAA receptor is expressed in human neurons derived from a teratocarcinoma cell line

    Biochem. Biophys. Res. Commun.

    (1997)
  • P. McCaffery et al.

    Regulation of retinoic acid signaling in the embryonic nervous system: a master differentiation factor

    Cytokine Growth Factor Rev.

    (2000)
  • I.E. Misiuta et al.

    The transcription factor Nurr1 in human NT2 cells and hNT neurons

    Brain Res. Dev. Brain Res.

    (2003)
  • M. Modo et al.

    Transplantation of neural stem cells in a rat model of stroke: assessment of short-term graft survival and acute host immunological response

    Brain Res.

    (2002)
  • K. Nakajima et al.

    GDNF is a major component of trophic activity in DA-depleted striatum for survival and neurite extension of DAergic neurons

    Brain Res.

    (2001)
  • P.T. Nelson et al.

    Clonal human (hNT) neuron grafts for stroke therapy: neuropathology in a patient 27 months after implantation

    Am. J. Path.

    (2002)
  • H. Nishino et al.

    Restoration of function by neural transplantation in the ischemic brain

    Prog. Brain Res.

    (2000)
  • S. Saporta et al.

    Neural transplantation of human neuroteratocarcinoma (hNT) neurons into ischemic rats. A quantitative dose-response analysis of cell survival and behavioral recovery

    Neuroscience

    (1999)
  • Y. Shimano et al.

    Tissue extract from dopamine-depleted striatum enhances differentiation of cultured striatal type-1 astrocytes

    Neurosci. Lett.

    (1996)
  • M. Bauer et al.

    Nonviral glial cell-derived neurotrophic factor gene transfer enhances survival of cultured dopaminergic neurons and improves their function after transplantation in a rat model of Parkinson's disease

    Hum. Gene. Ther.

    (2000)
  • S. Behrstock et al.

    Human neural progenitors deliver glial cell line-derived neurotrophic factor to parkinsonian rodents and aged primates

    Gene. Ther.

    (2006)
  • T. Bliss et al.

    Cell transplantation therapy for stroke

    Stroke

    (2007)
  • T.M. Bliss et al.

    Transplantation of hNT neurons into the ischemic cortex: cell survival and effect on sensorimotor behavior

    J. Neurosci. Res.

    (2006)
  • C.V. Borlongan et al.

    Viability and survival of hNT neurons determine degree of functional recovery in grafted ischemic rats

    Neuroreport

    (1998)
  • C.V. Borlongan et al.

    Cerebral ischemia and CNS transplantation: differential effects of grafted fetal rat striatal cells and human neurons derived from a clonal cell line

    Neuroreport

    (1998)
  • C.V. Borlongan et al.

    Neural transplantation for neurodegenerative disorders

    Lancet

    (1999)
  • C.V. Borlongan

    Motor activity-mediated partial recovery in ischemic rats

    Neuroreport

    (2000)
  • C.V. Borlongan et al.

    Glial cell survival is enhanced during melatonin-induced neuroprotection against cerebral ischemia

    FASEB J.

    (2000)
  • Borlongan, C.V., Isacson, O., Sanberg, P.R., 2000b. Immunosuppressant Analogs for Neuroprotection. New Jersey: Human...
  • C.V. Borlongan et al.

    Intracerebral transplantation of porcine choroid plexus provides structural and functional neuroprotection in a rodent model of stroke

    Stroke

    (2004)
  • C.V. Borlongan et al.

    Central nervous system entry of peripherally injected umbilical cord blood cells is not required for neuroprotection in stroke

    Stroke

    (2004)
  • C.V. Borlongan et al.

    Gene therapy, cell transplantation and stroke

    Front Biosci.

    (2006)
  • C.V. Borlongan et al.

    Stem cells and neurological diseases

    Cell Prolif.

    (2008)
  • P. Brundin et al.

    Transplanted dopaminergic neurons: more of less?

    Nat. Med.

    (2001)
  • R.R. Buchner et al.

    Anti-human kappa opioid receptor antibodies: characterization of site-directed neutralizing antibodies specific for a peptide kappa R(33–52) derived from the predicted amino terminal region of the human kappa receptor

    J. Immunol.

    (1997)
  • Y.J. Cao et al.

    Hypoxia-inducible transgene expression in differentiated human NT2N neurons—a cell culture model for gene therapy of postischemic neuronal loss

    Gene Ther.

    (2001)
  • T. Carlsson et al.

    Serotonin neuron transplants exacerbate L-DOPA-induced dyskinesias in a rat model of Parkinson's disease

    J. Neurosci.

    (2007)
  • J.E. Carroll et al.

    Is nuclear factor-kappaB a good treatment target in brain ischemia/reperfusion injury?

    Neuroreport

    (2000)
  • J. Chen et al.

    Therapeutic benefit of intravenous administration of bone marrow stromal cells after cerebral ischemia in rats

    Stroke

    (2001)
  • M. Chopp et al.

    Neurogenesis, angiogenesis, and MRI indices of functional recovery from stroke

    Stroke

    (2007)
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