Blastula-stage stem cells can differentiate into dopaminergic and serotonergic neurons after transplantation (original) (raw)
Related papers
Proceedings of the National Academy of Sciences, 2002
behavioral restoration of DA-mediated motor asymmetry. Behavioral recovery paralleled in vivo positron emission tomography and functional magnetic resonance imaging data demonstrating DA-mediated hemodynamic changes in the striatum and associated brain circuitry. These results demonstrate that transplanted ES cells can develop spontaneously into DA neurons. Such DA neurons can restore cerebral function and behavior in an animal model of Parkinson's disease. P arkinson's disease (PD) is a degenerative disorder characterized by a loss of midbrain dopamine (DA) neurons with a subsequent reduction in striatal DA (1). Pharmacological treatment with L-DOPA works initially, but reduced efficacy and development of motor complications requires treatment alternatives such as deep brain stimulation and fetal DA neuron transplantation (2). There is evidence both from animal models and clinical investigations showing that fetal DA neurons can produce symptomatic relief (3-6). Technical and ethical difficulties in obtaining sufficient and appropriate donor fetal brain tissue have limited the application of this new therapy.
Experimental Neurology, 2002
The ability to differentiate neural stem cells (NSCs) into dopamine neurons is fundamental to their role in cell replacement therapies for neurodegenerative disorders such as Parkinson's disease. We show here that when a clonal line (C17.2) of undifferentiated NSCs is transplanted into the intact or 6-hydroxydopaminelesioned striatum, cells withdraw from the cell cycle (BrdU ؊), migrate extensively in the host striatum, and express markers associated with neuronal (-tubulin III ؉ , NSE ؉ , NeuN ؉) but not glial (GFAP ؊ , MBP ؊ , A2B5 ؊) differentiation. Importantly, by 2-5 weeks postgrafting, in the majority of these transplants, nearly all engrafted cells express the dopamine-synthesizing enzymes tyrosine hydroxylase and aromatic L-amino decarboxylase, sometimes resulting in changes in motor behavior. In contrast, no NSCs stain for dopamine-hydroxylase, choline acetyltransferase, glutamic acid decarboxylase, or serotonin. We conclude that, following transplantation into the intact or 6-hydroxydopamine-lesioned rat, the adult brain contains intrinsic cues sufficient to direct the specific expression of dopaminergic traits in immature multipotential neural stem cells.
Isolation and transplantation of dopaminergic neurons generated from mouse embryonic stem cells
Neuroscience Letters, 2004
Embryonic stem (ES) cells differentiate into dopamine (DA)-producing neurons when co-cultured with PA6 stromal cells, but the resulting cultures contain a variety of unidentified cells. In order to label live DA neurons in mixed populations, we introduced a GFP reporter under the control of the tyrosine hydroxylase (TH) gene promoter into ES cells. GFP expression was observed in TH-immunoreactive cells that differentiated from the ES cells that carried the TH-GFPreporter gene. DA neurons expressing GFP were sorted from the mixed cell population by fluorescence-activated cell sorting of cells exhibiting GFP fluorescence, and the sorted GFP þ cells obtained were transplanted into a rat model of Parkinson's disease. Some of these cells survived and innervated the host striatum, resulting in a partial recovery from parkinsonian behavioral defects. This strategy of isolation and transplantation of ES-cell-derived DA neurons should be useful for cellular and molecular studies of DA neurons and for clinical application in the treatment of Parkinson's disease.
Stem Cells, 2004
The use of human embryonic stem cells (hESCs) as a source of dopaminergic neurons for Parkinson's disease cell therapy will require the development of simple and reliable cell differentiation protocols. The use of cell cocultures, added extracellular signaling factors, or transgenic approaches to drive hESC differentiation could lead to additional regulatory as well as cell production delays for these therapies. Because the neuronal cell lineage seems to require limited or no signaling for its formation, we tested the ability of hESCs to differentiate to form dopamine-producing neurons in a simple serum-free suspension culture system. BG01 and BG03 hESCs were differentiated as suspension aggregates, and neural progenitors and neurons were detecz after 2–4 weeks. Plated neurons responded appropriately to electrophysiological cues. This differentiation was inhibited by early exposure to bone morphogenic protein (BMP)-4, but a pulse of BMP-4 from days 5 to 9 caused induction of peripheral neuronal differentiation. Real-time polymerase chain reaction and whole-mount immunocytochemistry demonstrated the expression of multiple markers of the midbrain dopaminergic phenotype in serum-free differentiations. Neurons expressing tyrosine hydroxylase (TH) were killed by 6-hydroxydopamine (6-OHDA), a neurotoxic catecholamine. Upon plating, these cells released dopamine and other catecholamines in response to K+ depolarization. Surviving TH+ neurons, derived from the cells differentiated in serum-free suspension cultures, were detected 8 weeks after transplantation into 6-OHDA–lesioned rat brains. This work suggests that hESCs can differentiate in simple serum-free suspension cultures to produce the large number of cells required for transplantation studies.
Stem Cells, 2004
Objective. Transplantation of fetal mesencephalic cells into the striatum has been performed in about 350 patients with Parkinson's disease and has been intensively studied in rat models of Parkinson's disease. Limited access to this material has shifted the focus toward embryonic stem (ES) cells. The grafting of undifferentiated ES cells to 6-hydroxy-dopamine (6-OHDA)-lesioned rats leads to behavioral improvements but may induce teratoma-like structures. This risk might be avoided by using more differentiated ES cells. In this study, we aimed to investigate differentiated mouse ES cells regarding their in vivo development and fate after transplantation in the striatum in the 6-OHDA rat model and the behavioral changes induced after transplantation. Methods. Mouse ES cells were differentiated on PA6 feeder cells for 14 days before grafting. Twenty to twenty-five percent of the neurons obtained were positive for tyrosine-hydroxylase (TH). PKH26-labeled cells were transplanted in the striata of unilaterally 6-OHDA-lesioned rats. Results. Direct PKH26 fluorescence visualization and TH staining proved the existence of cell deposits in the striata of all grafted animals, indicating cell survival for at least 5 weeks posttransplantation. There was no evidence of tumor formation. Immunocytochemical staining showed glial immunoreactivity surrounding the grafted cell deposits, probably inhibiting axonal outgrowth into the surrounding host tissue. There was a significant reduction in amphetamine-induced rotational behavior seen in grafted animals, which was not observed in sham-operated animals. Conclusions. The findings of this study suggest that the amphetamine-induced rotational behavioral test without histological confirmation is not proof of morphological integration with axonal outgrowth within the first 4 weeks posttransplantation. Stem Cells 2004;22:396-404
Neuroscience, 1993
Abatraet-An important issue in clinical neural grafting is whether a second instriatial allograft can survive well in a patient who has received an allograft before. In this study, the survival, i~uno~ni~ity and function of intrastriatal grafts of allogeneic or syngeneic embryonic dopa~ne-~ch tissue in rats which had previously received either an intrastriatal allo-or syn-graft or sham injections were examined. The first graft tissue was taken from inbred Lewis or Sprague-Dawley rat embryos and grafted into an intact straitum of adult Sprague-Dawley rats subjected to a unilateral 6-hydroxydopamine lesion on the contralateral side. Eight weeks after the first transplantation, either allogeneic or syngeneic tissue was grafted as dissociated tissue into the dopamine depleted striatum. The function of the second grafts was assessed by rotational asymmetry at two different time points, i.e. eight and I4 weeks after the second transplantation. There were significant reductions of rotational asymmetry in all groups over time, but no significant dilTerence between groups. Tyrosine hydroxylase immunocytochemistry was used to assess dopamine cell survival and graft size. Statistical analysis revealed no significant difference in the mean number of tyrosine hydroxylase immunoreactive cells or the mean volume of the second grafts placed on the right side (lesioned side) between groups. Monoclonal antibodies were used to evaluate cellular immune reactions and the major hist~ompati~i~y complex class I and class II expression in and around grafts. No major ~istocom~tibi~ty complex class I expression was seen in any of the graft ~mbinations.
Neuroscience Letters, 2005
Usefulness of the in vitro and in vivo generation of neural precursors from embryonic stem (ES) cells has been widely discussed, but functional recovery in animal models of Parkinson's disease (PD) is not fully understood. The aim of this study was to investigate a transplantation strategy for PD by assessing whether double-transplants in the striatum (ST) and substantia nigra (SN), or ST and subthalamic nucleus (STN) induce functional recovery in 6-hydroxydopamine-lesioned rats. Methamphetamine-induced rotation was significantly reduced by transplantation of mouse ES cell-derived neurons into the ST, but not the STN or SN alone. Double-transplantation was also effective at recovering rotational behavior. Although immunoreactivity for tyrosine hydroxylase (TH) was almost completely lost in the ipsilateral striatum in hemiparkinsonian rats, TH immunoreactivity was detected in transplanted cells and sprouting fibers in the ST, STN and SN. These results suggest that both the involvement of ST as a place of transplantation and the number of ES cell-derived neurons are essential factors for efficacy on hemiparkinsonian behaviors.
Specific Reinnervation of Lesioned Mouse Striatum by Grafted Mesencephalic Dopaminergic Neurons
European Journal of Neuroscience, 1991
Selective lesions of the dopaminergic nigrostriatal system and embryonic neuron grafts were used to study the mechanism by which exogenous neurons can restore transmitter function and to examine CNS development and plasticity. C57BL mice treated with acetaldehyde/l-methyI-4-phenyl-l,2,3,6-tetrahydropyridine show irreversible loss of substantia nigra dopaminergic neurons. Implants of embryonic mesencephalic dopaminergic neurons functionally reinnervate the striatum and form a dense network of fibres;-20% of the implanted dopaminergic cells survive for several months. However, dopaminergic fibre outgrowth and mesencephalic graft development appear lower in control, non-lesioned, animals. Moreover, implants of embryonic hypothalamic dopaminergic neurons show little or no survival. These results indicate that interactions between embryonic and adult neurons are selective. We suggest that this specificity may be sustained by the action of still unknown trophic andlor tropic factors, possibly produced by the lesioned striatum and by putative inhibitory mechanisms of cell migration and neuritic outgrowth. DA, occurring 2-3 months after MPTP treatment (Chiueh et al.,
Characterization and transplantation of two neuronal cell lines with dopaminergic properties
Neurochemical Research, 1996
Immortalized rat mesencephalic cells (1RB3AN27) produced dopamine (DA) at a level that was higher than produced by undifferentiated or differentiated murine neuroblastoma cells (NBP2) in culture. Treatment of 1RB3AN27 and NBP2 cells with a cAMP stimulating agent increased tyrosine hydroxylase (TH) activity and the intensity of immunostaining for the DA transporter protein (DAT). 1RB3AN27 cells were labelled with primary antibodies to neuron specific enolase (NSE) and nestin and exhibited very little or no labeling with anti-glial fibrillary acidic protein (GFAP). 1RB3AN27 cells exhibited [3-and c~-adrenoreceptors, and prostaglandin El receptors, all of which were linked to adenylate cyclase (AC). Dopamine receptor (D~) and cholinergic muscarinic receptors linked to AC were not detectable. The levels of PKCa and PKC[3 isoforms were higher than those of PKC~ and PKC~ in 1RB3ANz7 cells. The 1RB3AN:7 cells were more effective in reducing the rate of methamphetamine-induced turning in rats with unilateral 6-OHDA lesion of the nigrostriatal system than differentiated NBP 2 cells. The grafted lRB3AN27 were viable as determined by DiI labelling, but they did not divide and did not produce T-antigen protein; however, when these grafted cells were cultured in vitro, they resumed production of T-antigen and proliferated after the primary glia cells and neurons of host brain died due to maturation and subsequent degeneration. Examination of H&E stained sections of the grafted sites revealed no evidence of infiltration of inflammatory cells in the grafted area suggesting that these cells were not immunogenic. They also did not form tumors.