| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
|
FJ
EXPRESS SUMMARY ARTICLE The Full-length version of this article is also available, published online June 21, 2005 as doi:10.1096/fj.04-3657fje. |
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
* Laboratory of Clinical Science, National Institute of Mental Health, NIH; and
National Institute of Neurological Disorders and Stroke, NIH, Bethesda, Maryland, USA
1Correspondence: Laboratory of Clinical Science, National Institute of Mental Health, Building 10, Room 3D41, Bethesda, MD 20892-1264, USA. E-mail: renpatr{at}intra.nimh.nih.gov
SPECIFIC AIMS
During mammalian brain development serotonin (5-HT) contributes to neuronal proliferation, migration and differentiation in cortex and other brain regions. In adulthood the brain serotonergic system thus participates in the coordination of complex sensory and motor functions that are associated with different behavioral states and are implicated in multiple neuropsychiatric disorders. The aim of the present study was to propagate serotonergic-like progenitor cells from mouse neural stem cells, to evaluate their properties [e.g., serotonin transporter (SERT) -mediated reuptake activity and receptor-mediated electrophysiological responses], then to implant these progenitor cells into the brains of mice with a targeted disruption of SERT, to investigate whether these implanted progenitors would survive, maintain the same phenotype and express SERT in the adult brain of SERT knockout mice.
PRINCIPAL FINDINGS
1. Differentiation of serotonergic-like progenitors after sonic hedgehog (Shh) and fibroblast growth factor-8 (FGF-8) treatment
We first investigated undifferentiated neural stem cells (NSCs) that were isolated from the midbrain region of embryonic (E14) mice. These NSCs were initially cultured with bFGF and N2 in serum-free knockout DMEM. Shh and FGF-8 were added every day to induce cells to differentiate into serotonergic-like neuronal cells. We studied phenotypical expression in primary spheres, using specific neuronal antibodies. At 2 wk, 95 ± 5% of these cells were stained with Nestin, 15 ± 2% with GFAP, 3 ± 1% with A2B5, and 0.5 ± 0.3% with tyrosine hydroxylase (TH) markers. The cells that had positive staining with 5-HT antibody increased from 0.4 ± 0.2% (2 wk) to 65 ± 6% (8 wk) during the period of differentiation (Fig. 1
A). To select a majority of serotonergic-like progenitor cells, we used a limiting dilution and subcloning culture procedure to isolate these multipotency cells. We found that individual clone cells grew rapidly and exhibited a round shape with a cell size similar to each other, and with the cell numbers increasing without showing any axon extensions. After the clone cells were repassaged to yield consistent cultures, the expanded cells exhibited axonal extensions with intercell connections at 12 wk. We further tested these serotonergic-like progenitor cells electrophysiologically by examining cells patch-clamped at –70 mV. Fast inward currents were induced by both glutamate and GABA, indicating that these serotonergic-like progenitor cells expressed glutamate-sensitive and GABA-sensitive ion channels with features of a pluripotent capacity during their differentiated stage. Glutamate-gated and GABA-gated ion channels have been reported in serotonergic neurons.
|
2. Characterization of the serotonergic-like progenitors and verification showing double-staining with 5-HT and tryptophan hydroxylase (TPH) plus En1, Pax3, and Pax5 mRNA expression
We next studied the expanded spheres, which were dispersed into suspensions containing single cells and cultured on well plates at different densities per well by limiting dilution. These cells proceeded to grow in the well, and single spheres were marked. Thereafter, the single sphere cells were expanded and repassaged into 2 plates. The first plate cells were consistent cultures. The second plate of cells was grown on the coverslips before coating with poly-D-lysine. The selected serotonergic-like single clones (Fig. 1B
) were double immunofluorescence stained with 5-HT and TPH antibodies, revealing that both 5-HT and TPH were colocalized in the cytoplasm of these serotonergic-like progenitor cells. These 5-HT/TPH-positive progenitor cells were also stained with the specific neuronal nuclear protein NeuN antibody (Fig. 1C
). RT-PCR results showed that these serotonergic-like progenitor cells expressed the mRNA of several genes associated with serotonergic neurons at early stages of CNS development: Pax3, Pax5, and the homeobox transcription factor En1 (Fig. 1D
).
3. Expression and function of SERT verified in serotonergic-like progenitors using [125I]-RTI-55 ligand binding and [3H]-5-HT uptake
We evaluated whether 10-wk-old progenitor cells expressed SERT using [125I]-RTI-55, a sensitive ligand for the 5-HT transporter. Since [125I]-RTI-55 binds to both SERT and the dopamine transporter (DAT), SERT-specific binding was determined using 10 µM GRB 12909 to block DAT; nonspecific binding was determined using 10 µM fluoxetine. The serotonergic-like progenitor cells exhibited a high SERT [125I]-RTI-55 specific binding. We also directly studied SERT function in these serotonergic-like progenitor cells, measuring [3H]-5-HT uptake in confluent monolayer cells. Uptake was temperature- and Na+ ion-dependent and was reduced 95% by fluoxetine and citalopram, selective serotonin reuptake inhibitors (SSRIs). The tricyclic imipramine also caused a profound inhibition (> 90%) (Fig. 2
A).
|
4. Expression of SERT after implantation of serotonergic-like progenitors into the brains of mice lacking the serotonin transporter
The above in vitro studies demonstrated that these stable lineages of progenitor cells expressed SERT sites and showed 5-HT uptake function. To determine whether the lineage serotonergic progenitor cells might survive and keep their phenotype in vivo, we implanted them or control vehicle into the brains of mice lacking SERT. We examined the survival of these progenitor cells after implantation of EGFP-lentiviral vector infected with progenitor cells in the hippocampus. Eight wk after implantation, brain sections showed that the most dense localization of the surviving implanted cells were in hippocampal areas adjacent to the lateral ventricle. Some surviving cells showed neuronal morphology with axonal extensions (Fig. 2B
). Western blot assays demonstrated that the expression of SERT protein was significantly increased in these hippocampal areas of the SERT knockout mice relative to vehicle implant controls (Fig. 2 C, D
). When the same blot was restained with VMAT2 and ß-actin antibodies as control proteins, the expression of VMAT2 protein was somewhat increased at 8 wk, while ß-actin protein remained unchanged. When these progenitor cells were pretreated with BrdU and implanted into the hippocampus, we observed that the cells began to migrate and integrate into surrounding brain tissues during the 2–8 wk of study. We found that anti-BrdU and anti-5-HT double-stained cells were localized as follows: 5% (125±70) in the dentate gyrus (DG) of the hippocampus, 77% (1839±329) in hippocampal areas adjacent to the lateral ventricular (LV) areas, and 18% (437±53) just under the corpus callosum (CC) after 8 wk implantation. All of these findings suggest that some of the implanted progenitor cells survived, kept their phenotype, and expressed SERT protein for at least 8 wk after implantation.
CONCLUSIONS AND SIGNIFICANCE
In the present study we investigated how to derive serotonergic-like progenitor cells from mouse brain neural stem cells in vitro for eventual implantation in vivo (Fig. 2
). We found that the specific growth factors bFGF, FGF-8, and Shh plus N-2 supplement medium were required to induce neural stem cell differentiation into neuronal progenitors with a serotonergic rather than a glial phenotype. Specifically, the serotonergic neuron-like phenotype of these cells comprised: 1) expression of the primary serotonin synthetic enzyme, TPH, colocalized with serotonin, as demonstrated using immunocytochemical double-staining beginning at 3 wk and considerably increased by 8 wk of culture; 2) expression of electrophysiologically active glutamate and GABA-sensitive ion channels; 3) expression of SERT protein identified by specific [125I]-RTI-55 binding; and 4) SERT function verified by specific Na+-dependent [3H]-5HT uptake inhibited by the serotonin reuptake inhibitors (SSRIs) fluoxetine and citalopram. When these differentiated serotonin progenitor cells expressing SERT were implanted into the brains of mice with a targeted disruption of SERT, the cells survived and maintained their phenotype for at least 8 wk, with some cells showing axonal extension. The expression of SERT protein in the brain significantly increased after implantation in these mice lacking SERT. This study thus advances cell culture biotechnology and developmental cell therapy strategies in which knocked out genes may be compensated for in adult brains.
FOOTNOTES
To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.04-3657fje;
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
