HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Full Text (PDF) All Versions of this Article: 17/14/2100 03-0118fjev1    most recent Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by JÜNGLING, K. Articles by GOTTMANN, K. Search for Related Content PubMed PubMed Citation Articles by JÜNGLING, K. Articles by GOTTMANN, K.

Purification of embryonic stem cell-derived neurons by immunoisolation¹

Department of Cell Physiology, Ruhr-Universität Bochum, D-44780 Bochum, Germany; and
^* Max Planck/CNRS Group, Centre de Neurochimie, CNRS UPR 2356, 67084 Strasbourg Cedex, France

²Correspondence: Department Cell Physiology, Ruhr-Universität Bochum, Universitätsstr. 150, D-44780 Bochum, Germany. E-mail: kurt.gottmann{at}ruhr-uni-bochum.de

SPECIFIC AIMS

After in vitro differentiation of embryonic stem (ES) cells, purification of ES cell-derived neurons is essential to obtain a homogeneous population of neurons devoid of undifferentiated and non-neuronal cells that can be used for biomedical research and cell replacement therapies. We developed a one-step immunoisolation procedure based on the neuron-specific expression of the cell adhesion molecule L1 and functionally characterized electrical excitability and synapse formation in the L1-selected cells.

PRINCIPAL FINDINGS

1. Cell yield of mouse ES cell-derived neurons immunoisolated with L1 antibody
We induced neuronal differentiation of mouse ES cells within embryoid bodies (EBs) using retinoic acid. After enzymatic and mechanical dissociation of EBs, the cell suspension was subjected to immunoisolation using a L1-specific monoclonal antibody. During immunoisolation, L1-expressing cells adhered to the antibody-coated dish. We counted the total number of cells used and the number of cells selected by the L1 antibody. In 13 independent experiments, the mean fraction of L1-selected cells was 3.7 + 0.01% (10⁶ cells), indicating a reliable selection by the immunoisolation procedure.

2. Immunocytochemical characterization of L1-selected cells revealed expression of a neuronal marker protein (neurofilament200)
L1-selected cells were short-term cultured for 2 days to allow for the regeneration of neuritic processes. We performed immunocytochemical staining (n=6) using antibodies against cell type-specific marker proteins including neurofilament200 (neurons), GFAP (astrocytes), O4 (oligodendrocytes), and fibronectin (non-neural cells). As positive controls, we cultured and stained cells that were nonadherent in the L1 immunoisolation (Fig. 1 ). The vast majority of these control cells were positive for fibronectin. Neurofilament-positive neurons and GFAP-positive astrocytes with typical morphologies were frequently detected. Strikingly, the L1-selected cells were almost exclusively neurofilament 200-positive (93.4+3.4%) and showed a clear neuronal morphology. A small fraction of cells (6.4+1.1%) was positive for GFAP, but did not show a typical astrocytic morphology. These cells presumably represent neuronal precursors, which have been shown to express this astrocytic marker.

View larger version (60K): [in this window] [in a new window]   Figure 1. Immunocytochemical characterization of cells purified by L1 immunoisolation. A) Immunostaining of cells contained in EBs and cultured for 2 days on poly-D-lysine/laminin-coated culture dishes. Micrographs show neurofilament 200-positive neurons (left), a GFAP-positive astrocyte (middle), and a fibronectin-positive cell (right). Scale bars: 25 µm. B) Micrograph showing ES cell-derived neurons purified by L1 immunoisolation and cultured for 2 days on poly-D-lysine/laminin-coated culture dishes in NB+ medium. Propidium-iodide staining was used to distinguish intact cells showing a typical neuronal morphology from dead cells (arrows). Scale bar: 40 µm. C) Immunostainings of cells purified by L1 immunoisolation. Neurofilamant 200-positive neuron (middle) and GFAP-positive cell (right). Scale bars: 25 µm. D) Mean fraction of cell types isolated by L1 immunoisolation. Note that >93% of cells were neurons, whereas fibronectin and O4 positive cells were completely absent. Error bars represent SE.

3. Electrical excitability of L1-selected cells
For electrophysiological (patch clamp) recordings, L1-selected cells were long-term cultured on micro islands of glial cells. After 10–12 days in vitro, each ES cell-derived neuron tested (n=29) fired TTX-sensitive action potentials upon depolarization by current injection (Fig. 2 ).

View larger version (24K): [in this window] [in a new window]   Figure 2. ES cell-derived neurons purified by L1 immunoisolation are electrically excitable and form functional synapses. A) Phase contrast micrograph of ES cell-derived neurons purified by L1 immunoisolation and cultured on a glial micro island for 11–13 days. Note the typical neuronal morphology. Scale bar: 75 µm. B) ES cell-derived neurons fire action potentials upon current injection. Upper panel shows membrane potential recordings. Lower panel indicates injected current pulses. C) Fluorescence micrograph showing synapsin I-positive puncta indicating the formation of presynaptic boutons on the soma (arrowhead) and the dendrites (arrow) of a postsynaptic ES cell-derived neuron. Scale bar: 20 µm. D) Glutamate receptor-mediated miniature postsynaptic currents (mPSCs) recorded from ES cell-derived neurons (upper traces) were completely blocked by 10 µM DNQX (lower traces). E) GABAA receptor-mediated mPSCs recorded from ES cell-derived neurons (upper traces) were completely blocked by 25 µM bicuculline (lower traces). F) Pair recordings in ES cell-derived neurons revealed AMPA and NMDA receptor-mediated PSCs at -60 mV and at +40 mV holding potential, respectively (middle and lower traces). Note the block of the slowly decaying, NMDA receptor-mediated PSC component by 1 mM Mg2+ at -60 mV. The presynaptic cell was stimulated with depolarizing voltage steps at a stimulus interval of 50 ms to elicit presynaptic action potentials (upper trace). Stimulation artifacts have been truncated.

4. L1-selected cells form functional glutamatergic and GABAergic synapses
The formation of synaptic specializations was shown by immunocytochemical staining of synapsin I-positive presynaptic boutons on the somata and dendrites (12.9+1.1 per 50 µm dendrite; n=15) of L1-selected neurons after 10 days in vitro (Fig. 2) . To demonstrate the formation of functional synapses, we recorded spontaneous miniature postsynaptic currents (mPSCs) mediated by GABA_A and glutamate receptors, respectively (Fig. 2) . At 11–13 days in vitro, GABA_A receptor-mediated mPSCs were observed at a mean frequency of 0.5 ± 0.1 Hz (n=11; mean amplitude: 11±2 pA) and were reversibly blocked by bicuculline (25 µM, n=3). Similarly, non-NMDA (AMPA/kainate) receptor-mediated mPSCs were observed at a mean frequency of 1.7 ± 0.4 Hz (n=5; mean amplitude: 12±1 pA) and were reversibly blocked by DNQX (10 µM, n=3). In addition, postsynaptic currents (PSCs) were evoked by eliciting action potentials in the presynaptic neuron in paired recordings. At a strongly negative holding potential (–60 mV), the fast decaying PSCs were mediated exclusively by AMPA receptors (completely blocked by 10 µM DNQX or 100 µM SYM2206). Intriguingly, upon depolarization to +40 mV an additional, much more slowly decaying and Mg²⁺-sensitive PSC component became apparent, indicating the presence of NMDA receptors (blocked by 40 µM MK-801).

CONCLUSIONS

Future therapeutic applications of stem cell-derived neurons require a purification step to ensure the absence of undifferentiated cells that may lead to tumor formation after transplantation. We have developed an efficient immunoisolation method (Fig. 3 ) that allows us to purify large numbers of neurons. In coculture with glial cells, purified ES cell-derived neurons showed typical neuronal features, including electrical excitability and the formation of functional glutamatergic and GABAergic synapses with basic properties similar to primary cultured cortical neurons. This new procedure to separate ES cell-derived neurons from non-neuronal and undifferentiated cells will strongly reduce the risk for side effects in transplantation studies. In principle, immunoisolation based on endogenous markers could be extended to purify other stem cell-derived cell types without the need for genetic modification.

View larger version (16K): [in this window] [in a new window]   Figure 3. Schematic diagram.

FOOTNOTES

¹ To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.03-0118fje; doi: 10.1096/fj.03-0118fje

This article has been cited by other articles:

				J. Ladewig, P. Koch, E. Endl, B. Meiners, T. Opitz, S. Couillard-Despres, L. Aigner, and O. Brustle Lineage Selection of Functional and Cryopreservable Human Embryonic Stem Cell-Derived Neurons Stem Cells, July 1, 2008; 26(7): 1705 - 1712. [Abstract] [Full Text] [PDF]

				G. Lalatta-Costerbosa, M. Mazzoni, P. Clavenzani, G. Di Guardo, G. Mazzuoli, G. Marruchella, L. De Grossi, U. Agrimi, and R. Chiocchetti Nitric Oxide Synthase Immunoreactivity and NADPH-d Histochemistry in the Enteric Nervous System of Sarda Breed Sheep With Different PrP Genotypes in Whole-mount and Cryostat Preparations J. Histochem. Cytochem., April 1, 2007; 55(4): 387 - 401. [Abstract] [Full Text] [PDF]

				C. Walz, K. Jungling, V. Lessmann, and K. Gottmann Presynaptic Plasticity in an Immature Neocortical Network Requires NMDA Receptor Activation and BDNF Release J Neurophysiol, December 1, 2006; 96(6): 3512 - 3516. [Abstract] [Full Text] [PDF]

				K. Jungling, V. Eulenburg, R. Moore, R. Kemler, V. Lessmann, and K. Gottmann N-cadherin transsynaptically regulates short-term plasticity at glutamatergic synapses in embryonic stem cell-derived neurons. J. Neurosci., June 28, 2006; 26(26): 6968 - 6978. [Abstract] [Full Text] [PDF]

				A. Copi, K. Jungling, and K. Gottmann Activity- and BDNF-Induced Plasticity of Miniature Synaptic Currents in ES Cell-Derived Neurons Integrated in a Neocortical Network J Neurophysiol, December 1, 2005; 94(6): 4538 - 4543. [Abstract] [Full Text] [PDF]

				W. -L. Kuan and R. A. Barker New Therapeutic Approaches to Parkinson's Disease Including Neural Transplants Neurorehabil Neural Repair, September 1, 2005; 19(3): 155 - 181. [Abstract] [PDF]

This Article Full Text (PDF) All Versions of this Article: 17/14/2100 03-0118fjev1    most recent Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by JÜNGLING, K. Articles by GOTTMANN, K. Search for Related Content PubMed PubMed Citation Articles by JÜNGLING, K. Articles by GOTTMANN, K.