Functional motor neurons differentiating from mouse multipotent spinal cord precursor cells in culture and after transplantation into transected sciatic nerve

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Object. One of the current challenges in neurobiology is to ensure that neural precursor cells differentiate into specific neuron types, so that they can be used for transplantation purposes in patients with neuron loss. The goal of this study was to determine if spinal cord precursor cells could differentiate into motor neurons both in culture and following transplantation into a transected sciatic nerve.

Methods. In cultures with trophic factors, neurons differentiate from embryonic precursor cells and express motor neuronal markers such as choline acetyltransferase (ChAT), Islet-1, and REG2. Reverse transcription—polymerase chain reaction analysis has also demonstrated the expression of Islet-1 in differentiated cultures. A coculture preparation of neurospheres and skeletal myocytes was used to show the formation of neuromuscular connections between precursor cell—derived neurons and myocytes both immunohistochemically and electrophysiologically. Following various survival intervals, precursor cells transplanted distal to a transection of the sciatic nerve differentiated into neurons expressing the motor neuron markers ChAT and the α11.2 (class C, L-type) voltage-sensitive Ca++ channel subunit. These cells extended axons into the muscle, where they formed cholinergic terminals.

Conclusions. These results demonstrate that motor neurons can differentiate from spinal cord neural precursor cells grown in culture as well as following transplantation into a transected peripheral nerve.

Article Information

Address reprint requests to: Robert M. Brownstone, M.D., Ph.D., Division of Neurosurgery, 1796 Summer Street, Room 3809, Halifax, Nova Scotia B3H 3A7, Canada. email: Rob.Brownstone@dal.ca.

© AANS, except where prohibited by US copyright law.

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Figures

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    Immunohistochemical detection of motor neuron antigens in tissue culture. Phase-contrast micrograph demonstrating clusters of proliferative neural precursor cells grown in tissue culture (A). After they were plated on a permissive substrate in the absence of mitogens, some precursor cells differentiate into large-diameter ChAT-positive neurons (B and C1 [arrow]). Immunohistochemical analysis was used to identify more specific markers of motor neuron differentiation and growth. A subpopulation of ChAT-positive neurons expresses the LIM homeobox transcription factor, Islet-1 (arrow in C2). An example of an REG2-immunoreactive neuron (arrow in D1), which also expresses Islet-1 (arrow in D2), is also shown. Bars = 100 µm (A); 15 µm (B); and 50 “m (C1–D2).

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    Expression of Islet-1 in differentiated neuroepithelial cultures. The Islet-1 RT-PCR product was electrophoresed on an agarose gel and visualized under ultraviolet illumination. The first lane corresponds to the 100-bp marker. The second lane displays a strong 350-bp band derived from spinal cord RNA. The RNA that was harvested from a 1-week-old culture displays a similar band. A faint band is visible for the RNA obtained from a 3-week-old culture, but no band was visible for ovary RNA or for RNA obtained from a 2-week-old culture.

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    Development of neuromuscular junctions in a precursor cell—myocyte coculture. An example of a cholinergic axon and terminal (arrow in A1) in apposition to a cluster of nicotinic ACh receptors (NICR; arrow in A2) is demonstrated using ChAT—VAChT and nicotinic receptor immunohistochemical analysis. Phase-contrast micrograph (B1) of a precursor cell cluster—myocyte coculture depicting a myocyte in close apposition to a differentiating cluster (white arrow). A contraction profile (B2) showing that the myocyte contracted in response to a perfusion of kainate or ACh (black arrows). Kainate application caused contraction only in the innervated myocyte, whereas an ACh perfusion caused contraction in all neighboring myocytes as well.

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    Electrophysiological responsiveness of a skeletal myocyte in coculture. A: Phase-contrast micrograph showing a precursor cell—myocyte coculture. The differentiating neurosphere is seen on the left side of the panel and is marked by the letter n. Long healthy myocytes are seen running along the substrate, and the recording electrode is seen on the targeted myocyte. This particular cell was seen to contract in response to 50 µM kainate and ceased to contract after the agonist was washed out. Bar = 50 µm. B: End-plate potentials in response to a low concentration of kainate in the recording medium. No spontaneous end-plate potentials were observed in the absence of kainate.

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    Expression of nestin and MAP-2 in transplanted sciatic nerves shown in sections of adult mouse sciatic nerve after transplantation of neural precursor cells. Neural precursor cells were traced using DiO (arrows) during the transplantation procedure (A2 and B2). One day after transplantation of precursor cells, intense nestin immunoreactivity is seen in cells at the graft site (arrows in A1). As early as 3 days posttransplantation, MAP-2-positive neurons are seen at the graft site (arrows in B1). Bars = 15 µm (A1 and A2) and 25 µm (B1 and B2).

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    Expression of motor neuron markers in transplanted sciatic nerve shown in sections of adult mouse sciatic nerve after cell transplantation. The DiO labeling is marked by arrows (A2 and B2). Expression of ChAT (arrow in A1) at 12 weeks and the α11.2 (class C) subunit of the L-type Ca++ channel (arrow in B1) at 4 weeks give evidence that motor neurons are differentiating in the cell-transplanted sciatic nerve. Bars = 20 µm (A1 and A2) and 10 µm (B1 and B2).

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    Precursor cells sending axons to distal muscles. A: At 12 weeks after transplantation, a DiO-labeled axon (arrow) runs along the musculature attached to the distal end of a sciatic nerve section. B: Cholinergic terminals labeled with VAChT antibodies (arrow) are seen on muscle tissue at the distal end of the sciatic nerve section at 16 weeks posttransplantation. Bars = 25 µm (A) and 5 µm (B).

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