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. 1999 Sep 1;19(17):7529-36.
doi: 10.1523/JNEUROSCI.19-17-07529.1999.

Polysialylated Neural Cell Adhesion Molecule-Positive CNS Precursors Generate Both Oligodendrocytes and Schwann Cells to Remyelinate the CNS After Transplantation

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Free PMC article

Polysialylated Neural Cell Adhesion Molecule-Positive CNS Precursors Generate Both Oligodendrocytes and Schwann Cells to Remyelinate the CNS After Transplantation

H S Keirstead et al. J Neurosci. .
Free PMC article

Abstract

Transplantation offers a means of identifying the differentiation and myelination potential of early neural precursors, features relevant to myelin regeneration in demyelinating diseases. In the postnatal rat brain, precursor cells expressing the polysialylated (PSA) form of the neural cell adhesion molecule NCAM have been shown to generate mostly oligodendrocytes and astrocytes in vitro (Ben-Hur et al., 1998). Immunoselected PSA-NCAM+ newborn rat CNS precursors were expanded as clusters with FGF2 and grafted into a focal demyelinating lesion in adult rat spinal cord. We show that these neural precursors can completely remyelinate such CNS lesions. While PSA-NCAM+ precursor clusters contain rare P75+ putative neural crest precursors, they do not generate Schwann cells in vitro even in the presence of glial growth factor. Yet they generate oligodendrocytes, astrocytes, and Schwann cells in vivo when confronted with demyelinated axons in a glia-free area. We confirmed the transplant origin of these Schwann cells using Y chromosome in situ hybridization and immunostaining for the peripheral myelin protein P0 of tissue from female rats that had been grafted with male cell clusters. The number and distribution of Schwann cells within remyelinated tissue, and the absence of P0 mRNAs in donor cells, indicated that Schwann cells were generated by expansion and differentiation of transplanted PSA-NCAM+ neural precursors and were not derived from contaminating Schwann cells. Thus, transplantation into demyelinated CNS tissue reveals an unexpected differentiation potential of a neural precursor, resulting in remyelination of CNS axons by PNS and CNS myelin-forming cells.

Figures

Fig. 1.
Fig. 1.
Cluster transplantation results in remyelination.A, Phase micrograph of PSA-NCAM+ neural clusters after 10 d in vitro on a nonadherent surface (immediately before transplant). Grafts consisted of ∼60,000 cells in clusters.B, Toluidine blue-stained transverse section of an x-irradiated ethidium bromide lesion in the dorsal funiculus after 30 d. Besides macrophages containing myelin debris (M), the lesion contains naked axons tightly apposed to each other with no evidence of remyelination or glial cell nuclei. C, Toluidine blue-stained transverse section of an x-irradiated ethidium bromide lesion 30 d after transplantation of PSA-NCAM+ neural clusters. Macrophages containing myelin debris (M), oligodendrocytes (O), astrocytes (A), and Schwann cells (S) are present, and virtually all axons are remyelinated. D, Electron micrograph of an x-irradiated ethidium bromide lesion 30 d after transplantation of PSA-NCAM+ neural clusters. An oligodendrocyte (O) is present among remyelinated axons, characterized by thin myelin sheaths. Schwann cells (S), with their nuclei closely apposed to myelin sheaths, are also evident. E,Electron micrograph of an x-irradiated ethidium bromide lesion 30 d after transplantation of PSA-NCAM+ neural clusters. The myelin sheaths produced by Schwann cells have a greater periodicity than oligodendrocyte myelin and are surrounded by basement membranes (BM). Magnification: A, 400×;B, C, 600×; D, 12,000×;E, 25,000×.
Fig. 2.
Fig. 2.
Remyelinating Schwann cells are transplant-derived. A, P0-immunostained transverse section of an x-irradiated, ethidium bromide-lesioned dorsal funiculus 30 d after transplantation of PSA-NCAM+ neural clusters. P0 immunostaining demonstrates many foci of Schwann cell myelination scattered throughout the lesion. B, Computerized overlay of serial tissue sections hybridized with Y chromosome probe (blue) and immunostained with P0 antibodies for Schwann cell myelin (yellow) in an x-irradiated, ethidium bromide-lesioned female rat 30 d after transplantation of PSA-NCAM+ neural clusters prepared from male donors. The foci of Schwann cell myelin correspond to regions of high density of transplanted cells. C, P0 and Y chromosome double-labeled transverse section of an x-irradiated, ethidium bromide-lesioned female rat 30 d after transplantation of PSA-NCAM+ neural clusters prepared from male donors. Intimate apposition of P0-immunostained Schwann cell myelin with Y chromosome-hybridized cell bodies is shown. Magnification:A, 200×; B, 300×; C, 400×.
Fig. 3.
Fig. 3.
P0 expression is not detected in the graft preparations. A, RT-PCR of total RNAs extracted from newborn rat sciatic nerve and PSA-NCAM+ cells just after immunopanning (Day 0), after 2 weeks of culture on nonadherent surface in FGF2 (2 wk Cl.), or after differentiation of growing clusters obtained by transfer to poly-d-ornithine-coated surfaces (Diff. Cl.). P0 expression is absent in PSA-NCAM+ cells in all three experimental conditions. A molecular size marker (174 DNA digested withHaeIII) was run on the right andleft. Controls for RT-PCR (RT) correspond to cDNA synthesized without reverse transcriptase.B, Immunopurified precursors were treated for 14 din vitro with both FGF2 and GGF2. Cell clusters were then transferred to adherent surfaces for differentiation (as inA) for 5 d without any growth factor. RNA extraction and the positive controls are as in A. Although GGF treatment was performed for 14 d, no P0 expression was observed during in vitro growth and differentiation of the PSA-NCAM precursors.
Fig. 4.
Fig. 4.
P0 and P75 transcripts are detected in purified Schwann cells and in Schwann cells mixed with PSA-NCAM clusters. RT-PCR of total RNAs extracted from purified Schwann cells (A) or PSA-NCAM CNS clusters to which increasing small numbers of Schwann cells were added (B) is shown. A, Signals for P0 and P75 were detected when 1 million Schwann cells were grown in FGF2, GGF2, or defined medium for 5 d. The most intense signal was seen for P75, and the P0 signal was weaker in defined medium alone. P0 signal is observed in Schwann cells cultivated with FGF2 or GGF. B, P0 is observed clearly in the mix of 4 million PSA-NCAM CNS precursors with 100 Schwann cells. These results mean that RT-PCR could detect 0.0025% Schwann cell contamination in the PSA-NCAM+ clusters. P75 shows a faint signal in the absence of Schwann cells, and that signal increases in intensity proportionally with the number of Schwann cells added. In contrast, the actin signal (seen at 800 MW) displays equal intensity when 10, 50, or 100 Schwann cells were added.
Fig. 5.
Fig. 5.
Immunofluorescence staining of a P75+ precursor cell in a PSA-NCAM cluster. Immunopurified PSA-NCAM+ cells were grown for 10 d in FGF2 and GGF2 on a nonadherent surface. After adherence they were immunolabeled with P75 antibody, followed by FITC-conjugated secondary antibody. Shown here is one (or two?) P75+ cells with short processes located inside an adherent cluster (delineated by white arrows). These cells did not co-stain with O4 or GFAP. Magnification, 400×.

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