Methods

Setting up brain slice cultures:

Establish brain slice cultures 8-12 d prior to transplantation.

1 Anaesthetize a rat with Forene®.

2 Swiftly remove the head using a decapitator, and carefully remove intact brains.

3 Use the vibroslicer, placed under the horizontal flow hood, to prepare 400 mm-thick brain slices, according to the manufacturer's instructions (39).

Protocol 6 continued

4 Incubate slices as interface cultures (39) on the polyester membranes with slice culture medium containing 20% normal horse serum, at 35 °C and with 5% CO2 in air. The day of explantation is designated 'day 0'.

5 Change medium on day 1 of culture, and then on every other day.

6 Gradually replace initial slice culture medium with serum-free medium by day 5.

Preparation of donor cells:

7 On the day of transplantation, aspirate medium from ESNP or ESGP cultures, and wash cells three times with HBSS.

8 Harvest cells from the dish with trypsin/EDTA solution, and stop trypsinization with trypsin inhibitor (Protocol 3, Step 2).

9 Triturate cells to a single-cell suspension with a flame-polished Pasteur pipette, and count viable cells.

10 Centrifuge cells for 5 min at 1000 rpm and 4 °C, and resuspend cells in HBSS/Hepes or DMEM/F12 at a concentration of 7.5 x 105 cells/ml, for transplantation into hip-pocampal slice cultures; or 2-4 x 105 cells in 4-8 ml for in utero transplantation (see Protocol 7).

11 Transfer cells into a 500 ml Eppendorf tube, and keep on ice until transplantation.

Donor cell transplantation:

Perform transplantation of neural cells at day 10±2.

12 Use a glass capillary connected to the manipulator of a stereotactic frame to deposit donor cells on the surface of the slice preparation at the desired location. Resume culture of the transplanted slices.

13 Two days after deposition, wash the slices thoroughly with medium to remove non-adherent donor cells, replenish medium and continue incubation. During culture, the migration and differentiation of EGFP-labelled donor cells may be monitored directly by epifluorescence microscopy. To avoid phototoxicity, keep observation times as short as possible.

14 Fix slices 10-20 d after transplantation with 4% paraformaldehyde, and use whole or cryosectioned slices for further immunfluorescence analysis. For cryoprotection, fixed slices are incubated in 30% saccharose until they sink (43). Store fixed slices at 4 °C in 0.1% sodium azide solution.

a For real-time tracing of the donor cells, use ESNPs or ESGPs expressing EGFP. (For details see

in vivo. To address this question with respect to the nervous system, the derivative cells can be introduced into the ventricular zone of the fetal brain via intrauterine transplantation ((27, 44-46); and see Figure 7). This procedure involves direct injection of donor cells through the uterine wall into the lateral ventricle

Figure 7 (see Plate 9) Transplantation as a tool to study the developmental potential of ES cell-derived neural precursors. (A) Schematic representation of the transplant approach. Following injection into the cerebral ventricles of rat embryos, the donor cells distribute throughout the ventricular system. (B) Frequent integration sites observed after transplantation of neural precursors into E15-E17 rat embryos include cortex, hippocampus, corpus callosum, septum, striatum, thalamus, hypothalamus, and tectum. (C) Xenografted mES cell-derived neural precursors can be reliably detected by in situ hybridization with a mouse-specific DNA probe. The example depicts a hybridized donor-derived oligodendrocyte double-labelled with an antibody to proteolipid protein. (D) Transplanted mES cell-derived astrocytes can be detected with an antibody to the mouse-specific antigen, M2 (red). The astrocytic identity is confirmed by double labelling with an antibody to glial fibrillary acidic protein (green). (E) Human donor cells transplanted into a neonatal mouse brain can be visualized with an antibody to human-specific nuclear antigen (HuNu, green). Double labelling with an antibody to p-III-tubulin (red) confirms the neuronal identity of this hES cell-derived donor cell. Scale bar = 20mm in C, 10mm in D, E. See refs (45, 65, 66). Copyright, National Academy of Sciences, U.S.A., and kind permission of Springer Science + Business Media.

of embryonic rats or mice (Figure 7A, B). From there, the grafted cells have been shown to distribute widely throughout the host ventricular zone, and to integrate into a variety of host brain regions (7, 44-47). Intrauterine transplantation is particularly advantageous for xenografts, which, due to the early time point of transplantation, can be conducted without the need for immunosuppression. The following protocol summarizes the key steps of this procedure; a detailed description has been reported elsewhere (48). Either ESNPs proliferating in the presence of FGF-2, or ESGPs proliferating in the presence of FGF-2 and EGF, (see Figure 1 and Protocol 1) are appropriate donor cell populations.

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