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First published online October 6, 2003
doi: 10.1242/10.1242/dev.00714

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Functional network integration of embryonic stem cell-derived astrocytes in hippocampal slice cultures

Björn Scheffler1,*, Tanja Schmandt1,2, Wolfgang Schröder3, Barbara Steinfarz1,2, Leila Husseini3, Jörg Wellmer4, Gerald Seifert3, Khalad Karram1,2, Heinz Beck4, Ingmar Blümcke1, Otmar D. Wiestler1, Christian Steinhäuser3 and Oliver Brüstle1,2,{dagger}

1 Department of Neuropathology, University of Bonn Medical Center, Sigmund-Freud-Strasse 25, D-53105 Bonn, Germany
2 Institute of Reconstructive Neurobiology, University of Bonn Medical Center, Sigmund-Freud-Strasse 25, D-53105 Bonn, Germany
3 Department of Neurosurgery, University of Bonn Medical Center, Sigmund-Freud-Strasse 25, D-53105 Bonn, Germany
4 Department of Epileptology, University of Bonn Medical Center, Sigmund-Freud-Strasse 25, D-53105 Bonn, Germany



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  		Fig. 1. Morphological and functional integrity of hippocampal slice cultures used as recipient tissue for ESGPs. (A) A 400 µm-thick slice one day after explantation. Dentate gyrus (DG), pyramidal-cell layer (CA3-CA1), entorhinal cortex (EC) and adjacent regions of the temporal cortex (TC) are well delineated. SC, Schaffer collaterals; MF, mossy fibers; PP, perforant path. (B) Cryostat section (10 µm) of a slice preparation maintained in culture for 31 days. Note the morphological preservation of the key anatomic structures (Hematoxylin and Eosin staining). The inset shows typical field potentials following orthodromic stimulation of the perforant path (PP-DG) and the Schaffer collaterals (SC-CA1) at the end of the culture period (stimulation artifacts blanked). (C) Anterograde tracing with rhodamine-conjugated dextran (Micro-Ruby®) confirms the integrity of the perforant path at 33 days in culture. (D) TIMM stain (performed according to Zimmer and Gähwiler, 1984Go) demonstrating the histological preservation of the MF system of a slice culture maintained for 33 days (20 µm cryostat section). Scale bars: 1 mm.

 


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  		Fig. 2. In vitro transplantation of ESGPs in hippocampal slice cultures. (A) Slice culture one day after deposition of 20,000 GFP-labeled ESGPs onto the entorhinal cortex. (B) 18 days after implantation, the donor cells have migrated along the pyramidal cell layer and the Schaffer collaterals into the hilar region and the dentate granule cell layer (dashed line) where they developed complex 3D morphologies (inset, digital reconstruction of a grafted GFP-expressing cell composed of 32 individual planes). (C) At this same stage, cross sections through the entire slice preparation revealed donor cells incorporated at various depths in the host tissue. Double labeling with an antibody to GFAP (red). (D) Although some grafted cells retained an immature morphology and expressed NG2 (red), the majority exhibited differentiated glial phenotypes. (E) About 30% of the ESGPs were immunolabeled for nestin, including cells with astrocytic morphology. (F) In addition to GFAP, donor-derived astrocytes expressed S100ß. (G) An ES cell-derived oligodendrocyte (in the EC region) identified using an antibody to CNP. (H) A GFP+ oligodendrocyte that expressed MBP (red) and had tubular processes that indicate myelin formation (stratum oriens, CA1). Scale bars: in A,B, 1 mm; in C, 100 µm; in D-H, 25 µm.

 


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  		Fig. 3. Patch clamp analysis of ESGPs before and after transplantation. (A-C) Cultured ESGPs exhibit IK(A) and/or IK(D) but lack IKir and IK(P). The current pattern during propagation in EGF/FGF (A), PDGF/FGF (B), and 9 days after growth factor withdrawal (C) are shown. (D-F) After transplantation, ESGPs have three distinct current patterns. Whereas some cells retain the immature pattern observed during monolayer culture (D), others display IKir (E). The majority of engrafted ESGPs displayed prominent IK(P) (F). Current phenotypes shown in E,F are characteristic of mature hippocampal astrocytes in vivo. The pulse protocol (see text) used in A,B also applies to (C, left) and (D-F).

 


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  		Fig. 4. Glial network integration of ES cell-derived astrocytes. (A) Epifluorescence microscopy during a 30-minute iontophoretic injection of LY into a GFP-labeled donor cell, located at the border of EC and subiculum (arrow, 25 days after transplantation; both LY and GFP signals are recorded in the FITC channel). Note the extensive dye coupling to adjacent host cells. (B) Confocoal microscopy and 3D reconstruction of (A), reveals a cluster of 50 coupled cells with total volume of 5.6x10–3 mm3. The picture is taken following fixation and depicts the 3D nature of the endogenous glial network. (C-D) Subsequent serial sectioning and double labeling with an antibody to the mouse-specific antigen M2 (red) confirms the donor origin of the injected astrocyte depicted in A. Note the extensive arborization (arrowheads) and the perforation of the cell body (arrow). (E) Overlay of C and D (boxed area delineated in C), triple labeled with an antibody to connexin43 (blue). Patches of connexin43 immunoreactivity (arrowheads) are detected at the contact zones between the LY/M2+ donor cell and two adjacent LY-filled host-cell processes. (F) Prominent connexin43 expression (blue) is also observed on LY-filled processes connecting individual host cells of the same cluster (confocal 3D reconstruction). Note that figures A-F are from the same donor-host cell cluster. Scale bars: in A, 200 µm; in B, 50 µm; in C-F, 25 µm.

 


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  		Fig. 5. Regional distribution and time course of cell-cell coupling. (A) Schematic representation of endogenous and incorporated ESGP-derived astrocytes used for analysis of network integration. (B-D) Examples of host-cell clusters coupled to individual GFP+ donor cells as seen by confocal analysis (each shown as overlay of 32 individual scans). After engraftment for 22-25 days, donor-derived astrocytes integrated equally well in glial networks of different anatomical regions of the recipient slice [molecular layer of the DG in B, EC/subiculum in C (as in Fig. 4B), TC region in D]. Both LY and GFP signals are recorded in the FITC channel. (E) Cell coupling of endogenous and engrafted ESGPs in three hippocampal subregions at different times in culture. Endogenous astrocytes reveal an increasing complexity of gap junction coupling during the culture period. After transplantation for 2-3 weeks, coupling ratios between donor and host cells in DG and EC equal those observed between endogenous astrocytes (engrafted slices were analyzed 12-25 days after donor cell deposition described in A). DG, dentate gyrus; dic, days in culture; dTx, days after deposition; EC, entorhinal cortex; TC, temporal cortex. Scale bars: 50 µm.

 




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