Thursday, 21 August 2003
This presentation is part of : Thursday Poster Sessions

PD-061 Can Transplanted Human Neural Stem Cells Migrate Positionally into the Cholinergic Lesioned Areas and Differentiate into Cholinergic Cells?

Tingyu Qu and Kiminobu Sugaya. Psychiatry, University of Illinois at Chicago, Chicago, IL, USA

Objective: Cholinergic degeneration inthe basal forebrain has been documented as an important factor underlying cognitive deficits found in Alzheimer's disease. Human neural stem cells (hNSCs) may provide an effective cell source for replacing degenerating cholinergic neurons in AD brain since they are able to differentiate into various brain cell types in vitro. To see if in vitro expanded hNSCs are able to migrate and to replace the degenerating cholinergic neurons in vivo, we will transplant these hNSCs into the cerebroventricles of a rat AD model with selective cholinergic lesions in the nucleus basalismagnocellularis (nBM).

Design: 192-IgG-saporin (Sap) is an immunotoxinthat targets cholinergic neurons in the basal forebrain. Administration of Sap to the basal cholinergic system can result in selective cholinergic apoptosis that mimics AD-associated neuropathological and behavioral changes. We have established a rat model with selective cholinergic lesions by injecting Sap into nBM, and then transplanted these hNSCs into the cerebroventricle of this rat model to investigate if hNSCs can migrate to the lesioned sites and differentiate into cholinergic neurons.

Materials and Methods: Selective cholinergic lesion: Under deep anesthesia with sodium pentobarbital (SPB), 4-month old male Sprague-Dawley rats were mounted in a Kopf apparatus. 1ug of Sap dissolved in 1ul of sterilized saline was injected into the left nBM using a Hamilton microsyringe.

hNSC transplantation: Fetal hNSCs were expanded in a serum-free culture medium containing bFGF and EGF. Before transplantation, hNSCs were incubated with 1ug BrdU for 48 hr to label the nuclei in order to distinguish them from the host cells. One day after Sap injections, rats were deeply anesthesized with SPB and mounted in a Kopf apparatus. Rats were injected with 100,000 hNSCs into the right cerebroventricles with cholinergic lesions of the left nBM.

Immunofluorescent histochemistry: Rats transplanted with hNSCs were sacrificed by an overdose of SPB and perfused with 4% paraformaldehyde (PFA) 4 weeks after hNSC transplantation. Brains were removed, placed into 20% sucrose in 4% PFA overnight, and sliced into 30um coronal sections using a cryo-microtome. The sections were subjected to double-immunofluorescent staining using rat anti-BrdU or mouse anti-human nuclei, combined with mouse anti-NeuN or goat anti-choline acetyltransferase or mouse anti-GFAP antibodies. Secondary antibodies conjugated to rhodamine or FITC were used to recognize their corresponding primary antibodies. DAPI were used for nuclei counterstaining. Fluorescent microscopy was used to capture these images.

Results: Our results showed that a small dose of Sap (1ug/1ul) produced selective cholinergic lesions without significant tissue damage. Under this condition, subpopulations of transplanted hNSCs migrated over the total distance through the cerebroventricle into the lesioned nBM areas and differentiated into ChAT-positive, NeuN-positive cells, and GFAP-positive astrocytes, respectively.

Conclusion: hNSCs transplanted into the cerebroventicles of a rat model with selective basal cholinergic lesions can migrate to the lesioned areas and can differentiate into cholinergic cells.

Back to PD Thursday Poster Sessions
Back to The Eleventh International Congress