Research PaperCellular and molecular mechanisms of the restoration of human APP transgenic mouse cognitive dysfunction after transplant of human iPS cell-derived neural cells
Introduction
Cholinergic neurons and acetylcholine play important roles in learning and memory functions. Cholinergic neuron activity and acetylcholine production are downregulated in patients with Alzheimer's disease (AD) (Davis et al., 1999), and downregulation of alpha7 nicotinic acetylcholine receptors (α7nAChRs) has been reported as one of the hallmarks of AD (Court et al., 2001). Immunohistochemical studies demonstrated that GABAergic neuronal deficits were more severe in AD patients than previously estimated (Schwab et al., 2013). Furthermore, the expressions of several subunits of GABAA receptor (GABAR) were shown to be frequently altered in AD (Rissman and Mobley, 2011).
It has been shown that cell transplantation improved learning and memory function in AD models (Babaei et al., 2012, Bissonnette et al., 2011, Blurton-Jones et al., 2009, Ma et al., 2013, Wang et al., 2006). We have previously reported that transplantation of human induced pluripotent stem (hiPS) cell-derived neurons improved cognitive deficits of PDGF promoter-driven amyloid precursor protein (PDAPP) transgenic dementia model mice (Fujiwara et al., 2013). Choline acetyltransferase (ChAT)-positive cholinergic neurons derived from hiPS cells were shown to be able to survive in the PDAPP mouse hippocampus (Fujiwara et al., 2013).
Here, we transplanted hiPS cell-derived neurons into the hippocampus of PDAPP mice. Cognitive function of the mouse improved significantly by transplantation, confirming our previous finding. After transplantation, we investigated the localization of ChAT, vesicular GABA transporter (VGAT), α7nAChR, and GABAR-expressing cells as well as the activation status of their associated pathways in the brain of dementia model mice.
Section snippets
Cell culture and neural differentiation
The hiPS cell lines, 201B7 and 253G1 (Nakagawa et al., 2008), were provided by RIKEN BRC through the National Bio-Resource Project of the MEXT, Japan, and were used in this study. Because both cell lines demonstrated essentially the same results in this study, only the results obtained using 253G1 (reprogrammed by Oct3/4, Sox2, and Klf4) were presented. The hiPS cell lines were maintained according to the RIKEN cell preparation manual. Cells were cultured in differentiation medium consisting of
Induction of hiPS cell differentiation into cholinergic and GABAergic neurons
Neural cells derived from hiPS cells and control hiPS cells at day 5 (before the addition of RA, NOG and SHH) were stained simultaneously (Fig. 1A). Cells at days 8 (1 day after the second addition of RA, NOG, and SHH) expressed several neural markers, such as nestin (a neural stem/progenitor cell marker), βIII-tubulin (panneuron marker), ChAT (cholinergic neurons), and VGAT (GABAergic neurons). The cells expressed nestin by day 8 (93.6 ± 1.0%: mean ± s.e.m. from three independent experiments, Fig.
Discussion
In the present study we observed that human iPS cell-derived neural cells grafted to dementia model mice differentiated into both ChAT-positive cholinergic neurons and VGAT-positive GABAergic neurons in the cortex and hippocampus. The recipient neurons expressing receptors for the neurotransmitters emerged after transplantation. It is possible that both acetyl choline/α7nAChR interaction and GABA/GABAR interaction in both the cortex and hippocampus may have led to the alleviation of cognitive
Conclusion
The grafted human cells differentiated into both ChAT-positive cholinergic neurons and VGAT-positive GABAergic neurons in the cortex and hippocampus. Recipient neurons expressing receptors for the neurotransmitters emerged after transplantation. The restoration of both acetyl choline/α7nAChR interaction and GABA/GABAR interaction in both the cortex and hippocampus may have led to the alleviation of cognitive dysfunction in the dementia model mice.
Acknowledgments
This study was supported in part by a SRF grant and JSPS KAKENHI Grant Number 25505006. The authors declare no competing financial interest.
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