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. 2023 Nov 26;12(23):2711.
doi: 10.3390/cells12232711.

Repeated Intravenous Administration of Human Neural Stem Cells Producing Choline Acetyltransferase Exerts Anti-Aging Effects in Male F344 Rats

Jangbeen Kyung  1 , Dajeong Kim  1 , Kyungha Shin  1 , Dongsun Park  2 , Soon-Cheol Hong  3 , Tae Myoung Kim  4 , Ehn-Kyoung Choi  4 , Yun-Bae Kim  1   4
Affiliations

Repeated Intravenous Administration of Human Neural Stem Cells Producing Choline Acetyltransferase Exerts Anti-Aging Effects in Male F344 Rats

Jangbeen Kyung et al. Cells. .

Abstract

Major features of aging might be progressive decreases in cognitive function and physical activity, in addition to withered appearance. Previously, we reported that the intracerebroventricular injection of human neural stem cells (NSCs named F3) encoded the choline acetyltransferase gene (F3.ChAT). The cells secreted acetylcholine and growth factors (GFs) and neurotrophic factors (NFs), thereby improving learning and memory function as well as the physical activity of aged animals. In this study, F344 rats (10 months old) were intravenously transplanted with F3 or F3.ChAT NSCs (1 × 106 cells) once a month to the 21st month of age. Their physical activity and cognitive function were investigated, and brain acetylcholine (ACh) and cholinergic and dopaminergic system markers were analyzed. Neuroprotective and neuroregenerative activities of stem cells were also confirmed by analyzing oxidative damages, neuronal skeletal protein, angiogenesis, brain and muscle weights, and proliferating host stem cells. Stem cells markedly improved both cognitive and physical functions, in parallel with the elevation in ACh levels in cerebrospinal fluid and muscles, in which F3.ChAT cells were more effective than F3 parental cells. Stem cell transplantation downregulated CCL11 and recovered GFs and NFs in the brain, leading to restoration of microtubule-associated protein 2 as well as functional markers of cholinergic and dopaminergic systems, along with neovascularization. Stem cells also restored muscular GFs and NFs, resulting in increased angiogenesis and muscle mass. In addition, stem cells enhanced antioxidative capacity, attenuating oxidative damage to the brain and muscles. The results indicate that NSCs encoding ChAT improve cognitive function and physical activity of aging animals by protecting and recovering functions of multiple organs, including cholinergic and dopaminergic systems, as well as muscles from oxidative injuries through secretion of ACh and GFs/NFs, increased antioxidant elements, and enhanced blood flow.

Keywords: acetylcholine; aging; choline acetyltransferase; cognitive function; growth factor; human neural stem cell; neurotrophic factor; physical activity.

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Conflict of interest statement

Authors Tae Myoung Kim, Ehn-Kyoung Choi, and Yun-Bae Kim were employed by the company Designed Cells Co., Ltd. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be constructed as a potential conflict of interest. The authors declare that this study received funding from National Research Foundation of Korea and Korea Health Industry Development Institute. The funders were not involved in the study design, collection, analysis, interpretation of data, the writing of this article or the decision to submit it for publication.

Figures

Figure 1
Figure 1
Locomotor activity (resting, slow-moving, and fast-moving times) ((A), n = 8–10/group) and rota-rod performance ((B), n = 8–10/group) of rats at 11–20 months of age. Black: young animals (7 weeks old); red: aged animals; green: aged animals transplanted with F3 cells (1 × 106 cells every month); blue: aged animals transplanted with F3.ChAT cells (1 × 106 cells every month). * Significantly different from young animals (p < 0.05). # Significantly different from aged animals (p < 0.05).
Figure 2
Figure 2
Passive avoidance (A) and Morris water maze (B) performances (n = 8–10/group) and acetylcholine levels (n = 10/group) in the brain (C) and muscles (D) at 21 months of age. Black: young animals (7 weeks old); red: aged animals; green: aged animals transplanted with F3 cells (1 × 106 cells every month); blue: aged animals transplanted with F3.ChAT cells (1 × 106 cells every month). * Significantly different from young animals (p < 0.05). # Significantly different from aged animals (p < 0.05). + Significantly different from F3 treatment (p < 0.05).
Figure 3
Figure 3
Distribution of the transplanted human (hMito-positive) F3 ((A,B), n = 8–10/group) and F3.ChAT ((A,C), n = 8–10/group) cells in the brain tissues. Sale bar = 20 μm.
Figure 4
Figure 4
Restorations of cholinergic ((A,C), n = 8–10/group) and dopaminergic ((B,D), n = 8–10/group) nervous systems following F3 or F3.ChAT cell transplantation. RT-PCR (A,B) and quantitative analyses (C,D) of the mRNA expression of cholinergic and dopaminergic components. Black: young animals (7 weeks old); red: aged animals; green: aged animals transplanted with F3 cells (1 × 106 cells every month); blue: aged animals transplanted with F3.ChAT cells (1 × 106 cells every month). ChAT: choline acetyltransferase. VAChT: vesicular acetylcholine transporter. ChT1: choline transporter 1, m1-AChR: muscarinic 1 acetylcholine receptor. nAChR: nicotinic acetylcholine receptor. AChE: acetylcholinesterase. GAPDH: glyceraldehyde-3-phosphate dehydrogenase. TH: tyrosine hydroxylase, VMAT2: vesicular monoamine transporter 2. DAT: dopamine transporter, DR: dopamine receptor. * Significantly different from young rats (p < 0.05). # Significantly different from aged rats (p < 0.05). + Significantly different from F3 treatment (p < 0.05).
Figure 5
Figure 5
Restoration of growth factors (GFs) and neurotrophic factors (NFs) in the brain ((A), n = 8–10/group) and muscles ((B), n = 8–10/group). Black: young animals (7 weeks old); red: aged animals; green: aged animals transplanted with F3 cells (1 × 106 cells every month); blue: aged animals transplanted with F3.ChAT cells (1 × 106 cells every month). BDNF: brain-derived neurotrophic factor. NGF: nerve growth factor. GDNF: glial cell-derived neurotrophic factor. VEGF: vascular endothelial growth factor. IGF-1: insulin-like growth factor-1. * Significantly different from young rats (p < 0.05). # Significantly different from aged rats (p < 0.05). + Significantly different from F3 treatment (p < 0.05).
Figure 6
Figure 6
Microvessel (vWF-positive) density in the brain ((A,B), n = 8–10/group) and muscles ((D,E), n = 8–10/group) and relative brain (C) and muscle (F) weights. Arrow: vWF-positive blood vessel. Sale bar = 50 μm. * Significantly different from young rats (p < 0.05). # Significantly different from aged rats (p < 0.05). + Significantly different from F3 treatment (p < 0.05).
Figure 7
Figure 7
Concentration of thiobarbituric acid-reactive substances (TBARS) in the brain ((A), n = 8–10/group) and muscles ((B), n = 8–10/group). * Significantly different from young rats (p < 0.05). # Significantly different from aged rats (p < 0.05).
Figure 8
Figure 8
Host neural stem cell (nestin-positive: red) regeneration and proliferation (Ki-67-positive: green) ((A,B), n = 5/group); CCL11 mRNA downregulation ((C,D), n = 8–10/group) and neuronal integrity (MAP2) restoration ((C,E), n = 8–10/group). (A): immunohistochemical staining, (B): RT-PCR analysis, (C): western blot analysis. Double-positive cells (yellow in Merge) in A indicate proliferating neural stem cells. Sale bar = 20 μm. * Significantly different from young rats (p < 0.05). # Significantly different from aged rats (p < 0.05). + Significantly different from F3 treatment (p < 0.05).
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