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Effect of ciliary neurotrophic factor on neural differentiation of stem cells of human exfoliated deciduous teeth.

ABSTRACT: The stem cells of human exfoliated deciduous teeth (SHEDs) are considered to be one of the main sources of seed cells in stem cell therapy. The aim of this study was to examine the effect of ciliary neurotrophic factor (CNTF) on neurogenic differentiation of SHEDs. With the consent of parents, SHEDs from 6 to 8?year old children were isolated and cultured. The mesenchymal stemness and the potential of multidirectional (adipogenic and osteogenic) differentiation for the isolated SHEDs were firstly determined. The effect of CNTF on specific neurogenic differentiation of SHEDs was then examined by detecting the expression of marker genes and proteins via RT-PCR, immunoblotting, and immunofluorescence microscopy. The isolated SHEDs expressed specific surface markers of mesenchymal stem cells, and their potential of osteogenic and adipogenic differentiation were confirmed. CNTF promoted the differentiation of SHEDs into neuron-like cells with a high expression of acetylcholine transferase (CHAT), a marker of cholinergic neurons. The expression of other neuron markers including nestin, microtubule-associated protein 2 (MAP 2), and ?-tublin III was also detected. Interestingly, the expression of neurogenic markers was maintained at a high level after neurogenic induction. SHEDs can be induced by CNTF to differentiate into cholinergic neuron-like cells under appropriate culture conditions. Our findings have laid a foundation for future use of SHEDs to treat neurological diseases.

SUBMITTER: Zeng S

PROVIDER: S-EPMC7724848 | biostudies-literature | 2020 Dec

REPOSITORIES: biostudies-literature

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Effect of ciliary neurotrophic factor on neural differentiation of stem cells of human exfoliated deciduous teeth.

Zeng Sujuan S Zhao Xuedan X Zhang Lingling L Pathak Janak L JL Huang Wenyan W Li Yunyang Y Guan Hongbing H Zhao Wanghong W Ge Lihong L Shu Yan Y

Journal of biological engineering 20201209 1

The stem cells of human exfoliated deciduous teeth (SHEDs) are considered to be one of the main sources of seed cells in stem cell therapy. The aim of this study was to examine the effect of ciliary neurotrophic factor (CNTF) on neurogenic differentiation of SHEDs. With the consent of parents, SHEDs from 6 to 8 year old children were isolated and cultured. The mesenchymal stemness and the potential of multidirectional (adipogenic and osteogenic) differentiation for the isolated SHEDs were firstl ...[more]

PMID: 33298129

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Project description:BACKGROUND:Basic fibroblast growth factor (bFGF) regulates maintenance of stemness and modulation of osteo/odontogenic differentiation and mineralization in stem cells from human exfoliated deciduous teeth (SHEDs). Mineralization in the bones and teeth is in part controlled by pericellular levels of inorganic phosphate (Pi), a component of hydroxyapatite, and inorganic pyrophosphate (PPi), an inhibitor of mineralization. The progressive ankylosis protein (gene ANKH; protein ANKH) and ectonucleotide pyrophosphatase phosphodiesterase 1 (ENPP1/ENPP1) increase PPi and inhibit mineralization, while tissue-nonspecific alkaline phosphatase (ALPL; TNAP) is a critical pro-mineralization enzyme that hydrolyzes PPi. We hypothesized that regulation by bFGF of mineralization in SHEDs occurs by modulation of Pi/PPi-associated genes. METHODS:Cells were isolated from human exfoliated deciduous teeth and characterized for mesenchymal stem cell characteristics. Cells were treated with bFGF, and the osteogenic differentiation ability was determined. The mRNA expression was evaluated using real-time polymerase chain reaction. The mineralization was examined using alizarin red S staining. RESULTS:Cells isolated from primary teeth expressed mesenchymal stem cell markers, CD44, CD90, and CD105, and were able to differentiate into osteo/odontogenic and adipogenic lineages. Addition of 10?ng/ml bFGF to SHEDs during in vitro osteo/odontogenic differentiation decreased ALPL mRNA expression and ALP enzyme activity, increased ANKH mRNA, and decreased both Pi/PPi ratio and mineral deposition. Effects of bFGF on ALPL and ANKH expression were detected within 24?h. Addition of 20?mM fibroblast growth factor receptor (FGFR) inhibitor SU5402 revealed the necessity of FGFR-mediated signaling, and inclusion of 1??g/ml cyclohexamide (CHX) implicated the necessity of protein synthesis for effects on ALPL and ANKH. Addition of exogenous 10??m PPi inhibited mineralization and increased ANKH, collagen type 1a1 (COL1A1), and osteopontin (SPP1) mRNA, while addition of exogenous Pi increased mineralization and osterix (OSX), ANKH, SPP1, and dentin matrix protein 1 (DMP1) mRNA. The effects of PPi and Pi on mineralization could be replicated by short-term 3- and 7-day treatments, suggesting signaling effects in addition to physicochemical regulation of mineral deposition. CONCLUSION:This study reveals for the first time the effects of bFGF on Pi/PPi regulators in SHEDs and implicates these factors in how bFGF directs osteo/odontogenic differentiation and mineralization by these cells.

Project description:Retinal degeneration is characterized by the progressive loss of photoreceptors, and stem cell therapy has become a promising strategy. Many studies have reported that mesenchymal stem cell transplantation can sustain retinal structure and prolong retinal functions based on two mechanisms. One is cell replacement, and the other is the paracrine action of stem cells. Cells from human exfoliated deciduous teeth (SHEDs) show characteristics typical of mesenchymal stem cells. They are derived from the neural crest and are a potential cellular source for neural regeneration in stem cell therapy. In this study, we explored the potential of SHEDs to be induced towards the retinal photoreceptor phenotype and to be sustainable in an animal model of retinal degeneration. A factor-cocktail protocol was used to induce SHEDs towards retinal photoreceptors for 24 days, and the characteristics of the induced cells were identified in terms of morphological changes, biomarker expression and subcellular distribution, and calcium influx. SHEDs were labeled with firefly luciferase for in vivo tracking by bioluminescent imaging and then transplanted into the subretinal space of mice. Our results showed that SHEDs successfully transdifferentiated into photoreceptor-like cells, which displayed neuron-like morphology, and expressed specific genes and proteins associated with retinal precursors, photoreceptor precursors, and mature photoreceptors. In addition, calcium influx was significantly greater in the retinal-induced than in noninduced SHEDs. In vivo tracking confirmed at least 2 weeks of good survival by bioluminescent imaging and 3 months of sustainability of SHEDs by histological analysis. We conclude that SHEDs have the potential to transdifferentiate into retinal photoreceptor-like cells in vitro and maintain good viability in vivo after transplantation into mice with a normal immune system. This demonstrates preliminary success in generating photoreceptor-like cells from SHEDs and applying SHEDs in treating retinal degeneration.

Dataset Information

Effect of ciliary neurotrophic factor on neural differentiation of stem cells of human exfoliated deciduous teeth.

Publications

Effect of ciliary neurotrophic factor on neural differentiation of stem cells of human exfoliated deciduous teeth.

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