Dataset Information

Establishment of transgene-free induced pluripotent stem cells reprogrammed from human stem cells of apical papilla for neural differentiation.

ABSTRACT: Induced pluripotent stem cells (iPSCs) are a potent cell source for neurogenesis. Previously we have generated iPSCs from human dental stem cells carrying transgene vectors. These exogenous transgenes may affect iPSC behaviors and limit their clinical applications. The purpose of this study was to establish transgene-free iPSCs (TF-iPSCs) reprogrammed from human stem cells of apical papilla (SCAP) and determine their neurogenic potential.A single lentiviral 'stem cell cassette' flanked by the loxP site (hSTEMCCA-loxP), encoding four human reprogramming factors, OCT4, SOX2, KLF4, and c-MYC, was used to reprogram human SCAP into iPSCs. Generated iPSCs were transfected with plasmid pHAGE2-EF1?-Cre-IRES-PuroR and selected with puromycin for the TF-iPSC subclones. PCR was performed to confirm the excision of hSTEMCCA. TF-iPSC clones did not resist to puromycin treatment indicating no pHAGE2-EF1?-Cre-IRES-PuroR integration into the genome. In vitro and in vivo analyses of their pluripotency were performed. Embryoid body-mediated neural differentiation was undertaken to verify their neurogenic potential.TF-SCAP iPSCs were generated via a hSTEMCCA-loxP/Cre system. PCR of genomic DNA confirmed transgene excision and puromycin treatment verified the lack of pHAGE2-EF1?-Cre-IRES-PuroR integration. Transplantation of the TF-iPSCs into immunodeficient mice gave rise to teratomas containing tissues representing the three germ layers -- ectoderm (neural rosettes), mesoderm (cartilage and bone tissues) and endoderm (glandular epithelial tissues). Embryonic stem cell-associated markers TRA-1-60, TRA-2-49 and OCT4 remained positive after transgene excision. After neurogenic differentiation, cells showed neural-like morphology expressing neural markers nestin, ?III-tubulin, NFM, NSE, NeuN, GRM1, NR1 and CNPase.TF-SCAP iPSCs reprogrammed from SCAP can be generated and they may be a good cell source for neurogenesis.

SUBMITTER: Zou XY

PROVIDER: S-EPMC3580434 | biostudies-literature | 2012 Oct

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Establishment of transgene-free induced pluripotent stem cells reprogrammed from human stem cells of apical papilla for neural differentiation.

Zou Xiao-Ying XY Yang Hsiao-Ying HY Yu Zongdong Z Tan Xiao-Bing XB Yan Xing X Huang George T-J GT

Stem cell research & therapy 20121024 5

<h4>Introduction</h4>Induced pluripotent stem cells (iPSCs) are a potent cell source for neurogenesis. Previously we have generated iPSCs from human dental stem cells carrying transgene vectors. These exogenous transgenes may affect iPSC behaviors and limit their clinical applications. The purpose of this study was to establish transgene-free iPSCs (TF-iPSCs) reprogrammed from human stem cells of apical papilla (SCAP) and determine their neurogenic potential.<h4>Methods</h4>A single lentiviral ' ...[more]

PMID: 23095454

Similar Datasets

Project description:IntroductionStem cell transplantation of exogenous neural progenitor cells (NPCs) derived from mesenchymal stem cells (MSCs) has emerged as a promising approach for neurodegenerative disease. Human stem cells from apical papilla (hSCAPs) are derived from migratory neural crest stem cells and exhibit a potential of neuronal differentiation. However, their neuronal differentiation is low and unpredictable. Resveratrol has been described as a sirtuin 1 (SIRT1) activator which plays an important role in enhancing neuronal differentiation. In this study, we investigate the potential of resveratrol as an enhancer on neuronal differentiation through NPCs induction of hSCAPs.MethodsStem cells were isolated from human apical papilla and characterized as MSCs. The cellular toxicity of resveratrol treatment to the characterized hSCAPs was investigated by MTT assay. The non-cellular toxicity concentrations of resveratrol were assessed with various pre-treatment times to select the optimal condition that highly expressed the neural progenitor gene, NES. Consequently, the optimal condition of resveratrol pre-treatment was synergistically performed with a neuronal induction medium to trigger neuronal differentiation. The differentiated cells were visualized, the genes profiling was quantified, and the percentage of neuronal differentiation was calculated. Moreover, the intracellular calcium oscillation was demonstrated.ResultsThe cellular toxicity of resveratrol was not observed for up to 50 μM for 12 h. Interestingly, hSCAPs pre-treated with 10 μM resveratrol for 12 h (RSV-hSCAPs) significantly expressed NES, which is determined as the optimal condition. Under neuronal induction, both of hSCAPs and RSV-hSCAPs were differentiated (d-hSCAPs and RSV-d-hSCAPs) as they exhibited neuronal-like appearances with Nissl substance staining. The highest expression of NES and SOX1 was observed in RSV-d-hSCAPs. Additionally, the percentage of neuronal differentiation of RSV-d-hSCAPs was significantly higher than d-hSCAPs for 4 times. Importantly, the neuronal-like cells exhibited slightly increasing pattern of calcium intensity.ConclusionThis study demonstrated that pre-treatment of resveratrol strongly induces neural progenitor marker gene expression which synergistically enhances neural progenitor-like cells' induction with neuronal induction medium.

Dataset Information

Establishment of transgene-free induced pluripotent stem cells reprogrammed from human stem cells of apical papilla for neural differentiation.

Publications

Establishment of transgene-free induced pluripotent stem cells reprogrammed from human stem cells of apical papilla for neural differentiation.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets