Incurable neurological disorders such as Parkinson’s disease (PD), Huntington’s disease (HD), and Alzheimer’s disease (AD) are very common and can be life-threatening because of their progressive disease symptoms with limited treatment options. To provide an alternative renewable cell source for cell-based transplantation and as study models for neurological diseases, we generated induced pluripotent stem cells (iPSCs) from human dermal fibroblasts (HDFs) and then differentiated them into neural progenitor cells (NPCs) and mature neurons by dual SMAD signaling inhibitors. Reprogramming efficiency was improved by supplementing the histone deacethylase inhibitor, valproic acid (VPA), and inhibitor of p160-Rho associated coiled-coil kinase (ROCK), Y-27632, after retroviral transduction. We obtained a number of iPS colonies that shared similar characteristics with human embryonic stem cells in terms of their morphology, cell surface antigens, pluripotency-associated gene and protein expressions as well as their in
vitro and in
vivo differentiation potentials. After treatment with Noggin and SB431542, inhibitors of the SMAD signaling pathway, HDF-iPSCs demonstrated rapid and efficient differentiation into neural lineages. Six days after neural induction, neuroepithelial cells (NEPCs) were observed in the adherent monolayer culture, which had the ability to differentiate further into NPCs and neurons, as characterized by their morphology and the expression of neuron-specific transcripts and proteins. We propose that our study may be applied to generate neurological disease patient-specific iPSCs allowing better understanding of disease pathogenesis and drug sensitivity assays.
Cholangiocarcinoma (CCA) is a malignancy of the bile duct epithelium which is caused by liver fluke infection. The clinical symptoms of CCA were revealed as the disease progresses to advanced stage. Thus, specific diagnostic biomarkers are important for this fatal disease. We applied methylation-sensitive high-resolution melting (MS-HRM) to quantify DNA methylation levels of opioid binding protein/cell adhesion molecule-like gene (OPCML) and Secreted frizzled-related protein 1 (SFRP1) in 73 primary CCA and 10 adjacent normal tissues and evaluated the sensitivity, specificity, and accuracy of the assay. The median methylation level of OPCML in CCA was 38.7 % (ranged from 0 to 82.2 %) and of SFRP1 was 31.5 % (ranged from 0 to 86.2 %). Methylation cutoff values of OPCML and SFRP1 derived from adjacent normal tissue were 6.90 and 10.44 %, respectively. With these cutoff values, the area under curve (AUC) of OPCML was 0.932 (95 % CI 0.878-0.986) and of SFRP1 was 0.951 (95 % CI 0.905-0.996). The sensitivity, specificity, and accuracy of OPCML were 89.04, 100, and 90.36 %, respectively, and of SFRP1 were 83.56, 100, and 85.54 %, respectively. In conclusion, the DNA methylation levels of OPCML and SFRP1 could be potential biomarkers for diagnosis of CCA with high specificity, sensitivity, and accuracy, in particular for biopsy specimens. Further validation in noninvasive samples such as serum or plasma is warranted for clinical applicability, especially as early diagnostic biomarkers.
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