Dual Small-Molecule Targeting of SMAD Signaling Stimulates Human Induced Pluripotent Stem Cells toward Neural Lineages

Abstract

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.

One-step genetic correction of hemoglobin E/beta-thalassemia patient-derived iPSCs by the CRISPR/Cas9 system

“…Fibroblasts were subcultured once every 5 days or whenever they reached 80% confluency by incubation with 0.25% Trypsin for 2 min. Generation and characterization of Eβ-iPSCs from a HbE/β-thalassemic patient’s HDFs were performed as described previously [ 15 ]. iPSCs were maintained in mTeSR™1 medium (StemCell Technologies, Canada) on Matrigel™-coated (BD Bioscience, USA) plates and subcultured using 1 mg/ml Dispase (StemCell Technologies) according to the manufacturer’s instructions.…”

Genomic Alteration in Head and Neck Squamous Cell Carcinoma (HNSCC) Cell Lines Inferred from Karyotyping, Molecular Cytogenetics, and Array Comparative Genomic Hybridization

“…Dissociation melting curves, created by running a heat dissociation protocol after the PCR (81 cycles of 55.0°C– 95.0°C for 10 s, increase set point temperature by 0.5°C), confirmed the specific amplification of cDNA target and the absence of nonspecific products. The GAPDH gene was also used as the internal reference to normalize the expression levels between samples, using GAPDH_01_F (5′-GATAACGGATTTGGTCGTATTG-3′) and GAPDH_01_R (5′-CATGGGTGGAATCATATTGGAA-3′) primers [ 27 ]. Quantitative real time PCR reactions of all cell lines were performed in biological triplicates.…”

Noggin Over-Expressing Mouse Embryonic Fibroblasts and MS5 Stromal Cells Enhance Directed Differentiation of Dopaminergic Neurons from Human Embryonic Stem Cells

“…To improve efficiency, SMAD signaling has been targeted using the combination of noggin and SB431542 as a differentiation strategy [ 23 – 26 ]. The addition of noggin elevated the differentiation rates of neural lineages of hESCs [ 23 ] and hiPSCs [ 27 ]. We found that CHA13 and 15 hESCs did not form rosette-structure cells unlike H9 and HSF6 hESCs when cultured using a published stromal cell co-culture method [ 7 ].…”