HDAC6 Inhibitors Rescued the Defective Axonal Mitochondrial Movement in Motor Neurons Derived from the Induced Pluripotent Stem Cells of Peripheral Neuropathy Patients with HSPB1 Mutation

Figure 1

Generation of CMT2F patient and dHMN2B patient-derived iPSCs. (a) Experimental timeline for iPSC generation. KOSR, KnockOut™ serum replacement; bFGF, basic fibroblast growth factor. (b) Morphology of fibroblasts from normal individual and patients (original magnification, 50x). Scale bars: 200 μm. (c) iPSC colonies showed ESC-like morphology, such as a flat cobblestone-like appearance with individual cells having a high nucleus-to-cytoplasm ratio (original magnification, 50x). Scale bars: 200 μm. (d) CMT2F-iPSCs and dHMN2B-iPSCs had preserved point mutation sites in the HSPB1 gene, verified by sequencing of RT-PCR products. (e) CMT2F-iPSCs and dHMN2B-iPSCs maintained normal karyotype. (f) Expression of total and endogenous Klf4, Oct3/4, Sox2, and c-Myc in CMT2F-iPSCs and dHMN2B-iPSCs was verified by RT-PCR. Two clones from each of the patients-derived iPSCs were tested (clone 1 and clone 2). (g) ESCs and iPSCs expressed stem cell markers such as NANOG (in the nucleus; original magnification, 200x) and SSEA4 (in the cytoplasm; original magnification, 100x). Scale bars: 200 μm. (h) EB-mediated in vitro spontaneous differentiation of ESCs and iPSCs resulted in the expression of three-germ-layer markers such as AFP (endoderm), SMA (mesoderm), and nestin (ectoderm; original magnification, 200x). Scale bars: 100 μm. (i) ESCs and iPSCs showed in vivo pluripotency by forming teratomas 8 weeks after subcutaneous injection into NOD/SCID mice. Teratomas consisted of various three-germ-layer tissues including columnar gland epithelial cells with secretions [Gl] for endodermal tissue, muscle [M] and cartilage with calcification [Ca] for mesodermal tissue, and neuroectodermal tissue [NE] and nerve axonal bundle [N] for ectodermal tissue (original magnification, 400x). Scale bars: 100 μm.

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