Pluripotency tests included flow cytometry analysis

Pluripotency tests included flow cytometry analysis of protein stem cell markers SSEA3, SSEA4, Tra1-60, Tra1-81 and Oct3/4 (Fig. 2A), alkaline phosphatase staining (ALP) (Fig. 2B), and qRT-PCR analysis of endogenous pluripotent transcription factors OCT-4, SOX-2, NANOG and REX1 (Fig. 2C). To finally confirm pluripotency, BSS3-PBMC-iPS4F8 orthopoxvirus variola were in vitro spontaneously differentiated into the three germ layers by embryoid body (EB) formation. Fig. 2D shows expression of representative markers of ectoderm (β3-Tubulin), mesoderm (Vimentin) and endoderm (CK AE1-AE3) in BSS3-PBMC-iPS4F8-derived EBs.
Materials and methods
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Resource table Resource details Spinal and bulbar muscular atrophy (SBMA) is a neuromuscular disorder caused by the expansion of the CAG repeat region in the first exon of the Androgen Receptor gene (AR) (Spada et al., 1991). Healthy individuals have about 9–36 CAG repeats while SBMA patients have greater than 38 CAG repeats (Andrew et al., 1997). SBMA is characterized by the selective degeneration of lower motor neurons within the spinal cord and brainstem which results in gradual weakness in the bulbar and limb muscles. The disease mechanisms underlying SBMA are not fully understood. iPSC lines from SBMA patients would serve as excellent tools to model and understand SBMA disease mechanism. In addition, iPSC lines from healthy siblings of the SBMA patients could be used as appropriate wild-type controls with similar genetic background. We reprogrammed peripheral blood mononuclear cells (PBMCs) from two SBMA patients and their respective healthy sibling. We also generated iPSC line from a healthy individual who is not related to the SBMA patients and could serve as a non-related healthy control. Clinical information of the PBMC donors is summarized in Table 1. The PBMCs were reprogrammed to iPSCs using a protocol described previously (Seki et al., 2012) with modifications. Firstly, PBMCs were cultured over a period of five days in the presence of IL-2 to activate the T-cells to form cell clusters (Fig. 1A, Day 0–Day 5). At Day 5, the T-cells were infected with Sendai virus carrying transgenes for the four Yamanaka factors. 48h post-infection, the T-cells were replated onto MEF feeder layer. As early as seven days post-infection, small colonies were observed (Fig. 1A, Day 12). By 25days post-viral infection, tightly packed pluripotent colonies were formed (Fig. 1A, Day 25). Selected colonies were transferred onto MEF feeder layer (Fig. 1A, Day 26) and expanded further (Fig. 1A, Day 47). iPSC colonies were subsequently adapted to Matrigel (Fig. 1A, Day 54) and maintained in feeder-free conditions (Fig. 1A, Day 68). All lines were named according to the guidelines proposed in (Luong et al., 2011). The wild-type line GIS2014-WT2C15 and SBMA line GIS2014-SB2C20 were generated from siblings. The wild-type line GIS2014-WT3C16 and SBMA line GIS2014-SB3C16 were generated from siblings. The wild-type line GIS2014-WT4C10 was generated from a healthy individual who is not related to the SBMA patients. All iPSC lines express pluripotency markers including SOX2, OCT3/4 and NANOG at the protein (Fig. 1B) and mRNA level (Fig. 1C). Embryoid bodies (EBs) generated from all the iPSC lines differentiated into the three germ layers, mesoderm, endoderm and ectoderm (Fig. 1D). Sequencing of the AR CAG repeat region ensured that the number of CAG repeats were unaltered during reprogramming (Fig. 1E). Furthermore, all iPSC lines have normal male karyotypes (Fig. 1F), did not retain the Sendai virus transgenes (Fig. S1A), and are free of mycoplasma (Fig. S1B). DNA fingerprinting of 24 genomic loci including 22 short tandem repeat (STR) loci confirmed that each iPSC line was derived from its respective patient PBMC sample (Table S2).
Materials and methods
Author contributions
Conflict of interest