Abstract
Peripheral blood mononuclear cells (PBMCs) were collected from an 8-year old female patient affected by ischemic Moyamoya disease (MMD). Patient's PBMCs were reprogrammed using Sendai virus particles delivering the four Yamanaka factors. The footprint free hiPSC line expressed the major pluripotency markers and exhibited a normal karyotype. Cells were competent to give rise to progeny of differentiated cells belonging to the 3 germ layers. This hiPSC line represents a good tool to in vitro model MMD in order to shed light on the cellular and molecular mechanisms responsible for the occurrence of this syndrome.
Highlights
Peripheral blood mononuclear cells (PBMCs) were collected from an 8-year old female patient affected by ischemic Moyamoya disease (MMD)
Peripheral blood mononuclear cells (PBMCs) Biological reagent: induced pluripotent stem cell; derived from a sporadic ischemic Moyamoya disease (MMD) patient Induced pluripotent stem cells hOCT4, hSOX2, hc-Myc, hKLF4 (CytoTuneTM-iPS 2.0 Sendai Reprogramming Kit - Thermo Fisher Scientific) Identity and purity of the cell line was confirmed by the following tests: expression of pluripotency genes by
Blood sample was collected from an 8-year old female patient with Moyamoya disease (MMD; Papa Giovanni XXIII Hospital, Bergamo, Italy)
Summary
Blood sample was collected from an 8-year old female patient with Moyamoya disease (MMD; Papa Giovanni XXIII Hospital, Bergamo, Italy). One of the isolated clones gave rise to the MMD ISCH#6 iPSC line, characterized by homogeneous colonies, normal diploid 46, XX karyotype, without detectable abnormalities, and stable expression of the pluripotency markers OCT4, Nanog, SOX2 and TRA1-60, verified by both immunofluorescence, RT-PCR and western blot (Fig. 1A–C) (Itskovitz-Eldor et al, 2000; Carpenter et al, 2003). Absence of residual episomal Sendai virus was confirmed at passage 10 (Fusaki et al, 2009), suggesting that the expression of OCT4, SOX2, KLF4, and c-Myc was indicative of a successful complete reprogramming process (Fig. 1B). The differentiation competency of this cell line was equivalent to that observed for a commercial hiPSC line, as shown by the comparable expression levels of the 3 germ layers markers FGF5 (ectoderm), Nestin (neuro-ectoderm), T-Brachyury (mesoderm), SOX-17 (endoderm) assessed by qRT-PCR (Fig. 2B)
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