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Materials and methods
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Resource details Fanconi anemia (FA) is caused by genetic defects in the Fanconi anemia pathway (FA-pathway), which is involved in DNA inter-strand cross-link repair. The disease is characterized by progressive bone marrow failure with a significant number of patients developing additional complications including leukemia, myelodysplastic syndrome and solid tumours. Somatic (R)-PFI 2 hydrochloride from FA patients are highly refractory to reprogramming, and restoration of the functional FA-pathway was found to be essential for the generation of FA-iPSC lines with normal karyotypes (Chlon et al., 2016; Müller et al., 2012; Raya et al., 2009; Yung et al., 2013). For the derivation of an FA patient-specific hiPSC line capable of mimicking the disease phenotypes in vitro, we used inducible complementation strategy. Dermal fibroblasts were obtained from an individual diagnosed to have FA based on clinical features and a very high number of chromosome breakages in the peripheral blood lymphocytes after treatment with Mitomycin C. Complementation analysis (Casado et al., 2007; Chandra et al., 2005; Pinto et al., 2009) was performed by transducing the fibroblasts with a DOX-inducible lentiviral vector encoding FANCA protein (pINDUCER20-FANCA) (Fig. 1A), followed by western blot analysis for FANCD2 ubiquitination. Restoration of FANCD2 ubiquitination in these fibroblasts cultured in the presence of DOX, suggested that FANCA gene was defective in this patient. We reprogrammed the pINDUCER20-FANCA transduced fibroblasts using Sendai Viruses to express the reprogramming factors along with FANCA complementation in the presence of DOX (Fig. 1A). Six iPSC colonies were isolated based on the morphology and cultured in the presence of DOX. One of the iPSC lines that was extensively cultured for more than 40 passages to establish the inducible complementation iPSC line, CSCR19i-indCFANCA, is described in this article (Fig. 1B). This iPSC line expressed pluripotency markers at levels comparable to a control iPSC line, BC1-hiPSC line (Fig. 1C–D), could form teratomas with three germ layers (Fig. 1E) and had a normal karyotype (Fig. 1F). On DOX withdrawal, this cell line showed the absence of FANCA expression (Fig. 1G) and the features of FA cells, i.e. the lack of FANCD2 ubiquitination (Fig. 1G), lack of γ H2A.X FANCD2 colocalization on DNA damage sites (Fig. 1H), and cell cycle arrest at G2/M phase (Fig. 1I) leading to cell death and progressive exhaustion in culture (Fig. 1J). DNA fingerprinting analysis confirmed the genetic identify of this iPSC line and donor fibroblasts (Supplementary Fig. 1A). Cellular phenotype analysis of all the six clones showed the phenotypes depicted in Fig. 1G–J. Our results showed that the CSCR19i-indCFANCA line and the haematopoietic cells derived from this line, in the presence and the absence of DOX, can be used for understanding FA disease mechanisms.
Materials and methods
Acknowledgement We acknowledge the research funding from Department of Biotechnology, Ministry of Science and Technology, India (grant numbers BT/01/COE/08/03 and BT/PR17316/MED/31/326/2015).
Introduction The inaccessibility to collect oligodendrocytes from patients and the limited access to fetal tissue has until recently hampered studies on neurodegenerative diseases. To date, it is still not possible to obtain oligodendrocytes from patients along disease progression in order to perform mechanistic studies. Assessment of postmortem samples is difficult as adaptive processes are already initiated. In fact, there is a lack of knowledge regarding to the oligodendrocyte biology at the initiation and during the progression of brain diseases. The use of embryonic stem cells (ESCs) and the discovery of induced pluripotent stem cells (iPSCs) (Takahashi et al., 2007) together with the improvement of reprogramming technologies, more recently the direct conversion of both oligodendrocyte progenitor cells (OPC) and oligodendrocytes from fibroblasts (Najm et al., 2013; Yang et al., 2013), now offer an unprecedented opportunity to explore the functional ramifications of a particular genotype in a cell type-specific manner and open doors to large scale drug screening and cell replacement therapy. Nowadays, mouse ESCs (mESC) are used to study in vitro development of cell-therapies and developing drug assays, with the advantage that several pathways of the OPCs commitment lineage are highly conserved between mice and human. In this way, the insights obtained from the mouse developmental biology could be applied to human oligodendrocytes (Murry and Keller, 2008). Yet, the existent protocols for generating OPCs from mESC are not very efficient to produce high yield cultures, and consequently the potential of mESC has not been fully exploited.