Generation of an induced pluripotent stem cell line that mimics the disease phenotypes from a patient with Fanconi anemia by conditional complementation

Abstract

Generation of Fanconi anemia (FA) patient-specific induced pluripotent stem cells (iPSCs) has been reported to be technically challenging due to the defects in the FA-pathway in the patients' somatic cells. By inducible complementation of FA-pathway, we successfully reprogrammed the fibroblasts of an FA patient to iPSCs. CSCR19i-indCFANCA, one of the iPSC lines generated by the inducible complementation of FA-pathway, was extensively characterized for its pluripotency and karyotype. In the absence of doxycycline (DOX) and FANCA expression, this line showed the cellular phenotypes of FA, suggesting it is an excellent tool for FA disease modeling and drug screening.

Efficient gene editing in induced pluripotent stem cells enabled by an inducible adenine base editor with tunable expression

“…To overcome this challenge of generating isogenic wild-type and mutant iPSCs for recapitulating FA cellular phenotypes, we attempted base-editing to create mutations in the FANCA gene in AAVS1-iABE8e iPSCs. It has been reported earlier that iPSCs with FANCA homozygous mutations ( FANCA -/-) exhibit severe cell death, G 2 /M cell cycle phase arrest and loss of FANCD2 ubiquitination 50 – 52 . We performed ABE in the Dox-treated and untreated AAVS1-iABE8e iPSCs to create the FANCA L1082P mutation, which has been previously reported in some FA patients 53 (Fig.…”

“…6 A). Subsequently, we examined the FANCA homozygous mutant iPSCs for the previously described cellular phenotypes 50 – 52 .

Figure 6 Recapitulation of Fanconi anemia (FA) cellular phenotypes in FANCA base edited in AAVS1-iABE8e iPSCs.

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Modeling blood diseases with human induced pluripotent stem cells

“…Inherited bone marrow failure syndromes (IBMFS) are another group of inherited blood disorders that are characterized by the decreased production of mature blood cells of one or more lineages, often with a predisposition to leukemia development. iPSC models of several IBMFS have been created, including Fanconi anemia (FA) (Bharathan et al, 2017; Chlon et al, 2014; Liu et al, 2014; Muller et al, 2012; Raya et al, 2009; Rio et al, 2014; Suzuki et al, 2015; Yung et al, 2013; Navarro et al, 2014), Shwachman Diamond syndrome (Tulpule et al, 2013), Diamond Blackfan anemia (DBA) (Doulatov et al, 2017; Garcon et al, 2013), dyskeratosis congenita (DC) (Agarwal et al, 2010; Batista et al, 2011; Gu et al, 2015; Jose et al, 2018), amegakaryocytic thrombocytopenia (Hirata et al, 2013) and severe congenital neutropenia (Morishima et al, 2014; Nayak et al, 2015; Pittermann et al, 2017; Hiramoto et al, 2013). Because IBMFS are characterized by a decline in the number and function of HSCs, iPSC-based cell therapy approaches, bypassing the need to harvest a patient's own HSCs, could hold promise for the future treatment of these diseases.…”

“…FA patients are susceptible to the development of various cancers. Efforts to reprogram somatic cells from FA patients revealed that FA cells are highly refractory to reprogramming (Bharathan et al, 2017; Chlon et al, 2014; Raya et al, 2009; Rio et al, 2014; Yung et al, 2013; Muller et al, 2012). This is perhaps the most dramatic example of a human disease-related pathway with such a profound effect on the reprogramming ability of the cells.…”

“…This is perhaps the most dramatic example of a human disease-related pathway with such a profound effect on the reprogramming ability of the cells. In most studies, reprogramming was only possible after lentiviral gene complementation of FANCA or FANCD2 in the patient fibroblasts (Bharathan et al, 2017; Chlon et al, 2014; Muller et al, 2012; Raya et al, 2009; Rio et al, 2014). In cases in which iPSCs could be generated from uncorrected FA cells, the efficiency was very low and some, but not all, of these iPSCs had abnormal karyotypes or other genome instability marks (Muller et al, 2012; Liu et al, 2014).…”