Models of Pluripotent and Somatic Stem Cells to Study Tissue-Specific Sensitivities in Fanconi Anemia

The Fanconi Anemia (FA) DNA Repair pathway functions through homologous recombination for error-free repair of DNA interstrand crosslinks. Loss of function of this pathway causes a complex genetic disease that is characterized by congenital abnormalities, bone marrow failure (BMF), and extreme incidence of squamous cell carcinomas. BMF is caused by exhaustion of hematopoietic stem and progenitor cells (HSPCs) and is nearly 100% penetrant by age 40 in FA patients, indicating a profound sensitivity of HSPCs to FA pathway deficiency. In contrast, stem cells in other rapidly regenerating tissues, such as the skin and intestine, are not similarly exhausted. Interestingly, squamous epithelium is highly prone to transformation while intestinal epithelium is not. In order to explore the developmental origins of such striking tissue-specific phenotypes in FA patients, we have generated induced pluripotent stem cell (iPSC) lines conditional for FA pathway function (cFA-iPSCs) and used them to derive FA-proficient and deficient in vitro models of diverse tissues.

FA patient cells are refractory to reprogramming. To circumvent this defect and prevent the selection of FA-resistant iPSC clones, fibroblasts from 2 FANCA patients were inducibly complemented with a FANCA transgene under the control of a tetracycline-inducible promoter and then were reprogrammed to iPSC. In this way, the FA pathway was functional throughout reprogramming and could then be turned on or off in established iPSC lines by the addition or withdrawal of doxycycline (DOX) to the media. Here, we describe the effect of FA pathway loss on iPSCs, and present preliminary data on iPSC-derived equivalents of three lineages: hematopoietic, squamous, and intestinal.

First, functional consequences of FA pathway loss on iPSC pluripotency and self-renewal were examined. Upon withdrawal of DOX from the culture media, the complementing FA transgene was effectively silenced, resulting in loss of FA pathway function within 7 days. FA-deficient iPSCs maintained normal expression of OCT-3/4 and NANOG and formed teratomas in NSG mice, indicating that pluripotency was maintained. However, profound cell cycle arrest and apoptosis were observed under normal in vitro culture conditions within 7 days of DOX-withdrawal, and the iPSCs failed to expand by 2-3 passages. Thus, we concluded that iPSCs require an intact FA pathway for self-renewal in vitro. Mechanistic studies of FA pathway-deficient iPSCs revealed a 10-fold increase in gH2AX foci in the G2-M phase of the cell cycle. This correlated with activated DNA damage response signaling through ATR and CHK1. Inhibition of CHK1 completely restored the growth of FA-deficient iPSCs to that of their FA-proficient counterparts through a remarkable rapid bypass of the G2-M checkpoint. Unexpectedly, cells maintained in CHK1 inhibitor for over 40days accrued few karypotypic abnormalities (<5% of cells), of which most were trisomies, and only 1 cell out of 40 contained translocations. The rarity of deletions and translocations in CHK1 inhibited iPSC suggests that error-free repair still occurs by an unknown mechanism.

We next differentiated the cFA-iPSCs into 3D squamous epithelium and intestine with timed-withdrawal of the FA pathway to determine the effect of FA pathway loss on tissue development and homeostasis. Squamous epithelial rafts and intestinal organoids were generated in the presence and absence of DOX using established protocols. These demonstrated that FA pathway loss does not cause gross abnormalities in epithelial tissues, in line with patient phenotypes. We will present the latest results on proliferation, survival, and differentiation of stem and progenitor cells within each tissue. These will include an assessment of epithelial hyperplasia, which has been observed previously in immortalized FA patient keratinocyte-derived organotypic squamous epithelial rafts and in the oral epithelia of FANCD2 knockout mice. Finally, we will present our latest data on the effects of FA deficiency on hematopoietic progenitor cells, which are currently being generated from the cFA-iPSCs. Collectively, these experiments will quantify cell intrinsic sensitivity of pluripotent versus somatic stem cells that reside in diverse tissue types to loss of the FA pathway. The results may inform the development of novel therapeutics to treat FA BMF without increasing disease risk in other tissues.