Abstract

Fanconi anemia (FA) is an inherited bone marrow failure syndrome characterized by loss of function mutations in any of at least 22 FANC genes that are critical for DNA repair. Progressive loss of hematopoietic stem/progenitor cells (HSPCs) in FA patients has been attributed in part to a heightened p53/p21 axis that restricts the cell cycle to G0/G1 in response to accumulation of unresolved DNA damage and replicative stress. Silencing of p53 was previously shown to rescue HSPC defects in FA models in vitro and in vivo. Due to insufficient numbers of primary HSPCs in FA for disease modeling and cell therapies, the concept of generating induced pluripotent stem cells (iPSCs) from an individual patient, and differentiating the FA-iPSCs into a theoretically infinite supply of autologous HSPCs arose. However, reprogramming FA cells has proven to be challenging and mostly dependent on hypoxic culture conditions or genetic complementation of the defect by integrating lentiviral vectors expressing a normal FANC gene. A recent study reported low-efficiency generation of integration-free FA-iPSCs by electroporation of fibroblasts with episomal plasmid vectors expressing both reprogramming factors and p53 shRNA. Here we sought to develop an improved and streamlined reprogramming strategy for FA by transduction of readily accessible FA mononuclear cells (MNCs) with commercially available non-integrating reprogramming Sendai Viruses (SeV2), combined with a more adaptable inhibition of the p53 pathway by addition of variable concentrations of a small molecule during reprogramming. Approximately 100,000 MNCs from an FA patient with confirmed biallelic mutations in FANCA were cultured for 10 days under hypoxic conditions (5% O2) favoring either myeloid or erythroid expansion. While myeloid cultures led to minimal proliferation of FA MNCs, we observed a 6-fold increase in total cell numbers in the presence of EPO. Erythroid-expanded FA cells were transduced with SeV2 harboring four standard reprogramming transcription factors (SOX2, OCT4, KLF4 and c-MYC). Cells were subsequently cultured under hypoxia for at least 30 days in a reprogramming cocktail supplemented or not with 0.3uM, 0.6µM or 1.2µM cyclic pifthrin-α (cPFT), a reversible small molecule inhibitor of p53-dependent gene transcription. In the absence of cPFT, no FA-iPSC clones were obtained, consistent with reported reprogramming barriers in FA cells. In contrast, several iPSC colonies were generated with cPFT-treated FA cells, albeit at much slower kinetics (~30 days) compared to MNCs without FA mutations (~20 days). The overall efficiency of reprogramming was ~0.01%, with a trend toward enhanced efficiencies in cultures containing the highest concentration of cPFT (1.2µM). To investigate the need for p53 inhibition beyond the reprogramming period, half of the FA-iPSC clones were passaged and further cultured in 5% O2 with cPFT 1.2µM, whereas the remaining clones were similarly treated but without p53 inhibitor. None of the FA-iPSC clones that were maintained in cPFT-containing cultures following reprogramming survived further passaging. In contrast, when cPFT was removed after reprogramming, all iPSC clones could stably undergo repeated passages provided that hypoxia (5% O2) was maintained. The FA-iPSC clones expressed high levels alkaline phosphatase and were TRA 1-60- and NANOG-positive, preliminarily validating their pluripotent nature. Karyotyping, trilineage differentiation, and in vivo teratoma assays are ongoing. Overall, this study provides a practical approach for successful reprogramming of FA peripheral blood cells based on commercially available non-integrating SeV2 reprogramming strategies supplemented with p53 inhibition. Regulated addition of variable concentrations of a small molecule inhibitor of p53 at various steps of the reprogramming process will allow further enhancement of reprogramming efficiencies in FA.

Disclosures

No relevant conflicts of interest to declare.

Author notes

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Asterisk with author names denotes non-ASH members.