Knockdown of the Fanconi Anemia Gene FANCD2 Directly Affects Hematopoiesis in Human Embryonic Stem Cells.

Fanconi anemia (FA) is a rare autosomal recessive disease characterized by the progressive bone marrow failure, developmental anomalies and cancer susceptibility. Twelve distinct FA complementation groups have been identified (A–C, D1, D2, E–G, I, J, L, M) and 11 cDNAs cloned. A current working model proposes that eight FA proteins (A, B, C, E, F, G, L and M) assemble to form a multi-protein nuclear complex, involved the repair of damaged DNA. The FA complex facilitates the monoubiquitination of the FANCD2 protein following exposure to DNA damaging agents. Ubiquinated FANCD2 co-localizes with the key DNA repair proteins RAD51, BRCA2 (know as FANCD1), BRCA1, and FANCJ (BACH1) to promote homologous recombination. The hallmark of FA patients is marrow failure due to defective hematopoietic stem cells. The study of human FA stem cell biology is severely restricted due to the few CD34+ cells that can be isolated from FA patients compared with normal individuals. We generated human embryonic stem cells (hESC) exhibiting FA phenotype by introducing small double strand RNA species to ablate FANCD2 gene function in hESC. Human ES cells were then differentiated into hematopoietic cells to investigate FA hematopoiesis. FANCD2-specific small hairpin RNAs (shRNAs) were designed and cloned into a self-inactivated lentiviral vector with a GFP cDNA. High titer virus (2×10⁸ iu/ml) was used for transduction of the hESC line H1. More than 90% of H1 cells were transduced with lentivirus that was observed by GFP expression. The FANCD2 protein expression was analyzed by Western blotting; FANCD2 shRNA targeted cells expressed 1–3% of the FANCD2 protein compared with control H1 cells. Functional assessment of the H1-FANCD2 was performed by incubation with DNA cross-linking agent, mitomycin C (MMC). The H1-FANCD2 cells were nearly 10 times more sensitive to MMC than untreated or scrambled shRNA ES cells. The effective dose 50 (ED50) of MMC required to induce apoptosis was only 4 ng/ml in H1-FANCD2 cells compared with 30 ng/ml in mock-infected of scrambled shRNA controls. Cytogentic abnormalities are the hallmark of Fanconi anemia. When H1-FANCD2 cells were exposed to MMC, 57% of cells had abnormal cytogenetics compared with 2% of scrambled shRNA transduced H1 cells. In addition, 75.34±0.69% of H1-FANCD2 cells were arrested at the G2/M phase of the cell cycle after MMC exposure compared with 60.63±2.5% of H1-scramble cells. MMC hypersensitivity, cell cycle defects and chromosomal abnormalities of H1-FANCD2 confirmed the FA phenotype. Differentiation of H1 and H1-scramble cells through a blastocyst intermediate produced a discrete number of KDR+/CD31+ hemangioblast cells that give rise to erythroid and myeloid hematopoietic colonies in methylcellulose culture. H1-FANCD2 ES cells produced blastocyst and hemangioblast cells without significant development of end-stage hematopoietic lineages. In conclusion, we successfully converted, using shRNAs, hESC H1 cells that exhibit the FA phenotype including abnormal hematopoiesis. Generation of mutant FA hES cells will be a valuable model to study FA pathophysiology and treatment.