Fancd2-/- mice have hematopoietic defects that can be partially corrected by resveratrol

Abstract

Progressive bone marrow failure is a major cause of morbidity and mortality in human Fanconi Anemia patients. In an effort to develop a Fanconi Anemia murine model to study bone marrow failure, we found that Fancd2(-/-) mice have readily measurable hematopoietic defects. Fancd2 deficiency was associated with a significant decline in the size of the c-Kit(+)Sca-1(+)Lineage(-) (KSL) pool and reduced stem cell repopulation and spleen colony-forming capacity. Fancd2(-/-) KSL cells showed an abnormal cell cycle status and loss of quiescence. In addition, the supportive function of the marrow microenvironment was compromised in Fancd2(-/-) mice. Treatment with Sirt1-mimetic and the antioxidant drug, resveratrol, maintained Fancd2(-/-) KSL cells in quiescence, improved the marrow microenvironment, partially corrected the abnormal cell cycle status, and significantly improved the spleen colony-forming capacity of Fancd2(-/-) bone marrow cells. We conclude that Fancd2(-/-) mice have readily quantifiable hematopoietic defects, and that this model is well suited for pharmacologic screening studies.

Figure 1

Fancd2^−/− mice have a smaller KSL stem and progenitor pool. (A) FACS profiles after KSL staining of the bone marrow cells from Fancd2 mutant mice and wild-type littermate controls. The percentage of the KSL gate is referring to the proportion of KSL cells in the whole nucleated bone marrow. To confirm the purity of double-sorted KSL cells, 3000 cells were analyzed for each genotype. (B) Quantification of hematopoietic KSL stem and progenitor frequencies in the bone marrow of FA mice (Fancd2^−/− or Fancc^−/−) and wild-type controls. All the mice used were between 3 and 8 months of age. In the Fancd2^−/− group, n = 19 for Fancd2^−/− mice and n = 12 for Fancd2^+/+ littermate controls; in the Fancc^−/− group, n = 6 for Fancc^−/− mice and n = 4 for Fancc^+/+ littermate controls. NS denotes not significant. (C) Quantification of CD34⁻KSL hematopoietic stem-cell frequencies in the bone marrow of Fancd2^−/− mice or wild-type controls. N = 6 for Fancd2^−/− mice and n = 7 for Fancd2^+/+ mice. (D) Quantification of CLP frequencies in the bone marrow of Fancd2^−/− mice or wild-type controls. N = 5 for Fancd2^−/− mice and n = 3 for Fancd2^+/+ mice.

Figure 2

Hematopoietic defects in Fancd2^−/− mice. (A) In vivo competitive repopulation of mixed FA (Fancc^−/− or Fancd2^−/−) and ROSA26^Tg/O bone marrow cells. Quantitative real-time PCR (qPCR) analyses were performed to evaluate donor contribution to the peripheral blood cells from each donor (7 or 9 months posttransplantation for Fancd2^−/− or Fancc^−/− donors, respectively). Three independent qPCR analyses were performed for each sample, and results from 5 animals were pooled together for each experimental group. P values were calculated by the 2-tailed, paired Student t test. Error bars represent SEM. (B) Representative picture for CAFC assay. The arrow indicates cobblestone colony. The image was acquired on an Axiovert 200 inverted microscope with an AxioCam MRc color camera at room temperature using AxioVision Release 4.8 software (Carl Zeiss MicroImaging). Original magnification ×100 (with 10× objective lens). (C) Quantification of CAFC results. P values were calculated by 2-tailed, paired Student t test.

Figure 3

Fancd2^−/− KSL cells have abnormal cell cycle status. (A) Representative cell-cycle profiles of KSL cells from a Fancd2^−/− mouse and its wild-type littermate control. DNA content was measured with Hoechst 33342. Data were analyzed with FlowJo (TreeStar) using the Dean-Jett-Fox model for the quantification of each cell-cycle phase. (B) Pooled results from cell cycle analysis on KSL cells. N = 9 for each group. (C) Quantification of the cell cycle analysis on CD34⁻KSL cells. N = 3 for each group. (D) Representative picture after costaining for DNA content (Hoechst 33342) and Ki67 expression in KSL cells from a Fancd2^−/− mouse and its wild-type littermate control. Percentages for each gate were denoted.

Figure 4

Resveratrol partially corrects hematopoietic defects in Fancd2^−/− mice. (A) KSL frequency was not significantly changed in either Fancd2^−/− or Fancd2^+/+ mice after resveratrol (RV) treatment. (B) Statistical quantification of the cell-cycle analysis on KSL cells in resveratrol- versus placebo-treated mice. N = 3 for each group. (C) Representative cell cycle profiles of a placebo-treated Fancd2^−/− mouse, its resveratrol-treated Fancd2^−/− littermate control, and placebo-treated wild-type control. Cells are stained for KSL and DNA content (Hoechst 33342). Data were analyzed with FlowJo using Dean-Jett-Fox model for the quantification of each cell cycle phase. (D) Representative picture after a costaining for DNA content (Hoechst 33342) and Ki67 expression in KSL subsets of the bone marrow cells from placebo- or resveratrol-treated Fancd2^−/− mice and a placebo-treated wild-type littermate control. Percentages for each gate were denoted. (E) Resveratrol treatment significantly improved the CFU-S–forming capacity of Fancd2^−/− bone marrow cells. Data represent 3 donors for each group with 3 (or 2 in 1 case) recipients for each donor.

Figure 5

ROS levels, apoptosis, and cytokine response in Fancd2^−/− and wild-type bone marrow cells. (A) Fluorescence intensity in KSL subsets of Fancd2^−/− and wild-type bone marrow after carboxy-H2DCFDA staining for intracellular ROS. Oxidation of carboxy-H2DCFDA by ROS generated fluorescence detectable by flow cytometry. (B) Quantification of early apoptotic KSL cells after 7-AAD, annexin V, and KSL staining. Data represent 7 mice for each group. NS denotes not significant. (C) Resveratrol suppressed R848-induced TNF-α overproduction in Fancc-shRNA–treated THP1 human leukemia cells. The experiments were done in triplicates on a 96-well plate.