The Apc(min) mouse has altered hematopoietic stem cell function and provides a model for MPD/MDS

Abstract

Apc, a negative regulator of the canonical Wnt signaling pathway, is a bona-fide tumor suppressor whose loss of function results in intestinal polyposis. APC is located in a commonly deleted region on human chromosome 5q, associated with myelodysplastic syndrome (MDS), suggesting that haploinsufficiency of APC contributes to the MDS phenotype. Analysis of the hematopoietic system of mice with the Apc(min) allele that results in a premature stop codon and loss of function showed no abnormality in steady state hematopoiesis. Bone marrow derived from Apc(min) mice showed enhanced repopulation potential, indicating a cell intrinsic gain of function in the long-term hematopoietic stem cell (HSC) population. However, Apc(min) bone marrow was unable to repopulate secondary recipients because of loss of the quiescent HSC population. Apc(min) mice developed a MDS/myeloproliferative phenotype. Our data indicate that Wnt activation through haploinsufficiency of Apc causes insidious loss of HSC function that is only evident in serial transplantation strategies. These data provide a cautionary note for HSC-expansion strategies through Wnt pathway activation, provide evidence that cell extrinsic factors can contribute to the development of myeloid disease, and indicate that loss of function of APC may contribute to the phenotype observed in patients with MDS and del(5q).

Figure 1

Apc^min mice have normal hematopoiesis at steady state. (A) Full blood count examination at 4, 8, and 12 weeks. (B) Peripheral blood morphology demonstrating lymphocytes (top) and neutrophils (bottom). (C) LKS⁺ (lineage^lowcKit^highSca-1⁺ enriched for hematopoietic stem cells) numbers as a percentage of total bone marrow cells. (D) GMP numbers. (E) CMP numbers. (F) MEP numbers. (G) Hematopoietic stem and progenitor cell numbers expressed as a percentage of LKS⁺ cells. Multipotent progenitors (MPP) LKS⁺CD34⁺Flk2⁺, short-term hematopoietic stem cells (ST-HSC) LKS⁺CD34⁺Flk2⁻, and long-term HSC (LT-HSC) LKS⁺CD34⁻Flk2⁻. (H) Long-term HSC, CD150⁺CD48⁻LKS⁺ expressed as a percentage of LKS⁺ cells. (I) Colony-forming ability of 10⁴ bone marrow cells in cytokine-enriched methylcellulose (M3434; StemCell Technologies) and scored for colony (> 50 cells) number and morphology after 7 days. Megakaryocyte colonies (CFU-Mk), megakaryocyte-erythroid colonies (CFU-EMk), mixed multilineage colonies (CFU-GEMM), granulocyte-monocyte colonies (CFU-GM), and blast forming units-erythroid colonies (BFU-E). A total of n = 3 is shown for each condition.

Figure 2

Apc^min bone marrow has enhanced repopulating potential. (A) Whole-blood chimerism (expressed as the fraction of CD45.2-positive donor cells divided by CD45.2 and CD45.1/2 competitor cells) 16 weeks after transplantation into lethally irradiated 45.1 recipients. Representative data of 2 independent experiments. Apc^min 73.1% versus WT 55.9% chimerism, P = .001 (B) Whole-blood chimerism of transplant recipients at 4, 8, and 16 weeks after transplantation. Mean ± SEM (C) Bone marrow chimerism, gated on LKS⁺ cells 16 weeks after transplantation into lethally irradiated recipients. Apc^min 79.8% versus 58.2% WT, P = .015

Figure 3

Apc^min bone marrow has impaired repopulating potential in secondary recipients. (A) Whole-blood chimerism 16 weeks after transplantation of Apc^min or WT bone marrow into lethally irradiated secondary CD45.1 recipients. CD45.2 chimerism 16.9% Apc^min versus 66% WT P < .01, representative data from 2 experiments. (B) Cell-cycle analysis on CD45.2⁺LKS⁺ cells 16 weeks after transplantation. Hoechst 33342 (HoechstA, x-axis) and pyronin Y (PyY, y-axis) were used to resolve G0 (quiescent, Hoe^lowPyY^low), G1 (Hoe^lowPyY^high), and SG2M (cycling Hoe^highPyY^high) populations. (C) Histogram representation of cell-cycle data, expressed as the percentage of CD45.2LKS⁺ cells in each gate. Quiescent fraction 42.8% Apc^min versus 53.4% WT, P = .01, G1 phase 32.6% Apc^min versus 22.1% WT, P = .03.

Figure 4

Aged Apc^min mice develop MDS/MPD. (A) White cell count (WCC × 10³/μL) and spleen weight (mg) from Apc^min mice with MDS/MPD. WCC 19.2 × 10³/μL Apc^min versus 7.4 × 10³/μL WT, P < .01; neutrophil count 5.7 × 10³/μL Apc^min versus 1.6 × 10³/μL WT, P < .01; and spleen weight 0.69 g Apc^min versus 0.09 g WT, P < .01. (B) Peripheral blood parameters, including red cell count (RCC × 10⁶/μL), hematocrit (HCT%), mean corpuscular volume (MCV fl), monocyte count (×10³/μL), red cell distribution width (RDW%), and platelet count (×10³/μL). Red cell count: 2.7 × 10⁶/μL Apc^min versus 9.7 × 10⁶/μL WT, P < .01; HCT: 17.4% Apc^min versus 42.8% WT, P < .01; MCV: 66.5 fl Apc^min versus 45 fl WT P < .01; monocyte count: 1.2 × 10³/μL Apc^min versus 0.3 × 10³/μL WT, P = .03; RDW: 23.7% Apc^min versus 17.3% WT, P < .01. (C) Hematopoietic stem and progenitor cell numbers expressed as a percentage of total bone marrow. LKS⁺ (lineage^lowcKit^highSca-1⁺ enriched for HSCs), LKS⁺SLAM (LKS⁺CD150⁺CD48⁻ highly enriched for long-term HSC), GMPs, CMPs, and MEPs. (D) Peripheral blood films (magnification ×40) and splenic histology (magnification ×10) demonstrating macrocytic anemia with anisopoikilocytosis in Apc^min mice and effacement of normal splenic architecture by extramedullary hematopoiesis (images taken with Nikon Eclipse E400 microscope and digital camera; SPOT Diagnostic Instruments, model 2.2.1). (E) Colony-formation assays in cytokine-enriched methylcellulose from 10⁴ bone marrow cells and (F) 10⁵ spleen cells. Megakaryocyte colonies (CFU-Mk), megakaryocyte-erythroid colonies (CFU-EMk), mixed multilineage colonies (CFU-GEMM), granulocyte-monocyte colonies (CFU-GM), and blast forming units-erythroid colonies (BFU-E). Bone marrow: 41.5 Apc^min versus 36.8 colonies/10⁴ cells WT, P = .35; BFU-E: 10.2 Apc^min versus 3.3 colonies/10⁴ cells WT, P < .01; spleen: 84.2 Apc^min versus 35.8 colonies/10⁵ cells WT, P < .01; CFU-GM: 58.8 Apc^min versus 30.8 colonies/10⁵ cells WT, P < .01; BFU-E: 13.2 Apc^min versus 1.8 colonies/10⁵ cells WT, P < .01). (G) Erythroid maturation flow cytometric analysis. Ery1 (CD71^high, Ter119^mid), Ery2 (CD71^high, Ter119^high), Ery3 (CD71^mid, Ter119^high), and Ery4 (CD71^low, Ter119^high). (H) Graphical representation of erythroid maturation demonstrating shift toward immature (Ery1/2) development. 2.5% and 34.8% Apc^min versus 0.2% and 8.0% WT for Ery1 and Ery2, respectively, P < .01; 3.1% and 0.6% Apc^min versus 11.4% and 3.2% WT for Ery3 and Ery4, respectively, P < .01.

Figure 5

Apc^min MDS/MPD is predominantly cell extrinsic. (A) Transplantation of 10⁶ bone marrow cells from Apc^min MDS/MPD into sublethally irradiated CD45.1 recipients. White cell count (WCC × 10³/μL), hematocrit (HCT%), and donor cell chimerism (%)16 weeks after transplantation. WCC: 9.9 × 10³/μL Apc^min versus 11.5 × 10³/μL WT, P = .12; HCT: 42.5% Apc^min versus 42.9% WT, P = .81; 51.4% Apc^min versus 53.4% WT, P = .75. (B) Bone marrow transplantation of 1 × 10⁶ CD45.1 congenic bone marrow cells into sublethally irradiated Apc^min or WT recipients. Sublethal irradiation was used because of previous reports describing intolerance of lethal irradiation in Apc^min mice. WCC × 10³/μL, HCT%, spleen weight (g) WCC: 8.64 × 10³/μL Apc^min versus 7.53 × 10³/μL WT, P = .59; HCT: 17.3% Apc^min versus 31.8% WT, P = .01; spleen: 0.41 g Apc^min versus 0.11 g WT, P < .01. (C) Peripheral blood parameters, including red cell count (RCC × 10⁶/μL), hematocrit (HCT%), mean corpuscular volume (MCV fl), mean corpuscular hemoglobin (pg), red cell distribution width (RDW%), and platelet count (×10³/μL).