Impaired differentiation and apoptosis of hematopoietic precursors in a mouse model of myelodysplastic syndrome

Expression of a NUP98-HOXD13 (NHD13) fusion gene, initiallyidentified in a patient with myelodysplastic syndrome, leadsto a highly penetrant myelodysplastic syndrome in mice thatrecapitulates all of the key features of the human disease.Expansion of undifferentiated lineage negative (lin^neg) hematopoieticprecursors that express NHD13 was markedly inhibited (30-fold)in vitro. Decreased expansion was accompanied by decreased productionof terminally differentiated cells, indicating impaired differentiationof NHD13 precursors. Rather than differentiate, the majority(80%) of NHD13 lin^neg precursors underwent apoptotic cell deathwhen induced to differentiate. These findings demonstrate thatNHD13 lin^neg cells provide a tractable in vitro system for studiesof myelodysplastic syndrome.

Key words: myelodysplastic syndromes, mouse model, NUP98, HOXD13, apoptosis.

Introduction

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Introduction

Myelodysplastic syndrome (MDS) is a clonal stem-cell disordercharacterized by ineffective hematopoiesis in one or more hematopoieticcell lineages. Despite peripheral blood cytopenias, the bonemarrow (BM) of patients with MDS usually shows normal or increasedcellularity.¹ This paradox can be explained by an increase inapoptosis, and several studies have demonstrated an increasedrate of apoptosis in the BM of patients with early-stage MDS.^2–5In addition to increased cell death or apoptosis, failure ofdifferentiation contributes to the lack of terminally differentiatedblood cells in patients with MDS.⁶ In vitro colony assays demonstrateabnormal colony formation by granulocyte-macrophage (CFU-GM),erythroid (BFU-E, CFU-E) and megakaryocyte (CFU-Meg) progenitorsin many patients with MDS.^7–9

Important clues to the etiology of many hematologic malignancies,including MDS have come from cytogenetic studies. Although rare,translocations of the NUP98 gene, especially those leading tofusions of NUP98 with clustered homeobox (HOX) genes have beenassociated with both MDS and AML.¹⁰ Recently, we developed amouse model of MDS that recapitulates all of the critical featuresof the human disease by expressing a NUP98-HOXD13 (hereafterNHD13) fusion gene in hematopoietic tissues under control ofthe Vav promoter.¹¹ In our initial studies, we showed that unmanipulatedBM from NHD13 and wild type (WT) mice gave rise to similar numbersof colony forming units (CFU) in vitro, and that BM from theNHD13 mice showed increased replating potential.¹¹ However,those studies did not assess the growth, differentiation, anddeath of the hematopoietic stem cell (HSC) population. In thisstudy, we demonstrate that NHD13 lineage negative (lin^neg) murinebone marrow cells, which contain the hematopoietic stem andprogenitor population, show increased apoptosis and differentiationfailure, similar to the human disease.

Design and Methods

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Design and Methods

Mice
All of the NHD13 transgenic mice were 7–9 months old ona C57Bl6 background. Bone marrow nucleated cells (BMNC) wereobtained by flushing the femur and tibia. Diagnosis of MDS wasconfirmed by complete blood counts (CBCs) and cytospin examinationof BM cells. CBCs were determined using a HEMAVET MultispeciesHematology Analyzer (CDC Technologies, Oxford, CT, USA). Thesestudies were approved by the National Cancer Institute AnimalCare and Use Committee.

Selection of lineage-negative (lin^neg) cells
We used the StemCep^TM Mouse Progenitor Enrichment Kit (StemcellTechnologies, Canada) following the manufacturer’s recommendations.The lineage cocktail contained the following antibodies: CD5(Ly-1), Mac-1, B220, Gr-1, and Ter119. Purity of the purifiedlin^neg population was assessed by fluorescence activated cellsorting (FACS).

Liquid cultures and colony forming cell (CFC) assays
Lin^neg cells were cultured at 37°C/5% CO₂ in 6-well cultureclusters (Corning Incorporated, NY, USA) in 3 mL of Iscove’sModified Dulbecco’s Medium (Invitro-gen, CA, USA) supplementedwith 15% fetal bovine serum and the following cytokines: recombinantmouse stem cell factor (SCF, 100 ng/mL), recombinant mouse interleukin3 (rmIL-3 , 6 ng/mL) and recombinant human interleukin 6 (rhIL-6,10 ng/mL) (all from R&D Systems, MN, USA). On days 2, 4and 7, cells were harvested, counted, and assayed for apoptosis.For CFC assays, 4x10⁴ whole BM or 2x10³ lin^neg cells were platedonto 35-mm Petri dishes in Methocult M3434 methyl-cellulosemedium (Stemcell Technologies, Canada) respectively. MethocultM3434 is 2% methylcellulose supplemented with cytokines (SCF[50 ng/mL], rmIL-3 [10 ng/mL], rhIL-6 [10 ng/mL], and rhEpo[3 U/mL]). The CFC plates were incubated at 37°C in a 5%CO₂ incubator, and the number of colonies was counted 12 daysafter plating.

Apoptosis assay
Harvested cells from suspension culture were stained with FITCAnnexin V (BD Biosciences, CA, USA) and propidium iodide (PI)(Sigma-Aldrich, MO, USA) using the manufacturer’s recommendedprotocol. FACS analyses were performed using a dual laser FACScan(BD Biosciences, CA, USA).

Statistical analysis
Data are expressed as the mean ± standard errors of themean (SEM). Differences between groups were analyzed by Student’st-test. p-values less than or equal to 0.05 were consideredto be significant.

Results and Discussion

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Results and Discussion

To determine whether the NHD13 bone marrow nucleated cells (BMNC)were impaired in their ability to differentiate, we performeda standard colony forming cell (CFC) assay in methylcellulose.Although there was no dramatic difference in the numbers ofcolonies produced from whole BM, there was a clear differencein the type of colonies generated (Figure 1A). Whereas mostof the colonies generated from WT BMNC were CFU-GM and CFU-GEMM,most of the NHD13 colonies were abnormally small BFU-E and CFU-Ecolonies. To assess the differentiation potential of more primitivehematopoietic progenitors, we isolated lin^neg BMNC using magneticbeads as described above, and repeated the CFC assay with purifiedlin^neg BMNC. In these experiments there was an almost 10-folddecrease in the number of colonies produced by the NHD13 lin-^negBMNC compared to WT lin^neg BMNC, suggesting a marked inhibitionof growth and/or differentiation of the lin^neg progenitors.These results are similar to studies of MDS patients comparingin vitro CFC results using CD34⁺ purified cells instead of wholeBM.^12,13 We, therefore, focused our subsequent studies on lin^negBMNC.

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Figure 1. In vitro expansion and differentiation of lin^neg cells. (A) CFC assay of whole BM and lin^neg BM cells from NHD13 or WT mice. Colony counts represent total number from two plates for each sample, n=3 NHD13 and WT mice each. CFU-E, colony forming unit-erythroid; BFU-E, burst forming unit-erythroid; CFU-GM, colony forming unit-granulocyte, macrophage; CFU-GEMM, colony forming unit-granulocyte, erythroid, macrophage, megakaryocyte; **p<0.01. (B) Expansion of lin^neg cells from WT and NHD13 mice grown in liquid culture. Cell counts represent pooled data from 3 independent experiments. (C) Percent lin^neg cells according to the culture day, pooled data from 3 independent experiments. (*p=0.051, ***p<0.01).

On day 0, 6x10⁴ lin^neg cells were placed in liquid culture withSCF, IL-3, and IL-6 as described above. The total number ofviable cells was decreased in the NHD13 cultures on day 2; thereafter,the total cell number slowly increased. By contrast, the WTcells accumulated briskly after a brief lag phase, such thatby day 7 there was a 30-fold difference in cell number betweenthe NHD13 and WT cultures (Figure 1B).

As murine hematopoietic cells differentiate, they acquire cellsurface markers such as Mac1 (monocyte/granulocyte), Gr1 (granulocyte),Ter119 (erythroid), B220 (B-lymphoid), and CD5 (T-lymphoid).We evaluated acquisition of these markers to determine whetherthe cells had differentiated after in vitro culture in the presenceof SCF, IL-3, and IL-6. As early as day 2, 77.6±13.1%of the NHD13 cells remained lin^neg, whereas only 25.2±10.7% of the WT cells remained lin^neg (Figure 1C). This trend continued,and on day 7 the fraction of NHD13 lin^neg cells was almost 10-foldgreater than the fraction of WT lin^neg cells (29.2±1.58%vs. 3.1±0.88%, p<0.01) (Figure 1C). These resultsdemonstrate that NHD13 BM cells are impaired in their abilityto differentiate in vitro, consistent with the in vivo resultsdemonstrating peripheral blood cytopenias and increased immaturegranulocytes in the BM of NHD13 mice.¹¹ To determine whetherincreased apoptosis of NHD13 lin^neg BMNC might contribute tothe decreased number of NHD13 compared to WT cells, we usedannexin V and PI staining to evaluate apoptotic cells. Lin^negcells were again placed in liquid culture with SCF, IL-3, andIL-6. An increased proportion of apoptotic cells was evidentin the NHD13 culture as early as day 2 (Figure 2), and persistedfor up to seven days. However, there was no clear increase inapoptosis of freshly isolated NHD13 lin^neg cells (Figure 2).These results suggest apoptosis of the NHD13 cells is inducedin vitro after the cells receive a differentiation stimulus.Alternatively, increased apoptosis of NHD13 BM may be more difficultto detect in vivo as the apoptotic cells are quickly and efficientlycleared by phagocytes. In this context, it is important to notethat increased apoptosis in BM from MDS patients is often difficultto appreciate, and has been attributed to rapid clearance ofapoptotic hematopoietic cells in vivo, this hypothesis is supportedby studies which show marked apoptosis of BMNC cells from MDSpatients following culture in vitro.^14–16 The hypothesisthat myeloid cells which are blocked in their ability to terminallydifferentiate undergo apoptosis in response to differentiationsignals is supported by previous studies which showed that murineBMNC with enforced Myc expression had decreased terminal differentiationand increased apoptosis when treated with GM-CSF.¹⁷

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Figure 2. Apoptosis of lin^neg NHD13 cells grown in liquid culture. (A) Apoptosis (%) of lin^neg cells in liquid culture (n=3, *p=0.051, ***p<0.01). (B) Representative FACS profiles of Annexin V and PI on culture day 2.

In this report, we show that lin^neg hematopoietic precursorsfrom NHD13 mice are impaired in their ability to differentiate,and undergo apoptosis when induced to differentiate, similarto findings with human MDS. These findings are consistent withfindings that embryonic stem (ES) cells with a NHD13 "knock-in"allele were impaired in their ability to generate hematopoieticcolonies in vitro.¹⁸ Given that authenticated stable cell lineswhich recapitulate MDS characteristics in vitro and in vivoare not available,^2,6,19 we believe that BM obtained from anaccurate mouse model of MDS can provide a useful in vitro platformfor MDS studies. This in vitro approach is more amenable tohigh throughput studies than are in vivo models, and providean accessible, in vitro culture system with which to study apoptosisand impaired differentiation associated with MDS.

Acknowledgments

we would like to thank Dave Caudell, Helge Hartung, Sarah Beachy,Dwayne Barber, and Eli Estey for helpful discussion. This researchwas supported by the Intramural Research Program of the NIH,NCI.

Footnotes

Authorship and Disclosures

CWC: designed and conducted research and wrote the first draftof the manuscript; YJC: designed and conducted research; CS:designed and conducted research; PDA: designed research andwrote the final draft of the manuscript. The authors reportedno potential conflicts of interest.

Received for publication March 10, 2008. Revision received April 8, 2008. Accepted for publication April 10, 2008.

References

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