Development of a Patient-Derived Xenograft Model of Human Myelodysplastic Syndromes
Authors: Krevvata Maria , Dos Santos Cedric , Shan Xiaochuan , Secreto Anthony , Danet-Desnoyers Gwenn , Carroll Martin 
Published: November 2019

Background: Myelodysplastic syndromes (MDS) comprise a group of hematologic disorders characterized by ineffective hematopoiesis and cytopenias that may lead to acute myeloid leukemia (AML) or bone marrow failure. Although a few genetic mouse models have been generated, they cannot recapitulate the heterogeneity of the disease. The attempts for the development of a xenograft model have been challenging in the past, mainly due to poor engraftment of MDS hematopoietic cells. Recent scientific evidence pinpoint the importance of the niche in the establishment and progression of hematological malignancies and suggested a novel approach to establishing MDS xenotransplantation models. In this study we aim to develop a mouse xenotransplantation model of human MDS and to evaluate the role of mesenchymal stem cells (MSCs) in the engraftment process.

Methods: MSC's from normal donors and MDS patient samples were generated and characterized using standard culture methods. NOD.Cg-Prkdcscid Il2rgtm1Wjl Tg(CMV-IL3,CSF2,KITLG)1Eav/MloySzJ (NSGS) mice were used as recipients. Mice were injected with 1 x 106 bone marrow mononuclear cells (MNC) alone or in combination with 5 x 105 MSC's. MSC's were from normal donors, from same patient as MNC's (autologous) or from other MDS patients (MDS allogeneic). All cells were injected intrafemorally. Mice were assessed for engraftment by bone marrow aspiration in the ipsilateral bone at 6 weeks, in the contralateral bone at 10 weeks and at necropsy at 12-22 weeks (depending on initial assessment). Long-term engrafted samples were further assessed for presence of stem cells by secondary transplantation into NSGS mice. 1 x 106 human CD45 selected hematopoietic cells were passaged again without or with 5 x 105 MSC's. Secondary recipients were assessed at 12 weeks for engrafment. MDS samples represented both high risk (refractory anemia with excess blasts, RAEB)) and low risk (refractory anemia (RA), refractory cytopenia with myelodysplasia (RCMD)) disease. The overall degree of engraftment was assessed by bone marrow aspiration and measurement of human CD45+ cells. Lymphoid, myeloid and erythroid engraftment was assessed.

Results: Five out of five injected MDS samples showed persistent human cells when assessed at 6 weeks. For most samples, engraftment at 6 weeks was low (<2%) and was not consistently influences by the presence or absence of MSC. On week 10, only two out of five patients had increased engraftment and only one showed higher engraftment levels in the mice co-transplanted with MSCs. Both of these better engrafting samples were from patients with RAEB. Engrafting samples demonstrated both myeloid and erythroid engraftment. Molecular analysis to confirm engraftment of MDS clone will be presented. In total, 2 out of 5 patients showed long-term engraftment (> 12 weeks). One of these samples has been transferred to secondary animals. Secondary transplanted mice injected with selected hCD45+ cells with or without MSC's showed variable engraftment levels on week 10 after injections, with the majority of those not reaching long-term engraftment.

Conclusions: Passive transferof MDS hematopoietic cells as assessed at 6 weeks after intrabone injection is highly consistent and not dependent on MSC when total MNC fraction is used. However, long-term engraftment of MDS cells in xenotransplanted mice is uncommon suggesting that true MDS stem cells do not consistently engraft. Further analysis with injection into different mouse strains with purified CD34+ cells is under way.

Disclosures

Dos Santos: Amgen: Employment.