Use of Hematopoietic Stem Cell Transplantation to Assess the Origin of Myelodysplastic Syndrome

Summary

We describe the use of hematopoietic stem cell transplantation (HSCT) to assess the malignant potential of genetically engineered hematopoietic cells. HSCT is useful for evaluating various malignant hematopoietic cells in vivo as well as generating a large cohort of mice with myelodysplastic syndromes (MDS) or leukemia to evaluate novel therapies.

Abstract

Myelodysplastic syndromes (MDS) are a diverse group of hematopoietic stem cell disorders that are defined by ineffective hematopoiesis, peripheral blood cytopenias, dysplasia, and a propensity for transformation to acute leukemia. NUP98-HOXD13 (NHD13) transgenic mice recapitulate human MDS in terms of peripheral blood cytopenias, dysplasia, and transformation to acute leukemia. We previously demonstrated that MDS could be transferred from a genetically engineered mouse with MDS to wild-type recipients by transplanting MDS bone marrow nucleated cells (BMNC). To more clearly understand the MDS cell of origin, we have developed approaches to transplant specific, immunophenotypically defined hematopoietic subsets. In this article, we describe the process of isolating and transplanting specific populations of hematopoietic stem and progenitor cells. Following transplantation, we describe approaches to assess the efficiency of transplantation and persistence of the donor MDS cells.

Introduction

Myelodysplastic syndromes (MDS) represent a diverse set of clonal blood disorders characterized by ineffective hematopoiesis, morphologic evidence of dysplasia, and a propensity for transformation to acute myeloid leukemia (AML)^1,2,3,4. Ineffective hematopoiesis is recognized as a maturation arrest in bone marrow, and results in peripheral blood cytopenias despite a hypercellular bone marrow^1,3. The incidence of MDS has been variously estimated as 2-12 cases per 100,000 persons annua....

Protocol

The animal procedures described in this article were approved by the National Cancer Institute at Bethesda Animal Care and Use Committee, and conform to the policies contained within The Public Health Service Policy on Humane Care and Use of Laboratory Animals, The Animal Welfare Act, and the Guide for the Care and Use of Laboratory Animals.

1. Cell Preparation

1. Harvesting bone marrow nucleated cell (BMNC)
   1. Use only sterile materials. Sterilize re-usable instruments using a steam autoclave. Purchase needles, syringes, and plastic ware in single use sterile containers. Perform all animal and cell manipulations in a Clas....

Results

We show representative figures for results of several experiments. Figure 1 shows a representative flow cytometry sorting experiment. During normal hematopoietic differentiation, as cells become committed to a specific hematopoietic lineage, they acquire lineage-defining cell surface markers and lose the potential for self-renewal. Therefore, in wild-type mice, stem cell self-renewal is confined to lineage-negative BMNC. In this experiment, we sorted BMNC fro.......

Discussion

Although MDS are a clonal hematopoietic stem cell disorder, the MDS "stem", or initiating cells, have not yet been characterized. We previously demonstrated that MDS can be transplantable to WT mice using bone marrow from NHD13 mice by HSCT, characterized by macrocytic anemia, leukopenia, neutropenia, and morphologic evidence of dysplasia¹⁵. In addition, competitive repopulation assays identified a growth advantage of cells from the NHD13 MDS bone marrow. Taken together, these findings imp.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
14 mL round bottom tube	Falcon	352057
Hank's balanced salt solution	Lonza	10-527F
Anti-CD45.2 antibody	Southern Biotech	1800-15	LOT# A077-T044O
3 mL Syringe	Monoject	8881513934
27-G needle	BD	305109
20-G needle	BD	305176
Lineage Cocktail	Miltenyi	130-090-858	LOT# 5170418221
Anti-Biotin antibodies	Miltenyi	130-113-288	LOT# 5171109046
1 mL Syringe	Excelint	26027	Insulin Syringe
Heating Lamp	Thermo Fisher Scientific	E70001901
FACS machine	Cytec	FACScan	2 lasers, 5 color detectors
FACS sorting instrument	Beckman Coulter	MOFLO ASTRIOS	5 lasers, 23 parameters, 6 population sorting simulteneously
Propidium Iodide	Thermo Fisher Scientific	P3566
Gamma Irradiator	Best Theratronics	Gammacell 40
Blood collection tube	RAM scientific	76011
Recipient mice	Charles River	B6-LY5.1/Cr, CD45.1
NUP98-HOXD13 mice	n/a	C57Bl/6, CD45.2	Colony maintained at NIH
5 mL round bottom tube	Falcon	352058