Testing the cancer stem cell (CSC) hypothesis: Modeling chronic myeloid leukemia (CML) using human embryonic stem cells (hESC)
Authors: Jessica Rusert, Daniel Goff, Edward Kavalerchick, Annelie Abrahamsson, Jessica Flippin, Ifat Geron, Catriona Jamieson, Lawrence Goldstein
Journal: Cancer Research (2008)

LB-89  Compelling studies suggest that self-renewing CSC are resistant to current forms of therapy targeted at rapidly dividing, non-self-renewing populations, which represent a reservoir for relapse and metastasis. Characterizing and targeting CSC in various cancers therefore has important implications for reducing the risk of relapse and rates of therapeutic recalcitrance. CML is a cancer where a hematopoietic stem cell (HSC) has been identified and suggested to give rise to this cancer by acquisition of BCR-ABL, a fusion gene that produces a constitutively active protein tyrosine kinase product p210BCR-ABL. Efforts to model disease with human cells have been limited by the relative dearth of CML patient samples. hESCs may provide a limitless and consistent source of primitive progenitor cells and have therefore been used to this end to produce in vitro and in vivo model systems of CML CSC. GFP-Luciferase transduced hESC were intra-hepatically injected into neonatal RAG2-/γc-/- mice +/- cotransduction with BCR-ABL. Engraftment from 4-10.5 weeks was observed, without teratoma formation, analyzed by both bioluminescent imaging (IVIS 200 ®) and FACS of hematopoietic organs for human hematopoietic markers (n=8). CD45 staining was observed by FACS in livers (1.6-72.1%) where 14-51% and 3-38% of these were CD38+ and CD34+ respectively, spleen (14.9%) where 6.4% of these were CD90+, bone marrow (1.37-10.7%) where 14-15.8%, .6-1%, and .5-1% of these were CD38, CD34, and CD90+ respectively, and in thymus (2.36-5.7%) where 34%, 12-16.6%, and 6.4% of these were CD38, CD34, and CD90+ respectively compared to untransplanted controls and exclusion of mouse MHC and CD45.2+ cells. After transduction with BCR-ABL hESC engraftment became more robust by bioluminescence (n=8) and a trend toward greater myeloid lineage engraftment may be occurring based on CD45RA staining. Although engraftment by direct hESC injection has been successful, injection of hematopoietic progenitors derived from hESC may show greater engraftment potential. Thus, for modeling CML CSC in vitro and creating a source of CD34+ hematopoietic progenitor cells for engraftment studies, hESC differentiation on OP9 stromal cell lines is being utilized. Up to 7% of differentiating hESCs expressed CD34 allowing future studies of BCR-ABL transduction prior to or subsequent to differentiation. Existence of enhanced self-renewal, alteration of transcriptional repertoire, or skewing toward specific hematopoietic lineages due to BCR-ABL expression will be evaluated. These hematopoietic progenitors can then be used for subsequent transplantation into RAG2-/γc-/- mice for in vivo CSC modeling. Preliminary studies showed engraftment by bioluminescent imaging after CD34 selection and 7.23% of liver stained for CD45 in one mouse where 43% were also CD38+ (n=2). Future experiments aim to rigorously test this hypothesis of the CML CSC.