Irrespective of CD34 expression, lineage-committed cell fraction reconstitutes and re-establishes transformed Philadelphia chromosome-positive leukemia in NOD/SCID/IL-2Rgammac-/- mice

Abstract

Stem cells of acute myeloid leukemia (AML) have been identified as immunodeficient mouse-repopulating cells with a Lin(-)CD34(+)38(-) phenotype similar to normal hematopoietic stem cells. To identify the leukemia-propagating stem cell fraction of Philadelphia chromosome-positive (Ph(+)) leukemia, we serially transplanted human leukemia cells from patients with chronic myeloid leukemia blast crisis (n = 3) or Ph(+) acute lymphoblastic leukemia (n = 3) into NOD/SCID/IL-2Rgammac(-/-) mice. Engrafted cells were almost identical to the original leukemia cells as to phenotypes, IGH rearrangements, and karyotypes. CD34(+)CD38(-)CD19(+), CD34(+)38(+)CD19(+), and CD34(-)CD38(+)CD19(+) fractions could self-renew and transfer the leukemia, whereas the CD34(-)CD38(+)CD19(+) fraction did not stably propagate in NOD/SCID mice. These findings suggest that leukemia-repopulating cells in transformed Ph(+) leukemia are included in a lineage-committed but multilayered fraction, and that CD34(+) leukemia cells potentially emerge from CD34(-) populations.

Figure 1

Bone marrow cells of leukemia‐transplanted NOD/SCID/IL‐2Rγc^−/− (NOG) mice showed a similar human hematopoietic stem cell/progenitor profile to the original patient’s bone marrow. (a) CD34/CD38 pattern. (b) Interleukin‐3R (IL‐3R)/CD45RA pattern in a CD34⁺CD38⁺ fraction. Human hematopoietic stem cell/progenitor profiles, which were analyzed by FACSAria and FlowJo software, showed that the percentage of granulocyte/monocyte progenitor fraction is increased.

Figure 2

Both the CD34⁺ and CD34⁻ populations of chronic myelogenous leukemia blast crisis (INH) and Philadelphia chromosome‐positive acute lymphoblastic leukemia (OMR) cells from primary recipient mouse bone marrow (BM) developed disease. (a) INH cells from a primary recipient mouse of UPN1 were sorted into CD34⁺CD38⁻, CD34⁺CD38⁺, and CD34⁻CD38⁺ fractions. Sorted cells (5 × 10³ to 1 × 10⁴) were transplanted into the secondary NOD/SCID/IL‐2Rγc^−/− (NOG) mice. Tertiary and quaternary transplantations using the CD34⁻CD38⁺ population were carried out and CD34/CD38 profiles of leukemia cells from the BM and spleen are shown. (b) OMR cells from the primary recipient mouse spleen of UPN5 were sorted into CD34⁺CD38⁺ and CD34⁻CD38⁺ fractions and 5 × 10⁴ sorted cells were transplanted into NOG mice. Tertiary transplantation was also carried out. The CD34/CD38 pattern was almost the same between mice transplanted with CD34⁺CD38⁺ and CD34⁻CD38⁺ BM cells. (c) CD34⁺ (n = 3) or CD34⁻ (n = 2) BM cells, or total BM cells (n = 2) (all at 2.5 × 10⁵) from UPN6 were transplanted into NOG mice.

Figure 3

Human leukemia cells transplanted into NOD/SCID/IL‐2Rγc^−/− (NOG) mice were not efficiently transferred into NOD/SCID mice. (a) Chronic myelogenous leukemia blast crisis (INH) cells repopulated in NOG mouse bone marrow (BM) were sorted into CD34⁺CD38⁺ and CD34⁻CD38⁺ fractions and these sorted cells were secondarily transplanted into NOG mice. Cells from the secondary recipient mice BM were further separated according to CD34 expression and 10⁴ cells were transplanted into NOD/SCID mice. (b) Chimerism of human CD45⁺ (hCD45) leukemia cells in NOD/SCID BM was measured by flow cytometry 8 weeks after transplantation (n = 3; left panel). CD34/CD38 expression profiles of fractions (1) and (2)‐transplanted NOD/SCID mouse BM are shown (right panel). (c) Fractionated 10⁴ OMR cells from NOG mouse BM were transplanted into NOD/SCID mice (n = 3). mCD45, murine CD45.

Figure 4

In vitro cultured chronic myelogenous leukemia blast crisis (INH) cells were not transplantable into NOD/SCID mice. Unsorted (a) and sorted (b) INH cells from the bone marrow of primary recipient mice were cultured in serum‐free medium containing rhSCF, rhTPO, rhFLT3L, and rhIL‐7. Immunophenotypic analysis was carried out at the designated time points. (c) Engrafted INH cells were detected by anti‐human CD19 (hCD19) antibody. Cells cultured in vitro for more than 2 days were not transplantable into NOD/SCID mice.

Figure 5

Immunostaining analysis of leukemic mouse bone marrow. Each leukemic mouse was dissected 8 weeks after transplantation. (a) Sterna of leukemic mice transplanted with unfractionated original leukemia cells (Table 1) were immunohistochemically stained for human CD34. (b) A sternum obtained from a mouse transplanted with CD34⁻ chronic myelogenous leukemia blast crisis (INH) cells was stained for human CD45 (left panel) and CD34 (right panel).

Figure 6

Summary of Philadelphia chromosome‐positive (Ph⁺) leukemia stem cells. Differential distribution of the self‐renewal populations in normal hematopoiesis (Normal), chronic myelogenous leukemia chronic phase (CML‐CP), and acute myeloid leukemia (AML), and CML blast crisis (CML‐BC) and Ph⁺ acute lymphoblastic leukemia (ALL). In CML‐CP and AML, only the CD34⁺CD38⁻ population possesses self‐renewal capability but proliferative potential is increased along with shifting from CD34⁺CD38⁻ and CD34⁺CD38⁺ to CD34⁻CD38⁺. In contrast, in CML‐BC and Ph⁺ ALL, the CD34⁺CD38⁻ population is dominant and has greater self‐renewal capability but all compartments can self‐renew. Furthermore, leukemia cells of CML‐BC and Ph⁺ ALL reversibly shift between CD34⁺ and CD34⁻. Curved arrow, self‐renewal capability; straight arrow, shift between compartments.