Fluorescence in situ hybridization to determine engraftment status after murine bone marrow transplant.

The mouse Y-specific DNA sequence pY2 was used as a probe for fluorescence in situ hybridization (FISH) to evaluate murine hematopoietic tissues after sex-mismatched bone marrow transplant (BMT). The pY2 probe was localized to the long arm of the Y chromosome on BM metaphases. Hybridization of pY2 in FISH of interphase cells from BM, spleen, and thymus after BMT was compared with Southern blot analysis; both methods gave comparable results. Only FISH was able to analyze post-BMT peripheral blood (PB) samples successfully, and provides a useful method for following engraftment status in the mouse on an ongoing basis.     

A clinically relevant population of leukemic CD34(+)CD38(-) cells in acute myeloid leukemia

Relapse of acute myeloid leukemia (AML) is thought to reflect the failure of current therapies to adequately target leukemia stem cells (LSCs), the rare, resistant cells presumed responsible for maintenance of the leukemia and typically enriched in the CD34(+)CD38(-) cell population. Despite the considerable research on LSCs over the past 2 decades, the clinical significance of these cells remains uncertain. However, if clinically relevant, it is expected that LSCs would be enriched in minimal residual disease and predictive of relapse. CD34(+) subpopulations from AML patients were analyzed by flow cytometry throughout treatment. Sorted cell populations were analyzed by fluorescence in situ hybridization for leukemia-specific cytogenetic abnormalities (when present) and by transplantation into immunodeficient mice to determine self-renewal capacity. Intermediate (int) levels of aldehyde dehydrogenase (ALDH) activity reliably distinguished leukemic CD34(+)CD38(-) cells capable of engrafting immunodeficient mice from residual normal hematopoietic stem cells that exhibited relatively higher ALDH activity. Minimal residual disease detected during complete remission was enriched for the CD34(+)CD38(-)ALDH(int) leukemic cells, and the presence of these cells after therapy highly correlated with subsequent clinical relapse. ALDH activity appears to distinguish normal from leukemic CD34(+)CD38(-) cells and identifies those AML cells associated with relapse.     

A histological survey of green fluorescent protein expression in 'green' mice: implications for stem cell research

AIMS: The transgenic enhanced green fluorescent protein (EGFP) expressing 'green' mouse (C57BL/6-TgN(ACTbEGFP)1Osb) is a widely used tool in stem cell research, where the ubiquitous nature of EGFP expression is critical to track the fate of single or small groups of transplanted haematopoietic stem cells (HSC). Our aim was to investigate this assumed ubiquitous expression by performing a detailed histological survey of EGFP expression in these mice. METHODS: Fluorescent microscopy of frozen tissue sections was used to perform a detailed histological survey of the pattern of EGFP expression in these mice. Flow cytometry was also used to determine the expression pattern in blood and bone marrow. RESULTS: Three patterns of EGFP expression were noted. In most tissues there was an apparently stochastic variegation of the transgene, with individual cell types demonstrating highly variable rates of EGFP expression. Certain specific cell types such as pancreatic ductal epithelium, cerebral cortical neurones and glial cells and glomerular mesangial cells consistently lacked EGFP expression, while others, including pancreatic islet cells, expressed EGFP only at extremely low levels, barely distinguishable from background. Lastly, in the colon and stomach the pattern of EGFP expression was suggestive of clonal inactivation. Only cardiac and skeletal muscle showed near ubiquitous expression. CONCLUSIONS: These findings raise questions regarding the 'ubiquitous' expression of EGFP in these transgenic mice and suggest caution in relying overly on EGFP alone as an infallible marker of donor cell origin.     

Bryostatin 1, a unique biologic response modifier: anti-leukemic activity in vitro

Bryostatin 1, a macrocyclic lactone isolated from the marine bryozoan Bugula neritina, has demonstrated both antineoplastic activity against the murine P388 leukemia line in vivo and stimulatory activity against mouse and human hematopoietic progenitors. We studied the effects of bryostatin 1 on the growth of human leukemias in vitro. Bryostatin 1 inhibited 1 to 4 logs of clonogenic leukemia cell growth from three of four leukemia cell lines. Bryostatin 1 also inhibited, by at least 1 log, the proliferation of clonogenic acute nonlymphocytic leukemia (ANLL) cells from 10 to 12 patients with newly diagnosed or relapsed ANLL. Maximal inhibition of leukemic growth occurred at 10(-9) to 10(-7) mol/L bryostatin 1. Interestingly, bryostatin 1 also inhibited the growth of hematopoietic progenitors from eight patients with myelodysplastic syndromes (MDS). Leukemia cells exposed to bryostatin 1 for up to 96 hours and then washed, demonstrated no substantial inhibition of clonogenic growth, indicating that the anti-leukemic effect of bryostatin 1 is cytostatic. The phorbol ester 12-0-tetradecanoylphorbol-13-acetate (TPA) produced more potent inhibition of clonogenic leukemia growth, and this inhibition was blocked by bryostatin 1. Thus, the anti-leukemic activity of bryostatin 1 may be mediated through activation of protein kinase C. Bryostatin 1 inhibits clonogenic leukemia cells at concentrations that stimulate normal hematopoietic progenitors. The differential effects of bryostatin 1 on normal and abnormal hematopoiesis suggest that bryostatin 1 may have value in the treatment of leukemias and MDS.     

Hematopoietic stem cell tracking in vivo: a comparison of short-term and long-term repopulating cells

We have previously demonstrated that we could separate long-term repopulating stem cells from cells that provided radioprotection (short-term repopulating cells) on the basis of size and suggested that this might be due to the quiescent nature of long-term repopulating cells. To further define the activity of these populations, we used a dye (PKH26), which incorporates into the membrane of cells and is equally distributed to daughter cells when they divide. We developed an assay, which allowed us to retrieve PKH26(+) long-term and short-term repopulating cells in the hematopoietic tissues of the recipients posttransplant. We were able to recover the labeled cells and determine their cell cycle activity, as well as their ability to reconstitute secondary lethally irradiated hosts in limiting dilution. The results of our assay suggest that long-term repopulating cells are quiescent in the bone marrow (BM) 48 hours after transplant. We were able to detect only a few labeled cells in the peripheral blood posttransplant and even though cells homed to both the spleen and BM, more long-term repopulating cells homed to the marrow and only these cells, which homed to the marrow, were capable of reconstituting lethally irradiated secondary hosts long-term.     

Inhibition of apoptosis by BCR-ABL in chronic myeloid leukemia

BCR-ABL expression is presumed to effect clonal expansion in chronic myeloid leukemia (CML) by deregulation of cell proliferation. However, most studies have found that relative rates of cell proliferation are not increased in CML. Moreover, we found that CML progenitors display a normal proliferative response to growth factors and do not manifest greater proliferative potential than normal progenitors. Growth of malignancies depends on an imbalance between the rate of cell production and the rate of cell death. We found that BCR-ABL expression inappropriately prolongs the growth factor-independent survival of CML myeloid progenitors and granulocytes by inhibiting apoptosis, a genetically programmed process of active cell death; inhibition of BCR- ABL expression by antisense oligonucleotides reversed the suppression of apoptosis as well as the enhancement of survival. The decreased rate of programmed cell death appears to be the primary mechanism by which BCR-ABL effects expansion of the leukemic clone in CML.     

Effects of neuraminidase on the regulation of erythropoiesis: III: Characterization of carbohydrate moieties on the surface of thymic regulatory cells that interact with erythroid colony-forming cells

In this study we further define cell surface carbohydrate structures relevant to cellular interactions that regulate erythropoiesis. An analysis of thymocyte cell surface negativity was made using fluoresceinated poly-L-ornithine (FITC poly-L-ornithine) as a probe that binds to negatively charged sites (i.e., sialic acid residues) at the cell surface. Two distinct subpopulations are labeled, comprising both intensely as well as weakly fluorescent subpopulations of thymocytes. Prior treatment of thymocytes with Vibrio cholerae neuraminidase (VCN), which removes cell surface sialic acid residues, markedly reduced the FITC poly-L-ornithine surface labeling of these cells. Distinct enzymatic modifications of regulatory cell functions were also assessed by the ability of thymocytes to function as separate regulatory subpopulations. Confirming our previous observations, treating thymocytes with VCN impaired the enhancement activity but had little effect on thymocyte regulatory ability to suppress erythroid colony growth. In contrast, treatment of thymocytes with galactose oxidase (GAO) or beta-galactosidase (beta-GAL) removed suppressor activity either before or after VCN treatment. A further exposure of GAO-treated thymocytes to sodium borohydride or hydroxylamine, which reduce D-galactose residues, restores their suppressor function and prevents enhancement. These differential enzymatic effects on thymocyte regulatory cell functions suggest that different carbohydrate structures may be involved in helper and suppressor activities for erythroid colony formation. Sialic acid residues may be associated with certain cells that function to enhance erythropoiesis, and D-galactose residues may be associated with the suppressor subpopulation.     

Characterization of chronic myeloid leukemia stem cells

Although tyrosine kinase inhibitors have redefined the care of chronic myeloid leukemia (CML), these agents have not proved curative, likely due to resistance of the leukemia stem cells (LSC). While a number of potential therapeutic targets have emerged in CML, their expression in the LSC remains largely unknown. We therefore isolated subsets of CD34(+) stem/progenitor cells from normal donors and from patients with chronic phase or blast crisis CML. These cell subsets were then characterized based on ability to engraft immunodeficient mice and expression of candidate therapeutic targets. The CD34(+)CD38(-) CML cell population with high aldehyde dehydrogenase (ALDH) activity was the most enriched for immunodeficient mouse engrafting capacity. The putative targets: PROTEINASE 3, SURVIVIN, and hTERT were expressed only at relatively low levels by the CD34(+)CD38(-)ALDH(high) CML cells, similar to the normal CD34(+)CD38(-)ALDH(high) cells and less than in the total CML CD34(+) cells. In fact, the highest expression of these antigens was in normal, unfractionated CD34(+) cells. In contrast, PRAME and WT1 were more highly expressed by all CML CD34(+) subsets than their normal counterparts. Thus, ALDH activity appears to enrich for CML stem cells, which display an expression profile that is distinct from normal stem/progenitor cells and even the CML progenitors. Indeed, expression of a putative target by the total CD34(+) population in CML does not guarantee expression by the LSC. These expression patterns suggest that PROTEINASE 3, SURVIVIN, and hTERT are not optimal therapeutic targets in CML stem cells; whereas PRAME and WT1 seem promising.     

Dye-mediated photolysis of normal and neoplastic hematopoietic cells

The purpose of this study was to determine the sensitivity to merocyanine 540 (MC 540)-mediated photolysis of normal human hematopoietic progenitor cells and four leukemia cell lines (Daudi, Raji, K562 and HL-60). Late erythroid progenitors were the most sensitive normal cells. Early erythroid progenitors were of intermediate sensitivity. Granulocyte/macrophage progenitors and multipotent progenitors were the least sensitive normal marrow cells. A combination of dye concentration, serum concentration, and illumination that eliminated 50% of multipotent progenitor cells reduced the concentration of leukemic cells by greater than or equal to 4.5 log. It is conceivable that this difference in photosensitivity can be exploited for the extracorporeal purging of autologous remission marrow grafts.