scholarly journals Selective Expansion of Primitive Normal Hematopoietic Cells in Cytokine-Supplemented Cultures of Purified Cells From Patients With Chronic Myeloid Leukemia

Blood ◽  
1997 ◽  
Vol 90 (1) ◽  
pp. 64-69 ◽  
Author(s):  
A.L. Petzer ◽  
C.J. Eaves ◽  
M.J. Barnett ◽  
A.C. Eaves
Keyword(s):  
Chronic Myeloid Leukemia ◽  
Myeloid Leukemia ◽  
Hematopoietic Cells ◽  
Granulocyte Colony Stimulating Factor ◽  
Flt3 Ligand ◽  
Stem Cell Rescue ◽  
In Vitro Expansion ◽  
Stimulating Factor

We have previously reported that primitive normal hematopoietic cells detectable as long-term culture-initiating cells (Ph-LTC-IC) are present at high levels in the blood of some patients with chronic myeloid leukemia (CML). We now show that this population can be expanded several-fold when highly purified CD34+CD38− cells isolated from the blood of such patients are cultured for 10 days in a serum-free medium containing 100 ng/mL of Flt3-ligand and Steel factor and 20 ng/mL of interleukin-3 (IL-3) and IL-6, and granulocyte colony-stimulating factor. In similar cultures initiated with CD34+CD38− cells from CML blood samples in which all of the LTC-IC were leukemic (Ph+), Ph+ LTC-IC activity was rapidly lost both in the presence and absence of admixed CD34+CD38− cells isolated from normal marrow. Conversely, the ability of normal LTC-IC to expand their numbers was shown to be independent of the presence of Ph+LTC-IC and later types of Ph+colony-forming cell (CFC) progenitors. In contrast to the LTC-IC, CFC were consistently -a m p l i f i e d  i n  c u l t u r e s  i n i t i a t e d  w i t h  C M L - d e r i v e d -CD34+CD38− cells and the additional CFC present after 10 days were, like the starting population of CFC, almost exclusively Ph+ regardless of the genotype(s) of the LTC-IC in the original CML samples. Amplification of the Ph+CFC population in these cultures showed the same factor dependence as previously demonstrated for the in vitro expansion of CFC from normal marrow CD34+CD38− cells. Ph+LTC-IC disappeared regardless of the cytokines present. Taken together these findings support a model of CML in which the leukemic stem cells are characterized by a decreased probability of self-renewal and an increased probability of differentiation. In addition, they suggest new opportunities for improving the treatment of CML using strategies that require autologous stem cell rescue.

scholarly journals Selective Expansion of Primitive Normal Hematopoietic Cells in Cytokine-Supplemented Cultures of Purified Cells From Patients With Chronic Myeloid Leukemia

Blood ◽  
1997 ◽  
Vol 90 (1) ◽  
pp. 64-69 ◽  
Author(s):  
A.L. Petzer ◽  
C.J. Eaves ◽  
M.J. Barnett ◽  
A.C. Eaves
Keyword(s):  
Chronic Myeloid Leukemia ◽  
Myeloid Leukemia ◽  
Hematopoietic Cells ◽  
Granulocyte Colony Stimulating Factor ◽  
Flt3 Ligand ◽  
Stem Cell Rescue ◽  
In Vitro Expansion ◽  
Stimulating Factor

Abstract We have previously reported that primitive normal hematopoietic cells detectable as long-term culture-initiating cells (Ph-LTC-IC) are present at high levels in the blood of some patients with chronic myeloid leukemia (CML). We now show that this population can be expanded several-fold when highly purified CD34+CD38− cells isolated from the blood of such patients are cultured for 10 days in a serum-free medium containing 100 ng/mL of Flt3-ligand and Steel factor and 20 ng/mL of interleukin-3 (IL-3) and IL-6, and granulocyte colony-stimulating factor. In similar cultures initiated with CD34+CD38− cells from CML blood samples in which all of the LTC-IC were leukemic (Ph+), Ph+ LTC-IC activity was rapidly lost both in the presence and absence of admixed CD34+CD38− cells isolated from normal marrow. Conversely, the ability of normal LTC-IC to expand their numbers was shown to be independent of the presence of Ph+LTC-IC and later types of Ph+colony-forming cell (CFC) progenitors. In contrast to the LTC-IC, CFC were consistently -a m p l i f i e d  i n  c u l t u r e s  i n i t i a t e d  w i t h  C M L - d e r i v e d -CD34+CD38− cells and the additional CFC present after 10 days were, like the starting population of CFC, almost exclusively Ph+ regardless of the genotype(s) of the LTC-IC in the original CML samples. Amplification of the Ph+CFC population in these cultures showed the same factor dependence as previously demonstrated for the in vitro expansion of CFC from normal marrow CD34+CD38− cells. Ph+LTC-IC disappeared regardless of the cytokines present. Taken together these findings support a model of CML in which the leukemic stem cells are characterized by a decreased probability of self-renewal and an increased probability of differentiation. In addition, they suggest new opportunities for improving the treatment of CML using strategies that require autologous stem cell rescue.

scholarly journals Detection of breakpoint cluster region- negative and nonclonal hematopoiesis in vitro and in vivo after transplantation of cells selected in cultures of chronic myeloid leukemia marrow

Blood ◽  
1990 ◽  
Vol 76 (11) ◽  
pp. 2404-2410 ◽  
Author(s):  
AG Turhan ◽  
RK Humphries ◽  
CJ Eaves ◽  
MJ Barnett ◽  
GL Phillips ◽  
...  
Keyword(s):  
Chronic Myeloid Leukemia ◽  
Myeloid Leukemia ◽  
Hematopoietic Cells ◽  
Breakpoint Cluster Region ◽  
Cluster Region ◽  
Differentiated Cells ◽  
The One

Abstract Philadelphia (Ph1) chromosome-positive clonogenic progenitors usually disappear within 4 to 6 weeks in long-term cultures established from the marrow of patients with chronic myeloid leukemia (CML). In contrast, coexisting chromosomally normal hematopoietic cells are relatively well maintained. Thus, even though normal cells are initially undetectable, they may become the predominant population. Recently, we have begun to explore the potential of such cultures as a strategy for preparing CML marrow for autografting, and based on cytogenetic studies of the differential kinetics of Ph1-positive and Ph1-negative clonogenic cells, have chosen a 10-day period in culture to obtain maximal numbers of selectively enriched normal stem cells. Here we present the results of molecular analyses of the cells regenerated in vivo for the initial three CML patients to be treated using this approach by comparison with the differentiated cells generated by continued maintenance of an aliquot of the autograft in vitro (using a slightly modified culture feeding procedure to enhance the production and release of cells into the nonadherent fraction after 4 weeks) for the one patient whose genotype made molecular analysis of clonality status also possible. These analyses showed that cells with a rearranged breakpoint cluster region (BCR) gene were not detectable by Southern blotting in either in vitro or in vivo populations of mature cells that might be assumed to represent the progeny of primitive cells present at the end of the initial 10 days in culture. Production of BCR- negative cells was also shown to be temporally correlated with the appearance of nonclonal hematopoietic cells both in culture and in vivo. These findings provide support for the view that prolonged maintenance of CML marrow cells in long-term culture may allow molecular characterization of both the BCR-genotype and clonality status of cells with in vivo regenerative potential.

scholarly journals Detection of breakpoint cluster region- negative and nonclonal hematopoiesis in vitro and in vivo after transplantation of cells selected in cultures of chronic myeloid leukemia marrow

Blood ◽  
1990 ◽  
Vol 76 (11) ◽  
pp. 2404-2410
Author(s):  
AG Turhan ◽  
RK Humphries ◽  
CJ Eaves ◽  
MJ Barnett ◽  
GL Phillips ◽  
...  
Keyword(s):  
Chronic Myeloid Leukemia ◽  
Myeloid Leukemia ◽  
Hematopoietic Cells ◽  
Breakpoint Cluster Region ◽  
Cluster Region ◽  
Differentiated Cells ◽  
The One

Philadelphia (Ph1) chromosome-positive clonogenic progenitors usually disappear within 4 to 6 weeks in long-term cultures established from the marrow of patients with chronic myeloid leukemia (CML). In contrast, coexisting chromosomally normal hematopoietic cells are relatively well maintained. Thus, even though normal cells are initially undetectable, they may become the predominant population. Recently, we have begun to explore the potential of such cultures as a strategy for preparing CML marrow for autografting, and based on cytogenetic studies of the differential kinetics of Ph1-positive and Ph1-negative clonogenic cells, have chosen a 10-day period in culture to obtain maximal numbers of selectively enriched normal stem cells. Here we present the results of molecular analyses of the cells regenerated in vivo for the initial three CML patients to be treated using this approach by comparison with the differentiated cells generated by continued maintenance of an aliquot of the autograft in vitro (using a slightly modified culture feeding procedure to enhance the production and release of cells into the nonadherent fraction after 4 weeks) for the one patient whose genotype made molecular analysis of clonality status also possible. These analyses showed that cells with a rearranged breakpoint cluster region (BCR) gene were not detectable by Southern blotting in either in vitro or in vivo populations of mature cells that might be assumed to represent the progeny of primitive cells present at the end of the initial 10 days in culture. Production of BCR- negative cells was also shown to be temporally correlated with the appearance of nonclonal hematopoietic cells both in culture and in vivo. These findings provide support for the view that prolonged maintenance of CML marrow cells in long-term culture may allow molecular characterization of both the BCR-genotype and clonality status of cells with in vivo regenerative potential.

scholarly journals Phenotypic heterogeneity of primitive leukemic hematopoietic cells in patients with chronic myeloid leukemia

Blood ◽  
1992 ◽  
Vol 80 (10) ◽  
pp. 2522-2530 ◽  
Author(s):  
C Udomsakdi ◽  
CJ Eaves ◽  
PM Lansdorp ◽  
AC Eaves
Keyword(s):  
Chronic Myeloid Leukemia ◽  
Myeloid Leukemia ◽  
Philadelphia Chromosome ◽  
Significant Proportion ◽  
Hematopoietic Cells ◽  
Chronic Phase ◽  
Leukemic Cells ◽  
Clonogenic Cell ◽  
Clonogenic Cells

Abstract The peripheral blood of chronic myeloid leukemia (CML) patients with chronic-phase disease and elevated white blood cell (WBC) counts typically contains markedly increased numbers of a variety of neoplastic pluripotent and lineage-restricted hematopoietic progenitors. These include cells detected in standard colony assays as well as their more primitive precursors. The latter are referred to as long-term culture-initiating cells (LTC-IC) because of their ability to generate clonogenic cell progeny detectable after a minimum of 5 weeks incubation on competent fibroblast feeder layers. In this study, we have investigated a number of the properties of the LTC-IC and clonogenic cells present in the blood of such CML patients with high WBC counts. This included an analysis of the light scattering properties of these progenitors, as well as their expression of CD34 and HLA-DR, Rhodamine-123 staining, and in vitro sensitivity to 4- hydroperoxycyclophosphamide. In the case of LTC-IC, the production of different types of lineage-restricted and multipotent progeny was also analyzed. Most of the circulating LTC-IC and clonogenic cells in the CML patients studied (on average approximately 70% and approximately 90%, respectively) showed features of proliferating or activated cells. This is in marked contrast to the majority of progenitors in the blood of normal individuals and most of the LTC-IC in normal marrow, all of which exhibit a phenotype expected of quiescent cells. Interestingly, a significant proportion of the circulating clonogenic cells and LTC-IC in the CML samples studied (on average approximately 10% and approximately 30%, respectively) appeared to be phenotypically similar to normal circulating progenitors, although their absolute numbers were indicative of a neoplastic origin. Both phenotypes of circulating CML clonogenic cells and LTC-IC could be obtained at approximately 10% to 20% purity by differential multiparameter sorting. These findings suggest that expansion of the Philadelphia chromosome-positive clone at the level of the earliest types of hematopoietic cells results from the activation of mechanisms that enable some, but not all, signals that block the cycling of normal stem cells to be bypassed or overcome. In addition, they provide strategies for purifying these primitive leukemic cells that should facilitate further analysis of the mechanisms underlying their abnormal proliferative behavior.

scholarly journals Cytogenetic studies of early myeloid progenitor compartments in Ph1- positive chronic myeloid leukemia. II. Long-term culture reveals the persistence of Ph1-negative progenitors in treated as well as newly diagnosed patients

Blood ◽  
1984 ◽  
Vol 63 (5) ◽  
pp. 1172-1177 ◽  
Author(s):  
ID Dube ◽  
DK Kalousek ◽  
L Coulombel ◽  
CM Gupta ◽  
CJ Eaves ◽  
...  
Keyword(s):  
Chronic Myeloid Leukemia ◽  
Myeloid Leukemia ◽  
Chronic Phase ◽  
Rapid Decline ◽  
Newly Diagnosed ◽  
A Minor ◽  
Positive Population ◽  
Marrow Cultures

We recently showed that long-term marrow cultures can be used to demonstrate the presence of Philadelphia (Ph1) negative progenitors in patients with newly diagnosed Ph1-positive chronic myeloid leukemia (CML). We now report results for 6 chronic phase patients studied 5–83 mo postdiagnosis and an additional 3 newly diagnosed patients. Marrow metaphases were exclusively Ph1-positive. Clonogenic assays revealed a minor population of Ph1-negative progenitors in 3 cases (1 treated, 2 untreated). Long-term marrow culture adherent layers contained Ph1- negative progenitors in 6 cases (3 treated, 3 untreated). Whenever this occurred, the Ph1-negative population had become the only one detectable within 3–4 wk, and this was always associated with a rapid decline of the Ph1-positive population. For 2 of the 3 cases where Ph1- negative progenitors were not detected, there was a similar rapid decline in the Ph1-positive population in culture. In the other case, Ph1-positive progenitors were maintained at levels typically seen in normal long-term marrow cultures. These results suggest that chromosomally normal stem cells may persist for a considerable period in the marrow of some, but perhaps not all, patients with CML, even in the face of maintenance chemotherapy. In addition, they provide new evidence of heterogeneity in this disease, as shown by the variable ability of Ph1-positive progenitor populations to be maintained in vitro.

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