Antonieta Chávez-González

In vitro biology of human myeloid leukemia

For about 40 years, the biology of human myeloid leukemia (ML) has been studied in different in vitro systems. Throughout this time, semisolid colony assays, Dexter-type long-term cultures and liquid suspension cultures have contributed to our understanding of the mechanisms involved in the origin and progression of this hematological disorder. By using such systems, it has been possible to identify the cells in which leukemia originates; to recognize a functional hierarchy within the hematopoietic system of leukemia patients; to identify factors, soluble and cell-associated, that regulate leukemic growth; and to study the effects of different antineoplastic drugs. Furthermore, in vitro systems for purging of leukemic cells have been developed. Still, many questions and problems remain unsolved regarding the biology of myeloid leukemia in vitro. This article presents a comprehensive review on the behavior of leukemic stem and progenitor cells, both from acute and chronic myeloid leukemia, in the different culture systems mentioned above.

Comparative analysis of the in vitro proliferation and expansion of hematopoietic progenitors from patients with aplastic anemia and myelodysplasia

Aplastic anemia (AA) and myelodysplasia (MDS) show great similarities in their biology. To date, however, it is still unclear to what extent hematopoietic progenitor cells (HPCs) from AA and MDS share biological properties and what the functional differences are between them. In trying to address this issue, in the present study we have analyzed, in a comparative manner, the proliferation and expansion capacities of bone marrow (BM) progenitor cells from AA and MDS in response to recombinant cytokines. BM samples from normal subjects (NBM) and patients with AA and MDS were enriched for HPC by immunomagnetic-based negative selection. Selected cells were cultured in the absence (control) or in the presence of early-acting cytokines (Mix I), or early-, intermediate- and late-acting cytokines (Mix II). Proliferation and expansion were assessed periodically. In NBM and MDS cultures apoptosis was also determined. In NBM cultures, Mix I induced a nine-fold increase in total cell numbers and a 3.6-fold increase in colony-forming cell (CFC) numbers. In Mix II-supplemented cultures, total cells were increased 643-fold, and CFC 12.4-fold. In AA cultures, no proliferation or expansion were observed in Mix I-supplemented cultures, whereas only a four-fold increase in total cell numbers was observed in the presence of Mix II. In MDS cultures, a 12-fold increase in total cells and a 2.9-fold increase in CFC were observed in the presence of Mix I; on the other hand, Mix II induced a 224-fold increase in total cells and a 5.9-fold increase in CFC. Apoptosis was reduced in cytokine-supplemented cultures from NBM. In contrast, Mix II induced a significant increase in the rate of apoptosis in MDS cultures. Our results demonstrate that, as compared to their normal counterparts, AA and MDS progenitors are deficient in their proliferation and expansion potentials. Such a deficiency is clearly more pronounced in AA cells, which seem to be unable to respond to several cytokines. MDS progenitors, on the other hand, are capable to proliferate and expand in response to cytokines; however, their rate of apoptosis is increased by intermediate- and late-acting cytokines, so that the overall proliferation and expansion are significantly lower than those of normal progenitor cells.

Expression of CD90, CD96, CD117, and CD123 on Different Hematopoietic Cell Populations from Pediatric Patients with Acute Myeloid Leukemia

BACKGROUND AND AIMS In trying to contribute to our knowledge on the biology of hematopoietic stem cells (HSC) and hematopoietic progenitor cells (HPC) from pediatric acute myeloid leukemia (AML), in the present study we analyzed the expression of four cell surface antigens relevant to human hematopoiesis-CD90, CD96, CD117, and CD123-in bone marrow from pediatric AML patients and normal control subjects. METHODS CD34(+) CD38(-) cells (enriched for HSC) and CD34(+) CD38(+) cells (enriched for HPC) were resolved on the basis of CD34 and CD38 expression. Concomitantly, expression of CD90 and CD96 or CD117 and CD123 was assessed by multicolor flow cytometry in each cell population. RESULTS CD90 and CD117 were expressed in a low proportion of CD34(+) CD38(-) and CD34(+) CD38(+) cells and no significant differences were observed between normal marrow and AML at diagnosis. In contrast, CD96(+) cells and CD123(+) cells were found at significantly higher levels in both cell populations from AML at diagnosis, as compared to normal marrow. Levels of both cell surface markers after treatment remained higher than in normal marrow. DISCUSSION These results show an increased frequency of CD96(+) and CD123(+) cells within the CD34(+) cell population from pediatric AML; this is consistent with the findings reported previously for adult AML. Our study supports the notion that expression of such antigens should be explored for their use as markers for diagnosis and prognosis.

Adult, but not neonatal, human lymphoid progenitors respond to TLR9 ligation by producing functional NK-like cells

Remarkable progress has been made in characterizing factors controlling lineage fate decisions of primitive progenitors that initiate the lymphoid program in bone marrow. However, the understanding of neonatal/adult differences in environmental signals that influence differentiation pathway stability is still incomplete. Our recent findings suggest that Toll-like receptors provide a mechanism for producing cells of the innate immune system from early stages of lymphoid development in mice. We now show that both human early multilymphoid progenitors and more differentiated lymphoid progenitors from normal adult bone marrow express TLR9. Furthermore, they respond to its ligation by upregulating the expression of IL-15Rβ (CD122) and accelerating the production of functional natural killer (NK)-like cells. Proliferation of the presumed equivalent progenitor cells from umbilical cord blood was stimulated by CpG-containing oligonucleotides or herpes simplex virus, but the already robust NK-cell formation was unchanged. This new information adds to other known differences between neonatal and adult lymphoid progenitors and suggests only the latter replenish innate NK-like cells in response to Toll-like receptor agonists.

In vitro cell cycle dynamics of primitive hematopoietic cells from human umbilical cord blood.

Abstract The goal of the present study was to investigate the specific way in which recombinant stimulatory cytokines modulate the cell cycle dynamics of primitive hematopoietic cells in vitro. A human cord blood-derived cell population, enriched for CD34+ Lin− cells, was obtained by negative selection and cultured in liquid cultures, in the absence or presence of recombinant stimulatory cytokines. The proportion of cells in each phase of the cell cycle, as well as the expression of cyclin D3, cyclin-dependent kinase-4 (cdk4), p16, p21 and p27, was determined at different time points. At the onset of culture, the vast majority of the cells were in the G0/G1 phase of the cell cycle. In the absence of cytokines, most cells remained in such a phase and no cell cycle activity was detected throughout the culture period, which correlated with the absence of population doublings. In the presence of cytokines, approximately four cell cycles, with a proportionate population doubling, were observed within the first 4 days of culture. In cultures incorporating cytokines, expression levels of cyclin D3 and cdk4 were higher than in their absence; in contrast, the levels of the cell cycle inhibitors p16 and p21 were higher in cultures without cytokines. Levels of p27 were also higher in the presence of cytokines. Our results indicate that the proliferation of primitive hematopoietic cells in liquid culture is promoted by recombinant cytokines via the induction of specific positive regulators of the cell cycle and down-regulation of particular cell cycle inhibitors.

Endothelial colony-forming cells: Biological and functional abnormalities in patients with recurrent, unprovoked venous thromboembolic disease

INTRODUCTION Endothelial cells (ECs) are an important component of the blood coagulation system because it maintains blood fluid. Because in patients with venous thromboembolic disease (VTD) a thrombophilic condition is not found sometimes, we investigated if endothelial colony-forming cells (ECFCs) from these patients have biological and functional abnormalities. PATIENTS AND METHODS Human mononuclear cells (MNCs) were obtained from peripheral blood from patients with VTD and controls to obtain ECFCs. These cells were assayed for their immunophenotype and electron microscopy characteristics and their ability to form capillary-like structures and to produce pro-inflammatory and pro-angiogenic cytokines and reactive oxygen species (ROS). RESULTS ECFCs appeared at 7 and 21 days of culture in VTD patients and controls, respectively. ECFCs increased 8-fold in patients and emerged 1 week earlier. No differences in the size of the colonies of ECFCs were found. Numbers and time of appearance of ECFCs was different between groups. ECFC-derived ECs (ECFC-ECs) of both groups expressed CD31, CD34, CD146, and CD-309 but none expressed CD45, CD14, or CD90. Interest CD34 was highly expressed in ECFC-ECs from patients. In both groups, ECFC-ECs showed similar capacity to form capillary-like structures but ECFC-ECs from patients had significant abnormalities in the mitochondrial membrane. We found a significant increase in ROS production in ECFC-ECs from patients. There were significant differences in cytokine profiles between VTD patients and controls. CONCLUSIONS We found a dysfunctional state in ECFC from VTD patients resembling some characteristics of dysfunctional ECs. These findings may help to understand some pathophysiological aspects of VTD.

Defective in vitro growth of primitive hematopoietic cells from pediatric patients with acute myeloid leukemia

Acute myeloid leukemia (AML) is a neoplastic hematologic disorder that arises at the level of a primitive stem/progenitor cell. Most studies on the biology of the hematopoietic system in AML have focused on cells from adult patients; much less is known about hematopoietic cells from childhood AML.

CDKIs p18(INK4c) and p57(Kip2) are involved in quiescence of CML leukemic stem cells after treatment with TKI.

ABSTRACT Chronic Myeloid Leukemia (CML) is sustained by a small population of cells with stem cell characteristics known as Leukemic Stem Cells that are positive to BCR-ABL fusion protein, involved with several abnormalities in cell proliferation, expansion, apoptosis and cell cycle regulation. Current treatment options for CML involve the use of Tirosine Kinase Inhibitor (Imatinib, Nilotinib and Dasatinib), that efficiently reduce proliferation proliferative cells but do not kill non proliferating CML primitive cells that remain and contributes to the persistence of the disease. In order to understand the role of Cyclin Dependent Kinase Inhibitors in CML LSC permanence after TKI treatment, in this study we analyzed cell cycle status, the levels of several CDKIs and the subcellular localization of such molecules in different CML cell lines, as well as primary CD34+CD38−lin− LSC and HSC. Our results demonstrate that cellular location of p18INK4c and p57Kip2 seems to be implicated in the antiproliferative activity of Imatinib and Dasatinib in CML cells and also suggest that the permanence of quiescent stem cells after TKI treatment could be associated with a decrease in p18INK4c and p57Kip2 nuclear location. The differences in p18INK4cand p57Kip2activities in CML and normal stem cells suggest a different cell cycle regulation and provide a platform that could be considered in the development of new therapeutic options to eliminate LSC.

Hematopoietic Stem Cells in Chronic Myeloid Leukemia

Chronic myeloid leukemia (CML) is a lethal hematological malignancy characterized by the abnormal amplification of the myeloid (mainly granulocityc) compartment of the hemato‐ poietic system. It originates from the transformation of a primitive hematopoietic cell that suffers a t(9;22) (q34; q11) balanced reciprocal translocation that results in the generation of the Philadelphia chromosome (Ph). Ph produces BCR-ABL, a constitutively active tyrosine kinase that drives a wide variety of physiological alterations [1].

Casiopeina III-Ea, a copper-containing small molecule, inhibits the in vitro growth of primitive hematopoietic cells from chronic myeloid leukemia

Several novel compounds have been developed for the treatment of different types of leukemia. In the present study, we have assessed the in vitro effects of Casiopeina III-Ea, a copper-containing small molecule, on cells from patients with Chronic Myeloid Leukemia (CML). We included primary CD34+ Lineage-negative (Lin-) cells selected from CML bone marrow, as well as the K562 and MEG01 cell lines. Bone marrow cells obtained from normal individuals - both total mononuclear cells as well as CD34+ Lin- cells- were used as controls. IC50 corresponded to 0.5μM for K562 cells, 0.63μM for MEG01 cells, 0.38μM for CML CD34+ lin- cells, and 1.0μM for normal CD34+ lin- cells. Proliferation and expansion were also inhibited to significantly higher extents in cultures of CML cells as compared to their normal counterparts. All these effects seemed to occur via a bcr-abl transcription-independent mechanism that involved a delay in cell division, an increase in cell death, generation of Reactive Oxygen Species and changes in cell cycle. Our results demonstrate that Casiopeina III-Ea possesses strong antileukemic activity in vitro, and warrant further preclinical (animal) studies to assess such effects in vivo.