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It is well recognized that most cancers derive and progress from transformation and clonal expansion of a single cell that possesses stem cell properties, i.e., self-renewal and multilineage differentiation capacities. Such cancer stem cells (CSCs) are usually present at very low frequencies and possess properties that make them key players in tumor development. Indeed, besides having the ability to initiate tumor growth, CSCs drive tumor progression and metastatic dissemination, are resistant to most cancer drugs, and are responsible for cancer relapse. All of these features make CSCs attractive targets for the development of more effective oncologic treatments. In the present review article, we have summarized recent advances in the biology of CSCs, including their identification through their immunophenotype, and their physiology, both in vivo and in vitro. We have also analyzed some molecular markers that might become targets for developing new therapies aiming at hampering CSCs regeneration and cancer relapse.

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.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).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.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.

Background: The gastrointestinal parasite Giardia lamblia causes giardiasis. Its treatment with standard drugs produces side effects and improper treatment can generate resistant strains. New antigiardial compounds are needed. An analysis was done to identify the antigiardial activity of Morinda royoc, a plant used in traditional Mayan medicine to treat stomach and bowel pain. We aimed to assess the efficacy of M. royoc roots against G. lamblia and their effect on cells viability. Methods: A methanol extract was done of the root and then fractionated. The extract and fractions were tested in vitro on G. lamblia trophozoites and their effect on cell viability was quantified by flow cytometry. The active extract and fractions were analyzed by gas chromatography–mass spectrometry and high-performance liquid chromatography. Results: The hexane fraction exhibited potent activity against G. lamblia (IC50 = 0.08 µg/mL). Its principal component was an anthraquinone-type compound. None of the fractions were toxic to human promyelocytic leukemia, chronic myelogenous leukemia and human mononuclear cells. Conclusion: The medicinal plant M. royoc contains promising bioactive agents with antigiardial activity and deserves further research.

Introduction: Medicinal plants have been used in traditional medicine practices since ancient times. Mayan traditional medicine uses herbs as treatment to illness that can be related to cancer disease such as Phoradendron wattii. As part of the search for novel cancer treatment two triterpene compounds were isolated from P. wattii: 3-epi-betulinic acid and lupenone with preliminary activity on leukemias. We investigated the cancer treatment abilities of 3-epi-betulinic acid and lupenone on human chronic myelogenous leukemia (K562) cell line and monocytes isolated from peripheral blood using flow cytometry methods. Results: Lupenone showed low effect on both cells with an IC50 of 177.60 ± 5.07 µg/mL and a selectivity index (SI) of 0.10 times more on K-562 cell line than on monocytes. Nevertheless, 3-epi-betulinic acid showed a moderate anti-CML activity with an IC50 of 38.74 ± 5.07 µg/mL, having a SI of 4.10 on K-562 cells when compared to monocytes. These results indicate that 3-epi-betulinic acid was 41 times more selective for K-562 cell line than lupenone and it also was 7.45 more selective than etoposide as positive control. Additionally, 3-epi-betulinic acid increased cell population in the G0/G1 phase of the cell cycle and apoptotic cells, and it inhibited cell proliferation in a dose-dependent manner. Furthermore, the relative expression of genes associated with apoptosis Bax and caspase 3 increased compared to control. Conclusion: Combined, these results suggest that 3-epi-betulinic acid plays an important regulatory role of cell cycle and apoptosis. Finally, the 3-epi-betulinic acid investigated in this work is potentially a cheap, safe, and effective alternative to chemotherapy in cancer treatment. Citation Format: Rosa E. Moo-Puc, Shirley A. Calderón Sauri, Jairo R. Villanueva-Toledo, Sergio R. Peraza-Sánchez, Antonieta Chávez-González. Apoptotic and cell cycle effects of triterpenes isolated from Phoradendron wattii on a chronic myelogenous leukemia cell line. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3832.

Background COVID-19 counts 46 million people infected and killed more than 1.2 million. Hypoxaemia is one of the main clinical manifestations, especially in severe cases. HIF1α is a master transcription factor involved in the cellular response to oxygen levels. The immunopathogenesis of this severe form of COVID-19 is poorly understood. Methods We performed scRNAseq from leukocytes from five critically ill COVID-19 patients and characterized the expression of hypoxia-inducible factor1α and its transcriptionally regulated genes. Also performed metanalysis from the publicly available RNAseq data from COVID-19 bronchoalveolar cells. Results Critically-ill COVID-19 patients show a shift towards an immature myeloid profile in peripheral blood cells, including band neutrophils, immature monocytes, metamyelocytes, monocyte-macrophages, monocytoid precursors, and promyelocytes-myelocytes, together with mature monocytes and segmented neutrophils. May be the result of a physiological response known as emergency myelopoiesis. These cellular subsets and bronchoalveolar cells express HIF1α and their transcriptional targets related to inflammation (CXCL8, CXCR1, CXCR2, and CXCR4); virus sensing, (TLR2 and TLR4); and metabolism (SLC2A3, PFKFB3, PGK1, GAPDH and SOD2). Conclusions The up-regulation and participation of HIF1α in events such as inflammation, immunometabolism, and TLR make it a potential molecular marker for COVID-19 severity and, interestingly, could represent a potential target for molecular therapy.Key messagesCritically ill COVID-19 patients show emergency myelopoiesis.HIF1α and its transcriptionally regulated genes are expressed in immature myeloid cells which could serve as molecular targets.HIF1α and its transcriptionally regulated genes is also expressed in lung cells from critically ill COVID-19 patients which may partially explain the hypoxia related events.

Endothelial colony-forming cells (ECFCs) contribute to postnatal vasculogenesis. In venous thromboembolic disease (VTD), they are functionally abnormal and produce high concentrations of TNF-α.To analyze the TNF-α signaling pathway and its relationship with the expression of cell-cycle regulators.Mononuclear cells (MNCs) were collected from the peripheral blood of 20 healthy human volunteers (controls) and 30 patients with VTD matched by age (20-50 years) and sex to obtain ECFCs. We analyzed the relative quantification of the gene transcripts of TNF, NFkB1, PLAU, HMOX1, GSS, eNOS, CDKN1A, and CDKN1B through quantitative RT-PCR (qRT-PCR assays). Identification of NF-κB and activated targets of each pathway: NF-κB (Ser536); IκBα (Ser32/Ser36); p38 (Thr180/Tyr182) JNK (Thr183/Tyr185), p53 and cell-cycle regulators: p16, p18, p21, p27, p57, Cyclin D, Cyclin E, Cyclin A, Cyclin B, CDK2, CDK4; cell-cycle status was determined by KI-67 and 7-AAD. Cells were analyzed with flow cytometry and the FlowJo vX software.In ECFCs from VTD patients, TNF-α receptor and NFkB were overexpressed and hyper-phosphorylated; eNOS and HMOX1 were down-regulated; cell-cycle regulators (p53, p18, p21) were elevated. In addition, the cell cycle was locked in the G2 phase.Our results strongly suggest that these molecular alterations in the pathway of TNF-α and cell cycle regulation induce endothelial dysfunction, reduced proliferation potential and vascular regeneration, and consequently, the occurrence of new thrombotic events.

During the last two decades, three different epidemics, caused by three different coronaviruses, have affected humankind. The most recent, known as COVID-19, has caused in only five months, more than 340,000 deaths worldwide. Knowing the biology of coronavirus is key, not just to face the current pandemic, but to prepare ourselves for future epidemics. With this in mind, this article is focused on the biology of coronaviruses emphasizing SARS-CoV-2, the agent that causes COVID-19. This is a comprehensive review article, which covers different topics, from the biology and taxonomy of viruses, to the molecular biology of SARS-CoV-2, its mechanisms of action, and the immune response this virus elicits. We have also addressed clinical aspects of COVID-19, its methods of detection, treatment, and vaccines.Durante las últimas dos décadas, tres epidemias de gran magnitud, causadas por tres distintos tipos de coronavirus, han impactado a la humanidad. La más reciente, conocida como COVID-19, ha provocado en tan solo cinco meses, más de 340 000 muertes en todo el mundo. Conocer la biología de los coronavirus es fundamental, tanto para enfrentar la pandemia actual, como para prepararnos para futuras epidemias. En este contexto, el presente artículo está enfocado en la biología de los coronavirus con énfasis en el SARS-CoV-2, agente causal de COVID-19. La temática que se incluye es muy amplia, abarca desde la biología general de los virus y su taxonomía, hasta aspectos muy puntuales de la biología molecular de SARS-CoV-2, así como de sus mecanismos de acción y la respuesta inmune. También presentamos distintos aspectos clínicos de COVID-19, de los métodos para su detección y algunos enfoques terapéuticos, incluyendo tratamientos antivirales y vacunas.

Since the second half of the 20th century, our knowledge about the biology of cancer has made extraordinary progress. Today, we understand cancer at the genomic and epigenomic levels, and we have identified the cell that starts neoplastic transformation and characterized the mechanisms for the invasion of other tissues. This knowledge has allowed novel drugs to be designed that act on specific molecular targets, the immune system to be trained and manipulated to increase its efficiency, and ever more effective therapeutic strategies to be developed. Nevertheless, we are still far from winning the war against cancer, and thus biomedical research in oncology must continue to be a global priority. Likewise, there is a need to reduce unequal access to medical services and improve prevention programs, especially in countries with a low human development index.

The P2RY8-CRLF2 and IGH-CRLF2 rearrangements induce the overexpression of cytokine receptor-like factor 2 (CRLF2) and have been associated with relapse and poor prognosis in B-cell acute lymphoblastic leukemia (B-ALL). Additionally, they are frequently documented in high-risk Hispanic populations. To better understand the potential causes of the adverse prognosis of childhood B-ALL in Mexico, we analyzed these rearrangements and the CRLF2 mRNA and protein levels in 133 Mexican children with B-ALL. We collected bone marrow samples at diagnosis and evaluated the CRLF2 gene expression by qRT-PCR and the total CRLF2 protein by flow cytometry. P2RY8-CRLF2 and IGH-CRLF2 were detected by RT-PCR and FISH, respectively. The median time of follow-up to determine the prognostic significance of the CRLF2 abnormalities was three years. In 82% of the participants, the mRNA levels correlated with the cell-surface and intracellular CRLF2 protein levels. The P2RY8-CRLF2 rearrangement was present in 31.5% (42/133) of the patients, while the IGH-CRLF2 rearrangement was detected in 13.5% (9/67) of patients with high expression of CRLF2 (6.8% of the total sample). CRLF2 copy number variations (gain) were also detected in 7.5% (5/67) of patients with high protein levels. The overall survival (OS) presented significantly lower rates in patients with high white blood cell count (≥50x10

From the knowledge that hematopoiesis does not occur randomly in the bone marrow but is regulated by the different components of the microenvironment, the use of in vitro coculture systems has been used as a powerful tool in the analysis of different processes that are involved in the maintenance of blood cells. In this chapter, we describe a methodological strategy to perform a coculture between primitive hematopoietic cells and endothelial cells to evaluate cell cycle, an aspect of relevant importance in the permanence of primitive leukemic cells.

From the knowledge that hematopoiesis does not occur randomly in the bone marrow but is regulated by the different components of the microenvironment, the use of in vitro coculture systems has been used as a powerful tool in the analysis of different processes that are involved in the maintenance of blood cells. In this chapter, we describe a methodological strategy to perform a coculture between primitive hematopoietic cells and endothelial cells to evaluate cell cycle, an aspect of relevant importance in the permanence of primitive leukemic cells.

Chronic myeloid leukemia (CML) is a myeloproliferative disease that results from the BCR-ABL gene-induced transformation of a primitive hematopoietic cell. This disease has been extensively studied, and, as a result, a very effective therapy has been developed: the tyrosine kinase inhibitors. Although, there is a significant knowledge about the intrinsic biology of CML cells, alterations in their bone marrow microenvironment are not yet completely understood. In this concise review, we summarized recent findings on the composition and function of the bone marrow microenvironment in CML, and their importance in the progression of the disease and treatment resistance.

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.

Tyrosine kinase inhibitors (TKI) have become a first‐line treatment for chronic myeloid leuakemia (CML). TKIs efficiently target bulk CML cells; however, they are unable to eliminate the leukaemic stem cell (LSC) population that causes resistance and relapse in CML patients. In this study, we assessed the effects of parthenolide (PTL) and dimethyl amino parthenolide (DMAPT), two potent inhibitors of LSCs in acute myeloid leukaemia (AML), on CML bulk and CML primitive (CD34+lin−) cells. We found that both agents induced cell death in CML, while having little effect on the equivalent normal hematopoietic cells. PTL and DMAPT caused an increase in reactive oxygen species (ROS) levels and inhibited NF‐κB activation. PTL and DMAPT inhibited cell proliferation and induced cell cycle arrest in G0 and G2 phases. Furthermore, we found cell cycle inhibition to correlate with down‐regulation of cyclin D1 and cyclin A. In summary, our study shows that PTL and DMAPT have a strong inhibitory effect on CML cells. Given that cell cycle arrest was not dependent on ROS induction, we speculate that this effect could be a direct consequence of NF‐κB inhibition and if this mechanism was to be evaded, PTL and DMAPT induced cell death would be potentiated.

In this study, we determined the gene expression profiles of bone marrow‐derived cell fractions, obtained from normal subjects and Chronic Myeloid Leukemia (CML) patients, that were highly enriched for hematopoietic stem (HSCs) and progenitor (HPCs) cells. Our results indicate that the profiles of CML HSCs and HPCs were closer to that of normal progenitors, whereas normal HSCs showed the most different expression profile of all. We found that the expression profiles of HSCs and HPCs from CML marrow were closer to each other than those of HSCs and HPCs from normal marrow. The major biologic processes dysregulated in CML cells included DNA repair, cell cycle, chromosome condensation, cell adhesion, and the immune response. We also determined the genomic changes in both normal and CML progenitor cells under culture conditions, and found that several genes involved in cell cycle, steroid biosynthesis, and chromosome segregation were upregulated, whereas genes involved in transcription regulation and apoptosis were downregulated. Interestingly, these changes were the same, regardless of the addition of Imatinib (IM) to the culture. Finally, we identified three genes—PIEZO2, RXFP1, and MAMDC2‐ that are preferentially expressed by CML primitive cells and that encode for cell membrane proteins; thus, they could be used as biomarkers for CML stem cells.

Background Venous thromboembolic disease (VTD) is a public health problem. We recently reported that endothelial colony-forming cells (ECFCs) derived from endothelial cells (EC) (ECFC-ECs) from patients with VTD have a dysfunctional state. For this study, we proposed that a dysfunctional status of these cells generates a reduction of its proliferative ability, which is also associated with senescence and reactive oxygen species (ROS). Methods and results Human mononuclear cells (MNCs) were obtained from peripheral blood from 40 healthy human volunteers (controls) and 50 patients with VTD matched by age (20-50 years) and sex to obtain ECFCs. We assayed their proliferative ability with plasma of patients and controls and supernatants of cultures from ECFC-ECs, senescence-associated β-galactosidase (SA-β-gal), ROS, and expression of ephrin-B2/Eph-B4 receptor. Compared with cells from controls, cells from VTD patients showed an 8-fold increase of ECFCs that emerged 1 week earlier, reduced proliferation at long term (39%) and, in passages 4 and 10, a highly senescent rate (30±1.05% vs. 91.3±15.07%, respectively) with an increase of ROS and impaired expression of ephrin-B2/Eph-4 genes. Proliferation potential of cells from VTD patients was reduced in endothelial medium [1.4±0.22 doubling population (DP)], control plasma (1.18±0.31 DP), or plasma from VTD patients (1.65±0.27 DP). Conclusions As compared with controls, ECFC-ECs from individuals with VTD have higher oxidative stress, proliferation stress, cellular senescence, and low proliferative potential. These findings suggest that patients with a history of VTD are ECFC-ECs dysfunctional that could be associated to permanent risk for new thrombotic events.

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.

In vitro, Imatinib inhibits the proliferation and stimulates the osteogenic and adipogenic differentiation of mesenchymal stromal cells (MSC). However, it is unknown whether Imatinib affects the biology of MSC in vivo. We asked whether MSC from long-term Imatinib-treated CML patients were affected by the in vivo treatment. MSC from untreated and Imatinib-treated patients displayed normal functional properties (i.e. proliferation, immunophenotype, differentiation and hematopoietic supportive capacity) - but a decreased frequency. In vitro, Imatinib lost its effect when discontinued; which suggest that it has a reversible effect on MSC. Therefore it might lose its effect on MSC after discontinuation in vivo.

Cancer stem cells (CSCs), also known as tumor-initiating cells (TICs), are characterized by high self-renewal and multi-lineage differentiation capacities. CSCs are thought to play indispensable roles in the initiation, progression and metastasis of many types of cancer. Leukemias are thought to be initiated and maintained by a specific sub-type of CSC, the leukemia stem cell (LSC). An important feature of LSCs is their resistance to standard therapy, which may lead to relapse. Increasing efforts are aimed at developing novel therapeutic strategies that selectively target LSCs, while sparing their normal counterparts and, thus, minimizing adverse treatment-associated side-effects. These LSC targeting therapies aim to eradicate LSCs through affecting mechanisms that control their survival, self-renewal, differentiation, proliferation and cell cycle progression. Some LSC targeting therapies have already been proven successful in pre-clinical studies and they are now being tested in clinical studies, mainly in combination with conventional treatment regimens.A growing body of evidence indicates that the selective targeting of LSCs represents a promising approach to improve disease outcome. Beyond doubt, the CSC hypothesis has added a new dimension to the area of anticancer research, thereby paving the way for shaping a new trend in cancer therapy.

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 0.5 μM for K562 cells, 0.63 μM for MEG01 cells, 0.38 μM for CML CD34+ lin− cells, and 1.0 μM 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.

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.

Background 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. Procedure By using a negative immunoselection system, we have obtained a primitive cell population (enriched for CD34+ Lin− cells) from the bone marrow (BM) of 17 pediatric AML patients and characterized its proliferation, expansion, and differentiation potentials in liquid cultures supplemented with a mixture of 8 different recombinant stimulatory cytokines. Results The proportion of CD34+ cells in AML patients was extremely heterogeneous, ranging from 0% to 74%. Regardless of their CD34+ cell content, and in contrast to normal cells, AML cells showed a deficient capacity to proliferate even in the presence of the stimulatory cytokines. AML progenitors were unable to generate new progenitor cells, indicating their inability to expand. Interestingly, AML cells were able to differentiate in culture, giving rise to morphologically recognizable precursors. A major difference, however, as compared to hematopoietic progenitors from normal subjects, was the fact that whereas in cultures of normal cells both myeloid and erythroid precursors were produced, in AML cultures the vast majority of the cells generated corresponded to myeloid cells, mostly mature macrophages. Conclusion As compared to their normal counterparts, primitive hematopoietic cells from pediatric patients with AML possess impaired proliferation, expansion, and differentiation potentials in vitro. Pediatr Blood Cancer 2008;51:741–746. © 2008 Wiley-Liss, Inc.

In this study, we have assessed the in vitro growth of hematopoietic progenitor cells (HPC) from chronic myeloid leukemia (CML) patients that have recovered after different treatments. Bone marrow cells were obtained from 33 CML patients, including patients at diagnosis, before treatment (n=12), and patients that have achieved hematological remission (and in most cases a major cytogenetic response) after different therapeutic procedures (n=21), including patients treated with Interferon-alpha (IFN; n=5), imatinib mesylate (IMATINIB; n=8) and patients that received an allogeneic hematopoietic cell transplant (HCT; n=8). Marrow cells were enriched for CD34(+) cells and cultured in a serum- and stroma-free liquid culture system, supplemented with a combination of 8 recombinant cytokines. Normal samples were studied as controls. HPC from CML patients before therapy showed deficient proliferation and expansion potentials in culture (140-fold increase in nucleated cell number and 1.3-fold increase in colony-forming cell number) as compared to normal progenitors (1200-fold increase in nucleated cell number and 25-fold increase in colony-forming cell number). In contrast, HPC from patients treated with IMATINIB showed growth potentials similar to those of normal progenitors. Progenitors from patients after HCT also showed significant proliferation and expansion capacities. Interestingly, progenitors from IFN-treated patients showed proliferation and expansion kinetics similar to those of cells from untreated patients. These results indicate that, although treatment of CML patients with IFN, IMATINIB or HCT resulted in complete hematological remission (and a major cytogenetic response), only patients treated with IMATINIB and, to a lesser extent, with HCT showed a full hematopoietic recovery, as determined by the in vitro growth of HPC in our culture system.

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.

Chronic Myeloid Leukemia (CML) is a malignant hematologic disease characterized by the presence of a chromosomal alteration known as the Philadelphia Chromosome (Ph). Ph originates in Leukemia Stem Cells (LSC) and is caused by the reciprocal translocation between chromosome 9 and 22, giving rise to the Bcr-Abl oncogene, which produces a constitutively active tyrosine kinase -the BCR-ABL oncoprotein. This CML feature has made it possible the development of Tyrosine Kinase Inhibitors (TKI's) Imatinib, Dasatinib, and Nilotinib, which are able to inhibit BCR-ABL and take patients into molecular remission. However TKI's are not able to eliminate the population of LSC, which cause patients to relapse if treatment is discontinued. This has made it necessary search for alternative ways to eliminate LSC in CML patients. Parthenolide (PTL), a sesquiterpene lactone extracted from the feverfew plant (Thanacethum partenium), which has been used in folk medicine to treat inflammatory symptoms, has shown activity against LSC in Acute Myeloid Leukemia (AML). Recently Guzman and collaborators have developed an analog of PTL, named DMAPT, which is orally bioavailable. Both molecules have shown to be active inhibitors of NFkB and induce high levels of Reactive Oxygen Species (ROS) in LSC of AML. In the present study we asked whether CML cells (LSC, Progenitors and CML cell lines) are sensitive to PTL and DMAPT. Cell lines K562, Kcl-22 and HL60 (as an AML control) were cultured in liquid suspension cultures with increasing concentrations of PTL or DMAPT at a cell density of 300,000 cells per ml. At 24 hours of exposure, PTL and DMAPT showed significant induction of apoptosis at 7.5 µM and 10 µM, respectively. To determine whether NFkB activity is inhibited by PTL and DMAPT, K562, Kcl-22, HL60 cell lines, as well as CD34+ cells from a CML patient in Chronic phase where exposed to 7.5 µM PTL or 10 µM DMAPT. After 6 hours, protein lysis was induced and Western blots were performed with protein extracts from 250,000 cells per lane. Membranes where probed for Phosphorilated-p65, total p65 and b-actin as a loading control. We observed a decrease in the levels of phosphorylated p65 in cells treated with PTL and DMAPT, compared with untreated cells, while levels of total p65 remained constant. We had previosly used DCFDA stain and HMOX expression, to observe ROS induction in CML cells exposed to PTL. To asses if ROS induction is necessary for CML cell death we pretreated cells with N-acetyl cysteine (NAC), precursor of glutathione, for 1 hour before treating cells with 7.5 µM PTL or 10 µM DMAPT, and evaluated ROS levels by staining with Cell rox deep red, Mitosox-PE, and also assessing cell viability with YoPro-1 and 7AAD stain. After 6 hours of PTL treatment, we found that NAC pretreatment diminished ROS levels in cytoplasm and mitochondrial dysfunction when cells were treated with PTL. Pretreatment with NAC was able to rescue the majority of cells from apoptosis induced by PTL. Similar results were observed in cultures treated with DMAPT. Taken together, our results indicate that PTL and DMAPT were able to induce cell death via apoptosis in CML cells. This process seems to involve inhibition of p65 phosphorylation and an increase in ROS levels, which we found to be necessary for cell death to occur. Disclosures No relevant conflicts of interest to declare.

Chronic myeloid leukemia (CML) is a clonal myeloproliferative neoplasia associated with the t(9,22)(q34:q11) reciprocal translocation, also known as Philadelphia chromosome (Ph). As a result of such abnormality, a chimeric gene (bcr-abl) is produced that is translated into a chimeric protein (BCR-ABL), a constitutively activated tyrosine kinase. Major cell dysfunctions result from this abnormal kinase activity, including increased proliferation and reduced apoptosis. Based on the structure of BCR-ABL, several molecules have been designed that inhibit its kinase activity. Five such molecules have already been brought into the clinic for the treatment of Ph+ CML patients. Good results have been obtained in terms of patients' remission rates and quality of life. Some major problems, however, have been observed. Firstly, a significant proportion of patients develop resistance to the drugs; secondly, it is clear that such drugs affect most of the leukemic cells, but do not eliminate leukemia stem cells. Thus, important CML-related challenges remain to be solved in the near future.

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.

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].

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 G(0)/G(1) 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.

Chronic Myeloid Leukaemia (CML) is a clonal disease, originated at the level of Hematopoietic Stem Cells (HSC) and characterized by the presence of the Philadelphia (Ph) chromosome and its oncogenic product p210(BcrAbl). Such a protein has been shown to be essential for malignant transformation, since it is capable of altering cell adhesion, proliferation and apoptosis. Historically, CML has been treated by using different approaches: arsenic (in the early days), a variety of chemical agents (busulfan, hydroxyurea, cytarabine), cytokines (IFN-alpha, IFNalpha-PEG), hematopoietic cell transplant (HCT), and more recently drugs generated by design (imatinib, nilotinib, dasatinib). All these molecules exert specific effects on HSC and lead to a variety of clinical and biological responses. In this article, we present an overview about hematopoiesis in CML and its implications in the treatment of this disease.

Lineage-negative (Lin(-)) cell populations, obtained by negative selection from umbilical cord blood (UCB) and adult mobilized peripheral blood (aMPB), were cultured in serum-free liquid cultures supplemented with a mixture of seven stimulatory cytokines. On specific days, proliferation potential was assessed and cell cycle status was determined by DNA content. Expression of the cell cycle regulators cyclin D3 (cD3), cyclin-dependent kinase 4 (cdk4), p21(cip1/waf1) (p21), and p27(kip1) (p27) was also determined. As expected, UCB cells showed significantly higher proliferation potentials than aMPB cells, particularly during the first 7 days of culture. During this period of time, higher numbers of cell cycles were observed in UCB cells (7-9 cycles), as compared to aMPB cells (5-6 cycles). Higher levels of cD3, cdk4, and p27 were also detected in UCB cells. Our results confirm that UCB cells possess an intrinsically higher proliferation potential, as compared to aMPB cells, and suggest that such a biological difference is due, at least in part, to differences in cell cycle status. This, in turn, seems to result from the differential expression of cell cycle regulatory molecules.

Chronic myeloid leukemia (CML) arises from the malignant transformation of a hematopoietic stem cell (HSC) that gives rise to functionally defective progeny, including primitive and relatively mature progenitor cells (HPC). Both HSC and HPC are comprised within the population of CD34 + cells, normally present in bone marrow (BM). In the present study, we have separated two different subpopulations of CD34 + cells from CML marrow: Population I, enriched for CD34 + Lin − cells; and Population II, enriched for CD34 + CD36 − CD38 − CD45RA − Lin − cells, and assessed their progenitor cell content as well as their capacity to proliferate and expand in response to a combination of hematopoietic cytokines in serum- and stroma-free long-term liquid cultures. The absolute cell numbers recovered in Population I from normal and CML samples were similar; in contrast, we found that Population II from CML was amplified four-fold, as compared to normal. In spite of this latter observation, no significant differences were observed in terms of the absolute number of CFC when comparing Populations I and II from CML patients and normal subjects. Interestingly, the proliferation and expansion potentials of CML cells were clearly deficient as compared to their normal counterparts. Indeed, in cultures of Population I cells the maximum fold increase in total and progenitor cell numbers corresponded to 30 and 8%, respectively, of those observed in cultures of normal marrow-derived Population I cells. Such functional deficiencies were even more evident in Population II cells in which the maximum fold increase in total and progenitor cell numbers corresponded to 3 and 0.5%, respectively, of the levels found in cultures of Population II cells from normal marrow. The present study demonstrates that bone marrow-derived CD34 + cells from CML patients possess functional abnormalities, clearly evident in the in vitro system used by us. Among the two CML subpopulations studied here, the more immature one (Population II; enriched for CD34 + CD36 − CD38 − CD45RA − Lin − cells) was the one that showed the most severe abnormalities, as compared to its relatively more mature counterpart (Population I; enriched for CD34 + Lin − cells).