Maintenance Of Hematopoietic Cells Research Articles

The noncanonical NFκB pathway: Regulatory mechanisms in health and disease.

The noncanonical NFκB signaling pathway mediates the biological functions of diverse cell survival, growth, maturation, and differentiation factors that are important for the development and maintenance of hematopoietic cells and immune organs. Its dysregulation is associated with a number of immune pathologies and malignancies. Originally described as the signaling pathway that controls the NFκB family member RelB, we now know that noncanonical signaling also controls NFκB RelA and cRel. Here, we aim to clarify our understanding of the molecular network that mediates noncanonical NFκB signaling and review the human diseases that result from a deficient or hyper-active noncanonical NFκB pathway. It turns out that dysregulation of RelA and cRel, not RelB, is often implicated in mediating the resulting pathology. This article is categorized under: Immune System Diseases > Molecular and Cellular Physiology Cancer > Molecular and Cellular Physiology Immune System Diseases > Stem Cells and Development.

Pou5f3.3 is involved in establishment and maintenance of hematopoietic cells during Xenopus development

Three POU family class V gene homologues are expressed in the development of Xenopus. In contrast to the expression of Pou5f3.1 and Pou5f3.2 in organogenesis, Pou5f3.3 is expressed during oogenesis in ovary. We investigated the expression and function of Pou5f3.3 in organogenesis of Xenopus laevis. RT-PCR and immunohistochemical analysis indicated that Pou5f3.3 was expressed in a small number of adult liver cells and blood cells. Immunocytochemical investigation proved that Bmi1, a marker for hematopoietic progenitor cells, was co-expressed in Pou5f3.3-expressing small spherical cells in the peripheral blood. In anemic induction by intraperitoneal injection of phenyl hydrazine, the number of Pou5f3.3-expressing cells significantly increased within 3 days after phenyl hydrazine injection. In CRISPR/Cas mutagenesis of Pou5f3.3, Bmi1-positive hematopoietic progenitor cell count decreased in the hematopoietic dorsal-lateral plate (DLP) region, resulting in a considerable reduction in peripheral blood cells. CRISPR/Cas-induced hematopoietic deficiency was completely rescued by Pou5f3.3 supplementation, but not by Pou5f3.1 or Pou5f3.2. Transplantation experiments using the H2B-GFP transgenic line demonstrated that DLP-derived Pou5f3.3-positive and Bmi1-positive cells were translocated into the liver and bone through the bloodstream. These results suggest that Pou5f3.3 plays an essential role in the establishment and maintenance of hematopoietic progenitor cells during Xenopus development.

Thrombopoietin maintains cell numbers of hematopoietic stem and progenitor cells with megakaryopoietic potential.

Thrombopoietin has long been known to influence megakaryopoiesis and hematopoietic stem and progenitor cells, although the exact mechanisms through which it acts are unknown. Here we show that MPL expression correlates with megakaryopoietic potential of hematopoietic stem and progenitor cells and identify a population of quiescent hematopoietic stem and progenitor cells that show limited dependence on thrombopoietin signaling. We show that thrombopoietin is primarily responsible for maintenance of hematopoietic cells with megakaryocytic differentiation potential and their subsequent megakaryocyte differentiation and maturation. The loss of megakaryocytes in thrombopoietin knockout mouse models results in a reduction of megakaryocyte-derived chemokine platelet factor 4 (CXCL4/PF4) in the bone marrow and administration of recombinant CXCL4/PF4 rescues the loss of quiescence observed in these mice. CXCL4/PF4 treatment does not rescue reduced hematopoietic stem and progenitor cell numbers, suggesting that thrombopoietin maintains hematopoietic stem and progenitor cell numbers directly.

Bone marrow niches in haematological malignancies.

Haematological malignancies were previously thought to be driven solely by genetic or epigenetic lesions within haematopoietic cells. However, the niches that maintain and regulate daily production of blood and immune cells are now increasingly being recognized as having an important role in the pathogenesis and chemoresistance of haematological malignancies. Within haematopoietic cells, the accumulation of a small number of recurrent mutations initiates malignancy. Concomitantly, specific alterations of the niches, which support haematopoietic stem cells and their progeny, can act as predisposition events, facilitating mutant haematopoietic cell survival and expansion as well as contributing to malignancy progression and providing protection of malignant cells from chemotherapy, ultimately leading to relapse. In this Perspective, we summarize our current understanding of the composition and function of the specialized haematopoietic niches of the bone marrow during health and disease. We discuss disease mechanisms (rather than malignancy subtypes) to provide a comprehensive description of key niche-associated pathways that are shared across multiple haematological malignancies. These mechanisms include primary driver mutations in bone marrow niche cells, changes associated with increased hypoxia, angiogenesis and inflammation as well as metabolic reprogramming by stromal niche cells. Consequently, remodelling of bone marrow niches can facilitate immune evasion and activation of survival pathways favouring malignant haematopoietic cell maintenance, defence against excessive reactive oxygen species and protection from chemotherapy. Lastly, we suggest guidelines for the handling and biobanking of patient samples and analysis of the niche to ensure that basic research identifying therapeutic targets can be more efficiently translated to the clinic. The hope is that integrating knowledge of how bone marrow niches contribute to haematological disease predisposition, initiation, progression and response to therapy into future clinical practice will likely improve the treatment of these disorders.

The KDM4/JMJD2 histone demethylases are required for hematopoietic stem cell maintenance

AbstractKDM4/JMJD2 are H3K9- and H3K36-specific demethylases, which are considered promising therapeutic targets for the treatment of acute myeloid leukemia (AML) harboring MLL translocations. Here, we investigate the long-term effects of depleting KDM4 activity on normal hematopoiesis to probe potential side effects of continuous inhibition of these enzymes. Utilizing conditional Kdm4a/Kdm4b/Kdm4c triple-knockout mice, we show that KDM4 activity is required for hematopoietic stem cell (HSC) maintenance in vivo. The knockout of the KDM4 demethylases leads to accumulation of H3K9me3 on transcription start sites and the corresponding downregulation of expression of several genes in HSCs. We show that 2 of these genes, Taf1b and Nom1, are essential for the maintenance of hematopoietic cells. Taken together, our results show that the KDM4 demethylases are required for the expression of genes essential for the long-term maintenance of normal hematopoiesis.

Mesenchymal stromal cells in bone marrow express adiponectin and are efficiently targeted by an adiponectin promoter-driven Cre transgene.

Stromal cells in bone marrow (BM) constitute a specific microenvironment supporting the development and maintenance of hematopoietic cells. Adiponectin is a cytokine secreted by adipocytes. Besides its anti-diabetic and anti-atherogenic roles, adiponectin reportedly regulates the development and function of hematopoietic cells in BM. However, it remains unclear whether mesenchymal stromal cells in BM express adiponectin. Here, we show that PDGFRβ+VCAM-1+ stromal cells express adiponectin. Lineage tracing revealed that a majority of PDGFRβ+VCAM-1+ cells were targeted by an adiponectin promoter-driven Cre (Adipoq-Cre) transgene. Additionally, the Adipoq-Cre transgene targets a minority of osteoblasts at a younger age but larger populations are targeted at an older age. Furthermore, the Adipoq-Cre transgene targets almost all CXCL12-abundant reticular (CAR) cells and most of the stromal cells targeted by the Adipoq-Cre transgene are CAR cells. Finally, deletion of interleukin-7 (IL-7) by the Adipoq-Cre transgene resulted in severe impairment of B lymphopoiesis in BM. These results demonstrate that PDGFRβ+VCAM-1+ stromal cells in BM express adiponectin and are targeted by the Adipoq-Cre transgene, suggesting a broader specificity of the Adipoq-Cre transgene.

Dermatopontin in Bone Marrow Extracellular Matrix Regulates Adherence but Is Dispensable for Murine Hematopoietic Cell Maintenance.

SummaryThe hematopoietic marrow microenvironment is composed of multiple cell types embedded in an extracellular matrix (ECM). We have explored marrow ECM using mass spectrometry and found dermatopontin (DPT), a small non-collagenous ECM protein, to be present. We found that DPT cooperates with other ECM proteins to promote hematopoietic cell adherence in vitro on plastic as well as OP9 stromal cells. We generated constitutional DPT−/− mice that were viable and had no peripheral lympho-hematopoietic abnormalities. The composition of the marrow of wild-type and DPT−/− mice was equivalent in terms of cellularity, CFU-C, LSK (Lineage−, SCA-1+, KIT+), and LSK-SLAM (LSK, CD48−, CD150+) frequencies. These data suggest that DPT fosters adherence but is not required for steady-state hematopoiesis in vivo. There are likely overlapping cellular adhesion mechanisms that can compensate to maintain the hematopoietic niche in the absence of DPT.

Histone acetyltransferase activity of MOF is required for adult but not early fetal hematopoiesis in mice

AbstractK(lysine) acetyltransferase 8 (KAT8, also known as MOF) mediates the acetylation of histone H4 at lysine 16 (H4K16ac) and is crucial for murine embryogenesis. Lysine acetyltransferases have been shown to regulate various stages of normal hematopoiesis. However, the function of MOF in hematopoietic stem cell (HSC) development has not yet been elucidated. We set out to study the role of MOF in general hematopoiesis by using a Vav1-cre–induced conditional murine Mof knockout system and found that MOF is critical for hematopoietic cell maintenance and HSC engraftment capacity in adult hematopoiesis. Rescue experiments with a MOF histone acetyltransferase domain mutant illustrated the requirement for MOF acetyltransferase activity in the clonogenic capacity of HSCs and progenitors. In stark contrast, fetal steady-state hematopoiesis at embryonic day (E) 14.5 was not affected by homozygous Mof deletion despite dramatic loss of global H4K16ac. Hematopoietic defects start manifesting in late gestation at E17.5. The discovery that MOF and its H4K16ac activity are required for adult but not early and midgestational hematopoiesis supports the notion that multiple chromatin regulators may be crucial for hematopoiesis at varying stages of development. MOF is therefore a developmental-stage–specific chromatin regulator found to be essential for adult but not early fetal hematopoiesis.

Bone Marrow Stromal Cells Derived at Diagnosis and Remission Preferentially Support AML Cell Proliferation in Vitro

Introduction: Acute myeloid leukemia (AML) is a heterogeneous clonal disorder of hematopoietic progenitor cells characterized by excessive proliferation of early progenitor cells in the bone marrow (BM). The BM niche is a specialized type of microenvironment (ME) that actively supports growth, maturation and maintenance of hematopoietic stem and progenitor cells. They include a complex network of extracellular matrix proteins, soluble growth factors, and cytokines. The information regarding the clinical significance of leukemic microenvironment for AML initiation and propagation is scarce. The aim of the current study was to characterize the AML BM ME and its crosstalk with leukemic progenitor cells and define the role of BM stromal cells (SCs) in supporting leukemic cell growth in active disease as compared to the complete remission (CR) state.Methods: We developed a unique model of patient own stroma (POS) by growing SCs obtained at diagnosis, CR and relapse in a specific medium, including cytokines and proteins derived from human plasma solution. The growth rate and morphological changes of AML SCs were assessed and their ability to support AML cell expansion was studied by monitoring immunophenotyping alterations of CD34 and CD45 expression using FACS analysis. Levels of secreted MCP-1, IL6, GM-CSF, G-CSF and TNF in the co-culture medium were measured using the bead-based immunoassay CBA (BD Biosciences). In addition, real-time PCR (RT-PCR) amplification was performed in the co-culture to quantify changes aiming to assess the expression of specific genes associated with the maintenance of hematopoietic cells, self-renewal, quiescence regulation and mobilization. The following genes were amplified: CXCL12 (SDF1), transforming growth factor beta 1 (TGFβ), secreted phosphoprotein 1 (SPP1) and angiopoietin 1 (ANGP1).Results: The developed POS model allowed revealing high diversity in the morphological appearance of leukemic SCs obtained from different patients and at various disease stages. Patient SCs were found to grow slower than those of healthy volunteers (fig. 1A). Moreover, the growth rate differed between the patients and disease stages, being highest at relapse and lowest at diagnosis (fig. 1A). Levels of SDF-1, TGF-1 and SPP1 were elevated (X50, X50 and X 300 fold, respectively) in CR stroma compared to that of healthy volunteers. Of note, the level of MCP-1 was increased (2200 pg/ml) in the stroma derived at active disease stages (diagnosis and relapse) compared to CR or normal SCs (1050 pg/ml). Additionally, the expansion of CD34+ leukemic progenitor cells was higher in the presence of CR-derived stroma compared to healthy SCs (fig. 1B). In the co-culture, we observed selective ability of POS to support CD34+ leukemic cell expansion, as detected by flow cytometry (1.5 million cells more in POS than in healthy stroma), as well as differential expression of MCP-1 (x3000 pg/ml) and IL-6 (x9000 pg/ml) cytokines in co-cultures of AML cells and POS. These cytokines are known to be involved in the regulation of cell migration and inflammation.Conclusions: AML BM SCs are heterogeneous and differ from healthy BM SCs in their growth rate, morphology, immunophenotype features and the ability to support expansion of early progenitor CD34+ cells. Moreover, even an AML CR-derived stroma, which is considered "healthy", can provide an advantageous ME to leukemogenesis, as evident by increased proliferation of CD34+ leukemic cells in a co-culture with CR SCs compared to healthy SCs. Additionally, POS was found to selectively support the expansion of AML cells of the same patient. These findings, suggesting a cross-talk between leukemic progenitor cells and their specific POS, may lead to better understanding of the mechanisms underlying AML initiation and relapse. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

LPS-stimulated human bone marrow stroma cells support myeloid cell development and progenitor cell maintenance.

The nonhematopoietic bone marrow (BM) microenvironment provides a functional niche for hematopoietic cell maintenance, recruitment, and differentiation. It consists of multiple cell types including vasculature, bone, adipose tissue, and fibroblast-like bone marrow stromal cells (BMSC), which can be summarized under the generic term niche cells. BMSC express Toll-like receptors (TLRs) and are capable to respond to TLR-agonists by changing their cytokine expression pattern in order to more efficiently support hematopoiesis. Here, we show that in addition to enhanced myeloid colony formation from human CD34+ cells, lipopolysaccharide (LPS) stimulation retains overall higher numbers of CD34+ cells in co-culture assays using BMSC, with eightfold more CD34+ cells that underwent up to three divisions as compared to non-stimulated assays. When subjected to cytokine-supplemented myeloid colony-forming unit (CFU) assays or transplanted into newborn RAG2(-/-) γc (-/-) mice, CD34(+) cells from LPS-stimulated BMSC cultures give rise to the full spectrum of myeloid colonies and T and B cells, respectively, thus supporting maintenance of myeloid and lymphoid primed hematopoietic progenitor cells (HPCs) under inflammatory conditions. Collectively, we suggest that BMSC enhance hematopoiesis during inflammatory conditions to support the replenishment of innate immune effector cells and to prevent the exhaustion of the hematopoietic stem and progenitor cell (HSPC) pool.

Ciliary neurotrophic factor has intrinsic and extrinsic roles in regulating B cell differentiation and bone structure.

The gp130 receptor and its binding partners play a central role in cytokine signalling. Ciliary neurotrophic factor (CNTF) is one of the cytokines that signals through the gp130 receptor complex. CNTF has previously been shown to be a negative regulator of trabecular bone remodelling and important for motor neuron development. Since haematopoietic cell maintenance and differentiation is dependent on the bone marrow (BM) microenvironment, where cells of the osteoblastic lineage are important regulators, we hypothesised that CNTF may also have important roles in regulating haematopoiesis. Analysis of haematopoietic parameters in male and female Cntf−/− mice at 12 and 24 weeks of age revealed altered B lymphopoiesis. Strikingly, the B lymphocyte phenotype differed based on sex, age and also the BM microenvironment in which the B cells develop. When BM cells from wildtype mice were transplanted into Cntf−/− mice, there were minimal effects on B lymphopoiesis or bone parameters. However, when Cntf−/− BM cells were transplanted into a wildtype BM microenvironment, there were changes in both haematopoiesis and bone parameters. Our data reveal that haematopoietic cell-derived CNTF has roles in regulating BM B cell lymphopoiesis and both trabecular and cortical bone, the latter in a sex-dependent manner.

Melanoma stem cells and metastasis: mimicking hematopoietic cell trafficking?

Malignant melanoma is a highly metastatic cancer that bears responsibility for the majority of skin cancer-related deaths. Amidst the research efforts to better understand melanoma progression, there has been increasing evidence that hints at a role for a subpopulation of virulent cancer cells, termed malignant melanoma stem or initiating cells (MMICs), in metastasis formation. MMICs are characterized by their preferential ability to initiate and propagate tumor growth and their selective capacity for self-renewal and differentiation into less tumorigenic melanoma cells. The frequency of MMICs has been shown to correlate with poor clinical prognosis in melanoma. In addition, MMICs are enriched among circulating tumor cells in the peripheral blood of cancer patients, suggesting that MMICs may be a critical factor in the metastatic cascade. Although these links exist between MMICs and metastatic disease, the mechanisms by which MMICs may advance metastatic progression are only beginning to be elucidated. Recent studies have shown that MMICs express molecules critical for hematopoietic cell maintenance and trafficking, providing a possible explanation for how circulating MMICs could drive melanoma dissemination. We therefore propose that MMICs might fuel melanoma metastasis by exploiting homing mechanisms commonly utilized by hematopoietic cells. Here we review the biological properties of MMICs and the existing literature on their metastatic potential. We will discuss possible mechanisms by which MMICs might initiate metastases in the context of established knowledge of cancer stem cells in other cancers and of hematopoietic homing molecules, with a particular focus on selectins, integrins, chemokines and chemokine receptors known to be expressed by melanoma cells. Biological understanding of how these molecules might be utilized by MMICs to propel the metastatic cascade could critically impact the development of more effective therapies for advanced disease.

Anti-Obese Hormone Adiponectin Regulates Emergency Hematopoiesis and Antibacterial Response Through Downregulation Of Socs3 In Hematopoietic Progenitor Cells

Anti-obesity countermeasures attract rising attention because obesity induces multiple health complications such as metabolic syndrome, predisposition to cancer, heart diseases, stroke, infections, etc. One of the key features of obesity is decreased adiponectin, a critical anti-diabetic hormone secreted by adipocytes to modulate a number of metabolic processes including glucose regulation and fatty acid oxidation. Hematopoietic stem and progenitor cells (HSPCs) expand in size against infections, especially bacterial infections through immune response. In spite of the emerging clinical and basic reports of adiponectin, precise mechanisms of adiponectin for infectious control, especially for hematopoietic response, are largely unknown.As adipocytes are one of the major cellular components of bone marrow microenvironment which is critical for the proliferation and maintenance of hematopoietic cells, we hypothesized that obesity-related dysfunction of adipocytes in bone marrow might cause compromised hematopoietic response leading to increased susceptibility to infection. With the use of high-fat-fed mice, a well-known obesity model, we revealed that obesity had no apparent impact on steady-state hematopoiesis except for a slightly hypercellular bone marrow. Upon G-CSF administration to mimic harmful infection in vivo, high-fat-fed mice showed impaired expansion of HSPCs and myeloid cells in the bone marrow, along with the reduction of myeloid cells in peripheral circulation, i.e. diminished “emergency hematopoiesis”. In a Listeria monocytogenes peritonitis model, high-fat-fed mice also confirmed reduced expansion of HSPCs and myeloid cells in the bone marrow, attenuated leukocytosis and infiltration of leucocytes into the peritoneal cavity, resulting in a delayed bacterial clearance in vivo. Interestingly, the concentration of adiponectin was significantly reduced in the bone marrow of high-fat-fed mice compared to control mice, possibly reflecting a high relevance of adiponectin to hematopoietic response. To assess the exact role of adiponectin in emergency hematopoiesis, we utilized adiponectin-deficient (adipo-/-) mice. Genetic ablation of adiponectin caused no change in steady-state hematopoiesis, whereas G-CSF administration in adipo-/- mice showed attenuated HSPC expansion in the bone marrow, consistent with a series of high-fat-fed mice. Reciprocal transplants with adipo+/+ and adipo -/- mice demonstrated that adiponectin from bone marrow environment of the recipient, not from the donor hematopoietic cells, is essential for the efficient expansion of HSPCs in response to G-CSF. Furthermore, adipo-/- mice showed reduced hematopoietic response against Listeria monocytogenes infection, similar to the phenotype of high-fat-fed mice. Importantly, we confirmed that intravenous administration of recombinant adiponectin restored the responsiveness to G-CSF and the bacterial clearance on Listeria infection, both in adipo-/- and high-fat-fed mice, which affirm the importance of adiponectin treatment against infection. In HSPCs and myeloid cells from adipo-/- mice or high-fat-fed mice, phosphorylation of Stat3, a key component for emergency hematopoiesis, was suppressed, underscoring a role of adiponectin in emergency hematopoiesis at the molecular level. We revealed that those cells aberrantly expressed Socs3, a major inhibitory factor of Stat3, and that was reverted by intravenous administration of adiponectin, indicating that adiponectin potentiated G-CSF-dependent Stat3 phosphorylation in vitro and in vivo. These data revealed that adiponectin regulates hematopoietic response against infections, highlighting adiponectin as a legitimate target against infectious diseases in obese patients. From our findings breakthrough drugs to increase adiponectin are highly warranted. Disclosures:No relevant conflicts of interest to declare.

MicroRNAs - Key Players in Haematopoiesis

Since the initial identification of microRNAs (miRNA) in C. elegans, functional characterisation of these small non-coding RNAs has demonstrated their critical involvement in numerous biological processes. Typically 20-22 nucleotides in length, miRNAs act as negative gene regulators at the post-transcriptional level. A large body of evidence points to the role of miRNAs in haematopoiesis. miRNAs provide a network of signals important for haematopoietic cell maintenance, lineage differentiation and cell maturation. The mechanism by which they mediate their effects in haematopoiesis is complex, demonstrating cell-specific, tissue-specific and maturation-specific expression. Understanding haematopoietic expression and key targets of miRNAs is important to further elucidate the functional roles of miRNAs. Furthermore, an appreciation of their role in normal physiological processes sheds light on the possibility that aberrations of miRNA expression may perturb these processes and cause disease. Indeed, deregulation of miRNAs has been demonstrated in oncogenesis and in haematological malignancies in particular. This review focuses on miRNA expression in normal haematopoiesis and provides brief insight into how deregulation of these miRNA may contribute to cancer pathogenesis.

MicroRNAs - Key Players in Haematopoiesis

Since the initial identification of microRNAs (miRNA) in C. elegans, functional characterisation of these small non-coding RNAs has demonstrated their critical involvement in numerous biological processes. Typically 20-22 nucleotides in length, miRNAs act as negative gene regulators at the post-transcriptional level. A large body of evidence points to the role of miRNAs in haematopoiesis. miRNAs provide a network of signals important for haematopoietic cell maintenance, lineage differentiation and cell maturation. The mechanism by which they mediate their effects in haematopoiesis is complex, demonstrating cell-specific, tissue-specific and maturation-specific expression. Understanding haematopoietic expression and key targets of miRNAs is important to further elucidate the functional roles of miRNAs. Furthermore, an appreciation of their role in normal physiological processes sheds light on the possibility that aberrations of miRNA expression may perturb these processes and cause disease. Indeed, deregulation of miRNAs has been demonstrated in oncogenesis and in haematological malignancies in particular. This review focuses on miRNA expression in normal haematopoiesis and provides brief insight into how deregulation of these miRNA may contribute to cancer pathogenesis.

Zebrafish stromal cells have endothelial properties and support hematopoietic cells

The goal of this study was to determine if we could establish a mesenchymal stromal line from zebrafish that would support hematopoietic cells. Such a coculture system would be a great benefit to study of the hematopoietic cell-stromal cell interaction in both invitro and invivo environments. Zebrafish stromal cells (ZStrC) were isolated from the "mesenchymal" tissue of the caudal tail and expanded in a specialized growth media. ZStrC were evaluated for phenotype, gene expression, and ability to maintain zebrafish marrow cells in coculture experiments. ZStrC showed mesenchymal and endothelial gene expression. Although ZStrC lacked the ability to differentiate into classic mesenchymal stromal cell lineages (i.e., osteocytes, adipocytes, chondrocytes), they did have the capacity for endotube formation on Matrigel and low-density lipoprotein uptake. ZStrC supported marrow cells for >2 weeks invitro. Importantly, marrow cells were shown to retain homing ability in adoptive transfer experiments. ZStrC were also shown to improve hematopoietic recovery after sublethal irradiation after adoptive transfer. As the zebrafish model grows in popularity and importance in the study of hematopoiesis, new tools to aid in our understanding of the hematopoietic cell-stromal cell interaction are required. ZStrC represent an additional tool in the study of hematopoiesis and will be useful in understanding the factors that mediate the stromal cell-hematopoietic cell interactions that are important in hematopoietic cell maintenance.

Wnt-expressing rat embryonic fibroblasts suppress Apo2L/TRAIL-induced apoptosis of human leukemia cells

Wnt signaling enhances cell proliferation and the maintenance of hematopoietic cells. In contrast, cytotoxic ligand Apo2L/TRAIL induces the apoptosis of various transformed cells. We observed that co-culture of human pre-B leukemia cells KM3 and REH with Wnt1- or Wnt3a-producing rat embryonic fibroblasts efficiently suppressed Apo2L/TRAIL-induced apoptosis of the lymphoid cells. This suppression occurs at the early stages of the Apo2L/TRAIL apoptotic cascade and, interestingly, the activation of the Wnt pathway alone in human leukemia cells is not sufficient for their full anti-apoptotic protection. We hypothesize that a stimulus emanating specifically from Wnt1- or Wnt3a-expressing rat fibroblasts is responsible for the observed resistance to Apo2L/TRAIL. This anti-apoptotic signaling was significantly hampered by the inhibition of the MEK1/ERK1/2 or NFkappaB pathways in KM3 and REH cells. Our results imply that paracrine Wnt-related signals could be important for the survival of pre-B cell-derived malignancies.

Assessment of cell viability and apoptosis in human umbilical cord blood following storage.

There is increasing use of human umbilical cord blood (UCB) stem cells for unrelated donor transplantation. Successful engraftment depends in large measure on the dose and quality of cells in the UCB unit. In the present study, we attempted to identify a simple and rapid technique for assessing the quality and recovery of UCB cells following laboratory manipulation. Mononuclear cells (MNC) from fresh (<48 h old) and thawed UCB units were stained with 7-amino-actinomycin D (7-AAD), revealing 2-3% dead cells. The frequencies of apoptotic cells in fresh and thawed sample were similar. However, UCB held for 72 h showed higher levels of cell deterioration. Interestingly, staining with 7-AAD was more sensitive to cellular damage than was uptake of Trypan Blue. 7-AAD staining of MNC also correlated with retention of hematopoietic function (progenitor assays) such that 7-AAD staining frequencies <20% predicted maintenance of hematopoietic cells. Importantly, hematopoietic precursors were less susceptible to storage injury than were UCB MNC as a whole. MNC showed higher levels of 7-AAD staining and apoptosis than did CD34(+) cells. This observation was confirmed in studies of caspase-3 activation, where MNC consistently showed higher frequencies of activation than did CD34(+) cells, especially after overnight storage. Furthermore, antiapoptotic proteins Bcl-2 and Bcl-x were expressed more consistently in CD34(+) cells than in total MNC. In contrast, Bax levels increased with MNC apoptosis. In conclusion, the data suggest that 7-AAD staining of UCB MNC provides a rapid and simple technique for assessing the viability, recovery, and hematopoietic functionality of stored UCB.