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Inside the July and August 2013 issues

Posted By Connections Editor, Monday, September 9, 2013
Updated: Thursday, August 22, 2013

Inside this issue July 2013

#Single-cell Raman spectroscopy demonstrates mechanochemical differences between normal, sickle, and cord red blood cells

See Liu et al., pages 656–661.

Sickle cell disease, a blood disorder characterized by abnormal rigid, sickle-shaped red blood cells (RBCs), can lead to various complications that can degrade a patient’s quality of life or be life-threatening. To better understand both the mechanical and biochemical properties of these cells, in this study Liu etal. applied laser tweezers Raman spectroscopy (LTRS), a novel label-free single-cell analytical tool, to measure the oxygenation response of RBCs to an applied mechanical force. LTRS was used to acquire the oxygenation-specific Raman spectra of individual optically trapped normal adult, sickle, and cord blood RBCs subjected to different forces by varying the laser power of the optical trap. For all cell types, an increase in laser power induced a greater deoxygenation of the cell. However, sickle RBCs deoxygenated more readily than normal RBCs when subjected to the same optical forces. Conversely, cord blood RBCs were able to maintain their oxygenation better than normal RBCs. These results suggest that differences in the chemical or mechanical properties of fetal, normal, and sickle cells affect the degree to which applied mechanical forces can deoxygenate the cell. Populations of normal, sickle, and cord RBCs were identified and discriminated based on this mechanochemical phenomenon. With its ability to characterize the functional properties (e.g., mechanical deformability, oxygen binding) of normal and diseased RBCs at the single-cell level, LTRS has the potential to be used for many applications, such as determining the efficacy of patient treatments in-vitro, assessing the effectiveness of sickle cell gene therapy approaches to restore normal RBC function, and in fundamental RBCresearch.

#Involvement of the aldehyde dehydrogenase 1 family member A2 in AraC resistance

See Kawasoe et al., pages 597–603.

1-β-D-arabinofuranosylcytosine (AraC) is one of the most effective drugs in the treatment of acute myeloid leukemia. It is also active against other hematologic malignancies, such as acute lymphoblastic leukemia and non-Hodgkin's lymphoma. However, AraC resistance remains a critical problem, underscoring the urgent need to clarify the underlying mechanisms. In this manuscript, Kawasoe etal. set out to identify candidate proteins associated with drug resistance. Using an unbiased proteomics-based approach to compare the protein expression profiles of K562 cells sensitive and resistant to AraC, the authors demonstrate that expression of aldehyde dehydrogenase 1 family member A2 (ALDH1A2), otherwise known as retinaldehyde dehydrogenase 2, is increased in resistant cells. They also show that the siRNA knockdown of ALDH1A2 in K562-resistant cells promoted the recovery of sensitivity to AraC, whereas ALDH1A2 overexpression in K562-sensitive cells induced AraC resistance. The role of ALDH1A2 in AraC resistance is still unclear; however, the enzyme catalyzes the synthesis of retinoic acid, which has been shown to have anti-apoptotic properties, and thus, may protect cells from the apoptotic-signaling trigger by AraC. This study provides useful insights into the mechanisms of AraC resistance and, if confirmed, points at ALDH1A2 as a potential therapeutic target in combination with AraC for the treatment of AraC-resistant neoplasms.

#New insights into the biology of endothelial selectins in hematopoietic stem/progenitor cell migration

See Nabors et al., pages 588–596.

It has been known for some time that endothelial (E- and P-) selectins are constitutively expressed in bone marrow (BM) sinusoidal endothelial cells, and that at least one of these molecules is critically involved in the rapid homing of hematopoietic stem/progenitor cells (HSPC) to BM in the context of BM transplantation. The distinct patterns of expression of E- versus P-selectin suggest the possibility that they mediate distinct functions. Moreover, it has never been established whether homing to BM requires both or only one of the two selectins. In this study, Nabors etal. examined the role of the individual endothelial selectins and found that either one was sufficient to rescue lethally irradiated mice that were transplanted with a limited number of cells. These data demonstrate, at least for homing and rescue, an "either/or” requirement for the endothelial selectins to support recruitment to BM. In addition, the authors found heterogeneity of expression of selectin ligands on HSPC, with higher levels of selectin ligands on progenitors (lineage-negative/sca-1+/c-kit-lo; LSK) than on stem cells (LSK CD48+CD150-negative). Acquisition of E- and P-selectin ligands did not appear to involve upregulation of glycoproteins known or proposed to function as selectin ligands, but rather, involved a gradual increase in expression of one or more glycosyltransferases responsible for biosynthesis of the glycans that function as selectin ligands. These results clarify the role of E- and P-selectin in HSPC migration to BM and document heterogeneity of selectin ligand expression on HSPC.

#Telomere shortening in Philadelphia-negative chronic myeloproliferative neoplasms

See Ruella et al., pages 627–634.

Among noncoding DNA structures, telomeres and the telomere length (TL) have been investigated in cancer cells with increasing interest. TL typically shortens with age and is therefore a marker of cellular aging. It also shortens as consequence of both DNA damages and oncogene activation. Moreover, telomere sequences are variably lost following exposure to chemotherapy. The observation that TL is shortened in most malignancies has pointed out the many biological links between telomere and neoplastic cell growth. In this manuscript, Ruella etal. investigated this connection further in Philadelphia-negative (Ph-neg) chronic myeloproliferative neoplasms (CMNs). Their study shows that among Ph-neg-CMNs, polycythemia vera (PV) and myelofibrosis (MF) presented a pronounced TL shortening, while essential thrombocytosis (ET) and secondary erythrocytosis had TL similar to the aged-matched normal population. On multivariate analysis, shortened TLs correlated with JAK2-V617F allele burden >50%, while no correlation was found with disease duration nor with Hydroxycarbamide therapy, which further supports the preferential use of this safe and cheap drug in patients requiring cytoreduction. Of note, in this study patients were not exposed to the new generation of JAK1-2 inhibitors, so these data may provide a useful historical control to understand the impact that these innovative drugs will have on the biology of telomeres. Altogether, these results support further studies to validate the use of TL in Ph-neg-CMNs to distinctly characterize PV and MF patients, to offer prognostic insights during the disease course, and to monitor DNA damage induced by potential therapeutic drugs under investigation. The study also shows the marked difference in TL between two apparently similar neoplasms such as PV and TE, suggesting the need for further biological studies to fully understand the pathogenetic steps leading to the various CMN subtypes.

Inside this issue August 2013

#Engraftable CD34+ cells from human embryonic stem cells

See Kim et al., pages 749–758.

Until recently, protocols for generating CD34+ cells from human embryonic stem cells (hESCs) have mostly relied on either embryoid body formation or coculture on mouse feeder cell lines, both of which pose significant challenges for their clinical utility. Moreover, the level of engraftment achieved after transplantation of hESC-derived CD34+ cells has remained low, raising concerns regarding their invivo potential. Based on a previous study from this group, Kim etal. hypothesized that a combination of mesenchymal stromal cells and macrophages could recapitulate a bone marrow microenvironment invitro, inductive to the generation of CD34+ cells from hESCs. In this study, these two types of human cells were used as feeder cells to direct the differentiation of hESCs into CD34+ cells. The hESC-derived CD34+ cells generated in this manner express cell surface markers and genes associated with hematopoietic stem cells. Most importantly, they demonstrate high levels of engraftment and development of multilineage blood cells following transplantation into the pre-immune fetal sheep model. To humanize the differentiation protocol further, the authors cultured undifferentiated hESCs in a human serum–based matrix to replace Matrigel, a mouse sarcoma tumor-derivative matrix. This study provides a clinically applicable methodology to generate engraftable CD34+ HSCs from hESCs and offers a framework for the design of more effective invitro platforms for efficient differentiation of hESCs.

#G6PD deficiency in a Zebrafish model

See Patrinostro et al., pages 697–710.

Glucose-6-phosphate dehydrogenase (G6PD) deficiency is the most common enzymopathy and genetic mutation worldwide. The zebrafish has been used in the modeling and discovery of new mechanisms involved with the development of the hematopoietic system, of which erythropoiesis is a foremost process. The zebrafish allows for unparalleled insight and visualization into the early developmental events governing the blood system because of its relatively rapid development that is external to any maternal environment. In this article by Patrinostro etal., the authors use morpholino-based downregulation of g6pd expression to reduce G6pd protein and activity levels significantly in the developing zebrafish. G6pd-deficient animals developed erythrocytic hemolysis when exposed to several prooxidant compounds, including naphthol, menthol, and primaquine, which typically lead to hemolytic crises in persons with G6PD deficiency. Animals showed high levels of reactive oxygen species (ROS) and subsequently developed significant red cell apoptosis, resulting in anemia and substantial cardiac edema, similar to the severe clinical presentation of G6PD-deficient individuals during a hemolytic crisis. This model system will be useful in trying to elucidate the nature of ROS and its effects on the developing organism, as well as the biological response to increased ROS. It can also be used to study the effects of ROS-induced hemolysis on the hematopoietic system. Finally, researchers can use this system to search for new and novel compounds or drugs to reduce ROS and its deleterious effects.

#Myelodysplastic syndrome and the genomic instability hypothesis

See Zhou et al., pages 665–674.

Myelodysplastic syndrome (MDS) has received increased attention from both the scientific community and the public, in part because the number of people who are most susceptible to developing this hematologic malignancy, in particular the elderly and cancer survivors, is expanding dramatically. Therefore, there is a pressing need to understand more about the molecular pathogenesis and underlying biology of this disease, so that unique or improved therapeutic strategies can be developed. In this review, Zhou etal. highlight recent advances in our understanding of MDS, including the identification and functional characterization of many novel gene perturbations that might be acquired by patients with the disease. As a result, the authors discuss the emerging notion that MDS is a genomic instability syndrome, possibly reflecting an underlying problem in DNA repair. They also summarize several seminal studies on the maintenance of genomic integrity within hematopoietic stem cells, believed to be the cell of origin in MDS, and their differentiating progeny. In addition to raising intriguing new questions, these studies provide rare insights into the response of hematopoietic stem cells to DNA damage. The review provides a balance between clinical findings and the appropriate mouse phenotypes, as well as from hematopoietic stem cell research with and without links to aging. The article is authored by specialists in all crucial areas under discussion and should be interesting reading for researchers in the field.

#Efficient platelet production from human pluripotent stem cell–derived megakaryocytes using a two-directional flow bioreactor

See Nakagawa et al., pages 742–748.

The underlying mechanisms of platelet biogenesis from megakaryocytes remain elusive. Under conditions recapitulating platelet release based on invivo behavior of thrombopoiesis within mouse bone marrow, shear stress induced by blood flow in capillary vessels of bone marrow appears to stimulate the adjacent megakaryocytes to promote platelet yield. Recently, a series of articles have reported the application of shear stress to invitro thrombopoiesis using one directional flow. In this study, Nakagawa etal. propose a novel bioreactor system to recapitulate the invivo situation of platelet production. They derived megakaryocytes successfully from human pluripotent stem cells by applying in a three-dimensional bioreactor a one-directional flow and a second flow-mediated fixation of megakaryocytes in appropriate positions. The pressure flow held the megakaryocytes in the chambers of the bioreactor, and the main flow produced shear stress on the megakaryocytes' surface. The authors concluded that the angle between the main flow direction and the megakaryocyte surface was one of the critical factors for efficient platelet production. In a 90-degree situation, platelet production seemed to be slightly better than in static culture; however, in a 60-degree situation, platelet production efficiency increased 3.6-fold compared with static cultures. This novel finding will definitively influence the future planning of exvivo platelet production systems from human pluripotent stem cell–derived megakaryocytes for transfusion medicine.

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