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Inside this Issue: September/October 2014

Posted By Connections Editor , Thursday, October 30, 2014
Updated: Tuesday, October 28, 2014


Furthering our understanding of the role of miRNAs in hematopoiesis

See Wojtowicz et al., pages 909-918.; Oh et al., pages 919-923., and Byon et al., pages 852-856.

MicroRNAs (miRNAs) are short, endogenous, noncoding RNA molecules that regulate the expression of crucial genes involved in regulatory functions related to cell growth, development, and differentiation, and they are associated with a wide variety of human diseases. MicroRNAs are transcribed by RNA polymerase II as large RNA precursors, which are processed in the nucleus by the microprocessor complex, consisting of the RNase III enzyme Drosha and the double-stranded-RNA-binding protein Pasha/DGCR8. The resulting pre-miRNAs are approximately 70 nucleotides in length and are transported into the cytoplasm, where they undergo an additional processing step by the RNAse III enzyme Dicer, generating the mature miRNA, a double-stranded RNA approximately 22 nucleotides in length. Dicer also initiates the formation of the RNA-induced silencing complex, which pairs the miRNAs with the 3' untranslated region of their target mRNA, leading to its destabilization and resulting in degradation or translational suppression.

A growing body of evidence indicates that miRNAs are vital for the proper functioning of hematopoietic stem and progenitor cells (HSPCs) and that they can influence erythroid lineage commitment and differentiation. The article by Wojtowicz et al. compares, side by side, the role in hematopoiesis of miR-125a, miR-125b1, and miR-125b2, which share the same seed sequence and are highly expressed in HSPCs. The authors could not detect any functional difference between them, showing that overexpression of each of the three miR-125 family members preserves HSPCs in a primitive state in vitro, results in a competitive advantage upon serial transplantation, and promotes skewing toward the myeloid lineage. Mechanistically, the study suggests that a seed-mutated version of miR-125 can rescue the effects of miR-125 overexpression, indicating that they likely operate in a seed-sequence-dependent manner.

In the brief communication by Byon et al., the authors describe a mouse model of conditional Dicer deletion limited to late erythroid cells (beyond proerythroblast). Under normal conditions, Dicer deletion did not affect any hematopoietic parameters; however, following stress with phenylhydrazine or 5-fluorouracil, the mice showed impaired recovery from anemia, suggesting that miRNAs primarily regulate stress erythropoiesis. This novel mouse model can be exploited to delineate miRNA function in late erythropoiesis, specifically under stress conditions, either by combining this model with other genetic models of stress erythropoiesis or by performing miRNA/shRNA rescue screens to identify specific miRNAs/mRNAs that restore the normal response to stress erythropoiesis.

Anti-T-cell globulin (ATG) mediates antitumor activity toward a variety of hematologic malignancies

See Westphal et al., pages 875-882.

Graft-versus-host disease (GVHD) is a major complication after allogeneic stem cell transplantation (allo-HSCT), leading to considerable morbidity and mortality. In vivo or in vitro depletion of T cells is effective to reduce the incidence of GVHD but has been demonstrated to be associated with increased relapse rates of malignancies after allo-HSCT.

More recently, clinical studies found that in vivo T-cell depletion with rabbit anti-T-cell globulin (ATG-F) lowered the incidence of GVHD without increasing tumor relapse rates. In this article, Westphal et al. examine the antitumor effect of ATG-F. They show that ATG-F binds to a variety of hematologic tumor cells, including acute myeloid leukemia and B-cell lymphoma. They demonstrate that ATG-F mediates antitumor effects in vitro by inducing antibody-dependent cellular cytotoxicity, complement-dependent cytotoxicity, and apoptosis. This is an interesting study that contributes to our understanding of antitumor mechanisms in the early phase after allo-HSCT. The findings described in the study may have clinical implications during allo-HSCT in situations when T-cell depletion is required but tumor relapse is a concern.

Understanding stem cell heterogeneity in disease

See Prick et al., pages 841-851.

Recent descriptions of heterogeneity in stem cells and cancers have emphasized the need to understand how single stem cells are subverted to cause tumors. Human myeloproliferative neoplasms arise from a transformed hematopoietic stem cell and provide a paradigm for studying the early stages of tumor establishment and progression.

This review postulates that the selective pressure placed on the first progeny in the initial clonal outgrowth is the source of substantial heterogeneity, which eventually manifests as distinct disease subtypes. It underscores the need to think about oncogenesis as a constantly evolving and highly interactive process, both with a tumor’s own clonal progeny and the tumor microenvironment. This review also details recent advances in our understanding of clonal evolution of the myeloproliferative neoplasms, including the molecular cataloguing of the genomic landscape and the current theories for how a single point mutation in JAK2 can be responsible for three distinct disease subtypes.



MSC-derived osteoblasts, a new source of feeder cells for the expansion of HSPC with enhanced thrombopoietic activity

See Dumont et al., pages 741-752.

The delay in platelet and neutrophil recovery following single and double umbilical cord blood transplantations limits their widespread utilization. Ex vivo expansion of hematopoietic stem and progenitor cells (HSPC) provides a mean to raise the dose of transplantable progenitors in order to promote early engraftment and prevent graft failure. Moreover, such procedure may one day allow for single cord blood unit transplant in patients that currently require two independent units. Multiple strategies have been reported to expand HSPC ex vivo, including co-culture of hematopoietic cells with mesenchymal stromal cells (MSC). Just recently, da Lima et al. reported that patients transplanted with 2 cord-blood units, 1 of which was expanded ex vivo in co-culture with MSC, had accelerated neutrophil recovery and also a significant improvement in platelet engraftment. In this issue, Dumont and colleagues compared the growth- and differentiation-modulatory activities of osteoblasts derived in vitro from human MSCs (i.e. M-OST) to that of the parental MSCs and found that M-OST supported greater expansion of HSPCs. More importantly, HSPCs expanded in M-OST conditioned medium yielded superior platelet engraftment than HSPCs expanded in MSC conditioned medium or in control cultures. Taken together, these data suggest that M-OST represent a new underappreciated source of feeder cells for the expansion of HSPC with enhanced thrombopoietic activity.

Bone marrow Th2 cells promote erythropoiesis at high altitude

See, Li et al., pages 804-815.

High-altitude hypoxia can lead to moderate or excessive increase of red blood cells in different individuals. The excessive erythropoiesis is termed high-altitude polycythemia (HAPC) and its mechanism remains largely obscure. The hypoxia-induced erythropoietin (EPO) is considered an underlying cause of HAPC; however, EPO levels usually do not correlate well with the severity of HAPC among individuals. A growing body of evidence has suggested that T lymphocytes, particularly bone marrow (BM) T cells, are involved in hematopoietic regulation. In this study, Li et al reported an association between altered BM Th2 cells and accelerated BM erythropoiesis at high altitude. Using a mouse model, they found that CXCR4-dependent Th2 cells trafficking to the BM during hypoxic exposure and their production of activin A and interleukin-9 contribute to erythropoiesis at high altitude. These findings provide a new insight into the EPO-independent mechanism underlying erythroid regulation at high altitude. More research is needed to reveal whether this pathway actually contributes to the pathology of excessive erythropoiesis at high altitude in humans. If this is the case, strategies to inhibit BM Th2 lymphocytes may be a new approach to cure HAPC patients.

CD45 regulatory elements facilitate efficient lentiviral tracking of transplanted cells

See Duong et al., pages 761-772.

Cell transplantation for the treatment of hematologic disease remains challenging due to a lack of suitably matched donors, and efforts are underway to use cellular reprogramming as a platform for attaining autologous blood cells for therapy. All studies to date employing defined factors to reprogram fibroblasts into induced pluripotent stem (iPS) cells or to an alternate cell lineage have utilized a reporter to document lineage respecification; however, a genetically tractable reporter system does not currently exist for marking the production of blood cells following differentiation or reprogramming. To construct a widely applicable hematopoietic delineation system, in this paper, Duang et al used transcription factor chromatin occupancy (ChIP-seq), promoter nuclease sensitivity (DNase-seq) and evolutionary conservation to define regulatory elements within the mouse and human blood surface gene CD45. The resulting lentiviral reporter enabled highly efficient and stable marking of lymphoid, myeloid and nucleated erythroid progenitor cells following long-term reconstitution in vivo. The CD45 reporter is hematopoietic restricted, and therefore not activated in fibroblasts or pluripotent cells. This specificity makes the system well-suited for following blood cell transplantation kinetics and persistence, isolating hematopoietic lineages from embryonic stem (ES) or induced pluripotent stem (iPS) cells, and for the development and facile monitoring of direct reprogramming strategies.

Induced pluripotent stem cells from myelofibrosis patients-a novel source of research material

See Hosoi et al., pages 816-825.

Induced pluripotent stem cells (iPSCs) derived from disease cells are expected to provide new experimental material, especially for the diseases for which the sample is difficult to obtain. Myelofibrosis (MF) is a rare and serious hematologic malignancy classified as a Philadelphia chromosome-negative myeloproliferative neoplasm (MPN). The disease is more common in males and in older individuals. Of the MPNs, MF presents with the most severe morbidity and greatest mortality. Although the cause of MF is unknown, it is thought to occur from acquired mutations that target the hematopoietic stem cell. The only curable treatment option is stem cell transplantation; however, it is warranted only to young patients without challenging complications. Although novel therapeutics to improve the outcome of MF patients are clearly needed, research material for this disease is often difficult to obtain from patients because of progressive scarring of the bone marrow. To overcome this problem, in this manuscript, Hosoi et al. established induced pluripotent stem cells (iPSCs) derived from primary and secondary MF patient samples. The authors confirmed that the disease specific genomic markers were sustained in those iPSCs, and also that those iPSCs were capable of differentiating into hematopoietic cells, such as megakaryocyte, erythrocyte and myelocyte. Megakaryocytes are considered to be responsible for generating effectors to myelofibrotic transformation in MF. These megakaryocytes are extremely difficult to harvest from MF patients, and thus this alternative source, through the differentiation of iPSCs, could potentially be a valuable tool to learn more about MF. Indeed, the authors used whole MF-iPSC derived megakaryocytes to study the expression level of IL8, a cytokine known to stimulate fibroblasts to produce collagen and extracellular matrix and which is highly elevated in patients with MF, especially in those with a poor prognosis. They showed that expression of IL8 in MF-iPSC was largely increased compared to normal iPSC derived megakaryocytes. Based on the data presented here, MF-iPSC provide a novel platform to investigate MF pathogenesis on the basis of patient-derived samples and should proved useful to accelerate the development of novel therapies which are urgently needed to help patients with this devastating disease.

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