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Inside this Issue: September and October

Posted By Connections Editor, Friday, November 6, 2015
Updated: Thursday, November 5, 2015


A new tool to identify the molecular and functional properties of individual stem cells at a single-cell resolution
Schulte et al., pages 803–811.


The recent discovery of significant heterogeneity in normal and malignant stem cell populations has driven the development of numerous tools to try and identify, at single-cell resolution, what the molecular and functional properties are of individual stem cells. Many groups have identified populations of hematopoietic stem cells that are based on a binary positive/negative gate and do not take into account the differing levels of proteins on the cell surface (i.e., how positive is a cell for marker X). To consider this, in this manuscript, Schulte et al. combine flow-cytometric index sorting (which quantifies the exact fluorescence of each cell surface marker form each single cell) with single-cell functional assays to determine whether or not different levels of cell surface molecules can enrich or deplete for functional activity. This technique is particularly useful when cell number is limited and can assess multiple marker intensities simultaneously in a single experiment. The technique has been already been usefully applied to uncover the molecular program of functional hematopoietic stem cells (HSCs; Wilson et al., Cell Stem Cell 2015) and can be applied to any cell-biological system with a defined single cell functional assay.

How the experimental setup of serial transplantations largely influences the interpretation of long-term HSC performance
Rundberg Nilsson et al., pages 812–817.

To study HSC self-renewal and longevity, serial transplantation has been considered the gold standard for many years. However, discrepancies in experimental procedures are widespread, including the choice to either transplant whole bone marrow (wBM) or fluorescence-activated cell sorting (FACS)-purified HSCs into secondary hosts. In this study, Rundberg Nilsson et al. show that this choice of experimental procedure significantly influences the readout following transplantation. The authors show that donor-derived HSCs can distribute highly unevenly among bones of the conditioned host, leading to serial transplantation of nonrepresentative HSC chimerism levels if wBM is isolated and transplanted from only one or a few bones. Moreover, by comparing mature donor cell output from transplanted wBM cells as opposed to FACS-purified HSCs, they found differences suggestive of cotransplantation of long-lived lymphoid progenitors and/or mature cells together with HSCs in the wBM, which could severely affect the interpretation of ongoing HSC activity. Furthermore, transplantation of different frequencies of HSCs using wBM transplantation between groups/recipients not only impeded the evaluation of the HSCs on a per cell basis, but also obscured the distinction between effects generated during the primary transplantation and those presenting during the secondary transplantation. Based on these data, the authors recommend serial transplantation of highly purified HSC populations using flow cytometry markers as opposed to unfractionated wBM. A standard experimental setup, such as the one suggested in this study, is highly important for the research community, both to aid comparison of results generated from different research groups and for optimal evaluation of HSC performance.

Targeting autophagy to promote differentiation in myeloid leukemia
Orfali et al., pages 781–793.

Acute myeloid leukemia (AML) affects predominantly an elderly population, who tend to tolerate intensive chemotherapy poorly. Differentiation therapy, a pharmacologic override of the differentiation block observed in this condition, is an attractive and potentially minimally toxic strategy. The use of all-trans-retinoic acid (ATRA) in the treatment of acute promyelocytic leukemia (APL) is the defining example of success in this field and a model for the study of differentiation pathways in myeloid cells. Recently published literature reports that ATRA treatment of APL cells induces autophagy, a lysosomal-dependent protein recycling pathway with defined roles in mammalian cell differentiation.
In this study, Orfali et al. investigate a functional role for autophagy in leukemic cell differentiation. The authors initially confirm the induction of autophagy by ATRA in
an APL cell line and in primary APL tissue cultured ex vivo. They proceed to demonstrate that autophagy blockade, using either pharmacologic agents (Chloroquine/3-methyladenine) or genetic inhibition of the critical autophagy regulator ATG7, impedes ATRA-mediated APL cell differentiation. Conversely, in an exciting experiment, the investigators show that promoting autophagy with the use of lithium chloride can enhance the differentiating effects of ATRA in AML cells previously resistant to differentiation.

In a time when the molecular heterogeneity of AML is increasingly understood, this work considers the endpoint at which these aberrancies converge: the differentiation block. If autophagy is of functional importance in leukemic cell differentiation, it is possible that defective autophagy could contribute to their malignant phenotype. Importantly, autophagy induction with readily available compounds may overcome ATRA-resistance in APL and potentiate the differentiation of non-APL leukemic subtypes.

Using cellular reprogramming to make blood
Singbrant et al., pages 756–759.

The current use of human HSCs to treat blood disorders is hampered by the need to find human leukocyte antigen (HLA)-matched donors and to obtain sufficient numbers of long-term engraftable HSCs. Despite the efforts of many researches, ex vivo expansion of HSCs to improve hematopoietic reconstitution and engraftment potential has been largely unsuccessful owing to the inability to generate sufficient HSC numbers and to excessive differentiation of the starting cell population. In search for an unlimited source of autologous HSCs, novel approaches, such as the reprogramming of somatic or pluripotent cells lines to HSCs, are now being pursued. Induced HSCs (iHSCs) hold much potential for regenerative medicine and, in the near future, could benefit patients through disease modeling and drug screening. The International Society of Experimental Hematology invited Drs. George Q. Daley and Derrick Rossi, two leaders in the field of cellular reprogramming, to present a webinar ith their most recent research on this topic. In this perspective, Singbrant et al. summarize the webinar (available online at and discuss the state of the field of hematopoietic specification.


Gene correction of a chronic granulomatous disease-causing mutation in iPS cells
Flynn et al., pages 838–848.

Chronic granulomatous disease (CGD) is a lifelong affliction causing serious morbidity and potential mortality, even in the present era of potent antimicrobials. Currently, the only curative treatment option involves allogenic bone marrow transplantation. However, a preferred option to avoid the inherent risks associated with this treatment, could be transplantation with genetically-modified, patient-derived cells (either from induced pluripotent stem [iPS] cell or the patient's bone marrow) would theoretically improve outcomes dramatically. To that end, in this study, Flynn et al. use the Cas9–clustered regularly interspaced short palindromic repeats (CRISPR) system to efficiently gene correct a CGD-causing point mutation in a patient-derived iPS cell line. This in situ modification of a single-point mutation within an intron of the main disease causing gene, CYBB, results in the correction of a splicing defect and restoration of the oxidative burst in macrophages derived from the iPS cells, a key antimicrobial response that is compromised in CGD sufferers. This work lays the foundation for future genetically clean (“footprintless”) gene therapy approaches to this, and other, life-threatening genetic diseases.

Encouraging advances in treating and curing globin disorders
Blobel et al., pages 821–837.

Throughout the era of molecular medicine, lessons learned from the globin gene disorders have led the way forward for research of many human genetic diseases. The pathway from understanding a human disease to developing an appropriate treatment or cure often takes decades. Thalassemia and sickle cell disease, which cause serious illness in millions of individuals worldwide, were arguably the first human genetic diseases to be understood at the molecular and cellular level, yet in more than 50 years of research, this increasingly detailed understanding has had only a modest impact on clinical treatment. However, recent, significant advances in our understanding of the developmental regulation of globin gene expression and in the technology for safely manipulating hematopoietic stem cells engender optimism that improved management for these disorders may be on the near horizon. Altering the normal developmental pattern of globin gene expression could be the most efficient way to treat the thalassemias, and over the past two decades, specific new pathways that regulate developmental switching have been identified, suggesting new ways to manipulate the activity of individual globin genes. Similarly, the promise of gene therapy to treat thalassemia, after three decades of meticulous study, has been achieved by lentiviral transduction of blood stem cells in affected patients. Genome editing of such cells may also soon be a reality. These advances, as well as many others, were presented at the 19th biennial Hemoglobin Switching Conference, held this past September in Oxford, United Kingdom, by the 160 participants who traveled from around the world to participate in this unique conference. This review by Blobel et al. highlights many of the most recent advances that were presented during the three days, and highlights why this meeting has become one of the most important international conferences examining the developmental biology, molecular genetics, cell biology, and epigenetics of human disease.

Interferon α promotes hematopoietic stem and progenitor division in humans
King et al., pages 912–918.

Named for their ability to interfere with viral transmission, interferons suppress the growth and survival of many cell types. Surprisingly, however, hematopoietic stem cells (HSCs) are activated to divide by the Type I interferon, interferon α, in murine in vivo studies. Similar effects have also been seen in response to Type II interferon. King et al. postulate in this study that interferon-mediated activation of HSC division could be a primitive mechanism for generating innate immune responses. To evaluate the effects of interferon α on human HSCs in vivo, serial bone marrow (BM) samples from patients with polycythemia vera or essential thrombocytosis were collected before and during treatment with pegylated interferon α. While the absolute number of CD34+CD38− hematopoietic stem and progenitor cells (HSPCs) did not change, the percentage of CD34+ cells undergoing cell division increased in most patients treated with pegylated interferon α. Furthermore, colony formation of whole BM cells in methylcellulose increased, indicative of enhanced differentiation. These findings were present even in patients with low or undetectable JAK2-mutant allele burden, suggesting that they reflect the activity of wild-type Jak2 in HSPCs. In contrast, patients treated with hydroxyurea, an antimetabolite, showed decreased cell division and decreased colony formation. This unique in vivo longitudinal study demonstrates for the first time in a human setting that interferon α can be used to promote division of quiescent HSPCs; this application may promote polyclonal hematopoiesis in patients with myeloproliferative neoplasms and improve chemosensitivity of hematologic malignancies.

MicroRNA-223 is a promoter of lineage commitment and differentiation and a modulator in AML
Gentner et al., pages 858–868.

Although the effect of miR-223 on granulopoiesis was recognized over 10 years ago, its role in hematopoietic stem and progenitor cells (HSCPs) and in acute myeloid leukemia (AML) is still not completely understood. In this study, Gentner et al. explore the role of miR-223 in various murine AML models and human CD34+ cells. Considering that high miR-223 levels were associated with favorable prognosis and that the expression of miR-223 was low in leukemia-initiating cell (LIC) fractions of AML patients, it is surprising that genetic depletion of miR-223 decreased the LIC frequency in an AML mouse model, but was not mandatory for rapid onset AML, indicating only a minor role in AML development. This correlates with their observations in CD34+ cells, where miR-223 fine tunes myeloid progenitor cells with respect to myeloerythroid differentiation and expansion. The findings of Gentner et al. close the gap between in vitro findings and the actual function of miR-223 in vivo as a regulator of the expansion/differentiation equilibrium in HSPCs cells and in AML, highlighting its role as rheostat and not on-or-off switch in granulopoiesis.

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