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The mouse hematopoietic system has served as a paradigm for analysis of developmental fate decisions in tissue homeostasis and regeneration. However, multiple immunophenotypic definitions of, and sometimes divergent nomenclatures used to classify, murine multipotent progenitors (MPPs) have emerged in the field over time. This has created significant confusion and inconsistency in the hematology field. To facilitate easier comparison of murine MPP phenotypes between research laboratories, a working group of four International Society for Experimental Hematology (ISEH) members with extensive experience studying the functional activities associated with different MPP phenotypic definitions reviewed the current state of the field with the goal of developing a position statement toward a simplified and unified immunophenotypic definition of MPP populations. In November of 2020, this position statement was presented as a webinar to the ISEH community for discussion and feedback. Hence, the Simplified MPP Identification Scheme presented here is the result of curation of existing literature, consultation with leaders in the field, and crowdsourcing from the wider experimental hematology community. Adoption of a unified definition and nomenclature, while still leaving room for individual investigator customization, will benefit scientists at all levels trying to compare these populations between experimental settings.

I am deeply honored to receive the International Society for Experimental Hematology (ISEH) 2020 Donald Metcalf Lecture Award. Although I am not a physician and have had no formal training in hematology, I have had the privilege of working with some of the top hematologists in the world, beginning in 1970 when Dr. David Nathan was a sabbatical visitor in my laboratory and introduced me to hematological diseases. And I take this award to be given not just to me but to an exceptional group of MD and PhD trainees and visitors in my laboratory who have cloned and characterized many proteins and RNAs important for red cell development and function. Many of these projects involved taking exceptionally large risks in developing and employing novel experimental technologies. Unsurprisingly, all of these trainees have gone on to become leaders in hematology and, more broadly, in molecular cell biology and molecular medicine. To illustrate some of the challenges we have faced and the technologies we had to develop, I have chosen several of our multiyear projects to describe in some detail: elucidating the regulation of translation of α- and β-globin mRNAs and the defect in beta thalassemia in the 1970s; cloning the Epo receptor and several red cell membrane proteins in the 1980s and 1990s; and more recently, determining the function of many microRNAs and long noncoding RNAs in red cell development. I summarize how we are currently utilizing single-cell transcriptomics (scRNAseq) to understand how dividing transit-amplifying burst-forming unit erythroid progenitors balance the need for more progenitor cells with the need for terminally differentiated erythroid cells, and to identify drugs potentially useful in treating Epo-resistant anemias such as Diamond Blackfan anemia. I hope that the lessons I learned in managing these diverse fellows and projects, initially without having grants to support them, will be helpful to others who would like to undertake ambitious and important lines of research in hematology.

The coordinated differentiation of hematopoietic stem and progenitor cells (HSPCs) into the various mature blood cell types is responsible for sustaining blood and immune system homeostasis. The cell fate decisions underlying this important biological process are made at the level of single cells. Methods to trace the fate of single cells are therefore essential for understanding the hematopoietic system activity in health and disease, and have made a major impact in how we understand and represent hematopoiesis. Here, we discuss the basic methodologies and technical considerations for three important clonal assays: single cell transplantation, lentiviral barcoding, and sleeping beauty barcoding. This Perspective is a synthesis of presentations and discussions from the 2019 International Society for Experimental Hematology (ISEH) Annual Meeting New Investigator Technology Session and the 2019 ISEH Winter Webinar.

The hematopoietic system is responsible for transporting oxygen and nutrients, fighting infections, and repairing tissue damage. Hematopoietic system dysfunction therefore causes a range of serious health consequences. Lifelong hematopoiesis is maintained by repopulating multipotent hematopoietic stem cells (HSCs) that replenish shorter-lived, mature blood cell types. A prokaryotic mechanism of immunity, the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9 nuclease system, has been recently "repurposed" to mutate mammalian genomes efficiently and in a sequence-specific manner. The application of this genome-editing technology to hematology has afforded new approaches for functional genomics and even the prospect of "correcting" dysfunctional HSCs in the treatment of serious genetic hematological diseases. In this Perspective, we provide an overview of three recent CRISPR/Cas9 methods in hematology: gene disruption, gene targeting, and saturating mutagenesis. We also summarize the technical considerations and advice provided during the May 2017 International Society of Experimental Hematology New Investigator Committee webinar on the same topic.

Controversy and context: two words that exemplified this year's International Society for Experimental Hematology meeting. Leaders in the field of hematology from around the world gathered in San Diego in August of 2016 to discuss cutting-edge research on diverse topics such as hemoglobin switching, hematopoietic stem cell emergence, leukemogenesis, and aging. Major questions discussed included the "when, where, and how" of hematopoietic emergence, bone marrow residence, and disease origination. This meeting summary covers some of the conference highlights.

Purpose of review The direct modification of RNA is now understood to be widespread, evolutionarily conserved and of consequence to cellular and organismal homeostasis. adenosine-to-inosine (A-to-I) RNA editing is one of the most common mammalian RNA modifications. Transcriptome-wide maps of the A-to-I editing exist, yet functions for the majority of editing sites remain opaque. Herein we discuss how hematology has been applied to determine physiological and malignant functions of A-to-I editing. Recent findings Functional studies have established that A-to-I editing and ADAR1, responsible for the majority of editing in blood cells, are essential for normal blood cell homeostasis. ADAR1 edits endogenous RNA and reshapes its secondary structure, preventing MDA5 from perceiving the cells own RNA as pathogenic. Roles for ADAR1 in human leukaemia, and most recently, cancer cell intrinsic and extrinsic functions of ADAR1 have been identified that highlight ADAR1 as a therapeutic target in cancer. Summary The studies reviewed have identified the key physiological function of ADAR1 and mechanistic basis for A-to-I editing in normal physiology and have now been extended to cancer. As our understanding of the biology and consequences of A-to-I editing evolve, it may be possible to target ADAR1 function advantageously in a number of settings.

// Jingya Wang 1, * , Xiaofei Ai 2, * , Tiejun Qin 3, * , Zefeng Xu 1, 3 , Yue Zhang 1, 3 , Jinqin Liu 1 , Bing Li 1, 3 , Liwei Fang 3 , Hongli Zhang 3 , Lijuan Pan 3 , Naibo Hu 3 , Shiqiang Qu 3 , Wenyu Cai 2 , Kun Ru 2 , Yujiao Jia 2 , Gang Huang 4 , Zhijian Xiao 1, 3 1 State Key Laboratory of Experimental Hematology, Institute of Hematology and Blood Diseases Hospital, Chinese Academy of Medical Sciences & Peking Union Medical College, Tianjin, China 2 Department of Pathology, Institute of Hematology and Blood Diseases Hospital, Chinese Academy of Medical Sciences & Peking Union Medical College, Tianjin, China 3 MDS and MPN Centre, Institute of Hematology and Blood Diseases Hospital, Chinese Academy of Medical Sciences & Peking Union Medical College, Tianjin, China 4 Divisions of Experimental Hematology and Cancer Biology, Cincinnati Children's Hospital Medical Center, Cincinnati, Ohio, USA * These authors have contributed equally to this work Correspondence to: Zhijian Xiao, email: zjxiao@medmail.com.cn Keywords: myelodysplastic syndromes, cytogenetic analysis, multiplex ligation-dependent probe amplification Received: August 26, 2016 Accepted: November 08, 2016 Published: November 29, 2016 ABSTRACT Cytogenetic analysis provides important diagnostic and prognostic information for patients with Myelodysplastic syndromes (MDS) and plays an essential role in the International Prognostic Scoring System (IPSS) and the revised International Prognostic Scoring System (IPSS-R). Multiplex ligation-dependent probe amplification (MLPA) assay is a recently developed technique to identify targeted cytogenetic aberrations in MDS patients. In the present study, we evaluated the results obtained using an MLPA assay in 437 patients with MDS to determine the efficacy of MLPA analysis. Using R-banding karyotyping, 45% (197/437) of MDS patients had chromosomal abnormalities, whereas MLPA analysis detected that 35% (153/437) of MDS cases contained at least one copy-number variations (CNVs) .2/5 individuals (40%) with R-band karyotype failures had trisomy 8 detected using only MLPA. Clonal cytogenetic abnormalities were detected in 20/235 (8.5%) MDS patients with a normal R-band karyotype, and 12/20 (60%) of those patients were reclassified into a higher-risk IPSS-R prognostic category. When sequencing and cytogenetics were combined, the fraction of patients with MDS-related oncogenic lesions increased to 87.3% (233/267 cases). MLPA analysis determined that the median OS of patients with a normal karyotype (n=218) was 65 months compared with 27 months in cases with an aberrant karyotype (P=0.002) in 240 patients with normal or failed karyotypes by R-banding karyotyping. The high-resolution MPLA assay is an efficient and reliable method that can be used in conjunction with R-band karyotyping to detect chromosomal abnormalities in patients with suspected MDS. MLPA may also provide more accurate prognostic information.

// Bing Li 1,2,* , Jinqin Liu 2,* , Shiqiang Qu 1,* , Robert Peter Gale 3 , Zhen Song 4 , Ruixian Xing 1 , Junxia Liu 5 , Yansong Ren 5 , Zefeng Xu 1,2 , Tiejun Qin 1 , Yue Zhang 1,2 , Liwei Fang 1 , Hongli Zhang 1 , Lijuan Pan 1 , Naibo Hu 1 , Wenyu Cai 6 , Peihong Zhang 6 , Gang Huang 7 and Zhijian Xiao 1,2 1 MDS and MPN Centre, Institute of Hematology and Blood Diseases Hospital, Chinese Academy of Medical Sciences & Peking Union Medical College, Tianjin, China 2 State Key Laboratory of Experimental Hematology, Institute of Hematology and Blood Diseases Hospital, Chinese Academy of Medical Sciences & Peking Union Medical College, Tianjin, China 3 Department of Medicine, Haematology Section, Division of Experimental Medicine, Imperial College, London, United Kingdom 4 Medical Service Division, Institute of Hematology and Blood Diseases Hospital, Chinese Academy of Medical Sciences & Peking Union Medical College, Tianjin, China 5 Cell Culture Laboratory, Institute of Hematology and Blood Diseases Hospital, Chinese Academy of Medical Sciences & Peking Union Medical College, Tianjin, China 6 Department of Pathology, Institute of Hematology and Blood Diseases Hospital, Chinese Academy of Medical Sciences & Peking Union Medical College, Tianjin, China 7 Divisions of Experimental Hematology and Cancer Biology, Cincinnati Children’s Hospital Medical Center, Cincinnati, Ohio, USA * Bing Li, Jinqin Liu and Shiqiang Qu have contributed equally to this study Correspondence to: Zhijian Xiao, email: // Keywords : myelodysplastic syndromes; colony-forming unit cell; prognosis Received : April 29, 2016 Accepted : August 25, 2016 Published : September 18, 2016 Abstract Background: In vitro colony-forming unit cell (CFU-C) assays are usually-used to detect the quantitative and qualitative features of haematopoietic stem cells (HSCs). We studies CFU-C assays in bone marrow samples from 365 consecutive subjects with newly-diagnosed myelodysplastic syndrome (MDS). Data were interrogated for associations with prognosis. Methods: CFU-C assays were performed according to the protocol of MethoCultTM H4435 Enriched. 365 consecutive newly-diagnosed, untreated subjects with MDS diagnosed from July, 2007 to April, 2014 were studied. All subjects were reclassified according to the 2008 WHO criteria. Subjects were observed for survival until July 31, 2015. Follow-up data were available for 289 (80%) subjects. Median follow-up of survivors was 22 months (range, 1-85) months. Erythroid and myeloid colonies were isolated from each subject with one cytogenetic abnormality such as del(5/5q), +8, del(7/7q) or del(20q). Cytogenetic abnormalities of each colony were analyzed by fluorescence in situ hybridization (FISH). SPSS 17.0 software was used to make statistical analysis. Results: The numbers of burst-forming units-erythroid (BFU-E), colony forming unit-erythroid (CFU-E) and colony forming unit-granulocytes/macrophages (CFU-G/M) were significantly lower than normals. A high ratio of cluster- to CFU-G/M was associated with poor-risk cytogenetics. In multivariable analyses a cluster- to CFU-G/M ratio >0.6 was an independent risk-factor for OS after adjusting for IPSS-R (HR 3.339, [95%CI 1.434-7.778]; P = 0.005) in very high-risk cohort. Conclusion: These data suggest abnormalities of proliferation and differentiation of erythroid and myeloid precursor cells in vitro parallel the ineffective hematopoiesis typical of MDS and may be useful in predicting outcomes of persons with higher-risk MDS.

// Linping Hu 1 , Xiuxiu Yin 1 , Jiangman Sun 1 , Anders Zetterberg 2 , Weimin Miao 1, 3 and Tao Cheng 1 1 State Key Laboratory of Experimental Hematology, Institute of Hematology, Blood Disease Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College, Tianjin China 2 The Department of Oncology-Pathology, Karolinska Cancer Institute, Karolinska Institute, Stockholm, Sweden 3 Union Stem Cell and Gene Engineering Co. Ltd, Tianjin China Correspondence to: Tao Cheng, email: chengtao@ihcams.ac.cn Weimin Miao, email: miaow@ihcams.ac.cn Keywords: molecular pathology, genotyping, sequential FISH, single cell, multi-gene detection Received: November 10, 2015 Accepted: May 01, 2016 Published: June 23, 2016 ABSTRACT Multi-gene detection at the single-cell level is desirable to enable more precise genotyping of heterogeneous hematology and oncology samples. This study aimed to establish a single-cell multi-gene fluorescence in situ hybridization (FISH) method for use in molecular pathology analyses. Five fluorochromes were used to label different FISH gene probes, and 5 genes were detected using a five-color FISH protocol. After the first hybridization, the previous FISH probe set was stripped, and a second set of five-color FISH probes was used for rehybridization. After each hybridization, the fluorescence signals were recorded in 6 fluorescence filter channels that included DAPI, Spectrum Green ™ , Cy3 ™ v1, Texas Red, Cy5, and PF-415. A digital automatic relocation procedure was used to ensure that exactly the same microscopic field was studied in each stripping and hybridization cycle. By using this sequential stripping and rehybridization strategy, up to 20 genes can be detected within a single nucleus. In conclusion, a practical molecular pathology method was developed for analyzing multiple genes at the single-cell level.

Since the end of the 20th century, novel approaches have emerged to manipulate experimental models of hematological disorders so that they more accurately mirror what is observed in the clinical setting. Despite these technological advances, the characterization of crucial genes for benign or malignant hematological disorders remains challenging, given the dynamic nature of the hematopoietic system and the genetic heterogeneity of these disorders. To overcome this limitation, genome-editing technologies have been developed to manipulate the genome specifically via deletion, insertion, or modification of targeted loci. These technologies have progressed swiftly, allowing their common use to investigate genetic function in experimental hematology. Among them, homologous-recombination-mediated targeting technologies have facilitated the manipulation of specific loci by generating knock-out and knock-in models. Despite promoting significant advances in our understanding of the molecular mechanisms involved in hematology, these inefficient, time-consuming, and labor-intensive approaches did not permit the development of cellular or animal models, recapitulating the complexity of hematological disorders. On October 26, 2016, Drs. Ben Ebert and Chad Cowan shared their knowledge of and experience with the utilization of CRISPR for models of myeloid malignancy, disease, and novel therapeutics in an International Society for Experimental Hematology webinar titled "Utility of CRISPR/Cas9 Systems in Hematology Research." Here, we provide an overview of the topics they covered, including their insights into the novel applications of the technique and its strengths and limitations.

(1991). Highlights of the 19th Annual Meeting of the International Society for Experimental Hematology (ISEH) Held in Seattle, Washington, USA, August 26–30, 1990. Leukemia & Lymphoma: Vol. 4, No. 5-6, pp. 425-428.

Summary Conditional knockout mice are commonly used to study the function of specific genes in hematopoiesis. Different promoters that drive Cre expression have been utilized, with the interferon-inducible Mx1-Cre still being the most commonly used “deleter strain” in experimental hematology. However, different pitfalls associated with this system could lead to misinterpretation in functional studies. We present here two of these issues related to the use of Mx1-Cre: first, a high spontaneous recombination rate when applying commonly used techniques in experimental hematology, and second, undesired short-term consequences of the use of polyinosinic:polycytidylic acid, including changes in cellular phenotypes that, however, resolve within days. Our studies emphasize therefore that proper controls are crucial when modeling gene deletion using the Mx1-Cre transgene., Graphical Abstract, Highlights • Common research methods induce uncontrolled activation of the Mx1-Cre system • The leakiness of the Mx1-Cre system can result in misinterpretation of results • pIpC alters the phenotype of HSPCs that resolves by day 8 • pIpC has limited effects on the proliferation of HSPCs, In this article, Cammenga, Velasco-Hernandez, and colleagues describe technical issues associated with the Mx1-Cre model in hematopoiesis, including a high level of spontaneous activation induced by multiple experimental procedures. These pitfalls can lead to misinterpretation of results so proper controls are needed to avoid these problems.

Amid the beauty of the Kyoto countryside, leaders in the field of hematology met at the 44th annual International Society for Experimental Hematology (ISEH) meeting in late September 2015. Led by ISEH President Paul Frenette and President-Elect David Traver, the meeting covered many aspects of hematopoiesis with a focus on technology. At the meeting, it became clear that the future of hematology is being shaped by innovations in single-cell "omics" and imaging approaches that will provide answers to age-old questions on cellular identity. In this meeting review, we highlight the advances presented in understanding the hematopoietic stem cell (HSC) niche, heterogeneity, stress response, epigenetics, and how these processes change from birth to old age.

Indolent nonfollicular B-cell lymphoma (INFBCL) has been classified in the World Health Organization 2008 system among the mature B-cell neoplasms and includes nodal and extranodal marginal zone lymphoma (MZL), lymphoplasmacytic lymphoma (LPL), and small lymphocytic lymphoma (SLL). Recently, the array and sequencing technologies have provided new insights into their molecular pathogenesis; the molecular discoveries, however, have not yet translated into consistent changes in their management. Thus, the therapy for INFBCL remains challenging. To promote widespread adoption of appropriate clinical practice, the Italian Society of Hematology and affiliate societies (Italian Society of Experimental Hematology and Italian Group for Bone Marrow Transplantation) reviewed the evidence regarding the management of these lymphomas to produce evidence-based recommendations aimed at contributing to therapy optimization and standardization. We used the Grades of Recommendation, Assessment, Development, and Evaluation system, which is based on a sequential assessment of the quality of evidence, followed by an analysis of the benefit/risk balance and subsequent judgment about the strength of recommendations. For issues without consistent evidence, we used the consensus technique. We have provided separate recommendations for diagnostic and staging requirements, first-line therapy, and postinduction therapy for the most frequent INFBCLs (ie, LPL, SLL, and nodal, splenic, and gastric MZL).