T cell acute lymphoblastic leukemia (T-ALL) is an aggressive malignancy of immature T cells. Recent next-generation sequencing efforts have exhaustively documented its genetic landscape. To understand the functional sequelae of various genetic inputs in this disease, we developed a de novo leukemia model by transducing human CD34+ cord blood cells with lentivirus to enforce expression of various combinations of known or suspected oncogenic drivers. Transcriptomic profiling of leukemias generated by transduction with activated NOTCH1, LMO2, TAL1, and BMI1 (NLTB) revealed two major clusters, which we have characterized as ‘Early’ and ‘Late’ types. The Early type fulfills diagnostic criteria as near ETP T-ALL, whereas the ‘Late’ type exhibits a CD4+ CD8+ (“DP”) phenotype with TCRαβ rearrangements. Differential gene expression analysis revealed HHEX, NFE2, MEF2C, SPI1, and MYCN are more highly expressed in Early than Late leukemias. ChIP-seq analyses performed on NLTB-transduced CB cells and their derived leukemias revealed a striking global increase in H3K4 trimethylation with particular emphasis over gene bodies. Although promoter-localized H3K4me3 marks are associated with active gene promoters, broadening of these peaks with extension into gene bodies has been associated with transcriptional consistency and cell “identity.” We observed broad H3K4me3 marks to extend into the body of highly expressed genes such as MYCN in Early leukemia cells, suggesting that H3K4me3 marks could be essential for supporting the Early (ETP-like) gene expression state. Indeed, shRNA-mediated knockdown of WDR5 or KMT2 paralogs A/B, C/D, and F/G significantly limited growth of Early leukemia cells. These findings suggest a model in which H3K4me3 marks are deposited and/or maintained by KMT2 complexes to support high levels of expression of oncogenes such as MYCN in ETP-like human leukemias.

Relapse remains a major challenge in acute myeloid leukemia (AML), often driven by therapy-resistant subclones. Although AML xenograft models have been widely used in studying disease mechanisms, most studies focus on preconditioned hosts. The resultant marrow damage and inflammatory sequelae likely have an impact on the dynamics of clonal evolution, potentially limiting our capacity to model relapse as it occurs in patients. To better model AML progression and relapse, we developed an unconditioned immunodeficient xenograft model combined with single-cell DNA-sequencing (scDNA-seq) to track clonal evolution before and after treatment.Bone marrow (BM) cells from four randomly selected diagnostic AML patient samples were injected into four congenic strains of female NRG mice: NRG, NRG-3GS, NRG-W41 and NRG-3GS-W41. Peripheral blood was monitored to track engraftment, and BM aspirates were collected when leukemic burden neared lethal thresholds (primary engraftment). Mice were then treated with 200 cGy total body irradiation and followed until moribund (relapse leukemia). All four samples demonstrated rapid engraftment in NRG-W41-3GS and NRG-3GS strains. In contrast, non-3GS strains showed patient-specific engraftment, with some failing to achieve detectable engraftment. scDNA-seq profiling across three timepoints—original sample, primary engraftment, and relapse—elucidated how specific subclones expand, persist, or are eliminated after therapy in different mice. This unconditioned xenograft model offers a powerful and biologically relevant system to study AML and its clonal evolution in vivo. High-resolution tracking of subclonal dynamics in response to therapy opens new avenues for studying treatment resistance and testing personalized therapeutic interventions.

ABSTRACT:Recent studies indicate the human lympho-myeloid restriction process to be a different and more heterogeneous one than historically inferred. Here we describe the development of bulk and clonal culture systems that efficiently support early B-lymphoid differentiation and its use to elucidate the biological and molecular changes that accompany their initial restriction from subsets of CD34+ human cord blood cells with lympho-myeloid-limited potential. Analyses of these changes revealed that the acquisition of B-lymphoid- and neutrophil/monocyte (NM)-restricted properties are accompanied by a concomitantly accelerated and lineage-shared cell cycling activity and loss of self-renewal potential. Single-cell transcriptome analysis identified reduced expression of multiple self-renewal-associated genes and an accompanying heterogeneous activation of lineage-regulatory modules during the production of B, NM, and dendritic cell precursors. By applying a novel culture system that supports early human lymphoid differentiation, we uncovered a shared mechanism of proliferation control, along with persistent biological and transcriptional heterogeneity in cells undergoing B- and NM-lineage restriction.

Background Understanding the interplay between genome variation and epigenomic structure is fundamental to the study of the development and mechanisms of disease. Previous studies have leveraged population-scale genotype surveys to associate alleles with epigenomic states in heterogenous tissue types. However, epigenomes are inherently cell type-specific, giving rise to unique genome-epigenome interactions that can influence distinct functional states and susceptibility to disease. Moreover, the extent of individual variation in cell type-specific epigenotypes remains poorly understood, posing additional challenges to accurately link genotypes with epigenomic features. Results We generated comprehensive genomic and epigenomic measurements in four functionally defined human breast cell types across eight individuals. We developed a method to measure histone modification variance, discovering significantly higher variation in repressive chromatin states marked by H3K27me3 compared to the active states marked by H3K27ac and H3K4me3. Genetic variation linked to variation in chromatin state was highly cell type-specific, with nearly 90% occurring uniquely in a single cell type, and active histone modifications were enriched in these variants relative to repressive modifications. Association with gene transcription allowed for the prioritization of functional candidates, and the regulatory impact of an ANXA1 -linked variant, rs75071948, was validated in vitro with CRISPR/Cas9-mediated HDR. Conclusions We define structures of epigenomic variability among breast cell types and present evidence of extensive cell type-specific genome-epigenome interactions, highlighting the critical role of cell type in mediating these associations in the breast. ### Competing Interest Statement The authors have declared no competing interest.

Identification of phenotypes of human hematopoietic cells that display long-term mature cell outputs in vitro and repopulating capability in immunodeficient mice has been important to anticipating the therapeutic potential of fresh harvests of bone marrow or cord blood before or after their physical or genetic manipulation. However, characterizing their key properties and strategies for their isolation from multiple sources at increasing cell purities and elucidating the mechanisms that regulate their ability to sustain mature blood cell production continues to be of major interest. Previous studies have shown that fetal and adult human cells with long-term blood cell output potential are highly enriched in their respective glycosylphosphatidylinositol (GPI)-anchored surface protein GPI80+ and CD49f+ subsets of a developmentally preserved CD45+CD34+CD38-CD45RA-CD90+ population. The so-called "GPI80" hematopoietic cells found in first-trimester human fetal liver are of particular interest because of their very high regenerative capability compared with their adult or even neonatal (cord blood) "CD49f" counterparts. Here, it was hypothesized that high regenerative activity of the GPI80+ cells could be further enriched within a CD49f+ subset. We now demonstrated that coexpression of CD49f within the GPI80+ population identifies a subset with reduced short-term myeloid colony-forming activity in semisolid medium and greater progeny outputs in both 12-week growth factor-supplemented stromal cocultures and in transplanted immunodeficient mice. These findings demonstrated that CD49f is a pervasive marker of human hematopoietic stem cells (HSCs) throughout ontogeny and aging.

Recent studies indicate the human lympho-myeloid restriction process to be a different and more heterogeneous one than historically inferred. Here we describe the development of bulk and clonal culture systems that efficiently support early B-lymphoid differentiation and their use to identify biological and molecular changes that accompany their initial restriction from subsets of CD34+ human cord blood cells with lympho-myeloid-limited potential. Analyses of the changes observed revealed the acquisition of B-lymphoid- and neutrophil/monocyte (NM)-restricted properties are accompanied by a concomitantly accelerated and lineage-shared cell cycling activity and loss of self-renewal properties. Parallel, single-cell transcriptome analysis identified reduced expression of multiple self-renewal-associated genes and an accompanying heterogeneous activation of lineage-regulatory modules during the production of B, NM and dendritic cell precursors. These results uncover a connected regulation of lineage-shared proliferation control with persistent heterogeneity in the biological and transcriptional changes in the same cells undergoing B and NM lineage restriction.### Competing Interest StatementThe authors have declared no competing interest.

Targeted therapeutics for high-risk cancers remain an unmet medical need. Here we report the results of a large-scale screen of over 11,000 molecules for their ability to inhibit the survival and growth in vitro of human leukemic cells from multiple sources including patient samples, de novo generated human leukemia models, and established human leukemic cell lines. The responses of cells from de novo models were most similar to those of patient samples, both of which showed striking differences from the cell-line responses. Analysis of differences in subtype-specific therapeutic vulnerabilities made possible by the scale of this screen enabled the identification of new specific modulators of apoptosis, while also highlighting the complex polypharmacology of anti-leukemic small molecules such as shikonin. These findings introduce a new platform for uncovering new therapeutic options for high-risk human leukemia, in addition to reinforcing the importance of the test sample choice for effective drug discovery.

Mechanisms that regulate cell survival and proliferation are important for both the development and homeostasis of normal tissue, and as well as for the emergence and expansion of malignant cell populations. Caspase-3 (CASP3) has long been recognized for its proteolytic role in orchestrating cell death-initiated pathways and related processes; however, whether CASP3 has other functions in mammalian cells that do not depend on its known catalytic activity have remained unknown. To investigate this possibility, we examined the biological and molecular consequences of reducing CASP3 levels in normal and transformed human cells using lentiviral-mediated short hairpin-based knockdown experiments in combination with approaches designed to test the potential rescue capability of different components of the CASP3 protein. The results showed that a ≥50% reduction in CASP3 levels rapidly and consistently arrested cell cycle progression and survival in all cell types tested. Mass spectrometry-based proteomic analyses and more specific flow cytometric measurements strongly implicated CASP3 as playing an essential role in regulating intracellular protein aggregate clearance. Intriguingly, the rescue experiments utilizing different forms of the CASP3 protein showed its prosurvival function and effective removal of protein aggregates did not require its well-known catalytic capability, and pinpointed the N-terminal prodomain of CASP3 as the exclusive component needed in a diversity of human cell types. These findings identify a new mechanism that regulates human cell survival and proliferation and thus expands the complexity of how these processes can be controlled.

Identification of phenotypes of human hematopoietic cells that are highly enriched in those with long-term repopulating activity in immunodeficient mice has been essential to elucidating mechanisms that regulate their properties. First trimester human fetal liver (hFL) has long been appreciated as a source of HSCs with remarkable in vivo regenerative capability as compared to their adult or even neonatal counterparts, although conditions to support the in vitro maintenance of this function, particularly of hFL HSCs, have remained elusive. In a first series of experiments, we found that expression of the CD49f integrin on first trimester GPI80+CD90+CD38-CD45RA-CD34+CD45+ hFL cells selectively depleted cells with initial myeloid colony-forming activity in methylcellulose cultures. At the same time, this phenotype became enriched for cells with immediate long-term repopulating capability in both 12-week growth factor (GF)-supplemented stromal co-cultures and in sublethally irradiated, transplanted immunodeficient NRG-W41 mice. Next, we compared the functional properties of this enriched HSC subset following their maintenance in vitro for 2 days under different culture conditions. The results demonstrated that FLT3-L, as a single GF in serum-free medium best maintained their ability to regenerate serially transplantable HSCs. In contrast, the same 2-day pre-transplant culture condition resulted in the poorest survival and division of the input cells. Subsequent cell cycle analysis confirmed the existence of a G0 population within the HSC-enriched subset of hFL cells that best maintains their growth potential in FLT3-L alone. These results demonstrate CD49f expression to be a pervasive marker of human HSCs in vivo and reinforce the concept that the maintenance of HSC properties may be adversely affected by factors that strongly promote survival and rapid cell cycle entry.

T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive leukemia of immature T-cells. The outcome for patients with intrinsic therapy resistance or disease relapse remains poor, and the biological bases for aggressive disease remain ill-defined. Using our synthetic leukemia model from human CD34+ cord blood (CB) cells, we performed RNA-Seq on 32 synthetic leukemias (SynLs) generated with Notch1 (N), LMO2 (L), TAL1 (T), and/or BMI1 (B) oncogenes. Genetic loss-of-function approaches were used to assess potential dependencies of oncogene-transduced CB cells and SynLs. Transcriptomic analysis of SynLs revealed two types of disease corresponding to early (ETP-like) and later (post beta-selection) T-cell development stages. Interestingly, early vs. late SynLs could be distinguished by MYCN expression level, the highest being in early SynLs. Notably, T-ALL patients with high MYCN expression present a shorter survival (TARGET cohort). To assess MYCN's contribution to T-ALL, we performed MYCN knockdown (KD)/knockout (KO) in NLTB-transduced CB or early SynLs and observed significantly reduced cell growth and clonogenic activity. Conversely, overexpression of MYCN along with Notch1 in CB cells increased clonogenic activity in vitro but was insufficient to generate leukemia in vivo. To explore mechanisms of MYCN gene regulation, we performed histone ChIP-Seq on NLTB-transduced CB and early SynLs. NLTB transduction resulted in global gain of H3K4me3, often distributed over gene bodies including MYCN. KD of the H3K4 methyltransferase complex components WDR5 and KMT2D led to reduced growth/survival of NLTB-transduced CB and early SynLs, while normal T-progenitors appeared unaffected by KMT2D KD. Our results reveal MYCN as a critical oncogene associated with immature tumors and suggest that epigenetic regulation may drive consistent oncogene expression and malignant identity.

Abstract Human breast cancers (BCs) are well recognized for the diagnostic and treatment challenges posed by their extensive and continuously evolving genetic and biologic heterogeneity. Although much progress has been made in identifying features that may serve as new targets, most of these have not been found to be usefully applied to metastatic BCs characterized by a lack of cellular expression of ER, PR, HER2 and a high proliferative activity. Accordingly, these generally incurable BCs, thus identified as “triple-negative” (TNBCs) are in particular need of strategies to model their development in ways that could facilitate the design and testing of effective treatments. Given the drawbacks historically encountered in using cell lines, mouse models, and patient-derived xenografts for this purpose, we launched experiments to assess the potential of creating a useful human model of TNBC de novo. We first discovered that forced expression of KRASG12D (K) alone is sufficient to produce low grade, but persistent human tumors in transplanted immunodeficient NRG mice from 2 of the 3 cell types that constitute and maintain the normal adult human female mammary gland. Histologically, these YB-1-dependent K–induced tumors were classified as variably human ER-, PR- and HER2-positive, invasive ductal carcinomas, also able to produce derivative small tumors in secondary hosts. Subsequent experiments to test whether the indolent properties of these tumors can be modified (i.e., by additional forced expression of human hepatocyte growth factor, or maintenance of transplanted mice on an obesity-inducing diet) showed that neither of these treatments enhance the growth of the K-induced tumors. However, in sharp contrast, transplantation of female NRG mice with the same types of freshly isolated normal adult female mammary cells transduced with lentivirally-encoded BMI1, MYC and TP53R273C and K (KBMT) produced growths containing an increased frequency of Ki67+ cells within a week, and subsequently, produced still YB-1-elevated, but continuously expanding TNBCs. Interestingly, K was required in addition to all 3 of these oncogenes to obtain aggressively expanding TNBCs and, when any one of the BMI1, MYC, or TP53R273C oncogenes was omitted, variably diminished outputs were obtained. FACS analyses showed the surface marker phenotypes of the cells present in any of the more rapidly growing tumors to be remarkably homogeneous with features of normal basal cells. In addition, despite any gross evidence of metastatic capability, cells isolated from all KBMT-induced tumors tested could be expanded in vitro and generated fast growing tumors in transplanted secondary mice. Taken together, these findings demonstrate the feasibility of rapidly and reproducibly creating a model of human TNBC de novo from initially normal human mammary cells, thus offering a new and powerful platform for potentially developing novel prevention, diagnostic, as well as treatment approaches applicable to this disease. Citation Format: Susanna Tan, Davide Pellacani, Sylvain Lefort, Amal El-Naggar, Shengsen Ding, Poul H Sorensen, Martin Hirst, Connie Eaves. Creation and preliminary analyses of a full spectrum of breast cancers initiated de novo from normal human mammary cells [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Breast Cancer Research; 2023 Oct 19-22; San Diego, California. Philadelphia (PA): AACR; Cancer Res 2024;84(3 Suppl_1):Abstract nr IA16.

Differences between mice and humans, such as variations in the major histocompatibility complex (MHC) and variances in immune system function, pose challenges to accurately model human biology in mice. Therefore, improving humanized mouse models, particularly models of human hematopoietic lineages (such as functional lymphocytes), is essential. This optimization is especially relevant to the study of the immune system for advancing treatments like CAR-T therapy and immune checkpoint inhibitors in cancer treatment.A novel mouse model with the essential immune components needed to generate mature B and T cells has been developed. The DRAGA mouse model has an NRG background and carries human HLA-A2 and HLA-DR4 transgenes. These transgenes have been shown to enhance hematopoietic engraftment and promote the development of fully functional T cells and antibody-secreting B cells (Casares, 2016).To test the ability of the DRAGA mouse strain to support the late-stage differentiation of human B- and T-cells from HLA-matched cord blood (CB) progenitors, we transplanted 10,000 matched or unmatched CD34+ CB cells into DRAGA mice. We then tracked the output of human hematopoietic cells for 20 weeks. Analysis showed higher T- and B-cell progenitors in the bone marrow of mice engrafted with matched cord blood progenitors compared to unmatched ones, indicating that HLA compatibility may enhance mature T-cell selection and early lymphopoiesis. In addition, comparing matched cord blood progeny with infant human thymus tissue reveals similar T-lineage phenotypes, suggesting this model replicates this aspect of human T cell development and maturation in mice. Our findings suggest that the DRAGA mouse holds promise as a model for creating a humanized immune system. This model may be valuable for studying immunotherapies and exploring immune cell functions across different cancer types.

Abstract Men normally develop and maintain bi-layered mammary glands that appear structurally and histologically similar to those that develop in women, but with reported deficient lobule formation. Men also develop breast cancer, mostly of the ER+ subtype and an age-associated increasing onset, but at a 100-fold lower incidence and overall worse outcome. However, the classification and treatment of male breast cancers are largely based on strategies developed for female patients, despite their known hormonal and other differences. This is likely due, at least in part, to the fact that adult male mice, unlike humans do not develop full mammary glands. Thus, experimental access to mouse models have been lacking and studies of “normal” male mammary tissue have thus been largely restricted to analysis of human male gynecomastia samples. We now report the utility of multiple systems for analyzing normal adult human female mammary cell properties to this source of freshly obtained and viably cryopreserved normal human male mammary tissue. Histological examination of male mammary tissues (3 donors) confirmed their reported bi-layered structure surrounded by fibroblasts and lack of gross evidence of lobules. Use of procedures created for analyzing female breast tissue indicates that the human male mammary glands contain the same subsets but in different relative proportions; i.e., the proportion of EpCAM+CD49f- mammary cells (Luminal Cells, LCs) is relatively increased and of the EpCAM+CD49f+ cells (Luminal Progenitors, LPs) and EpCAM-CD49f+ cells (Basal Cells, BCs) is correspondingly decreased, consistent with a lack of alveolae. However, the freshly isolated male LPs and BCs contain a similar frequency of EGF-responsive colony-forming cells (CFCs) as female LPs and BCs. Subcutaneous transplantation of the male mammary cells in both male and female immunodeficient mice resulted in the generation of hollow mammary structures 4 weeks later as previously shown for female cells, and these were also found to contain LC, LP and BC populations including some with detectable levels of CFCs suggesting their derivation from a male mammary stem cell population (Eirew et al Nat Med 2008). We have also found evidence from qPCR and RNA-seq analyses of reduced progesterone signaling and alveologenesis in male LPs and LCs. These findings suggest hormonal differences may play a major role in regulating the altered ratios of cell types present in the normal adult human male mammary gland in comparison to premenopausal female. Interestingly, preliminary experiments also indicate that normal human male mammary cells transduced with KRASG12D without, or with BMI1+MYC+TP53R273C , generate analogous slowly-growing ER+, and rapidly-growing, highly aggressive tumours, respectively, in male immunodeficient mice. Taken together, our findings demonstrate the potential of established cell culture, xenotransplantation and molecular analytical tools to reveal the altered mechanisms that account for the different structure and biology of human male mammary tissue. Citation Format: Shengsen Ding, Susanna Tan, Ebrahim Eskandari-Nasab, Shrinka Sen, Martin Hirst, Connie Eaves. Characterization of normal human male mammary cells and their de novo transformed derivatives [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Breast Cancer Research; 2023 Oct 19-22; San Diego, California. Philadelphia (PA): AACR; Cancer Res 2024;84(3 Suppl_1):Abstract nr A078.

Cells with sustained multi-lineage blood cell regenerative potential are referred to as hematopoietic stem cells (HSCs). Current findings indicate that they are responsible for lifelong blood production, are first detected within the first month of development in humans and are largely created prior to birth after which they expand their numbers in response to physiological demands for normal or enhanced blood cell output requirements. Interestingly, a number of the intrinsically determined functional properties of HSCs also change after birth. These include an apparent decline in their self-renewal potential. Thus, for example, in humans as in mice, cells with long-term (>6 month) HSC regenerative properties present in the developing fetal liver (FL) are a source of HSCs that possess a much higher in vivo regenerative capability than those from older donor sources, such as adult bone marrow or even cord blood (CB). Accordingly, it has been of longstanding interest to better understand the molecular regulation of this high regenerative capacity prevalent in fetal HSCs for potential future therapeutic as well as scientific exploitation. Here, we describe the results of experiments designed to answer the hypothesis that human FL HSCs with human-relevant self-renewal properties can be isolated as a quiescent CD49f+ subset of the GPI80+CD90+CD38-CD45RA-CD34+CD45+ population following their incubation in standard serum-free culture medium supplemented with FLT3-ligand (FLT3-L) alone. Initial experiments showed expression of the CD49f integrin on first trimester hFL cells selectively depleted cells able to produce colonies of granulocytes, macrophages or erythroid cells in standard 2-week methylcellulose cultures containing SCF, GM-CSF, IL6, IL3 and EPO. Conversely, expression of the CD49f integrin selectively enriched for cells with 12-week output capabilities in both growth factor (GF)-supplemented stromal co-cultures and in sublethally irradiated, transplanted immunodeficient NOD-Rag1 -/-IL2Rγc -/- W 41/41 (NRG-W) mice. Initial experiments designed to test the effect of multiple GF and small molecule additives on the maintenance over a 7-day period of this in vivo regenerative ability of the input FL HSC confirmed GPI80 expression to be a continuing positive selective phenotype. In addition, the result of transplant experiments showed that a 2-day incubation in FLT3-L alone maintained the 12-week serially transplantable activity of the HSCs (12-weeks/cycle) as fully equivalent to the unmanipulated input cells and significantly superior (P<0.05) to FLT3-L+IL3+SF (3GF) with or without addition of UM171, stemregenin, or eltrombopag. Interestingly, in vitro monitoring of GPI80+ cells showed FLT3-L alone maintained the survival of only 20% of the input GPI80+ cells compared to any of the 3GF-based conditions. In addition, the time to complete a first division and subsequent divisions of the input cells was delayed and prolonged, respectively, in the FLT3-L versus the 3GF conditions. Subsequent 7-day suspension cultures and 6-week GF-supplemented stromal co-cultures experiments have confirmed the existence of a G0 population within the HSC-enriched subset of human FL cells that best maintains their growth potential in FLT3-L alone. Together, these results demonstrate CD49f expression to be a pervasive marker of human HSCs throughout development and reveal the importance of different GF conditions to support the maintenance of viability and retention of self-renewal capability of human fetal HSCs in contrast to those required to activate/support a rapid initiation of cell division. These findings set the stage for future development of strategies to exploit human FL cells therapeutically and may also be critical to designing conditions that will best support the ex vivo maintenance of HSCs at and after birth.

Human hematopoietic stem cells (HSCs), like their counterparts in mice, comprise a functionally and molecularly heterogeneous population of cells throughout life that collectively maintain required outputs of mature blood cells under homeostatic conditions. In both species, an early developmental change in the HSC population involves a postnatal switch from a state in which most of these cells exist in a rapidly cycling state and maintain a high self-renewal potential to a state in which the majority of cells are in a quiescent state with an overall reduced self-renewal potential. However, despite the well-established growth factor dependence of HSC proliferation, whether and how this mechanism of HSC regulation might be affected by aging has remained poorly understood. To address this knowledge gap, we isolated highly HSC-enriched CD34+CD38-CD45RA-CD90+CD49f+ (CD49f+) cells from cord blood, adult bone marrow, and mobilized peripheral blood samples obtained from normal humans spanning 7 decades of age and then measured their functional and molecular responses to growth factor stimulation in vitro and their regenerative activity in vivo in mice that had undergone transplantation. Initial experiments revealed that advancing donor age was accompanied by a significant and progressively delayed proliferative response but not the altered mature cell outputs seen in normal older individuals. Importantly, subsequent dose-response analyses revealed an age-associated reduction in the growth factor-stimulated proliferation of CD49f+ cells mediated by reduced activation of AKT and altered cell cycle entry and progression. These findings identify a new intrinsic, pervasive, and progressive aging-related alteration in the biological and signaling mechanisms required to drive the proliferation of very primitive, normal human hematopoietic cells.

How time flies! Just over 50 years after the birth of the International Society of Experimental Hematology, we are announcing a new phase in the leadership and operation of Experimental Hematology, the life blood and historical flagship of the society. We are particularly proud and grateful for the expanding energy and involvement of the entire ISEH membership and its contributions to the continuing growth and increasing success of the journal. This has enabled a major shift upward of the journal's content and impact on the broad field of hematopoiesis, new relevant methodologies, and relevant clinical insights.

Identification of phenotypes of human cell populations with long-term repopulating capability in immunodeficient mice has been essential to elucidating the mechanisms that regulate their maintenance and loss of this defining hematopoietic stem cell (HSC) property. First trimester human fetal liver (hFL) has long been appreciated as a source of human HSCs with remarkable regenerative capability compared to their adult or even neonatal counterparts, but conditions to support the maintenance of this function in vitro have remained elusive. In a first series of experiments, we found that expression of the CD49f integrin on GPI80+CD90+CD38-CD45RA-CD34+CD45+ first trimester hFL cells selectively depleted cells with initial myeloid colony-forming activity in methylcellulose cultures but enriched for cells with long-term repopulating capability in 12-week growth factor (GF)-supplemented stromal co-cultures and in transplanted immunodeficient NRG-W41 mice. A comparison of the ability of different culture conditions to maintain this in vivo function then demonstrated that FLT3-L as a single GF supplement to a serum-free medium was optimal and sufficient to support serially transplantable hFL HSCs. Of interest, this single GF condition was poorly supportive of both GPI80+ hFL cell survival and division. This study points to CD49f as a pervasive marker of human HSCs throughout ontogeny and aging and reinforces the idea that the molecular maintenance of HSC properties may be adversely affected by rapid cell cycle transit times. Identification of phenotypes of human cell populations with long-term repopulating capability in immunodeficient mice has been essential to elucidating the mechanisms that regulate their maintenance and loss of this defining hematopoietic stem cell (HSC) property. First trimester human fetal liver (hFL) has long been appreciated as a source of human HSCs with remarkable regenerative capability compared to their adult or even neonatal counterparts, but conditions to support the maintenance of this function in vitro have remained elusive. In a first series of experiments, we found that expression of the CD49f integrin on GPI80+CD90+CD38-CD45RA-CD34+CD45+ first trimester hFL cells selectively depleted cells with initial myeloid colony-forming activity in methylcellulose cultures but enriched for cells with long-term repopulating capability in 12-week growth factor (GF)-supplemented stromal co-cultures and in transplanted immunodeficient NRG-W41 mice. A comparison of the ability of different culture conditions to maintain this in vivo function then demonstrated that FLT3-L as a single GF supplement to a serum-free medium was optimal and sufficient to support serially transplantable hFL HSCs. Of interest, this single GF condition was poorly supportive of both GPI80+ hFL cell survival and division. This study points to CD49f as a pervasive marker of human HSCs throughout ontogeny and aging and reinforces the idea that the molecular maintenance of HSC properties may be adversely affected by rapid cell cycle transit times. Erratum to '3117 − ENKURIN: A NOVEL MARKER FOR MYELOPROLIFERATIVE NEOPLASMS FROM VALIDATED PLATELET, MEGAKARYOCYTE, AND WHOLE BLOOD SPECIMENS' [VOLUME 111, SUPPLEMENT , S103, JANUARY 01, 2022]Experimental HematologyVol. 117PreviewThe publisher regrets that in the original abstract, Dr. Anandi Krishnan was listed as first author which was incorrect. The correct Author line is shown above. Full-Text PDF