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Clonal hematopoiesis (CH) arises when hematopoietic stem cells (HSCs) acquire mutations, most frequently in the DNMT3A and TET2 genes, conferring a competitive advantage through mechanisms that remain unclear. To gain insight into how CH mutations enable gradual clonal expansion, we used single-cell multi-omics with high-fidelity genotyping on human CH bone marrow (BM) samples. Most of the selective advantage of mutant cells occurs within HSCs. DNMT3A- and TET2-mutant clones expand further in early progenitors, while TET2 mutations accelerate myeloid maturation in a dose-dependent manner. Unexpectedly, both mutant and non-mutant HSCs from CH samples are enriched for inflammatory and aging transcriptomic signatures, compared with HSCs from non-CH samples, revealing a non-cell-autonomous effect. However, DNMT3A- and TET2-mutant HSCs have an attenuated inflammatory response relative to wild-type HSCs within the same sample. Our data support a model whereby CH clones are gradually selected because they are resistant to the deleterious impact of inflammation and aging., Competing Interests: Declaration of interests J.E.D. receives royalties from Trillium Therapeutics Inc./Pfizer and a commercial research grant from Celgene/BMS., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

Gene expression profiling and proteome analysis of normal and malignant hematopoietic stem cells (HSCs) point to shared core stemness properties. However, discordance between mRNA and protein signatures highlights an important role for post-transcriptional regulation by microRNAs (miRNAs) in governing this critical nexus. Here, we identify miR-130a as a regulator of HSC self-renewal and differentiation. Enforced expression of miR-130a impairs B lymphoid differentiation and expands long-term HSCs. Integration of protein mass spectrometry and chimeric AGO2 crosslinking and immunoprecipitation (CLIP) identifies TBL1XR1 as a primary miR-130a target, whose loss of function phenocopies miR-130a overexpression. Moreover, we report that miR-130a is highly expressed in t(8;21) acute myeloid leukemia (AML), where it is critical for maintaining the oncogenic molecular program mediated by the AML1-ETO complex. Our study establishes that identification of the comprehensive miRNA targetome within primary cells enables discovery of genes and molecular networks underpinning stemness properties of normal and leukemic cells., Competing Interests: Declaration of interests G.W.Y. is a cofounder, a member of the board of directors, on the scientific advisory board, an equity holder, and a paid consultant for Locanabio and Eclipse Bioinnovations. G.W.Y. is a visiting professor at the National University of Singapore. G.W.Y.’s interests have been reviewed and approved by the University of California, San Diego, in accordance with its conflict-of-interest policies. E.L.V.N. is a co-founder, a member of the board of directors, on the SAB, an equity holder, and a paid consultant for Eclipse Bioinnovations. E.L.V.N.’s interests have been reviewed and approved by the University of California, San Diego, in accordance with its conflict of interest policies., (Copyright © 2022. Published by Elsevier Inc.)

It is critical to understand how human quiescent long-term hematopoietic stem cells (LT-HSCs) sense demand from daily and stress-mediated cues and then transition into bioenergetically active progeny to differentiate and meet these cellular needs. However, the demand-adapted regulatory circuits of these early steps of hematopoiesis are largely unknown. Here we show that lysosomes, sophisticated nutrient-sensing and signaling centers, are regulated dichotomously by transcription factor EB (TFEB) and MYC to balance catabolic and anabolic processes required for activating LT-HSCs and guiding their lineage fate. TFEB-mediated induction of the endolysosomal pathway causes membrane receptor degradation, limiting LT-HSC metabolic and mitogenic activation, promoting quiescence and self-renewal, and governing erythroid-myeloid commitment. In contrast, MYC engages biosynthetic processes while repressing lysosomal catabolism, driving LT-HSC activation. Our study identifies TFEB-mediated control of lysosomal activity as a central regulatory hub for proper and coordinated stem cell fate determination., Competing Interests: Declaration of interests J.E.D. received research funding from Celgene and serves on the Advisory Board of Trillium Therapeutics., (Copyright © 2021 The Author(s). Published by Elsevier Inc. All rights reserved.)

Current treatments for acute myeloid leukemia (AML) are often ineffective in eliminating leukemic stem cells (LSCs), which perpetuate the disease. Here, we performed a metabolic drug screen to identify LSC-specific vulnerabilities and found that nicotinamide phosphoribosyltransferase (NAMPT) inhibitors selectively killed LSCs, while sparing normal hematopoietic stem and progenitor cells. Treatment with KPT-9274, a NAMPT inhibitor, suppressed the conversion of saturated fatty acids to monounsaturated fatty acids, a reaction catalyzed by the stearoyl-CoA desaturase (SCD) enzyme, resulting in apoptosis of AML cells. Transcriptomic analysis of LSCs treated with KPT-9274 revealed an upregulation of sterol regulatory-element binding protein (SREBP)-regulated genes, including SCD, which conferred partial protection against NAMPT inhibitors. Inhibition of SREBP signaling with dipyridamole enhanced the cytotoxicity of KPT-9274 on LSCs in vivo. Our work demonstrates that altered lipid homeostasis plays a key role in NAMPT inhibitor-induced apoptosis and identifies NAMPT inhibition as a therapeutic strategy for targeting LSCs in AML., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Lifelong blood production requires long-term hematopoietic stem cells (LT-HSCs), marked by stemness states involving quiescence and self-renewal, to transition into activated short-term HSCs (ST-HSCs) with reduced stemness. As few transcriptional changes underlie this transition, we used single-cell and bulk assay for transposase-accessible chromatin sequencing (ATAC-seq) on human HSCs and hematopoietic stem and progenitor cell (HSPC) subsets to uncover chromatin accessibility signatures, one including LT-HSCs (LT/HSPC signature) and another excluding LT-HSCs (activated HSPC [Act/HSPC] signature). These signatures inversely correlated during early hematopoietic commitment and differentiation. The Act/HSPC signature contains CCCTC-binding factor (CTCF) binding sites mediating 351 chromatin interactions engaged in ST-HSCs, but not LT-HSCs, enclosing multiple stemness pathway genes active in LT-HSCs and repressed in ST-HSCs. CTCF silencing derepressed stemness genes, restraining quiescent LT-HSCs from transitioning to activated ST-HSCs. Hence, 3D chromatin interactions centrally mediated by CTCF endow a gatekeeper function that governs the earliest fate transitions HSCs make by coordinating disparate stemness pathways linked to quiescence and self-renewal., Competing Interests: Declaration of Interests J.E.D. served on the SAB at Trillium Therapeutics, reports receiving a commercial research grant from Celgene, and has ownership interest (including patents) in Trillium Therapeutics., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Age-related clonal hematopoiesis is a major risk factor for myeloid malignancy and myeloid skewing is a hallmark of aging. However, while it is known that non-cell-autonomous components of the microenvironment can also influence this risk, there have been few studies of how the spatial architecture of human bone marrow (BM) changes with aging. Here, we show that BM adiposity increases with age, which correlates with increased density of maturing myeloid cells and CD34+ hematopoietic stem/progenitor cells (HSPCs) and an increased proportion of HSPCs adjacent to adipocytes. However, NGFR+ bone marrow stromal cell (NGFR+ BMSC) density and distance to HSPCs and vessels remained stable. Interestingly, we found that, upon aging, maturing myeloid cell density increases in hematopoietic areas surrounding adipocytes. We propose that increased adjacency to adipocytes in the BM microenvironment may influence myeloid skewing of aging HSPCs, contributing to age-related risk of myeloid malignancies., (Copyright © 2020 The Authors. Published by Elsevier Inc. All rights reserved.)

Cellular stress responses serve as crucial decision points balancing persistence or culling of hematopoietic stem cells (HSCs) for lifelong blood production. Although strong stressors cull HSCs, the linkage between stress programs and self-renewal properties that underlie human HSC maintenance remains unknown, particularly at quiescence exit when HSCs must also dynamically shift metabolic state. Here, we demonstrate distinct wiring of the sphingolipidome across the human hematopoietic hierarchy and find that genetic or pharmacologic modulation of the sphingolipid enzyme DEGS1 regulates lineage differentiation. Inhibition of DEGS1 in hematopoietic stem and progenitor cells during the transition from quiescence to cellular activation with N-(4-hydroxyphenyl) retinamide activates coordinated stress pathways that coalesce on endoplasmic reticulum stress and autophagy programs to maintain immunophenotypic and functional HSCs. Thus, our work identifies a linkage between sphingolipid metabolism, proteostatic quality control systems, and HSC self-renewal and provides therapeutic targets for improving HSC-based cellular therapeutics., (Copyright © 2019 The Authors. Published by Elsevier Inc. All rights reserved.)

Lifelong maintenance of the blood system requires equilibrium between clearance of damaged hematopoietic stem cells (HSCs) and long-term survival of the HSC pool. Severe perturbations of cellular homeostasis result in rapid HSC loss to maintain clonal purity. However, normal homeostatic processes can also generate lower-level stress; how HSCs survive these conditions remains unknown. Here we show that the integrated stress response (ISR) is uniquely active in HSCs and facilitates their persistence. Activating transcription factor 4 (ATF4) mediates the ISR and is highly expressed in HSCs due to scarcity of the eIF2 translation initiation complex. Amino acid deprivation results in eIF2α phosphorylation-dependent upregulation of ATF4, promoting HSC survival. Primitive acute myeloid leukemia (AML) cells also display eIF2 scarcity and ISR activity marks leukemia stem cells (LSCs) in primary AML samples. These findings identify a link between the ISR and stem cell survival in the normal and leukemic contexts., (Copyright © 2018 The Author(s). Published by Elsevier Inc. All rights reserved.)

Hematopoietic stem and progenitor cells (HSPCs) tightly couple maintenance of the bone marrow (BM) reservoir, including undifferentiated long-term repopulating hematopoietic stem cells (LT-HSCs), with intensive daily production of mature leukocytes and blood replenishment. We found two daily peaks of BM HSPC activity that are initiated by onset of light and darkness providing this coupling. Both peaks follow transient elevation of BM norepinephrine and TNF secretion, which temporarily increase HSPC reactive oxygen species (ROS) levels. Light-induced norepinephrine and TNF secretion augments HSPC differentiation and increases vascular permeability to replenish the blood. In contrast, darkness-induced TNF increases melatonin secretion to drive renewal of HSPCs and LT-HSC potential through modulating surface CD150 and c-Kit expression, increasing COX-2/αSMA + macrophages, diminishing vascular permeability, and reducing HSPC ROS levels. These findings reveal that light- and darkness-induced daily bursts of norepinephrine, TNF, and melatonin within the BM are essential for synchronized mature blood cell production and HSPC pool repopulation., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Umbilical cord blood (CB) is a convenient and broadly used source of hematopoietic stem cells (HSCs) for allogeneic stem cell transplantation. However, limiting numbers of HSCs remain a major constraint for its clinical application. Although one feasible option would be to expand HSCs to improve therapeutic outcome, available protocols and the molecular mechanisms governing the self-renewal of HSCs are unclear. Here, we show that ectopic expression of a single microRNA (miRNA), miR-125a, in purified murine and human multipotent progenitors (MPPs) resulted in increased self-renewal and robust long-term multi-lineage repopulation in transplanted recipient mice. Using quantitative proteomics and western blot analysis, we identified a restricted set of miR-125a targets involved in conferring long-term repopulating capacity to MPPs in humans and mice. Our findings offer the innovative potential to use MPPs with enhanced self-renewal activity to augment limited sources of HSCs to improve clinical protocols., (Copyright © 2016 Elsevier Inc. All rights reserved.)

MicroRNA (miRNA)-126 is a known regulator of hematopoietic stem cell quiescence. We engineered murine hematopoiesis to express miRNA-126 across all differentiation stages. Thirty percent of mice developed monoclonal B cell leukemia, which was prevented or regressed when a tetracycline-repressible miRNA-126 cassette was switched off. Regression was accompanied by upregulation of cell-cycle regulators and B cell differentiation genes, and downregulation of oncogenic signaling pathways. Expression of dominant-negative p53 delayed blast clearance upon miRNA-126 switch-off, highlighting the relevance of p53 inhibition in miRNA-126 addiction. Forced miRNA-126 expression in mouse and human progenitors reduced p53 transcriptional activity through regulation of multiple p53-related targets. miRNA-126 is highly expressed in a subset of human B-ALL, and antagonizing miRNA-126 in ALL xenograft models triggered apoptosis and reduced disease burden., (Copyright © 2016 Elsevier Inc. All rights reserved.)

To investigate miRNA function in human acute myeloid leukemia (AML) stem cells (LSC), we generated a prognostic LSC-associated miRNA signature derived from functionally validated subpopulations of AML samples. For one signature miRNA, miR-126, high bioactivity aggregated all in vivo patient sample LSC activity into a single sorted population, tightly coupling miR-126 expression to LSC function. Through functional studies, miR-126 was found to restrain cell cycle progression, prevent differentiation, and increase self-renewal of primary LSC in vivo. Compared with prior results showing miR-126 regulation of normal hematopoietic stem cell (HSC) cycling, these functional stem effects are opposite between LSC and HSC. Combined transcriptome and proteome analysis demonstrates that miR-126 targets the PI3K/AKT/MTOR signaling pathway, preserving LSC quiescence and promoting chemotherapy resistance., (Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.)

Chromosomal rearrangements are a hallmark of acute lymphoblastic leukemia (ALL) and are important ALL initiating events. We describe four different rearrangements of the erythropoietin receptor gene EPOR in Philadelphia chromosome-like (Ph-like) ALL. All of these rearrangements result in truncation of the cytoplasmic tail of EPOR at residues similar to those mutated in primary familial congenital polycythemia, with preservation of the proximal tyrosine essential for receptor activation and loss of distal regulatory residues. This resulted in deregulated EPOR expression, hypersensitivity to erythropoietin stimulation, and heightened JAK-STAT activation. Expression of truncated EPOR in mouse B cell progenitors induced ALL in vivo. Human leukemic cells with EPOR rearrangements were sensitive to JAK-STAT inhibition, suggesting a therapeutic option in high-risk ALL., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Mutations in the histone 3 variant H3.3 have been identified in one-third of pediatric glioblastomas (GBMs), but not in adult tumors. Here we show that H3.3 is a dynamic determinant of functional properties in adult GBM. H3.3 is repressed by mixed lineage leukemia 5 (MLL5) in self-renewing GBM cells. MLL5 is a global epigenetic repressor that orchestrates reorganization of chromatin structure by punctuating chromosomes with foci of compacted chromatin, favoring tumorigenic and self-renewing properties. Conversely, H3.3 antagonizes self-renewal and promotes differentiation. We exploited these epigenetic states to rationally identify two small molecules that effectively curb cancer stem cell properties in a preclinical model. Our work uncovers a role for MLL5 and H3.3 in maintaining self-renewal hierarchies in adult GBM., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Alterations of IKZF1, encoding the lymphoid transcription factor IKAROS, are a hallmark of high-risk acute lymphoblastic leukemia (ALL), however the role of IKZF1 alterations in ALL pathogenesis is poorly understood. Here, we show that in mouse models of BCR-ABL1 leukemia, Ikzf1 and Arf alterations synergistically promote the development of an aggressive lymphoid leukemia. Ikzf1 alterations result in acquisition of stem cell-like features, including self-renewal and increased bone marrow stromal adhesion. Retinoid receptor agonists reversed this phenotype, partly by inducing expression of IKZF1, resulting in abrogation of adhesion and self-renewal, cell cycle arrest, and attenuation of proliferation without direct cytotoxicity. Retinoids potentiated the activity of dasatinib in mouse and human BCR-ABL1 ALL, providing an additional therapeutic option in IKZF1-mutated ALL., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Regulated blood production is achieved through the hierarchical organization of dormant hematopoietic stem cell (HSC) subsets that differ in self-renewal potential and division frequency, with long-term (LT)-HSCs dividing the least. The molecular mechanisms underlying this variability in HSC division kinetics are unknown. We report here that quiescence exit kinetics are differentially regulated within human HSC subsets through the expression level of CDK6. LT-HSCs lack CDK6 protein. Short-term (ST)-HSCs are also quiescent but contain high CDK6 protein levels that permit rapid cell cycle entry upon mitogenic stimulation. Enforced CDK6 expression in LT-HSCs shortens quiescence exit and confers competitive advantage without impacting function. Computational modeling suggests that this independent control of quiescence exit kinetics inherently limits LT-HSC divisions and preserves the HSC pool to ensure lifelong hematopoiesis. Thus, differential expression of CDK6 underlies heterogeneity in stem cell quiescence states that functionally regulates this highly regenerative system., (Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.)

Genetic analyses have shaped much of our understanding of cancer. However, it is becoming increasingly clear that cancer cells display features of normal tissue organization, where cancer stem cells (CSCs) can drive tumor growth. Although often considered as mutually exclusive models to describe tumor heterogeneity, we propose that the genetic and CSC models of cancer can be harmonized by considering the role of genetic diversity and nongenetic influences in contributing to tumor heterogeneity. We offer an approach to integrating CSCs and cancer genetic data that will guide the field in interpreting past observations and designing future studies., (Copyright © 2014 Elsevier Inc. All rights reserved.)

The hematopoietic system sustains regeneration throughout life by balancing self-renewal and differentiation. To stay poised for mature blood production, hematopoietic stem cells (HSCs) maintain low-level expression of lineage-associated genes, a process termed lineage priming. Here, we modulated expression levels of Inhibitor of DNA binding (ID) proteins to ask whether lineage priming affects self-renewal of human HSCs. We found that lentiviral overexpression of ID proteins in cord blood HSCs biases myeloerythroid commitment at the expense of lymphoid differentiation. Conversely, reducing ID2 expression levels increases lymphoid potential. Mechanistically, ID2 inhibits the transcription factor E47 to attenuate B-lymphoid priming in HSCs and progenitors. Strikingly, ID2 overexpression also results in a 10-fold expansion of HSCs in serial limiting dilution assays, indicating that early lymphoid transcription factors antagonize human HSC self-renewal. The relationship between lineage priming and self-renewal can be exploited to increase expansion of transplantable human HSCs and points to broader implications for other stem cell populations., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Lifelong blood cell production is governed through the poorly understood integration of cell-intrinsic and -extrinsic control of hematopoietic stem cell (HSC) quiescence and activation. MicroRNAs (miRNAs) coordinately regulate multiple targets within signaling networks, making them attractive candidate HSC regulators. We report that miR-126, a miRNA expressed in HSC and early progenitors, plays a pivotal role in restraining cell-cycle progression of HSC in vitro and in vivo. miR-126 knockdown by using lentiviral sponges increased HSC proliferation without inducing exhaustion, resulting in expansion of mouse and human long-term repopulating HSC. Conversely, enforced miR-126 expression impaired cell-cycle entry, leading to progressively reduced hematopoietic contribution. In HSC/early progenitors, miR-126 regulates multiple targets within the PI3K/AKT/GSK3β pathway, attenuating signal transduction in response to extrinsic signals. These data establish that miR-126 sets a threshold for HSC activation and thus governs HSC pool size, demonstrating the importance of miRNA in the control of HSC function., (Copyright © 2012 Elsevier Inc. All rights reserved.)

There is increasing evidence that some cancers are hierarchically organized, sustained by a relatively rare population of cancer-initiating cells (C-ICs). Although the capacity to initiate tumors upon serial transplantation is a hallmark of all C-ICs, little is known about the genes that control this process. Here, we establish that ID1 and ID3 function together to govern colon cancer-initiating cell (CC-IC) self-renewal through cell-cycle restriction driven by the cell-cycle inhibitor p21. Regulation of p21 by ID1 and ID3 is a central mechanism preventing the accumulation of excess DNA damage and subsequent functional exhaustion of CC-ICs. Additionally, silencing of ID1 and ID3 increases sensitivity of CC-ICs to the chemotherapeutic agent oxaliplatin, linking tumor initiation function with chemotherapy resistance., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Despite its complexity, blood is probably the best understood developmental system, largely due to seminal experimentation in the mouse. Clinically, hematopoietic stem cell (HSC) transplantation represents the most widely deployed regenerative therapy, but human HSCs have only been characterized relatively recently. The discovery that immune-deficient mice could be engrafted with human cells provided a powerful approach for studying HSCs. We highlight 2 decades of studies focusing on isolation and molecular regulation of human HSCs, therapeutic applications, and early lineage commitment steps, and compare mouse and humanized models to identify both conserved and species-specific mechanisms that will aid future preclinical research., (Copyright © 2012 Elsevier Inc. All rights reserved.)

To identify FDA-approved agents targeting leukemic cells, we performed a chemical screen on two human leukemic cell lines and identified the antimicrobial tigecycline. A genome-wide screen in yeast identified mitochondrial translation inhibition as the mechanism of tigecycline-mediated lethality. Tigecycline selectively killed leukemia stem and progenitor cells compared to their normal counterparts and also showed antileukemic activity in mouse models of human leukemia. ShRNA-mediated knockdown of EF-Tu mitochondrial translation factor in leukemic cells reproduced the antileukemia activity of tigecycline. These effects were derivative of mitochondrial biogenesis that, together with an increased basal oxygen consumption, proved to be enhanced in AML versus normal hematopoietic cells and were also important for their difference in tigecycline sensitivity., (2011 Elsevier Inc. All rights reserved.)

Highly regenerative tissues such as blood must possess effective DNA damage responses (DDR) that balance long-term regeneration with protection from leukemogenesis. Hematopoietic stem cells (HSCs) sustain life-long blood production, yet their response to DNA damage remains largely unexplored. We report that human HSCs exhibit delayed DNA double-strand break rejoining, persistent gammaH2AX foci, and enhanced p53- and ASPP1-dependent apoptosis after gamma-radiation compared to progenitors. p53 inactivation or Bcl-2 overexpression reduced radiation-induced apoptosis and preserved in vivo repopulating HSC function. Despite similar protection from irradiation-induced apoptosis, only Bcl-2-overexpressing HSCs showed higher self-renewal capacity, establishing that intact p53 positively regulates self-renewal independently from apoptosis. The reduced self-renewal of HSCs with inactivated p53 was associated with increased spontaneous gammaH2AX foci in secondary transplants of HSCs. Our data reveal distinct physiological roles of p53 that together ensure optimal HSC function: apoptosis regulation and prevention of gammaH2AX foci accumulation upon HSC self-renewal., (Copyright 2010 Elsevier Inc. All rights reserved.)

Leukemia stem cells (LSCs) initiate and sustain the acute myeloid leukemia (AML) clonal hierarchy and possess biological properties rendering them resistant to conventional chemotherapy. The poor survival of AML patients raises expectations that LSC-targeted therapies might achieve durable remissions. We report that an anti-interleukin-3 (IL-3) receptor alpha chain (CD123)-neutralizing antibody (7G3) targeted AML-LSCs, impairing homing to bone marrow (BM) and activating innate immunity of nonobese diabetic/severe-combined immunodeficient (NOD/SCID) mice. 7G3 treatment profoundly reduced AML-LSC engraftment and improved mouse survival. Mice with pre-established disease showed reduced AML burden in the BM and periphery and impaired secondary transplantation upon treatment, establishing that AML-LSCs were directly targeted. 7G3 inhibited IL-3-mediated intracellular signaling of isolated AML CD34(+)CD38(-) cells in vitro and reduced their survival. These results provide clear validation for therapeutic monoclonal antibody (mAb) targeting of AML-LSCs and for translation of in vivo preclinical research findings toward a clinical application.

The major limitations of Moloney murine leukemia virus (MoMLV)-based vectors for human stem cell applications, particularly those requiring bone marrow (BM) stem cells, include their requirement for mitosis and retroviral receptor expression. New vectors based upon lentiviruses such as HIV-1 exhibit properties that may circumvent these problems. We report that novel third-generation, self-inactivating lentiviral vectors, expressing enhanced green fluorescent protein (EGFP) and pseudotyped with vesicular stomatitis virus G glycoprotein (VSV-G), can efficiently transduce primitive human repopulating cells derived from human BM and cord blood (CB) tested by the SCID-repopulating cell (SRC) assay. Highly purified CD34+ CD38- CB or BM cells were efficiently transduced (4-69%) and stably expressed in EGFP for 40 days in culture following infection for only 24 h without fibronectin, polybrene, or cytokines. Nonobese diabetic/severe combined immune-deficient (NOD/SCID) mice transplanted with transduced cells from either CB or BM donors were well engrafted, demonstrating maintenance of SRC during the infection procedure. Serially obtained femoral BM samples indicated that the proportion of EGFP+ cells within both myeloid and lymphoid lineages was maintained or even increased over time, averaging 42.3 +/- 6.6% for BM donors and 23.3 +/- 7.2% for CB at 12 weeks. Thus, the third-generation lentivectors readily transduce human CB and BM stem cells, under minimal conditions of ex vivo culture, where MoMLV-based vectors are ineffective. Since CB is inappropriate for most therapeutic applications, the efficient maintenance and transduction of BM-derived SRC during the short infection procedure are notable advantages of lentivectors.

We have used the random chromosomal integration sites of retrovirus vectors as unique clonal markers to analyze cell lineage relationships within the hemopoietic stem cell hierarchy. Using a high efficiency protocol for retrovirus-mediated gene transfer, anemic W/Wv mutant mice were reconstituted with bone marrow cells infected with a NEO vector. Analysis of the DNA from bone marrow, thymus, and spleen of these reconstituted W/Wv mice indicated insertion of the vector into primitive pluripotent stem cells capable of producing both myeloid and lymphoid progeny as well as into more committed stem cells apparently restricted to either the myeloid or lymphoid lineages. The neo gene was also expressed in these mice, as they contained a variety of G418 resistant in vitro colony-forming cells. These results demonstrate high-efficiency gene transfer and expression in primitive hemopoietic stem cells and provide a direct approach for analyzing the hemopoietic stem cell hierarchy.