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Summary 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., Highlights • Aging increases adipose, myeloid, and CD34+ HSPC density in the human bone marrow • Human CD34+ HSPC niche is reticular, perivascular, and periadipocytic in aging • Aging increases maturing myeloid cell density surrounding adipocytes, In this article, Dick, Flores-Figueroa, and colleagues show that aging alters the spatial organization between CD34+ hematopoietic stem/progenitor cells and adipocytes within human bone marrow. An increased mature myeloid output correlates with these findings, raising the idea that these age-related changes may influence myeloid skewing and the accompanying risk of myeloid malignancy.

Context: Acute leukemias of ambiguous lineage (ALAL) pose significant diagnostic and therapeutic challenges due to lack of clarity surrounding their cellular origins and driving genomic alterations, as well as poor long-term response to conventional chemotherapy. Most ALAL cases are diagnosed as mixed phenotype acute leukemia (MPAL) or acute undifferentiated leukemia (AUL); however, early T-cell precursor acute lymphoblastic leukemias (ETP-ALL, a subset of T-ALL), often express myeloid markers, suggesting a degree of lineage ambiguity. Biological distinctions between these diseases remain vague, as T/myeloid MPAL and ETP-ALL exhibit similar mutational and immunophenotypic profiles, highlighting the need for an improved understanding of ALAL etiology. Objective: To resolve biological heterogeneity of ALAL, we performed a large-scale genomic analysis, including whole-transcriptome and whole-exome/genome sequencing, of 1,114 primary leukemia samples spanning the stem, myeloid, and T lineages. Results: Transcriptional profiling identified a group of cases (N=61) with a distinct gene expression profile that included one-third of all T/myeloid MPAL and ETP-ALL cases examined, as well as subsets of acute myeloid leukemia (AML) and AUL. Whole-genome sequencing identified structural variants targeting the T-cell transcription factor gene BCL11B in 100% of cases and FLT3 mutations in 80% of cases. Most structural variants resulted in chromosomal translocations that placed the BCL11B gene in proximity to super-enhancers active in hematopoietic stem or early progenitor cells, a cell type where BCL11B is normally repressed. Additionally, 20% of cases harbored high-copy tandem amplification of a 2.5-kb non-coding element downstream of BCL11B that we term BCL11B enhancer tandem amplification or BETA. Analysis of 3D chromatin structure in primary leukemia samples demonstrated ectopic chromatin interactions between BCL11B and rearranged hematopoietic stem/progenitor cell super-enhancers, or between BCL11B and BETA, thereby demonstrating enhancer hijacking or de novo super-enhancer formation as the mechanism of aberrant BCL11B expression in this subtype. In vitro modeling in human CD34+ stem/progenitor cells demonstrated that ectopic BCL11B expression was sufficient to upregulate T-lineage gene expression programs and block myeloid differentiation, which was exacerbated by constitutive FLT3 signaling. Conclusions: BCL11B deregulation in primitive hematopoietic cells is an oncogenic driver of a subset of ALAL that transcends conventional immunophenotypic distinctions.

Current treatments for acute myeloid leukemia (AML) are often ineffective in eliminating leukemic stem cells (LSCs), which perpetuate the disease. Here, we performed a metabolically focused drug screen to identify LSC-specific vulnerabilities and found that inhibitors of nicotinamide phosphoribosyltransferase (NAMPT), a rate-limiting enzyme in the NAD+ salvage pathway, 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 lipotoxicity and apoptosis of AML cells. Transcriptomic analysis of LSCs treated with KPT-9274 revealed an upregulation of genes involved in lipid metabolism, including SCD . This transcriptional response conferred partial protection against NAMPT inhibitors, and its inhibition 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.

Leukemia stem cells (LSC) possess distinct self-renewal and arrested differentiation properties that are responsible for disease emergence, therapy failure and recurrence in acute myeloid leukemia (AML). Pleiotropic cytokines like interleukin (IL)-3 dimerize their receptors to signal multiple functional activities of AML cells, however, the mechanistic link between cytokine receptor dimerization and LSC fate regulation is unknown. Here, we show that the Class I heterodimeric IL3Rα/βc receptors (IL-3R) assemble as hexamers and dodecamers that segregate proliferation versus differentiation signals, respectively. Receptor stoichiometry varies across the individual cells that make up the AML hierarchy, with LSCs presenting with the highest IL3Rα/βc ratio thereby driving resultant hexamer-mediated stemness programs and poor patient survival. Our study establishes a new signaling mechanism of cytokine pleiotropy and the biased instruction of cell fate that directs the AML hierarchy; a signaling mechanism that may be generalizable to other normal and transformed cellular hierarchies.

Summary 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.

Inflammation underlies many chronic diseases of aging, involves dysregulated myeloid cells, and has emerged as a critical process regulating both normal hematopoietic stem cell (HSC) function and myeloid malignancy. However, few of the signaling pathways that underlie these proposed linkages are known. Here, we show that bioactive lipid signaling through sphingosine 1-phosphate receptor 3 (S1PR3) regulates human myeloid lineage determination via inflammatory programs and is dysregulated in acute myeloid leukemia (AML). S1PR3 is myeloid-restricted and not expressed by quiescent HSC, but upon overexpression (OE) promoted myelopoiesis and activated inflammatory signatures. S1PR3 is deficient in primitive AML, but enriched in more mature cases. Importantly, in functionally-validated primary leukemia stem cells (LSC) sorted for high S1PR3 expression or when differentiation was induced through S1PR3OE exhibited reduced xenograft repopulating ability. Treatment of recipient mice engrafted with the S1P prodrug FTY720 reduced patient AML LSC frequency. Thus, S1PR3 marks a subset of less primitive AML cells with a distinct inflammatory signature, and activation of S1PR3 may represent a novel therapeutic approach to disrupt LSC function through induction of differentiation.

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.

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.

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.

Deregulation of self-renewal and differentiation programs are central to the pathogenesis of hematologic malignancies. MicroRNAs (miRNAs) represent a large class of post-transcriptional regulators that mediate repression of multiple target mRNAs and are frequently deregulated in acute myeloid leukemia (AML). To identify miRNA(s) that play a functional role in human hematopoiesis, we performed an in vivo competitive repopulation screen in which candidate miRNAs were over-expressed (OE) in human CD34+CD38- umbilical cord blood (CB) cells and subsequently transplanted into immune-deficient mice for 24 weeks. miR-130a was shown to enhance long-term hematopoietic reconstitution and chosen for further investigation. Enforced expression of miR-130a conferred a competitive repopulation advantage and induced an expansion of HSPC in xenograft assays. Secondary transplantation at limiting dilution revealed a 10-fold increase in HSC frequency, consistent with a role of miR-130a in enhancing HSC self-renewal. Label-free semi-quantitative proteomics in human HSPC combined with GSEA identified chromatin remodeling as the most significant pathway repressed by miR-130a. TBL1XR1, a component of the nuclear receptor corepressor (NCoR) complex that mediates the exchange of corepressors for coactivators, and CBFb, the β subunit of RUNX1, were among the most repressed miR-130a targets. Interestingly, interrogation of AML datasets revealed elevated expression of miR-130a in t(8,21) AML characterized by the fusion of AML1(RUNX1) and ETO genes. High miR-130a expression was correlated with poor patient overall survival. AML1-ETO recruits the aforementioned NCoR complex to repress RUNX1 target genes, thus preventing myeloid differentiation. To test whether miR-130a controls the AML1-ETO molecular program via repression of TBL1XR1, we tested the effect of miR-130a knock-down in Kasumi cell line and patient samples with the t(8,21). Notably, miR-130a KD resulted in differentiation of transduced cells and increased protein levels of TBL1XR1. Immunoprecipitation of AML1-ETO following miR-130a KD revealed loss of NCoR and CBFβ. Thus, we have uncovered a novel mechanism whereby elevated expression of miR-130a in t(8,21) AML endows enhanced self-renewal and perpetuates the aberrant molecular programs driven by AML1-ETO through inhibition of TBL1XR1.

Inherited genetic variants substantially contribute to the risk of myeloproliferative neoplasms (MPNs), a type of blood cancer that arise in the hematopoietic stem cell (HSC) compartment. To identify the germline genetic factors and underlying mechanisms predisposing to MPNs, we conducted a genome-wide association study involving 3,797 MPN cases and 1,152,977 controls and identified 17 genome-wide significant loci. We find an enrichment for risk variants mapping to accessible chromatin in HSCs, an association between MPN risk and multi-lineage blood cell traits, an association with longer leukocyte telomere length, and a significant genetic correlation with predisposition to clonal hematopoiesis of indeterminate potential. Collectively, all of these findings implicate HSC function and self-renewal. In addition to these global studies, we performed in depth mechanistic studies at one locus located ∼12kb downstream of the transcription factor GFI1B. Fine-mapping analyses and reporter assays identified rs524137 as the likely causal variant with the risk allele decreasing hematopoietic reporter activity. Endogenous deletion of the 1.6 kb enhancer region harboring this variant with Cas9 ribonucleoproteins in adult human hematopoietic stem and progenitor cells (HSPCs) resulted in ∼65% editing with a 36% reduction in GFI1B expression. Furthermore, deletion of GFI1B enhancer improved the maintenance of phenotypic HSCs during a 7-day HSPC culture and increased progenitor self-renewal capacity in colony replating assays. Interestingly, deletion of this enhancer only affected HSPC function without disruption of erythropoiesis, an independent effect observed with GFI1B coding perturbation. These results and our ongoing studies demonstrate that the GFI1B MPN risk locus expands HSPCs and reinforces this general mechanism as a risk factor for MPN acquisition.

The mechanisms governing how human hematopoietic stem cells (HSC) balance their capacity for maintaining long-term (LT) regeneration with meeting daily and/or stress demands are unknown. We previously linked INKA1-mediated PAK4 inhibition along with reduction of H4K16 acetylation (H4K16ac) to quiescence in leukemia stem cells. Here, we uncover mutually exclusive immunostaining for INKA1 and H4K16ac in cord blood HSC; supported by BioID and PLA data showing INKA1 interacts with the H4K16 deacetylase SIRT1. The proteomic data also pointed us to CD112, that enabled isolation of LT-HSC into transcriptionally distinct subsets that respond differently to regenerative stress: a CD112low subset (H4K16aclow, CDK6low, ROSlow, INKA1high) exhibited a transient restraint before contributing to hematopoietic reconstitution and a primed CD112high subset (H4K16achigh, CDK6high, ROShigh, INKA1low) that responded rapidly. Protein and scRNAseq pseudotime analysis of in vitro and in vivo (G-CSF-) activated HSC, respectively, confirmed upregulation of H4K16ac, PAK4, CDK6 and CD112 coinciding with cell-cycle priming. INKA1-overexpression (OE) or PAK4 knock-down (KD) induced transient restraint in reconstitution (@4w) with 20w engraftment reaching control levels; HSC frequency increased 4-8 fold as measures in secondary transplants. Thus, INKA1-OE or PAK4-KD enables HSC to resist early activation, thereby protracting their regenerative potential (a phenomenon we term latency). By elevating hematopoietic demand with 5-FU, the INKA1-OE imposed restraint was released engendering even further increased HSC frequency; in controls regenerative capacity was abolished. Collectively, our data uncover a new molecular axis for HSC self-renewal regulation and point to latency as an orchestrated physiological response that integrates quiescence control with HSC fate choices to maintain LT-HSC pool size.

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.

Summary 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., Graphical Abstract, Highlights • Sphingolipid composition is diverse across the human hematopoietic hierarchy • DEGS1 is a sphingolipid enzyme required for hematopoietic stem cell (HSC) function • Modulating DEGS1 function activates autophagy and the unfolded protein response • Variations in sphingolipid homeostasis serve to regulate HSC fate, Lipid metabolism is distinctly regulated in human hematopoietic stem cells (HSCs) versus progenitors. Xie et al. profiled the sphingolipidome in human cord blood and show that modulating sphingolipids during the transition from quiescence to cellular activation in ex vivo culture induced proteostatic cellular stress programs to maintain HSC self-renewal.

Acute leukemia is a rapidly progressing blood cancer with low survival rates. Unfavorable prognosis is attributed to the insufficiently characterized subpopulations of Leukemia Stem Cells (LSCs) that drive chemoresistance and leukemia relapse. Here we utilized a genetic reporter which enables stemness assessment to enrich and functionally characterize LSCs. We revealed heterogeneous activity of the ERG+85 enhancer based fluorescent reporter in human leukemias. Cells with high reporter activity (tagBFPHigh) exhibited elevated expression of stemness and chemo-resistance genes, demonstrated increased clonogenicity and resistance to chemo- and radio- therapy as compared to their tagBFPNeg counterparts. Moreover, tagBFPHigh enriched fraction was capable of regenerating the original cellular heterogeneity and demonstrated increased invasion ability. Most importantly, tagBFPHigh fraction was enriched for leukemia initiating cells in a xenograft assay. We also identified USP9X deubiquitinase enzyme as a novel ERG transcriptional target that sustains ERG+85 positive cells by controlling ERG ubiquitination. Therapeutic targeting of USP9X led to the preferential inhibition of the ERG-dependent leukemias. In summary, we have developed a new strategy to characterize LSCs and propose ERG targeting via USP9X inhibition as a potential anti-leukemia treatment.

Summary 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., Graphical Abstract Highlights ► miR-126 is a novel regulator of the HSC quiescence/proliferation equilibrium ► Reduction in miR-126 induces an expansion of long-term HSC without exhaustion ► Constitutive miR-126 expression promotes HSC quiescence and progenitor proliferation ► miR-126 attenuates PI3K/AKT activation in response to cytokine stimulation, miR-126 regulates multiple targets within the PI3K/AKT/GSK3β pathway to promote HSC quiescence and progenitor proliferation.

SummaryThere 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.

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.

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. Hematopoietic stem cells balance apoptosis with survival to maintain lifelong integrity of the blood system. Van Galen et al. show that specific translation dynamics prime normal and leukemia stem cells to activate the integrated stress response, which can enhance survival in the presence of stressors such as amino acid deprivation.

It has long been appreciated that significant functional and morphologic heterogeneity can exist within the individual cells that comprise a tumor. Increasing evidence indicates that many solid tumors are organized in a hierarchical manner in which tumor growth is driven by a small subset of cancer stem cells (CSCs) or tumor-initiating cells. Although these cells represent a small percentage of the overall tumor population, they are the only cells capable of initiating and driving tumor growth. Emerging evidence indicates that these cells are also resistant to chemotherapy and radiation therapy, which has led to much speculation and interest surrounding the potential clinical applicability of CSCs.

Objective Although increased expression of CD38 on the surface of human CD34 + cells is associated with differentiation, we reported recently that both lineage-negative (Lin − ) CD34 + CD38 − and Lin − CD34 + CD38 lo fractions of cord blood contain primitive severe combined immunodeficient (SCID)–repopulating cells (SRC). Thus, it is important to determine if a hierarchical relationship exists between the SRC from these two populations or if CD38 is reversibly expressed. Materials and Methods To determine if SRC from the CD34 + CD38 − and CD34 + CD38 lo cell fractions could generate SRC of the same and/or alternate CD38 expression, cells from primary nonobese diabetic/SCID mice transplanted with CD34 + CD38 − cells were resorted into both CD34 + CD38 − and CD34 + CD38 lo fractions and injected into separate secondary recipients, which were evaluated for human cell engraftment 7 to 10 weeks later. As primary mice transplanted with CD34 + CD38 lo cells also contained cells of both immunophenotype, these cells were also resorted and transplanted into separate secondary recipients. The cell-cycle status of various CD34 + SRC fractions were evaluated using Hoechst 33342 and Pyronin Y staining in order to determine if CD38 expression was coordinated with divisional activation. Results Each cell fraction obtained from primary recipients was able to reconstitute secondary mice, indicating that CD38 expression reversibly oscillates between negative and low levels on CD34 + repopulating cells. CD38 expression on repopulating cells correlated with a transition between the G 0 and G 1 phases of the cell cycle. Conclusion CD38 is reversibly expressed on CD34 + SRC between negative and low levels and corresponds to a change in the cell-cycle state. These observations establish a foundation to uncover the molecular program of stem cell regulation and underscore the importance of functional assessments when isolating and characterizing human hematopoietic stem cells.

There is a great need in cell biology for the simultaneous detection of many intracellular and extracellular proteins within single cells. Current optical methods based on fluorescence activated flow cytometry are difficult to multiplex. We have developed a novel application of ICP-MS-linked metal-tagged immunophenotyping which has great potential for highly multiplexed proteomic analysis. Expression of intracellular oncogenic kinase BCR/Abl, myeloid cell surface antigen CD33, human stem cell factor receptor c-Kit and integrin receptor VLA-4 were investigated using model human leukemia cell lines. Antigens to which specific antibodies are available and are distinguishably tagged can be determined simultaneously, or multiplexed. Four commercially available tags (Au, Sm, Eu, and Tb) conjugated to secondary antibodies enable a 4-plex assay assuming that the primary antibodies are not cross-reactive. Results obtained by ICP-MS were compared with data from FACS. ICP-MS as an analytical detector possesses several advantages that enhance the performance of immunoassays, which are discussed in detail. Although multiplexing using metal-conjugated reagents is in a very early stage of research and feasibility studies, it is already apparent that more than four antigens could be accurately detected simultaneously using the ICP-MS instrument.

A fundamental problem in cancer research is identification of the cell type capable of initiating and sustaining growth of the tumor – the cancer stem cell (CSC). While the existence of CSCs was first proposed over 40 years ago, only in the past decade have these cells been identified and characterized in hematological malignancies. Recent studies have now described CSCs in solid tumors of the breast and brain, raising the possibility that such cells are at the apex of all neoplastic systems. An appreciation of the biological distinctness of CSCs is crucial not only for the design of studies to understand how tumorigenic pathways operate but also for the development of specific therapies that effectively target these cells in patients.

Fanconi anemia (FA) is a complex recessive genetic disease that causes bone marrow failure in children. The mechanism by which the gene for FA group C (Fancc) impinges on the normal hematopoietic program is unknown. Here we demonstrate that the bone marrow from Fancc-/- mice have reduced ability for primary and secondary long-term reconstitution of myeloablated recipients compared to wild-type or heterozygous mice, indicating that the Fancc gene product is required for the maintenance of normal numbers of hematopoietic stem cells. Long-term and secondary transplant studies suggested that there also were qualitative changes in their developmental potential. Consistent with the reduction in reconstitution, flow cytometric analysis of the primitive subfractions of hematopoietic cells obtained from the bone marrow of Fancc -/- mice demonstrated that they contained 40 to 70% fewer lineage-negative (Lin-)Thy1.2-/lowScal(+) c-Kit(+)CD34+ cells compared to controls. In contrast, the number of Lin Thy1.2-/ lowScal(+)c-Kit CD34(-)cells was comparable to that of wild-type mice. The differential behavior of Lin(-)Thy1.2-/lowScal+c-Kit+CD34+ and Lin(-)Thy1.2-/lowScal(+)c-Kit CD34 subfractions also was observed in mice treated with the DNA cross-linking agent mitomycin C(MMC). Fancc-/- mice treated with MMC had an 92% reduction of CD34 cells as compared to Fancc+/+ mice. The number of CD34 cells only was reduced about 20%. These results suggest that the Fancc gene may act at a stage of primitive hematopoietic cell development identified by CD34 expression.

In the hierarchy of human hematopoietic progenitors, long-term culture-initiating cells (LTC-IC) and extended LTC-IC belong to the earliest cell populations that can be assayed in vitro. We report the identification of a multipotential lymphomyeloid progenitor detected in a nonswitch culture system. We observed the emergence of CD33+ myeloid and CD19+ B-lymphoid cells following plating of lineage-depleted (Lin-) CD34 -enriched or purified CD34+ CD38- cord blood cells on MS-5 stroma in the absence of exogenous cytokines. Both CD19+ CD20- pro-B and CD19+ CD20+ pre-B lymphocytes coexist with myeloid cells in long-term culture. A limiting dilution approach was used to show that a single CD34+ CD38- cell can generate lymphomyeloid progeny in conventional (5-week) and extended (10-week) cultures. Most of the clones in long-term culture or extended long-term culture contained not only lymphoid and myeloid cells, but also myeloid clonogenic progenitors. A high proportion of CD34+ CD38- cells gave rise to lymphomyeloid clones after 5 and 10 weeks of culturing (up to 48% and 16%, respectively), which distinguishes the assay reported here from those using switch culture conditions. We performed retroviral gene transfer experiments involving 1-3 days of exposure of Lin CD34+ -enriched cells to virus encoding enhanced green fluorescent protein. Monitoring of gene transfer efficiency into LTC-IC by enhanced green fluorescent protein fluorescence showed that it is possible to achieve marking of lymphomyeloid LTC-IC, albeit to a lesser extent than myeloid-restricted LTC-IC.

The Ras pathway plays a central role in the control of cellular proliferation and differentiation. In order to examine the role of Ras signaling in hematopoietic regulation, we transduced CD34 + -enriched human cord blood cells with an MSCV-based retroviral vector encoding R12 activated H-Ras. The effects upon proliferation and differentiation were then examined in vitro. Compared to cells transduced with a control vector, H-Ras expressing cells had an elevated frequency of CFU-M (76 ± 16% vs. 22 ± 13%) and increased monocyte/macrophage cell frequency in long-term suspension cultures (69 ± 10% CD14 + vs. 28 ± 8% CD14 + ). H-Ras expression also impaired CFC survival/expansion in suspension culture relative to controls. To determine the effects of decreasing the levels of H-Ras signaling, the farnesyltransferase inhibitor 66177 (Schering-Plough) was used. At low doses, the inhibitor did not significantly reduce the frequency of monocyte/macrophage lineage cells, but led to the appearance of primitive/blastic monocytic colonies and cells with extended proliferative and self-renewal capacities. Upon removal of the inhibitor, these cells rapidly differentiated into large, adherent macrophages. These results suggest that the level of Ras pathway signaling is an important determinant of myeloid cell fate, and may illustrate a manner in which Ras activation in primitive hematopoietic cells contributes to leukemogenesis.

The levels of intracellular ribonucleotide reductase activity, a highly regulated ratelimiting step in DNA synthesis, were investigated during serial subculture of normal human diploid fibroblasts in vitro. This key enzyme activity was found to decline significantly during cellular senescence. This observation along with previous findings of a mutator gene associated with mammalian ribonucleotide reductase suggests a possible mutation mechanism for aging which involves changes in reductase activity during cellular senescence. Furthermore, in keeping with the decrease in enzyme activity, we show that cell resistance to the antitumor agent hydroxyurea, whose site of action is ribonucleotide reductase, decreases progressively with increasing passage numbers. This indicates that an important factor to be considered in drug therapy aimed at the reductase is the increased sensitivity of normal cells to drug with cell age, due to a decline in enzyme activity. Much remains to be determined about age-dependent factors involved in drug therapy; cultured normal human diploid fibroblasts provide a useful system in which to investigate these important parameters.

We are using a genetic approach to study the complex properties of mammalian ribonucleotide reductase activity. Cytotoxic drugs (hydroxyurea, guanzole and N-carbamoyloxyurea) whose intracellular site of action is ribnonucleotide reductase have been used as selective agents in culture to isolate drug resistant cell lines with specific alterations in reductase activity. In general, cell lines resistant to “hydroxyurea-like” drugs exhibit genetic properties satisfying the majority of the criteria for classification as authentic somatic cell mutants. To study the mammalian enzyme properties, in a situation resembling physiological conditions, we have developed a convenient assay system for ribonucleotide reductase activity in whole cells. The procedure is relatively easy to perform, can accurately determine enzyme activity in as few as 10 6 cells grown conveniently on the surface of a tissue culture plate, and unlike cell-free enzyme preparations, activity is linear at low enzyme concentrations. The procedure can be adapted to investigate activity in a relatively small number of intact cells, it is very useful for studying cultures like human diploid fibroblasts, where large quantities of cells containing high enzyme activity may be difficult to obtain. Using this inact cell assay procedure, we have characterized the reductase activity in CHO cells with respect to pyrimidine (CDP) and purine (ADP) substrates, negative and positive effectors, and drug inhibition with the three antitumor agents employed as selective agents for isolating drug resistant cell lines. Some differences in the mode of action of N-carbamoyloxyurea when compared to hydroxyurea or guanazole were noticed and indicate the potential usefulness of the intact cell assay system for examining the action of specific drugs (e.g., 65) on intracellular enzyme activity. Intracellular ribonucleotide reductase activity was examined in the drug resistant cell lines and three different mutant classes were identified. 1. Cell lines containing an apparent structural alteration to the enzyme which results in reductase activity being less sensitive to drug inhibition. 2. Mutants containing elevated levels of reductase activity with a wild type sensitivity to the drug. 3. Cell lines containing a combination of the first two alterations described above; these mutants apparently contain elevated levels of a ribonucleotide reductase enzyme which has a structural alteration rendering it less sensitive to drug inhibition. Furthermore, some recent preliminary work suggests that additional mutant types with alterations in reductase activity can be isolated. The significance of these mutations in providing a better understanding of the regulation of mammalian ribonucleotide reductase was discussed.

Retrovirus vectors are ideal for transferring new genetic material into the undifferentiated stem cells of the hematopoietic (blood-forming) system of mice and humans. This technology provides new ways of studying the molecular events associated with stem cell differentiation, and also offers prospects for the development of gene therapy to treat genetic diseases of the hematopoietic system.

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.

The plasma membrane is intimately involved in a variety of cellular phenomena and may play an important role in the aging of human cells in culture. Significant differences in human diploid fibroblast surface glycoproteins were observed with in vitro aging. Senescent cells bound more concanavalin A (Con A) than young cells and exhibited two distinct classes of binding sites. Cell surfaces of senescent cells incorporated less labelled mannose and more labelled fucose and glucosamine than young cell surfaces. Membranes prepared from older cells were also less effective than membrane preparations from young cells in incorporating mannose from GDP-mannose into a group of oligolipid intermediates, required for the synthesis of glycoproteins with asparagine-linked oligosaccharides. These results demonstrate novel quantitative changes in membrane structure and lectin reactivity in aging human diploid fibroblasts, which must reflect the fundamental physiological modifications in the cell that occur during senescence.

Changes in ribonucleotide reductase occur during the senescence of normal human diploid fibroblasts in culture. Enzyme activity is significantly lower in cells and extracts at high mean population doublings (MPD) as compared to fibroblasts at low MPD. Although many of the kinetic properties of the enzyme remain unaltered in cells at high MPD, changes in the extent and kinetic mechanism of inhibition of CDP and ADP reductase activity of dATP, the overall negative effector of ribonucleotide reductase, were observed. These results and a previous observation that the four deoxyribonucleotide pools are markedly altered during in vitro senescence of human diploid fibroblasts, provide evidence for a link between ribonucleotide reduction, doxyribonucleotide pools, and the establishment of the non-proliferative or senescent state.