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Machine learning and topological analysis methods are becoming increasingly used on various large-scale omics datasets. Modern high dimensional flow cytometry data sets share many features with other omics datasets like genomics and proteomics. For example, genomics or proteomics datasets can be sparse and have high dimensionality, and flow cytometry datasets can also share these features. This makes flow cytometry data potentially a suitable candidate for employing machine learning and topological scoring strategies, for example, to gain novel insights into patterns within the data. We have previously developed the Topological Score (TopS) and implemented it for the analysis of quantitative protein interaction network datasets. Here we show that the TopS approach for large scale data analysis is applicable to the analysis of a previously described flow cytometry sorted human hematopoietic stem cell dataset. We demonstrate that TopS is capable of effectively sorting this dataset into cell populations and identify rare cell populations. We demonstrate the utility of TopS when coupled with multiple approaches including topological data analysis, X-shift clustering, and t-Distributed Stochastic Neighbor Embedding (t-SNE). Our results suggest that TopS could be effectively used to analyze large scale flow cytometry datasets to find rare cell populations.

Transplantation of hematopoietic stem cells (HSCs) from human umbilical cord blood (hUCB) holds great promise for treating a broad spectrum of hematological disorders including cancer. However, the limited number of HSCs in a single hUCB unit restricts its widespread use. Although extensive efforts have led to multiple methods for ex vivo expansion of human HSCs by targeting single molecules or pathways, it remains unknown whether it is possible to simultaneously manipulate the large number of targets essential for stem cell self-renewal. Recent studies indicate that N6-methyladenosine (m6A) modulates the expression of a group of mRNAs critical for stem cell-fate determination by influencing their stability. Among several m6A readers, YTHDF2 is recognized as promoting targeted mRNA decay. However, the physiological functions of YTHDF2 in adult stem cells are unknown. Here we show that following the conditional knockout (KO) of mouse Ythdf2 the numbers of functional HSC were increased without skewing lineage differentiation or leading to hematopoietic malignancies. Furthermore, knockdown (KD) of human YTHDF2 led to more than a 10-fold increase in the ex vivo expansion of hUCB HSCs, a fivefold increase in colony-forming units (CFUs), and more than an eightfold increase in functional hUCB HSCs in the secondary serial of a limiting dilution transplantation assay. Mapping of m6A in RNAs from mouse hematopoietic stem and progenitor cells (HSPCs) as well as from hUCB HSCs revealed its enrichment in mRNAs encoding transcription factors critical for stem cell self-renewal. These m6A-marked mRNAs were recognized by Ythdf2 and underwent decay. In Ythdf2 KO HSPCs and YTHDF2 KD hUCB HSCs, these mRNAs were stabilized, facilitating HSC expansion. Knocking down one of YTHDF2′s key targets, Tal1 mRNA, partially rescued the phenotype. Our study provides the first demonstration of the function of YTHDF2 in adult stem cell maintenance and identifies its important role in regulating HSC ex vivo expansion by regulating the stability of multiple mRNAs critical for HSC self-renewal, thus identifying potential for future clinical applications.

Loss of sensory hair cells leads to deafness and balance deficiencies. In contrast to mammalian hair cells, zebrafish ear and lateral line hair cells regenerate from poorly characterized support cells. Equally ill-defined is the gene regulatory network underlying the progression of support cells to differentiated hair cells. scRNA-Seq of lateral line organs uncovered five different support cell types, including quiescent and activated stem cells. Ordering of support cells along a developmental trajectory identified self-renewing cells and genes required for hair cell differentiation. scRNA-Seq analyses of fgf3 mutants, in which hair cell regeneration is increased, demonstrates that Fgf and Notch signaling inhibit proliferation of support cells in parallel by inhibiting Wnt signaling. Our scRNA-Seq analyses set the foundation for mechanistic studies of sensory organ regeneration and is crucial for identifying factors to trigger hair cell production in mammals. The data is searchable and publicly accessible via a web-based interface.

SUMMARY Tumor-initiating stem cells (TSCs) are critical for drug resistance and immune escape. However, the mutual regulations between TSC and tumor microenvironment (TME) remain unclear. Using DNA-label retaining, single-cell RNA sequencing (scRNA-seq), and other approaches, we investigated intestinal adenoma in response to chemoradiotherapy (CRT), thus identifying therapy-resistant TSCs (TrTSCs). We find bidirectional crosstalk between TSCs and TME using CellPhoneDB analysis. An intriguing finding is that TSCs shape TME into a landscape that favors TSCs for immunosuppression and propagation. Using adenoma-organoid co-cultures, niche-cell depletion, and lineaging tracing, we characterize a functional role of cyclooxygenase-2 (Cox-2)-dependent signaling, predominantly occurring between tumor-associated monocytes and macrophages (TAMMs) and TrTSCs. We show that TAMMs promote TrTSC proliferation through prostaglandin E2 (PGE2)-PTGER4(EP4) signaling, which enhances β-catenin activity via AKT phosphorylation. Thus, our study shows that the bidirectional crosstalk between TrTSC and TME results in a pro-tumorigenic and immunosuppressive contexture., Graphical Abstract, In brief He et al. examined the nature of therapy-resistant tumor-initiating stem cells (TSCs) in murine intestinal adenoma using single-cell RNA sequencing (scRNA-seq) analysis and revealed a bidirectional crosstalk between TSC and its microenvironment, TME, in which TSC shapes TME, especially tumor-associated monocyte and macrophage (TAMM), into an immunosuppressive barrier and pro-tumorigenic niche.

The niche controls stem cell self-renewal and progenitor differentiation for maintaining adult tissue homeostasis in various organisms. However, it remains unclear whether the niche is compartmentalized to control stem cell self-renewal and stepwise progeny differentiation. In the Drosophila ovary, inner germarial sheath (IGS) cells form a niche for controlling germline stem cell (GSC) progeny differentiation. In this study, we have identified four IGS subpopulations, which form linearly arranged niche compartments for controlling GSC maintenance and multi-step progeny differentiation. Single-cell analysis of the adult ovary has identified four IGS subpopulations (IGS1-IGS4), the identities and cellular locations of which have been further confirmed by fluorescent in situ hybridization. IGS1 and IGS2 physically interact with GSCs and mitotic cysts to control GSC maintenance and cyst formation, respectively, whereas IGS3 and IGS4 physically interact with 16-cell cysts to regulate meiosis, oocyte development, and cyst morphological change. Finally, one follicle cell progenitor population has also been transcriptionally defined for facilitating future studies on follicle stem cell regulation. Therefore, this study has structurally revealed that the niche is organized into multiple compartments for orchestrating stepwise adult stem cell development and has also provided useful resources and tools for further functional characterization of the niche in the future.

Transplantation of hematopoietic stem cells (HSCs) from human umbilical cord blood (hUCB) holds great promise for treating a broad spectrum of hematological disorders including cancer. However, the limited number of HSCs in a single hUCB unit restricts its widespread use. Although extensive efforts have led to multiple methods for ex vivo expansion of human HSCs by targeting single molecules or pathways, it remains unknown whether it is possible to simultaneously manipulate the large number of targets essential for stem cell self-renewal. Recent studies indicate that N(6)-methyladenosine (m(6)A) modulates the expression of a group of mRNAs critical for stem cell-fate determination by influencing their stability. Among several m(6)A readers, YTHDF2 is recognized as promoting targeted mRNA decay. However, the physiological functions of YTHDF2 in adult stem cells are unknown. Here we show that following the conditional knockout (KO) of mouse Ythdf2 the numbers of functional HSC were increased without skewing lineage differentiation or leading to hematopoietic malignancies. Furthermore, knockdown (KD) of human YTHDF2 led to more than a 10-fold increase in the ex vivo expansion of hUCB HSCs, a fivefold increase in colony-forming units (CFUs), and more than an eightfold increase in functional hUCB HSCs in the secondary serial of a limiting dilution transplantation assay. Mapping of m(6)A in RNAs from mouse hematopoietic stem and progenitor cells (HSPCs) as well as from hUCB HSCs revealed its enrichment in mRNAs encoding transcription factors critical for stem cell self-renewal. These m(6)A-marked mRNAs were recognized by Ythdf2 and underwent decay. In Ythdf2 KO HSPCs and YTHDF2 KD hUCB HSCs, these mRNAs were stabilized, facilitating HSC expansion. Knocking down one of YTHDF2′s key targets, Tal1 mRNA, partially rescued the phenotype. Our study provides the first demonstration of the function of YTHDF2 in adult stem cell maintenance and identifies its important role in regulating HSC ex vivo expansion by regulating the stability of multiple mRNAs critical for HSC self-renewal, thus identifying potential for future clinical applications.

The mammalian imprinted Dlk1-Gtl2 locus produces multiple non-coding RNAs (ncRNAs) from the maternally inherited allele, including the largest miRNA cluster in the mammalian genome. This locus has characterized functions in some types of stem cell, but its role in hematopoietic stem cells (HSCs) is unknown. Here, we show that the Dlk1-Gtl2 locus plays a critical role in preserving long-term repopulating HSCs (LT-HSCs). Through transcriptome profiling in 17 hematopoietic cell types, we found that ncRNAs expressed from the Dlk1-Gtl2 locus are predominantly enriched in fetal liver HSCs and the adult LT-HSC population and sustain long-term HSC functionality. Mechanistically, the miRNA mega-cluster within the Dlk1-Gtl2 locus suppresses the entire PI3K-mTOR pathway. This regulation in turn inhibits mitochondrial biogenesis and metabolic activity and protects LT-HSCs from excessive reactive oxygen species (ROS) production. Our data therefore show that the imprinted Dlk1-Gtl2 locus preserves LT-HSC function by restricting mitochondrial metabolism.

Although it is established that some general transcription factors are inactivated at mitosis, many details of Mitotic Inhibition of Transcription (MIT) and its underlying mechanisms are largely unknown. We have identified Mitotic Transcriptional Activation (MTA) as a key regulatory step to control transcription in mitosis for genes with transcriptionally engaged RNA Polymerase II (Pol II) to activate and transcribe until the end of the gene to clear Pol II from mitotic chromatin, followed by global impairment of transcription reinitiation through MIT. Global nascent RNA sequencing and RNA fluorescence in situ hybridization demonstrate the existence of transcriptionally engaged Pol II in early mitosis. Both genetic and chemical inhibition of P-TEFb in mitosis lead to delays in the progression of cell division. Together, our study reveals a mechanism for MTA and MIT whereby transcriptionally engaged Pol II can progress into productive elongation and finish transcription to allow proper cellular division.

Transplantation of hematopoietic stem cells (HSCs) from human umbilical cord blood (hUCB) holds great promise for treating a broad spectrum of hematological disorders including cancer. However, the limited number of HSCs in a single hUCB unit restricts its widespread use. Although extensive efforts have led to multiple methods for ex vivo expansion of human HSCs by targeting single molecules or pathways, it remains unknown whether it is possible to simultaneously manipulate the large number of targets essential for stem cell self-renewal. Recent studies indicate that N

Errors in mitosis are a primary cause of chromosome instability (CIN), generating aneuploid progeny cells. Whereas a variety of factors can influence CIN, under most conditions mitotic errors are rare events that have been difficult to measure accurately. Here we report a green fluorescent protein−based quantitative chromosome transmission fidelity (qCTF) assay in budding yeast that allows sensitive and quantitative detection of CIN and can be easily adapted to high-throughput analysis. Using the qCTF assay, we performed genome-wide quantitative profiling of genes that affect CIN in a dosage-dependent manner and identified genes that elevate CIN when either increased (icCIN) or decreased in copy number (dcCIN). Unexpectedly, qCTF screening also revealed genes whose change in copy number quantitatively suppress CIN, suggesting that the basal error rate of the wild-type genome is not minimized, but rather, may have evolved toward an optimal level that balances both stability and low-level karyotype variation for evolutionary adaptation.

International audience; Aneuploid genomes, characterized by unbalanced chromosome stoichiometry (karyotype), are associated with cancer malignancy and drug resistance of pathogenic fungi. The phenotypic diversity resulting from karyotypic diversity endows the cell population with superior adaptability. We show here, using a combination of experimental data and a general stochastic model, that the degree of phenotypic variation, thus evolvability, escalates with the degree of overall growth suppression. Such scaling likely explains the challenge of treating aneuploidy diseases with a single stress-inducing agent. Instead, we propose the design of an ``evolutionary trap'' (ET) targeting both karyotypic diversity and fitness. This strategy entails a selective condition ``channeling'' a karyotypically divergent population into one with a predominant and predictably drugable karyotypic feature. We provide a proof-of-principle case in budding yeast and demonstrate the potential efficacy of this strategy toward aneuploidy-based azole resistance in Candida albicans. By analyzing existing pharmacogenomics data, we propose the potential design of an ET against glioblastoma.

Summary Hox genes modulate the properties of hematopoietic stem cells (HSCs) and reacquired Hox expression in progenitors contributes to leukemogenesis. Here, our transcriptome and DNA methylome analyses revealed that Hoxb cluster and retinoid signaling genes are predominantly enriched in LT-HSCs, and this coordinate regulation of Hoxb expression is mediated by a retinoid-dependent cis-regulatory element, distal element RARE (DERARE). Deletion of the DERARE reduced Hoxb expression, resulting in changes to many downstream signaling pathways (e.g., non-canonical Wnt signaling) and loss of HSC self-renewal and reconstitution capacity. DNA methyltransferases mediate DNA methylation on the DERARE, leading to reduced Hoxb cluster expression. Acute myeloid leukemia patients with DNMT3A mutations exhibit DERARE hypomethylation, elevated HOXB expression, and adverse outcomes. CRISPR-Cas9-mediated specific DNA methylation at DERARE attenuated HOXB expression and alleviated leukemogenesis. Collectively, these findings demonstrate pivotal roles for retinoid signaling and the DERARE in maintaining HSCs and preventing leukemogenesis by coordinate regulation of Hoxb genes.

Herpes simplex virus type 1 (HSV-1) is a significant human pathogen that infects a large portion of the human population. Cells deploy a variety of defenses to limit the extent to which the virus can replicate. One such factor is the promyelocytic leukemia (PML) protein, the nucleating and organizing factor of nuclear domain 10 (ND10). PML responds to a number of stimuli and is implicated in intrinsic and innate cellular antiviral defenses against HSV-1. While the role of PML in a number of cellular pathways is controlled by post-translational modifications, the effects of phosphorylation on its antiviral activity toward HSV-1 have been largely unexplored. Consequently, we mapped phosphorylation sites on PML, mutated these and other known phosphorylation sites on PML isoform I (PML-I), and examined their effects on a number of PML’s activities. Our results show that phosphorylation at most sites on PML-I is dispensable for the formation of ND10s and colocalization between PML-I and the HSV-1 regulatory protein, ICP0, which antagonizes PML-I function. However, inhibiting phosphorylation at sites near the SUMO-interaction motif (SIM) of PML-I impairs its ability to respond to HSV-1 infection. Overall, our data suggest that PML phosphorylation regulates its antiviral activity against HSV-1.

Piwi-interacting RNAs (piRNAs) are important for repressing transposable elements (TEs) and modulating gene expression in germ cells, thereby maintaining genome stability and germ cell function. Although they are also important for maintaining germline stem cells (GSCs) in theDrosophilaovary by repressing TEs and preventing DNA damage, piRNA expression has not been investigated in GSCs or their early progeny. Here, we show that the canonical piRNA clusters are more active in GSCs and their early progeny than late germ cells and also identify more than 3,000 new piRNA clusters from deep sequencing data. The increase in piRNAs in GSCs and early progeny can be attributed to both canonical and newly identified piRNA clusters. As expected, piRNA clusters in GSCs, but not those in somatic support cells (SCs), exhibit ping-pong signatures. Surprisingly, GSCs and early progeny express more TE transcripts than late germ cells, suggesting that the increase in piRNA levels may be related to the higher levels of TE transcripts in GSCs and early progeny. GSCs also have higher piRNA levels and lower TE levels than SCs. Furthermore, the 3′ UTRs of 171 mRNA transcripts may produce sense, antisense, or dual-stranded piRNAs. Finally, we show that alternative promoter usage and splicing are frequently used to modulate gene function in GSCs and SCs. Overall, this study has provided important insight into piRNA production and TE repression in GSCs and SCs. The rich information provided by this study will be a beneficial resource to the fields of piRNA biology and germ cell development.

The epigenetic regulation of imprinted genes by monoallelic DNA methylation of either maternal or paternal alleles is critical for embryonic growth and development. Imprinted genes were recently shown to be expressed in mammalian adult stem cells to support self-renewal of neural and lung stem cells; however, a role for imprinting per se in adult stem cells remains elusive. Here we show upregulation of growth-restricting imprinted genes, including in the H19-Igf2 locus, in long-term haematopoietic stem cells and their downregulation upon haematopoietic stem cell activation and proliferation. A differentially methylated region upstream of H19 (H19-DMR), serving as the imprinting control region, determines the reciprocal expression of H19 from the maternal allele and Igf2 from the paternal allele. In addition, H19 serves as a source of miR-675, which restricts Igf1r expression. We demonstrate that conditional deletion of the maternal but not the paternal H19-DMR reduces adult haematopoietic stem cell quiescence, a state required for long-term maintenance of haematopoietic stem cells, and compromises haematopoietic stem cell function. Maternal-specific H19-DMR deletion results in activation of the Igf2-Igfr1 pathway, as shown by the translocation of phosphorylated FoxO3 (an inactive form) from nucleus to cytoplasm and the release of FoxO3-mediated cell cycle arrest, thus leading to increased activation, proliferation and eventual exhaustion of haematopoietic stem cells. Mechanistically, maternal-specific H19-DMR deletion leads to Igf2 upregulation and increased translation of Igf1r, which is normally suppressed by H19-derived miR-675. Similarly, genetic inactivation of Igf1r partly rescues the H19-DMR deletion phenotype. Our work establishes a new role for this unique form of epigenetic control at the H19-Igf2 locus in maintaining adult stem cells.

Previous studies have shown that fibroblast growth factor (FGF) signaling promotes hematopoietic stem and progenitor cell (HSPC) expansion in vitro. However, it is unknown whether FGF promotes HSPC expansion in vivo. Here we examined FGF receptor 1 (FGFR1) expression and investigated its in vivo function in HSPCs. Conditional knockout (CKO) of Fgfr1 did not affect phenotypical number of HSPCs and homeostatic hematopoiesis, but led to a reduced engraftment only in the secondary transplantation. When treated with 5-fluorouracil (5FU), the Fgfr1 CKO mice showed defects in both proliferation and subsequent mobilization of HSPCs. We identified megakaryocytes (Mks) as a major resource for FGF production, and further discovered a novel mechanism by which Mks underwent FGF-FGFR signaling dependent expansion to accelerate rapid FGF production under stress. Within HSPCs, we observed an up-regulation of nuclear factor κB and CXCR4, a receptor for the chemoattractant SDF-1, in response to bone marrow damage only in control but not in Fgfr1 CKO model, accounting for the corresponding defects in proliferation and migration of HSPCs. This study provides the first in vivo evidence that FGF signaling facilitates postinjury recovery of the mouse hematopoietic system by promoting proliferation and facilitating mobilization of HSPCs.

Summary Wnt signaling is involved in self-renewal and maintenance of hematopoietic stem cells (HSCs); however, the particular role of noncanonical Wnt signaling in regulating HSCs in vivo is largely unknown. Here, we show Flamingo (Fmi) and Frizzled (Fz) 8, members of noncanonical Wnt signaling, both express in and functionally maintain quiescent long-term HSCs. Fmi regulates Fz8 distribution at the interface between HSCs and N-cadherin + osteoblasts (N-cad + OBs that enrich osteoprogenitors) in the niche. We further found that N-cad + OBs predominantly express noncanonical Wnt ligands and inhibitors of canonical Wnt signaling under homeostasis. Under stress, noncanonical Wnt signaling is attenuated and canonical Wnt signaling is enhanced in activation of HSCs. Mechanistically, noncanonical Wnt signaling mediated by Fz8 suppresses the Ca 2+ -NFAT- IFNγ pathway, directly or indirectly through the CDC42-CK1α complex and also antagonizes canonical Wnt signaling in HSCs. Taken together, our findings demonstrate that noncanonical Wnt signaling maintains quiescent long-term HSCs through Fmi and Fz8 interaction in the niche.

Jarid2 was recently identified as an important component of the mammalian Polycomb repressive complex 2 (PRC2), where it has a major effect on PRC2 recruitment in mouse embryonic stem cells. Although Jarid2 is conserved in Drosophila, it has not previously been implicated in Polycomb (Pc) regulation. Therefore, we purified Drosophila Jarid2 and its associated proteins and found that Jarid2 associates with all of the known canonical PRC2 components, demonstrating a conserved physical interaction with PRC2 in flies and mammals. Furthermore, in vivo studies with Jarid2 mutants in flies demonstrate that among several histone modifications tested, only methylation of histone 3 at K27 (H3K27), the mark implemented by PRC2, was affected. Genome-wide profiling of Jarid2, Su(z)12 (Suppressor of zeste 12), and H3K27me3 occupancy by chromatin immunoprecipitation with sequencing (ChIP-seq) indicates that Jarid2 and Su(z)12 have very similar distribution patterns on chromatin. However, Jarid2 and Su(z)12 occupancy levels at some genes are significantly different, with Jarid2 being present at relatively low levels at many Pc response elements (PREs) of certain Homeobox (Hox) genes, providing a rationale for why Jarid2 was never identified in Pc screens. Gene expression analyses show that Jarid2 and E(z) (Enhancer of zeste, a canonical PRC2 component) are not only required for transcriptional repression but might also function in active transcription. Identification of Jarid2 as a conserved PRC2 interactor in flies provides an opportunity to begin to probe some of its novel functions in Drosophila development.

In the initial published version of this article, there was an inadvertent omission from the Acknowledgements that this work was supported by Stowers Institute for Medical Research (SIMR-1004) and NIH National Cancer Institute grant to University of Kansas Cancer Center (P30 CA168524). This omission does not affect the description of the results or the conclusions of this work.

Within the bone marrow, three hematopoietic stem cell niches have been identified; the osteoblastic niche (Arai et al., 2004; Calvi et al., 2003; Nilsson et al., 2001; Zhang et al., 2003), the vascular niche (Kiel et al., 2005), and the CAR cell niche (Sugiyama et al., 2006). The adhesion molecule N-cadherin has been found associated with the osteoblastic and CAR cell niches, implicating N-cadherin’s function for hematopoietic stem cell (HSC) anchoring and signaling (Arai et al., 2004; Muguruma et al., 2006; Zhang et al., 2003). However, as of yet, a HSC population expressing N-cadherin has not been fully characterized. Therefore, we examined how N-cadherin expression in HSCs relates to their function and lifecycle. Unexpectedly, we found that doses of 5000 bone marrow cells expressing the highest level of N-cadherin (N-cadherinhi) failed to reconstitute hematopoietic lineages in irradiated recipient mice. An explanation for this engraftment failure came with detailed cell surface phenotyping which revealed that these N-cadherinhi cells were primarily Lineage+ and devoid of the characteristic hematopoietic stem pool, Lineage-Sca+cKit+ (LSK) cells. Instead, we found that Flk2-LSK HSCs express a gradient of N-cadherin which could be described as low (N-cadherinlo) to intermediate (N-cadherinint) levels (Figure1). FACS applications were used to isolate pure populations of these N-cadherinlo and N-cadherinint Flk2-LSK HSCs. Real time RT-PCR (N-cadherin primers crossed the intron between Exon 2 and 3). (Figure 2), microarray studies, and competitive reconstitution transplantation assays revealed that this N-cadherin division of Flk2- LSK HSCs formed two populations with very distinct properties. In transplantation assays the N-cadherinlo population more robustly reconstituted the hematopoietic system. Principle Component Analysis and gene ontology analysis of microarray data revealed that the N-cadherinlo cells expressed genes that may prime them to respond to signals and to mobilize. This data was confirmed with mobilization studies which showed that HSCs mobilized from bone marrow to spleen were predominantly N-cadherinlo. In contrast, the expression profile of N-cadherinint cells suggests they are more ‘reserved’ and this population was also maintained with HSCs spared by 5-fluouracil (5FU) treatment. Our results suggest that differential N-cadherin expression reflects important functional distinctions between HSC subpopulations. N-cadherinlo HSCs, with their robust reconstitution efficiency and properties similar to mobilized HSCs, may have clinical relevance. Figure Figure Figure Figure

Adult stem cells continuously undergo self-renewal and generate differentiated cells. In the Drosophila ovary, two separate niches control germ line stem cell (GSC) self-renewal and differentiation processes. Compared to the self-renewing niche, relatively little is known about the maintenance and function of the differentiation niche. In this study, we show that the cellular redox state regulated by Wnt signaling is critical for the maintenance and function of the differentiation niche to promote GSC progeny differentiation. Defective Wnt signaling causes the loss of the differentiation niche and the upregulated BMP signaling in differentiated GSC progeny, thereby disrupting germ cell differentiation. Mechanistically, Wnt signaling controls the expression of multiple glutathione-S-transferase family genes and the cellular redox state. Finally, Wnt2 and Wnt4 function redundantly to maintain active Wnt signaling in the differentiation niche. Therefore, this study has revealed a novel strategy for Wnt signaling in regulating the cellular redox state and maintaining the differentiation niche. DOI: http://dx.doi.org/10.7554/eLife.08174.001, eLife digest An animal or plant has many different types of cells that have specific roles in the life of the organism. These cells are organized into tissues. In most tissues in adult animals, small groups of cells called stem cells are responsible for replacing the other cells that have been lost due to disease, injury, or as part of normal body maintenance. The ‘germ line’ stem cells of female fruit flies—which produce female sex cells (or eggs)—are an effective system for studying how stem cells are regulated. These cells live in an area of the ovary called a stem cell niche. Each time a stem cell divides, it produces one stem cell and one other daughter cell. This daughter cell then moves into another niche called the ‘differentiation’ niche and undergoes a series of divisions that produce the egg cells. The differentiation niche is formed by escort cells and is crucial for producing the egg cells, but it is not clear how the escort cells promote this process, or how the niche is maintained. Wang et al. have now studied the differentiation niche in more detail. The experiments show that a cell communication system called Wnt signaling maintains the differentiation niche by controlling the ability of the escort cells to grow and divide. If Wnt signaling is defective, the differentiation niche is lost, which disrupts the formation of egg cells. Further experiments show that two proteins called Wnt2 and Wnt4 in the differentiation niche—which activate Wnt signaling—act as signals to regulate the niche, mainly by controlling the expression of four particular genes. These four genes encode enzymes that remove ‘reactive oxygen species’ from cells. Wang et al.'s findings have revealed an important role for Wnt signaling in maintaining the differentiation niche. The next step is to figure out the details of how this works. DOI: http://dx.doi.org/10.7554/eLife.08174.002

Haematopoietic stem cells (HSCs) are a subset of bone marrow cells that are capable of self-renewal and of forming all types of blood cells (multi-potential). However, the HSC 'niche'--the in vivo regulatory microenvironment where HSCs reside--and the mechanisms involved in controlling the number of adult HSCs remain largely unknown. The bone morphogenetic protein (BMP) signal has an essential role in inducing haematopoietic tissue during embryogenesis. We investigated the roles of the BMP signalling pathway in regulating adult HSC development in vivo by analysing mutant mice with conditional inactivation of BMP receptor type IA (BMPRIA). Here we show that an increase in the number of spindle-shaped N-cadherin+CD45- osteoblastic (SNO) cells correlates with an increase in the number of HSCs. The long-term HSCs are found attached to SNO cells. Two adherens junction molecules, N-cadherin and beta-catenin, are asymmetrically localized between the SNO cells and the long-term HSCs. We conclude that SNO cells lining the bone surface function as a key component of the niche to support HSCs, and that BMP signalling through BMPRIA controls the number of HSCs by regulating niche size.

Clinical trials evaluating the herpes simplex virus thymidine kinase (HSV-tk)/ganciclovir (GCV) suicide gene therapy system for the control of graft-versus-host disease (GVHD) have been limited by low transduction efficiencies and inefficient selection procedures. In this study, we designed and evaluated a novel chimeric suicide gene consisting of the extracellular and transmembrane domains of human CD34 and full-length HSV-tk (DeltaCD34-tk). High-efficiency transfer of DeltaCD34-tk to primary human T cells was accomplished after a single exposure to VSV-G-pseudotyped, Moloney murine leukemia virus-based retrovirus 48 h after activation of human PBMCs with anti-CD3 and anti-CD28 antibodies immobilized on magnetic beads. Using an optimized 5-day transduction and selection procedure, transduction efficiencies averaged 71%, with isolation purities greater than 95% and yields exceeding 90%. The immunoselected T cells were selectively eliminated by GCV (IC(50) approximately 3 nM), maintained a normal subset composition, exhibited a polyclonal TCR Vbeta family repertoire, and contained 5 or 6 vector copies per transduced cell when optimally transduced. No increase in GCV sensitivity was observed upon incorporation of highly active mutant HSV-tk enzymes into the DeltaCD34-tk suicide gene. T cells modified with the DeltaCD34-tk gene using the optimized protocol should improve the overall efficacy of the HSV-tk/GCV suicide gene therapy method of GVHD control.

Hematopoietic stem cells (HSCs) maintain hematopoiesis by giving rise to all types of blood cells. Recent reports suggest that HSCs also possess the potential to generate nonhematopoietic tissues. To evaluate the underlying mechanisms in the commitment of HSCs into multitissue and multihematopoietic lineages, we performed oligonucleotide array analyses targeting for prospectively purified HSCs, multipotent progenitors (MPPs), common lymphoid progenitors (CLPs), and common myeloid progenitors (CMPs). Here we show that HSCs coexpress multiple nonhematopoietic genes as well as hematopoietic genes; MPPs coexpress myeloid and lymphoid genes; CMPs coexpress myeloerythroid, but not lymphoid genes, whereas CLPs coexpress T-, B-, and natural killer–lymphoid, but not myeloid, genes. Thus, the stepwise decrease in transcriptional accessibility for multilineage-affiliated genes may represent progressive restriction of developmental potentials in early hematopoiesis. These data support the hypothesis that stem cells possess a wide-open chromatin structure to maintain their multipotentiality, which is progressively quenched as they go down a particular pathway of differentiation.

In unstimulated conditions, osteoclast (OC) formation is regulated by stromal cell production of the key osteoclastogenic factors receptor activator of nuclear factor kappaB ligand (RANKL) and macrophage colony-stimulating factor (M-CSF). However, the mechanisms of accelerated osteoclastogenesis and bone loss characteristic of inflammatory conditions are poorly understood but appear to involve T cells. In addition, the mechanism by which OCs arise spontaneously in cultures of peripheral blood mononuclear cells in the absence of stromal cells or added cytokines remains unclear. Using a stromal cell free human osteoclast generating system, we investigated the ability of activated T cells to support osteoclastogenesis. We show that when activated by phytohemagglutinin-P (PHA), T cells (both CD4+ and CD8+) stimulate human OC formation in vitro. Although both soluble M-CSF and RANKL were detected in activated T cell supernatants, the presence of M-CSF was not essential for macrophage survival or RANKL-dependent osteoclast formation, suggesting that other soluble T cell-derived factors were capable of substituting for this cytokine. We also found that saturating concentrations of osteoprotegerin (OPG) failed to neutralize 30% of the observed OC formation and that T cell conditioned medium (CM) could superinduce osteoclastogenesis in cultures of purified monocytes maximally stimulated by RANKL and M-CSF. Together, these data suggest that activated T cells support osteoclastogenesis via RANKL-dependent and -independent mechanisms. Although not relevant for T cell-induced osteoclastogenesis, secretion of soluble M-CSF is a previously undescribed property of activated T cells.

The role of B lymphocytes in osteoclast (OC) formation is controversial, because both stimulatory and inhibitory effects of B-lineage cells on osteoclastogenesis and life span have been reported. In this study, we have investigated the effects of mature B cells on human osteoclastogenesis using cultures of peripheral blood stem cells (PBSC), a system that generates functional OCs in the absence of stromal cells. We report that B cells inhibit the formation of OCs and shorten the life span of mature OCs by secreting transforming growth factor β (TGFβ), a factor that induces apoptosis in these cells. The antiosteoclastogenic effects of B cells are abolished by addition of anti-TGFβ antibody to osteoclast cultures and mimicked by treatment of B cell-deprived PBSC cultures with recombinant TGFβ, thus confirming TGFβ as the B cell produced antiosteoclastogenic activity. Thus, the ability of B cells to downregulate osteoclastogenesis by secretion of the apoptotic cytokine TGFβ provides new insights into the ability of immune cells to regulate OC formation under basal and inflammatory conditions. J. Cell. Biochem. 78:318–324, 2000. © 2000 Wiley-Liss, Inc.

Investigation of the molecular basis of megakaryocyte (MK) and platelet biology has been limited by an inadequate source of genetically manipulable cells exhibiting physiologic MK and platelet functions. We hypothesized that ex vivo cultured MKs would exhibit agonist inducible glycoprotein (GP) IIb-IIIa activation characteristic of blood platelets and that these cultured MKs would be capable of transgene expression. Microscopic and flow cytometric analyses confirmed that human hematopoietic stem cells cultured in the presence of pegylated recombinant human MK growth and development factor (PEG-rHuMGDF) differentiated into morphologic and phenotypic MKs over 2 weeks. Cultured MKs expressed functional GPIIb-IIIa receptors as assessed by agonist inducible soluble fibrinogen and PAC1 binding. The specificity and kinetics of fibrinogen binding to MK GPIIb-IIIa receptors were similar to those described for blood platelets. The reversibility and internalization of ligands bound to MK GPIIb-IIIa also shared similarities with those observed in platelets. Cultured MKs were transduced with an adenoviral vector encoding green fluorescence protein (GFP) or β-galactosidase (β-gal). Efficiency of gene transfer increased with increasing multiplicities of infection and incubation time, with 45% of MKs expressing GFP 72 hours after viral infection. Transduced MKs remained capable of agonist induced GPIIb-IIIa activation. Thus, ex vivo cultured MKs (1) express agonist responsive GPIIb-IIIa receptors, (2) are capable of expressing transgenes, and (3) may prove useful for investigation of the molecular basis of MK differentiation and GPIIb-IIIa function.

Deafness caused by the terminal loss of inner ear hair cells is one of the most common sensory diseases. However, nonmammalian animals (e.g., birds, amphibians, and fish) regenerate damaged hair cells. To understand better the reasons underpinning such disparities in regeneration among vertebrates, we set out to define at high resolution the changes in gene expression associated with the regeneration of hair cells in the zebrafish lateral line. We performed RNA-Seq analyses on regenerating support cells purified by FACS. The resulting expression data were subjected to pathway enrichment analyses, and the differentially expressed genes were validated in vivo via whole-mount in situ hybridizations. We discovered that cell cycle regulators are expressed hours before the activation of Wnt/β-catenin signaling following hair cell death. We propose that Wnt/β-catenin signaling is not involved in regulating the onset of proliferation but governs proliferation at later stages of regeneration. In addition, and in marked contrast to mammals, our data clearly indicate that the Notch pathway is significantly down-regulated shortly after injury, thus uncovering a key difference between the zebrafish and mammalian responses to hair cell injury. Taken together, our findings lay the foundation for identifying differences in signaling pathway regulation that could be exploited as potential therapeutic targets to promote either sensory epithelium or hair cell regeneration in mammals.

Osteoclastogenesis is a complex process that is facilitated by bone marrow stromal cells (SCs). To determine if SCs are an absolute requirement for the differentiation of human hematopoietic precursors into fully mature, osteoclasts (OCs), CD34+ cells were mobilized into the peripheral circulation with granulocyte colony-stimulating factor, harvested by leukapheresis, and purified by magnetic-activated cell sorting. This procedure yields a population of CD34+ cells that does not contain SC precursors, as assessed by the lack of expression of the SC antigen Stro-1, and that differentiates only into hematopoietic cells. We found that CD34+, Stro-1- cells cultured with a combination of granulocyte/macrophage colony-stimulating factor, interleukin 1, and interleukin 3 generated cells that fulfill current criteria for the characterization of OCs, including multinucleation, presence of tartrate-resistant acid phosphatase, and expression of the calcitonin and vitronectin receptors and of pp60c-src tyrosine kinase. These OCs also expressed mRNA for the noninserted isoform of the calcitonin receptor and excavated characteristic resorption pits in devitalized bone slices. These data demonstrate that accessory SCs are not essential for human osteoclastogenesis and that granulocyte colony-stimulating factor treatment mobilizes OC precursors into the peripheral circulation.

Chromosomal translocations of the mixed-lineage leukemia (MLL) gene with various partner genes result in aggressive leukemia with dismal outcomes. Despite similar expression at the mRNA level from the wild-type and chimeric MLL alleles, the chimeric protein is more stable. We report that UBE2O functions in regulating the stability of wild-type MLL in response to interleukin-1 signaling. Targeting wild-type MLL degradation impedes MLL leukemia cell proliferation, and it downregulates a specific group of target genes of the MLL chimeras and their oncogenic cofactor, the super elongation complex. Pharmacologically inhibiting this pathway substantially delays progression, and it improves survival of murine leukemia through stabilizing wild-type MLL protein, which displaces the MLL chimera from some of its target genes and, therefore, relieves the cellular oncogenic addiction to MLL chimeras. Stabilization of MLL provides us with a paradigm in the development of therapies for aggressive MLL leukemia and perhaps for other cancers caused by translocations.

Mechanism of the inhibitory effect of isoprenoid farnesol on cell proliferation has been studied in human acute leukemia CEM-C1 cells. Farnesol (20 microM) reduced the rate of radioactive label incorporation into cellular diacylglycerol (DAG) and phosphocholine, the products of degradation of phosphatidylcholine (PC), indicating inhibition of PC-specific phospholipase C after about 1 h of incubation. Inhibition of phospholipase D by farnesol at the later incubation time (about 2 h) was demonstrated by a decrease in synthesis of PC-derived phosphatidylethanol in the presence of ethanol. These effects of farnesol on PC degradation and formation of DAG were followed by apoptotic fragmentation of cellular DNA and inhibition of cell growth. Exogenous DAG reduced the level of DNA fragmentation and cell growth inhibition. Results are consistent with the involvement of cellular signal transduction in the mechanism of inhibition of cell proliferation by farnesol.

Previously reported observations have shown that trans-trans farnesol inhibits incorporation of choline into phosphatidylcholine and reduces the growth rate of the human acute leukemia CEM-C1 cell line (Melnykovych, G., Haug, J.S. and Goldner, C.M. (1992) Biochem. Biophys. Res. Commun. 186, 543-548). These findings have now been followed up in order to establish a relationship between the inhibition of phosphatidylcholine synthesis and the ensuing cell shrinkage and cell death which takes place at higher concentrations of farnesol or upon long incubation. The present results show that after incubation in the presence of farnesol the cells decrease in viability. Their nuclear DNA becomes fragmented at internucleosomal linker regions, showing characteristic pattern of bands at 180 to 200 base-pair intervals. This farnesol-induced effect was also demonstrated by flow cytometry by staining the cellular DNA with propidium iodide and was partially reversible with phosphatidylcholine.

THE setup and successful operation of a core facility requires specialized accounting practices that, to our knowledge, have not been described recently within the cytometry community. While yearly budgeted expenses such as employee’s salaries, supplies, and instrument service contracts are easily estimated, it is more difficult to calculate overhead costs and equitably amortize total yearly expenses through core facility charges. At the most basic level, a facility must maintain accurate cost information so that fees for services can be set at levels to recuperate known portions of operating expenses. The charges recovered by the facility should be calculated in a way that complies with applicable grant funding standards such as Federal cost principles (OMB Circular A-21 and A-122) for US federal funding or higher education institutions’ (HEI) transparent approaches to costing (TRAC) in the UK. However, more advanced methods also provide resource usage and resource availability data, which can provide a context in which cost data can be more fully understood (i.e., the manner in which a resource is used depends on an empirical understanding of its availability). Furthermore, the combination of cost and resource capacity data allow new opportunities for strategic planning so that a facility can grow and change over time. Recently, we evaluated an activity-based accounting method called time-driven activity-based costing (TD-ABC) (1,2). This method is an alternative to conventional activitybased accounting, and it is more simplified so that a typical non-accountant laboratory manager can collect data and calculate a useful model within just a few days. Moreover, TDABC provides accurate resource usage data so that strategic decisions can be made to maximize resource use. TD-ABC deviates from the conventional method in two significant ways. First, activities are measured by time intervals rather than proportions of work intervals. This greatly simplifies the data collection process since it is fundamentally easier to determine how much time is required to perform a particular activity than to survey employees on what fraction of their time is spent performing various activities. Second, the resources that support these activities are assigned practical capacity limits (hours). Therefore, as activities are performed, used and unused capacity hours of resources can be assessed. The primary goals of implementing a TD-ABC analysis in our facility were to: Devise a method that estimates the true cost of flow cytometry experiments regardless of the instrument or combination of facility resources used Determine where significant discrepancies in cost recovery occur Model new fee schedules with the goal of more accurately reflecting resource usage in fees while maintaining the ability to motivate users to make efficient use of facility resources (e.g., unassisted usage during off-peak hours)

Epigenetic regulation plays critical roles in the regulation of cell proliferation, fate determination, and survival. It has been shown to control self-renewal and lineage differentiation of embryonic stem cells. However, epigenetic regulation of adult stem cell function remains poorly defined. Drosophila ovarian germline stem cells (GSCs) are a productive adult stem cell system for revealing regulatory mechanisms controlling self-renewal and differentiation. In this study, we show that Eggless (Egg), a H3K9 methyltransferase in Drosophila, is required in GSCs for controlling self-renewal and in escort cells for regulating germ cell differentiation. egg mutant ovaries primarily exhibit germ cell differentiation defects in young females and gradually lose GSCs with time, indicating that Egg regulates both germ cell maintenance and differentiation. Marked mutant egg GSCs lack expression of trimethylated H3K9 (H3k9me3) and are rapidly lost from the niche, but their mutant progeny can still differentiate into 16-cell cysts, indicating that Egg is required intrinsically to control GSC self-renewal but not differentiation. Interestingly, BMP-mediated transcriptional repression of differentiation factor bam in marked egg mutant GSCs remains normal, indicating that Egg is dispensable for BMP signaling in GSCs. Normally, Bam and Bgcn interact with each other to promote GSC differentiation. Interestingly, marked double mutant egg bgcn GSCs are still lost, but their progeny are able to differentiate into 16-cell cysts though bgcn mutant GSCs normally do not differentiate, indicating that Egg intrinsically controls GSC self-renewal through repressing a Bam/Bgcn-independent pathway. Surprisingly, RNAi-mediated egg knockdown in escort cells leads to their gradual loss and a germ cell differentiation defect. The germ cell differentiation defect is at least in part attributed to an increase in BMP signaling in the germ cell differentiation niche. Therefore, this study has revealed the essential roles of histone H3K9 trimethylation in controlling stem cell maintenance and differentiation through distinct mechanisms., Author Summary Epigenetic regulation plays critical roles in the regulation of cell proliferation, fate determination, and survival. It has been extensively studied in embryonic stem cells for its roles in the control of self-renewal and lineage differentiation. However, epigenetic regulation of adult stem cell function remains poorly defined. In this study, we show that Eggless (Egg), a H3K9 methyltransferase in Drosophila, is required in germline stem cells (GSCs) for controlling self-renewal and in escort cells for regulating germ cell differentiation. egg mutant ovaries exhibit both germ cell differentiation defects and GSC loss, indicating that Egg regulates both germ cell maintenance and differentiation. Intrinsic inactivation of egg function in GSCs leads to loss of trimethylated H3K9 expression and rapid departure from the niche, indicating that Egg is required intrinsically to control GSC self-renewal. Our genetic results reveal that Egg intrinsically controls GSC self-renewal through repressing a Bam/Bgcn-independent pathway. Furthermore, RNAi-mediated egg knockdown in escort cells leads to germ cell differentiation defects due to increased BMP signaling. Therefore, this study has revealed essential roles of histone H3K9 trimethylation in controlling stem cell maintenance and differentiation through distinct mechanisms in the Drosophila ovary.

Although self-renewal is the central property of stem cells, the underlying mechanism remains inadequately defined. Using a hematopoietic stem and progenitor cell (HSPC)-specific conditional induction line, we generated a compound genetic model bearing both Pten deletion and β-catenin activation. These double mutant mice exhibit a novel phenotype, including expansion of phenotypic long-term hematopoietic stem cells (LT-HSCs) without extensive differentiation. Unexpectedly, constitutive activation of β-catenin alone results in apoptosis of HSCs. However, together, the Wnt/β-catenin and PTEN/PI3k/Akt pathways interact to drive phenotypic LT-HSC expansion by inducing proliferation while simultaneously inhibiting apoptosis and blocking differentiation, demonstrating the necessity of complementary cooperation between the two pathways in promoting self-renewal. Mechanistically, β-catenin activation reduces multiple differentiation-inducing transcription factors, blocking differentiation partially through up-regulation of Inhibitor of differentiation 2 (Id2). In double mutants, loss of Pten enhances the HSC anti-apoptotic factor Mcl-1. All of these contribute in a complementary way to HSC self-renewal and expansion. While permanent, genetic alteration of both pathways in double mutant mice leads to expansion of phenotypic HSCs, these HSCs cannot function due to blocked differentiation. We developed a pharmacological approach to expand normal, functional HSCs in culture using factors that reversibly activate both Wnt/β-catenin and PI3K/Akt signaling simultaneously. We show for the first time that activation of either single pathway is insufficient to expand primitive HSCs, but in combination, both pathways drive self-renewal and expansion of HSCs with long-term functional capacity.

To define the molecular regulators required for differential pattern of H3K79 methylation by Dot1, we performed a GPS screen and discovered that the components of the cell cycle-regulated SBF complex were required for normal levels of H3K79 di- but not trimethylation. Genome-wide mapping revealed that H3K79 di- and trimethylation to present a mutually exclusive pattern on chromatin with M/G1 cell-cycle-regulated genes significantly enriched for H3K79 dimethylation. Since H3K79 trimethylation requires prior monoubiquitination of H2B, we performed genome-wide profiling of H2BK123 monoubiquitination and showed that H2BK123 monoubiquitination is excluded from cell cycle regulated genes and sites containing H3K79me2 but not from H3K79me3 containing regions. A genome-wide screen for factors responsible for the establishment/removal of H3K79 dimethylation resulted in the identification of several genes including NRM1 and WHI3, which both impact the transcription by the SBF, and MBF complexes, further linking the regulation of H3K79’s methylation status to the cell cycle.

Caspase-2 is unique among all the mammalian caspases in that it is the only caspase that is present constitutively in the cell nucleus, in addition to other cellular compartments. However, the functional significance of this nuclear localization is unknown. Here we show that DNA damage induced by gamma-radiation triggers the phosphorylation of nuclear caspase-2 at the S122 site within its prodomain, leading to its cleavage and activation. This phosphorylation is carried out by the nuclear serine/threonine protein kinase DNA-PKcs and promoted by the p53-inducible death-domain-containing protein PIDD within a large nuclear protein complex consisting of DNA-PKcs, PIDD, and caspase-2, which we have named the DNA-PKcs-PIDDosome. This phosphorylation and the catalytic activity of caspase-2 are involved in the maintenance of a G2/M DNA damage checkpoint and DNA repair mediated by the nonhomologous end-joining (NHEJ) pathway. The DNA-PKcs-PIDDosome thus represents a protein complex that impacts mammalian G2/M DNA damage checkpoint and NHEJ.

Haematopoietic stem cells are found in a specialized regulatory environment or niche in bone marrow. The precise location and characteristics of this niche are now made clearer by two sophisticated imaging studies. Lo Celso et al. performed live-animal tracking of individual haematopoietic cells and find that osteoblasts are enmeshed in microvessels and that different haematopoietic cell populations are localized in different areas according to their stage of differentiation. Xie et al. used newly developed ex vivo real-time imaging technology and immunoassays to trace the homing of fluorescently labelled haematopoietic stem cells in response to irradiation. They report that the membrane lining the medullary cavity of bone forms a special zone that normally maintains haematopoietic stem cells, but promotes their expansion in response to bone marrow damage. Recently haematopoietic stem cell niches have been shown to comprise osteoblastic and vascular microenvironments. This study describes a newly developed ex vivo real-time imaging technology and immunoassaying to trace the homing of highly purified GFP-expressing haematopoietic stem cells in response to irradiation. Haematopoietic stem cell (HSC) niches, although proposed decades ago1, have only recently been identified as separate osteoblastic and vascular microenvironments2,3,4,5,6. Their interrelationships and interactions with HSCs in vivo remain largely unknown. Here we report the use of a newly developed ex vivo real-time imaging technology and immunoassaying to trace the homing of purified green-fluorescent-protein-expressing (GFP+) HSCs. We found that transplanted HSCs tended to home to the endosteum (an inner bone surface) in irradiated mice, but were randomly distributed and unstable in non-irradiated mice. Moreover, GFP+ HSCs were more frequently detected in the trabecular bone area compared with compact bone area, and this was validated by live imaging bioluminescence driven by the stem-cell-leukaemia (Scl) promoter–enhancer7. HSCs home to bone marrow through the vascular system. We found that the endosteum is well vascularized and that vasculature is frequently localized near N-cadherin+ pre-osteoblastic cells, a known niche component. By monitoring individual HSC behaviour using real-time imaging, we found that a portion of the homed HSCs underwent active division in the irradiated mice, coinciding with their expansion as measured by flow assay. Thus, in contrast to central marrow, the endosteum formed a special zone, which normally maintains HSCs but promotes their expansion in response to bone marrow damage.

Background: Applications of fluorescence-activated cell sorting (FACS) are ideally performed under aseptic conditions so that isolated cells can be successfully cultured, transplanted, or processed for the isolation of protein and nucleic acids. However, modern “off-the shelf” flow cytometers are suboptimally designed for these purposes because nonsterile instrument hardware components directly contact sample-harboring fluids, compromising their sterility. Methods: We have described the design and modular modification of a cytometer with a sterile and disposable FACS fluid handling system that meets requirements of high-speed FACS and good manufacturing practice. This system was tested for functionality and its ability to maintain a clean and sterile fluid environment. Results: Our data have shown that this new fluidic subsystem completely replicated the intended function of the manufacturer's standard fluid handling system, and isolates the fluid from contaminants such as bacteria and fungus, endotoxins, mycoplasma, and helicobacter. Conclusions: FACS has emerged as a powerful tool used to study and manipulate stem cells. However, if stem cell discoveries are to be fully utilized in clinical transplant medicine, aseptic instrument configurations must be developed. For this purpose, we have designed a disposable sterile fluid handling system. © 2006 International Society for Analytical Cytology

Suicide gene therapy is one approach being evaluated for the control of graft-vs-host disease (GVHD) after allogeneic bone marrow transplantation (BMT). We recently constructed a novel chimeric suicide gene in which the entire coding region of HSV thymidine kinase (HSV-tk) was fused in-frame to the extracellular and transmembrane domains of human CD34 (ΔCD34-tk). ΔCD34-tk is an attractive candidate as a suicide gene in man because of the ensured expression of HSV-tk in all selected cells and the ability to rapidly and efficiently purify gene-modified cells using clinically approved CD34 immunoselection techniques. In this study we assessed the efficacy of the ΔCD34-tk suicide gene in the absence of extended ex vivo manipulation by generating transgenic animals that express ΔCD34-tk in the peripheral and thymic T cell compartments using the CD2 locus control region. We found that ΔCD34-tk-expressing T cells could be purified to near homogeneity by CD34 immunoselection and selectively eliminated ex vivo and in vivo when exposed to low concentrations of GCV. The optimal time to administer GCV after allogeneic BMT with ΔCD34-tk-expressing transgenic T cells was dependent on the intensity of the conditioning regimen, the leukemic status of the recipient, and the dose and timing of T cell infusion. Importantly, we used a controlled graft-vs-host reaction to promote alloengraftment in sublethally irradiated mice and provide a graft-vs-leukemia effect in recipients administered a delayed infusion of ΔCD34-tk-expressing T cells. This murine model demonstrates the potential usefulness of ΔCD34-tk-expressing T cells to control GVHD, promote alloengraftment, and provide a graft-vs-leukemia effect in man.

Balanced regulation is essential for the long-term preservation of stem cells while providing for ongoing tissue maintenance. We and others have previously shown that these dichotomous functions are accomplished through the co-existence of at least two stem cell populations—reserve and primed stem cells. This balance has been shown previously to be regulated by protein-coding genes; however, the potential roles of noncoding RNAs (ncRNAs) and their relationships with protein-coding genes in regulating hematopoietic stem cells (HSCs) remain largely unknown. To systematically identify ncRNAs involved in the murine hematopoiesis, we used RNA sequencing and identified unique, differentially expressed (fingerprint) ncRNAs representing reserve HSCs, primed HSCs, and more active stem/progenitor cells. These were also compared with committed progenitors and all major mature hematopoietic lineages. Intriguingly, all of the fingerprint ncRNAs uniquely expressed in reserve HSCs were derived from the imprinted Dlk1-Gtl2 locus, which spans a 780kb region on the mouse chromosome 12qF1 and is precisely controlled by the Intergenic Germ line-derived Differentially Methylated Region (IG-DMR). The Gtl2 locus contains a large cluster of snoRNAs (23 snoRNAs) and the largest cluster of mammalian miRNAs (57 miRNAs) as part of a single transcript of long length ncRNA downstream of Gtl2. To determine the role of Dlk1-Gtl2 locus in hematopoiesis, we utilized the IG-DMR knockout mouse model and carried out phenotypic and functional assays in E15.0 fetal liver HSCs since the embryos loss of maternal IG-DMR are lethal after E16. We observed that deletion of the maternal IG-DMR (ΔmIG-DMR), but not the paternal one, leads to 2-fold reduction in CD93+ fetal liver HSC number and 4-fold decrease of reconstitution ability after tertiary transplantation. Further, we employed RNA-seq using fetal liver HSCs from wt and ΔmIG-DMR and found that several pathways involved in growth control, mitochondrial function and energy metabolism, such as mTOR, PI3K/Akt and Wnt, are significantly enhanced in ΔmIG-DMR HSCs. We also carried out small RNA-seq in both adult HSCs and fetal liver HSCs and identified 13 HSC-specific miRNAs, which are predominantly expressed in reserve HSCs and predicted to target multiple proteins in PI3K/Akt/mTOR pathway. Mechanistically, maternal IG-DMR deletion leads to down-regulation of Gtl2-derived 13 miRNAs and hyperactivation of PI3K/Akt/mTOR pathway, which further enhances mitochondrial activity and biogenesis, increases oxidative phosphorylation (OXPHOS) mediated ATP production and ROS levels, and eventually causes HSC exhaustion. Moreover, either pharmacological inhibition of the mTOR activity by rapamycin or overexpression of Gtl2-derived miRNAs could partially, if not all, rescues the defective HSC phenotype and bioenergetic activities caused by mIG-DMR deletion. Collectively, our work provides a global landscape of murine hematopoietic lncRNAs and demonstrates that Dlk1-Gtl2 locus is critical in maintaining primitive HSCs with a fundamentally epigenetic regulation of mitochondrial function, energy metabolism via repression of PI3K/Akt/mTOR pathway. Disclosures No relevant conflicts of interest to declare.

To investigate the role of signal transducer and activator of transcription (STAT) proteins in granulocyte colony-stimulating factor (G-CSF)-regulated biological responses, we generated transgenic mice with a targeted mutation of their G-CSF receptor (termed d715F) that abolishes G-CSF-dependent STAT-3 activation and attenuates STAT-5 activation. Homozygous mutant mice are severely neutropenic with an accumulation of immature myeloid precursors in their bone marrow. G-CSF-induced proliferation and granulocytic differentiation of hematopoietic progenitors is severely impaired. Expression of a constitutively active form of STAT-3 in d715F progenitors nearly completely rescued these defects. Conversely, expression of a dominant-negative form of STAT-3 in wild-type progenitors results in impaired G-CSF-induced proliferation and differentiation. These data suggest that STAT-3 activation by the G-CSFR is critical for the transduction of normal proliferative signals and contributes to differentiative signals.

GVHD is a significant cause of morbidity and mortality following allogeneic peripheral blood stem cell transplantation (AlloPBSC). CD34+ cell selection could reduce GVHD by negative selection of T cells. In an attempt to reduce the T cell content of alloPBSC we carried out a trial in which 11 patients with hematologic malignancies received alloPBSC from HLA-matched siblings following density gradient separation using an isotonic colloidal silica solution (BDS 60; Dendreon Corporation). Cyclosporine and methylprednisone were used for GVHD prophylaxis. The mean yield of CD34+ cells was 69 +/- 15.6% with a purity of 2.9 +/- 1.7%. The mean number of CD3+ cells infused was 1.0 +/- 1.2 x 107/kg, representing a 1.3 log depletion. A high risk of acute GVHD was observed: grade II-IV in 7/11 (64%) and grade III-IV GVHD in 5/11 (45%) patients. Nine of the 11 (82%) patients died with a median survival of 68 days. Cytokine expression in PBSC was compared pre and post processing. Interferon-gamma was detected only following density gradient separation while IL-8 expression increased 3- to 6-fold post processing. Therefore, processing with this device may augment production of pro-inflammatory cytokines. Bone Marrow Transplantation (2000) 25, 1223-1228.

Allogeneic bone marrow transplantation (BMT) is associated with prolonged periods of neutropenia and thrombocytopenia, which can lead to severe infections and bleeding complications. Transplantation-related side effects might be ameliorated by use of cytokine-mobilized peripheral blood progenitor cells (PBPC) Instead of bone marrow. We have studied PBPC mobilization and transplantation in more than 150 patients with high-risk hematologic malignancies. Normal donors can be sufficiently mobilized with granulocyte colony-stimulating factor (G-CSF), with 91% of G-CSF-stimulated normal donors producing more than 2 x 10(6) CD34+ cells/kg by a single apheresis. The combination of G-CSF plus granulocyte-macrophage colony-stimulating factor (GM-CSF) was more effective than mobilization with G-CSF alone. A clear relationship was seen between numbers of resting CD34+ cells premobilization and numbers of PBPC collected by apheresis, indicating that resting CD34+ cells might be used to predict mobilization results and identify donors who could benefit from more effective mobilization regimens. Transplantation of G-CSF-mobilized PBPC was associated with a more rapid engraftment than that observed for BMT. While engraftment was safe and acute graft-versus-host disease (aGvHD) rates were not increased over BMT, chronic GvHD rates were higher after PBPC transplantation. An additional PBPC infusion on day +3 resulted in a further shortening of neutropenia and thrombocytopenia. Incorporation of these innovative approaches with "minimal" conditioning regimens has resulted in near-complete elimination of fever, neutropenia, thrombocytopenia, and the need for antibiotics and RBC and platelet transfusions after allogeneic transplantation.

The piRNA pathway plays an important role in maintaining genome stability in the germ line by silencing transposable elements (TEs) from fly to mammals. As a highly conserved piRNA pathway component, Piwi is widely expressed in both germ cells and somatic cells in the Drosophila ovary and is required for piRNA production in both cell types. In addition to its known role in somatic cap cells to maintain germline stem cells (GSCs), this study has demonstrated that Piwi has novel functions in somatic cells and germ cells of the Drosophila ovary to promote germ cell differentiation. Piwi knockdown in escort cells causes a reduction in escort cell (EC) number and accumulation of undifferentiated germ cells, some of which show active BMP signaling, indicating that Piwi is required to maintain ECs and promote germ cell differentiation. Simultaneous knockdown of dpp, encoding a BMP, in ECs can partially rescue the germ cell differentiation defect, indicating that Piwi is required in ECs to repress dpp. Consistent with its key role in piRNA production, TE transcripts increase significantly and DNA damage is also elevated in the piwi knockdown somatic cells. Germ cell-specific knockdown of piwi surprisingly causes depletion of germ cells before adulthood, suggesting that Piwi might control primordial germ cell maintenance or GSC establishment. Finally, Piwi inactivation in the germ line of the adult ovary leads to gradual GSC loss and germ cell differentiation defects, indicating the intrinsic role of Piwi in adult GSC maintenance and differentiation. This study has revealed new germline requirement of Piwi in controlling GSC maintenance and lineage differentiation as well as its new somatic function in promoting germ cell differentiation. Therefore, Piwi is required in multiple cell types to control GSC lineage development in the Drosophila ovary.

Embryonic cells that migrate long distances must critically balance cell division in order to maintain stream dynamics and population of peripheral targets. Yet details of individual cell division events and how cell cycle is related to phases of migration remain unclear. Here, we examined these questions using the chick cranial neural crest (NC). In vivo time-lapse imaging revealed that a typical migrating NC cell division event lasted ∼1 hour and included four stereotypical steps. Cell tracking showed that dividing NC cells maintained position relative to non-dividing neighbors. NC cell division orientation and the time and distance to first division after neural tube exit were stochastic. To address how cell cycle is related to phases of migration, we used FACs analysis to identify significant spatiotemporal differences in NC cell cycle profiles. Two-photon photoconversion of single and small numbers of mKikGR-labeled NC cells confirmed that lead NC cells exhibited a nearly fourfold faster doubling time after populating the branchial arches. By contrast, Ki-67 staining showed that one out of every five later emerging NC cells exited the cell cycle after reaching proximal head targets. The relatively quiescent mitotic activity during NC cell migration to the branchial arches was altered when premigratory cells were reduced in number by tissue ablation. Together, our results provide the first comprehensive details of the pattern and dynamics of cell division events during cranial NC cell migration.