The traditional view of hematopoiesis has been that all the cells of the peripheral blood are the progeny of a unitary homogeneous pool of hematopoietic stem cells (HSCs). Recent evidence suggests that the hematopoietic system is actually maintained by a consortium of HSC subtypes with distinct functional characteristics. We show here that myeloid-biased HSCs (My-HSCs) and lymphoid-biased (Ly-HSCs) can be purified according to their capacity for Hoechst dye efflux in combination with canonical HSC markers.
Distinct hematopoietic stem cell subtypes are differentially regulated by TGF-beta1.
Sex, Specimen part
View SamplesAge-related defects in stem cells can limit proper tissue maintenance and hence contribute to a shortened life-span. Using highly purified hematopoietic stem cells from mice aged 2 to 21 months, we demonstrate a deficit in function yet an increase in stem cell number with advancing age. Expression analysis of more than 14,000 genes identified 1500 that were age-induced and 1600 that were age-repressed. Genes associated with the stress response, inflammation, and protein aggregation dominated the upregulated expression profile, while the downregulated profile was marked by genes involved in the preservation of genomic integrity and chromatin remodeling. Many chromosomal regions showed coordinate loss of transcriptional regulation, and an overall increase in transcriptional activity with aged, and inappropriate expression genes normally regulated by epigenetic mechanisms was observed. Hematopoietic stem cells from early-aging mice expressing a mutant p53 allele reveal that aging of stem cells can be uncoupled from aging at an organismal level. These studies show that HSC are not protected from aging. Instead, loss of epigenetic regulation at the chromatin level may drive both functional attenuation of cells, as well as other manifestations of aging, including the increased propensity for neoplastic transformation.
Aging hematopoietic stem cells decline in function and exhibit epigenetic dysregulation.
No sample metadata fields
View SamplesHSC (Sca+ SP) were isolated from 8-12 week C57B6 mice at various time points after treatment with 5-Fluorouracil. RNA was isolated from 50,000-100,000 FACS sorted cells and subjected to two rounds of T7 based linear amplification using Ambion's Message Amp kit. Two replicates from each time point were analyzed.
Molecular signatures of proliferation and quiescence in hematopoietic stem cells.
No sample metadata fields
View SamplesTET-family dioxygenases oxidize 5-methylcytosine (5mC) in DNA, and exert tumor suppressor activity in many types of cancers. Even in the absence of TET coding region mutations, TET loss-of-function is strongly associated with cancer. We show that acute elimination of TET function induces the rapid development of an aggressive, fully-penetrant and cell-autonomous myeloid leukemia in mice, pointing to a causative role for TET-loss-of-function in this myeloid malignancy. Phenotypic and transcriptional profiling showed aberrant differentiation of hematopoietic stem/ progenitor cells, impaired erythroid and lymphoid differentiation and strong skewing to the myeloid lineage, with only a mild relation to changes in DNA modification. We also observed progressive accumulation of DNA damage and strong impairment of DNA break repair, suggesting a key role for TET proteins in maintaining genomic integrity. Overall design: Jungeun, An
Acute loss of TET function results in aggressive myeloid cancer in mice.
Specimen part, Subject
View SamplesWe compared gene expression differences in Atxn1L knockout vs wildtype HSCs
Ataxin1L is a regulator of HSC function highlighting the utility of cross-tissue comparisons for gene discovery.
Specimen part
View SamplesWe demonstrate that dCas9-SunTag-DNMT3A dramatically increased CpG methylation at the HOXA5 locus in human embryonic kidney 293T cells (HEK293T). Furthermore, using a single sgRNA, dCas9-SunTag-DNMT3A was able to methylate a 4.5 kb genomic region and repress HOXA5 gene expression. Reduced representation bisulfite sequencing (RRBS) and RNA-seq showed that dCas9-SunTag-DNMT3A methylated regions of interest with minimal impact on the global DNA methylome and transcriptome. Overall design: I)PCR amplicon deep sequencing of dCas9-SunTag-DNMT3A treated HEK2937 samples using Illumina Nextseq sequencing system. II) Reduced representation bisulfited sequencing (RRBS) of plasmid transfected HEK 293T cells using Illumina Hiseq2000 sequencing system. III) Whole genome bisulfite sequencing of dCas9-SunTag-DNMT3A treated HEK2937 samples. IV) RNA sequencing of dCas9-SunTag-DNMT3A treated HEK2937 samples
DNA epigenome editing using CRISPR-Cas SunTag-directed DNMT3A.
Specimen part, Cell line, Subject
View SamplesGSM48315-GSM48332: Ten cells from C57Bl/6 male mouse bone marrow (SP or CD8 T cells) were sorted into individual wells of 96-well plates. The mRNA of these cells was amplified by the global single cell RT-PCR method and biotinylated targets were generated after optimal digestion with DNAse I.
Evidence for diversity in transcriptional profiles of single hematopoietic stem cells.
No sample metadata fields
View SamplesWe compared gene expression differences in Lyl-1 knockout vs wildtype LMPPs
The transcription factor Lyl-1 regulates lymphoid specification and the maintenance of early T lineage progenitors.
Specimen part
View SamplesA unique property of many adult stem cells is their ability to exist in a non-cycling, quiescent state. Although quiescence serves an essential role in preserving stem cell function until the stem cell is needed in tissue homeostasis or repair, defects in quiescence can lead to an impairment in tissue function, the extent to which stem cells can regulate quiescence is unknown. Here, we show that the stem cell quiescent state is composed of two distinct functional phases: G0 and an alert phase we term GAlert, and that stem cells actively and reversibly transition between these phases in response to injury-induced, systemic signals. Using genetic models specific to muscle stem cells (or satellite cells (SCs)), we show that mTORC1 activity is necessary and sufficient for the transition of SCs from G0 into GAlert and that signaling through the HGF receptor, cMet is also necessary. We also identify G0-to-GAlert transitions in several populations of quiescent stem cells. Quiescent stem cells that transition into GAlert possess enhanced tissue regenerative function. We propose that the transition of quiescent stem cells into GAlert functions as an 'alerting' mechanism, a novel adaptive response that positions stem cells to respond rapidly under conditions of injury and stress without requiring cell cycle entry or a cell fate commitment.
mTORC1 controls the adaptive transition of quiescent stem cells from G0 to G(Alert).
Specimen part, Treatment, Time
View SamplesTo assess gene expression changes in Irgm1 (Lrg-47) deficient HSCs
Irgm1 protects hematopoietic stem cells by negative regulation of IFN signaling.
No sample metadata fields
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