Metabolomics,Unknown,Transcriptomics,Genomics,Proteomics

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Gene expression in mouse hematopoietic stem and multi-potent progenitor cells with temporally defined divisional histories

ABSTRACT: Homeostatic hematopoietice stem cells (HSCs) with greater divisional history lose repopulating potential after very few cell divisions. Divisional history overrides both phenotype and immediate quiescence in determining functional activity. In GFP label retaining system GFP is progressively diluted when cells proceed through a cascade of divisions. We used a GFP label retaining system and performed microarray expression analyses to track the changes in the gene expression profile of bone marrow (BM) LSK cells that relates to divisional history during homeostasis. Chromatins are dynamically labeled in a double transgenic mouse (huCD34-tTA X Tet-O-H2B-GFP) by the expression of GFP tagged Histone2B. The expression is driven by a human CD34 promoter active specifically in all bone marrow (BM) hematopoietic stem and multi-potent progenitor cells. This is a pulse/chase Tet-off system where cells constantly label with GFP. Upon administration of Doxycyclin (Dox) to GFP labeled animals, further labeling is stopped during the chase period. With each round of cell division, the newly synthesized endogenous H2B will dilute H2B-GFP by one-half, whereas cells that don’t divide will retain GFP labeling. Therefore, the level of GFP reflects how many times cells have divided over the period of Dox treatment (chase). Animals were treated with Dox for 12 weeks starting at the age of 6-8 weeks. After chase, BM Lineage negative, Sca1+, Kit+ (LSK) cells with varying GFP levels were sorted for mRNA extraction. GFP levels were ranked from 0 to 4 based on the kinetic analysis of GFP fully labeled LSK cells and obvious peaks in the GFP histogram of LSK cells after Dox treatment. There is approximately 1-2 cell divisions between each level of GFP.

ORGANISM(S): Mus musculus

SUBMITTER: Kateri Moore

PROVIDER: E-GEOD-48261 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:In homeostasis of adult vertebrate tissues, stem cells are thought to self-renew by infrequent and asymmetric divisions that generate another stem cell daughter and a progenitor daughter cell committed to differentiate. This model is based largely on in vivo invertebrate or in vitro mammal studies. Here we examine the dynamic behaviour of adult hair follicle stem cells in their normal setting by employing mice with repressible H2B-GFP expression to track cell divisions and Cre inducible mice to perform long-term single cell lineage tracing. We provide direct evidence for the infrequent stem cell division model in intact tissue. Moreover, we find that differentiation of progenitor cells occurs at different times and tissue locations than self-renewal of stem cells. Distinct fates of differentiation or self-renewal are assigned to individual cells in a temporal-spatial manner. We propose that large clusters of tissue stem cells behave as populations, whose maintenance involves unidirectional daughter-cell fate decisions. We used microarrays to expression profile the stem cell progeny generated at distinct differentiation and self-renewal stages. Experiment Overall Design: We employed double transgenic mice, K5tTA x pTRE-H2B-GFP in which an epithelial Keratin 5 (K5) promoter drove repressible histone H2B-GFP expression. Repression is achieved by feeding the mice doxycycline for a period of time (chase), when the H2B-GFP dilutes in cells by 2-fold at division. This allowed us to quantify precise proliferation history in vivo, from the amount of H2B-GFP fluorescence retained in cells after chase. After various chase periods we sacrificed mice at different ages corresponding to distinct phases of hair cycle. We isolated skin cell suspensions from these mice and stained them for surface expression of stem cell niche bulge(Bu) markers CD34 and alpha 6-integrin. Fluorescence-activated cell sorting (FACS) revealed histograms with distinct peaks of 2-fold median H2B-GFP intensity corresponding to distinct divisions. For microarray, we FACS sorted bulge(Bu) and non-bulge(NonBu) cells based cell surface marker staining and their proliferation history with H2B-GFP intensity, after two doxycycline chase schemes (Postnatal Day 18-PD21 or PD22-PD25), then extract total RNA from each cell fractions. For microarrays 5 ng of high quality RNA (Bioanalyzer, Agilent) were amplified (Ovation Amplification; Nugene) in the Cornell Microarray Core Facility. GeneChip IVT labelling was followed by Gene Chip MOE 430 2.0 hybridization, GeneArray 3000 scanning, GCOS generation of present calls and signal values (Affymetrix).

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Gene expression in mouse hematopoietic stem and multi-potent progenitor cells with temporally defined divisional histories

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