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BACKGROUND
• During aging, the immune system becomes less ef5icient in deterring invaders, infections, and disease.
• It is currently known that aging of hematopoietic stem cells (HSCs) leads to an increased production of myeloid cells and a decreased production of T and B cells. How this clonal phenotype arises remains inconclusive.
• To better understand the aging process , it is important to examine the clonal behavior of the heterogeneous stem cell population over time.
Tracking Hematopoietic Stem Cells in vivo Over Time Keck School of Medicine of USC
Mike Chin, Rong Lu
METHODS
Figure 1. Genetic barcoding and tracking of HSC clones in vivo. Donor HSCs were isolated from the bone marrow of mice. HSCs (ckit+/lineage(CD3,CD4, CD8,B220,Gr1,Mac1,Ter119)-‐Sac1+/CD34-‐/CD150+/Flk2-‐) were infected with a lentiviral vector so that each HSC received a unique barcode. Mice were lethally irradiated and transplanted with barcoded HSCs. Peripheral blood was collected from recipient mice at several time points over 1 year. Genomic DNA from granulocytes, B, CD4T, and CD8T cells were quanti5ied with high throughput sequencing.
Figure 2. HSC production changes over time. The proportion of granulocytes, B, CD4T, and CD8T cells relative to total number of white blood cells was examined on Day 61, 117, 297, 362. Myeloid production is seen to increase while lymphoid production is seen to decrease over these 4 time points.
BLOOD PRODUCTION OVER TIME
REFERENCES Lu R, Neff NF, Quake SR, Weissman IL. (2011) Tracking single hematopoieCc stem cells in vivo using
high-‐throughput sequencing in conjuncCon with viral geneCc barcoding. Nat Biotechnol. 29, 928-‐33.
Leventhal J, Miller J, Abecassis M, Tollerud DJ, Ildstad ST. (2013) Evolving approachs to hematopoieCc stem cell-‐based therapies to induce tolerance to organ transplants: the long road to tolerance. Clin. Pharmacol Ther. 93, 36-‐45. Weissman IL, Shizuru JA. (2008) The origins of the idenCficaCon and isolaCon of hematopoieCc stem
cells, and their capability to induce donor-‐specific transplantaCon tolerance and treat autoimmune diseases. Blood. 112, 3543-‐53.
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Figure 3. Lineage bias of HSC clones varies over time. (A-‐D) Proportion of granulocyte, B, CD4T, CD8T clones from irradiated recipients on days 61, 117, 297, 362 were examined for clonal lineage bias. Lineage-‐biased clones are de5ined as those whose relative copy numbers in one lineage are more than 2.4142 (cotangent 22.5 degree) times their relative copy numbers in the other lineage. (E-‐H) Histograms indicate clonal lineage bias behavior over time. Clones are assigned a score from -‐1.0 to 1.0, indicating the relative bias.
Figure 4. Dominant clone production varies with cell type over time. (A-‐B) Clonal dominance is de5ined as the percentage of a cell population contributed by the top 3 or top 5 most abundant clones. Averages for top clones are plotted for all cell types over days 61, 117, 297, and 362. (C) Heat map examining clonal contribution over time for each cell type. Each horizontal line represents a clone. Bright red indicates high clonal production.
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Granulocyte Bias Balance CD8T Bias
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Granulocyte B CD4 T CD8T
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Granulocyte Biased
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Granulocyte Bias Balance CD4T Bias
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CLONAL LEVEL LINEAGE BIAS CLONAL DOMINANCE
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Gr Bias CD4T Bias Gr Bias CD4T Bias Gr Bias CD4T Bias Gr Bias CD4T Bias
Gr Bias CD8T Bias Gr Bias CD8T Bias Gr Bias CD8T Bias Gr Bias CD8T Bias
CD4T Bias CD8T Bias CD4T Bias CD8T Bias CD4T Bias CD8T Bias CD4T Bias CD8T Bias
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TOP 3 TOP 5
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Granulocyte CD4T B CD8T
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