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Blood, Vol. 111, Issue 12, 5553-5561, June 15, 2008
A new mechanism for the aging of hematopoietic stem cells: aging changes the clonal composition of the stem cell compartment but not individual stem cells
Blood Cho et al.
111: 5553
Supplemental materials for: Cho et al
Files in this Data Supplement:
- Figure S1. Detection of T and B lymphocytes and myeloid cells of donor origin by immune fluorescence (JPG, 134 KB)
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Shown are examples of flow cytometry plots of white blood cells from (A) an unmanipulated D2-CD45.1 mouse; (B) a clonally repopulated mouse (host D2, 56.1± 0.6 % donor D2-CD45.1); (C) a non-repopulated host (0.8 ± 0.1 % donor type cells). Cells are stained with a mAb specific for CD45.1 and co-stained with mAbs specific for Thy-1, B220, and Mac-1+Gr-1.
- Figure S2. Stability of lineage markers expressed on white blood cells of young and aged D2 mice (JPG, 110 KB)
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(A) Cells were co-stained with goat-anti-mouse and a mAb specific for B220. (B) Cells were co-stained with a mAb specific for the T cell receptor beta-chain and Thy-1. (C) Cells were stained with Mac-1+Gr-1+. Data are representative for 3 independent experiments. (D) Mac-1+8C5+ cells (in the gate indicated in panel C) were sorted from the blood of a young and an aged D2 mouse. Cells were spun unto a slide, stained with Wrights Giemsa, and the indicated cell types were counted by microscopy. Forty cells were counted from each sorted population. Low levels of contaminating lymphocytes were found in young but not in aged sorted cells. Low levels (∼ 1/100) of eosinophils were found. Presumably, the sort depleted these cells. Collectively, the data show the markers Thy-1, B220, or Mac-1/Gr-1 faithfully detect B and T lymphocytes and myeloid cells from both young and aged cells.
- Figure S3. Comparison of D2-CD45.1 and parental D2 mice (JPG, 42.4 KB)
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(A) the level of CD45.1 on peripheral blood lymphocytes of D2-CD45.1 gray), B6-CD45.1 (black) and parental D2 mice (white) as measured by flow cytometry. (B) Percent of lymphoid and myeloid cells in the blood of D2-CD45.1 (gray) and D2 (white) mice.
- Figure S4. Dynamics of the repopulation of myeloid and lymphoid cells after clonal HSC transplantation (JPG, 132 KB)
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Shown are two representative examples each of hosts repopulated by individual My-bi HSCs (A), Ly-bi HSCs (B), and Bala (C) HSC clones. Mice were bled at the times points indicated on the y-axis and the percent of donor type cells of the myeloid (black), B lymphoid (white) and T lymphoid (gray) cells was measured. Each bar represents the measurements in an individual mouse. Clonally repopulated mice were derived by transplanting limiting numbers of BM cells derived from young B6 mice. Mice in panel D are controls that were not transplanted but bled at the same time as the test mice.
- Figure S5. Myeloid bias of HSCs from aged D2 mice is stable through serial transplantation (JPG, 100 KB)
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Three My-bi HSCs donor type (D2-CD45.1) were identified in three primary recipients. Nine months after the first transplant, 5 × 106 BM cells from each primary host were transplanted into 2 to 3 secondary, lethally irradiated D2 hosts. The percent of myeloid (black) cells, B (white), and T (gray) lymphocytes was measured 3 to 5 months after the secondary transplant. As predicted by the high level of T cells seen in blood, mouse R2+R4 in the secondary transplant died from graft failure.
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