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. 2014 Mar 10;211(3):487–497. doi: 10.1084/jem.20131804

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© 2014 Verovskaya et al.

This article is distributed under the terms of an Attribution–Noncommercial–Share Alike–No Mirror Sites license for the first six months after the publication date (see http://www.rupress.org/terms). After six months it is available under a Creative Commons License (Attribution–Noncommercial–Share Alike 3.0 Unported license, as described at http://creativecommons.org/licenses/by-nc-sa/3.0/).

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Figure 1. — Old and young HSCs are asymmetrically distributed among various skeletal niches and migrate at a similar rate. (A) Experimental setup. BM cells were isolated from young and aged donors, and LSK48⁻150⁺ cells were separately purified by cell sorting and subsequently pooled. After transduction with a barcoded vector library, cells were transplanted into lethally irradiated animals. Recipient mice were sacrificed 6–11 mo after transplant. BM cells were isolated from four skeletal locations, and LSK48⁻150⁺ cells of young and old origin were single-cell sorted into cytokine-rich medium. Colonies expanded to ∼30,000 cells were harvested for barcode analysis. (B) Schematic representation of the four bone groups that were analyzed. Bones analyzed in the same group are shown in the same color. (C) Skeletal distribution of barcoded clones in young (left) and old (right) LSK48⁻150⁺ cells in a single mouse, analyzed 6 mo after transplantation. Clones that are colored white are unique, i.e., were found only once. The asterisks identify clones with distributions that are significantly different from random (P < 0.05 based on Monte Carlo simulations). The number of barcoded colonies analyzed for every skeletal location is indicated above the column. Clones found in four or more colonies and further used for distribution analysis are numbered. (D) Summary of all clones of young origin found in four or more colonies 6 mo after transplantation (n = 6; data were derived from two cohorts of mice). X axis shows the rank of clone size, based on mean value over all four locations, ranked from most frequent to the least frequent. Y axis shows the contribution of a certain clone to the total pool in one location. Dots on the same vertical line reflect the size of a particular clone in each of the four different locations. The mean contribution for a given clone is shown with a horizontal line. As in C, clones significantly deviating from equal distribution (P < 0.05 based on Monte Carlo simulations) are marked with an asterisk. For clarity, four numbered clones from C are colored according to the bone group colors as in B. (E) Same as D, but shown for old clones (summary of two independent cohorts of mice; n = 6). (F) Same as D, but for young clones from mice sacrificed 8.5–11 mo after transplantation (one cohort; n = 4). (G) Same as F, but for old clones (n = 4). (H) Summary of the RSDs for all clones shown in D–G. Horizontal lines indicate mean value.