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. 2012 Jan 15;481(7382):516-9.
doi: 10.1038/nature10734.

Multi-isotope Imaging Mass Spectrometry Quantifies Stem Cell Division and Metabolism

Free PMC article

Multi-isotope Imaging Mass Spectrometry Quantifies Stem Cell Division and Metabolism

Matthew L Steinhauser et al. Nature. .
Free PMC article


Mass spectrometry with stable isotope labels has been seminal in discovering the dynamic state of living matter, but is limited to bulk tissues or cells. We developed multi-isotope imaging mass spectrometry (MIMS) that allowed us to view and measure stable isotope incorporation with submicrometre resolution. Here we apply MIMS to diverse organisms, including Drosophila, mice and humans. We test the 'immortal strand hypothesis', which predicts that during asymmetric stem cell division chromosomes containing older template DNA are segregated to the daughter destined to remain a stem cell, thus insuring lifetime genetic stability. After labelling mice with (15)N-thymidine from gestation until post-natal week 8, we find no (15)N label retention by dividing small intestinal crypt cells after a four-week chase. In adult mice administered (15)N-thymidine pulse-chase, we find that proliferating crypt cells dilute the (15)N label, consistent with random strand segregation. We demonstrate the broad utility of MIMS with proof-of-principle studies of lipid turnover in Drosophila and translation to the human haematopoietic system. These studies show that MIMS provides high-resolution quantification of stable isotope labels that cannot be obtained using other techniques and that is broadly applicable to biological and medical research.


Figure 1
Figure 1. MIMS quantitation of stable isotope-labeled thymidine incorporation by dividing cells

Dividing fibroblast labeled with 15N-thymidine. The cell surface was sputtered to reach the nuclei. Left: Differential interference contrast reflection microscopy. Middle: MIMS 14N image revealing subcellular details including the nucleus (white arrows) with nucleoli. Right: Hue saturation intensity (HSI) image mapping the 15N/14N ratio. The rainbow scale ranges from blue, set to natural ratio (0.37%, expressed as 0% above natural ratio), to red, where the ratio is several fold above natural ratio (700%=8x natural ratio). 15N labeling is concentrated in the nucleus. Scale bars=10µm.

Fibroblast nuclei after serial or parallel DNA labeling with 15N-thymidine, 13C-thymidine, BrdU (81Br), or IdU (127I). Parallel labeling (top row): colocalization of label confirmed by the merged image (far right). Sequential labeling (bottom row): non-superimposable nuclear labeling. Scale bars=5µm.

Concentration-dependent nuclear 15N-labeling in 15N-thymidine treated fibroblasts. Pixel-by-pixel quantitation of 15N-incorporation. Data are derived from the mean of intranuclear pixels. Sigmoidal dose response curve: R2=0.99.

Nuclei from 15N-thymidine-labeled human foreskin fibroblasts after 24hr chase (cycle ~ 18 hr). Scale bars=5µm.

One group labeled similarly to control cells (undivided), the other with labeling that was approximately half that of control (divided).

15N/14N HSI image of the small intestine. 15N-thymidine labeling (one wk) of nuclei extends from the crypts to the tips of the villi. Mosaic: 8 tiles, 80µm each. Scale bar=30µm.

Sigmoidal dose response curve: 15N-thymidine labeling after single subcutaneous injection (R2=0.99).

Figure 2
Figure 2. No label-retaining stem cells in the small intestinal crypt

15N-thymidine administered from post-natal day 4 - week 8. After 4-wk chase, BrdU was administered (500 µg i.p. Every 6 h) for 24 hrs before sacrifice (See Supplemental Fig 8).

14N: crypt structure and intense signal in intracytoplasmic Paneth granules at the crypt base. 31P: intense intranuclear signal. 32S: intense signal within cytoplasmic Paneth cell granules. 81Br: direct measure of BrdU incorporation. 15N/14N HSI: 15N-thymidine labeling within 15N+/BrdU- Paneth cells (large arrow) and mesenchymal cells (small arrows). No other cells reveal 15N retention. Large arrow head: recently formed (15N/BrdU+) Paneth cell. Small hatched arrow (middle left side of the crypt): unlabeled Paneth cell, (15N/Br-). Scale bar=15µm.

Continued analysis of the same crypt after narrowing the acquisition field. High 15N-signal in a BrdU Paneth cell (large arrow) and mesenchymal cells closely associated with the crypt (small arrows). BrdU+ CBC (hatched arrow) and Paneth cell (arrow head) are 15N. Scale bar=5µm.

15N/14N HSI image of small intestinal crypt at the end of 15N-thymidine pulse. All nuclei are labeled. Nuclei with lesser degrees of labeling likely represent cells born during a period of lower circulating 15N-thymidine as expected given the different labeling protocols (Supplemental Fig 8). Scale bar=20µm.

Unlabeled mouse image. The entire crypt contains the natural abundance of 15N. Scale bar=10µm.

Mean % 15N+ cells at the completion of pulse and after pulse-chase (± standard error of CBC, TA, Paneth, and mesenchymal (Mes) cells (n=3 mice per group).

Figure 3
Figure 3. Label-dilution in adult mice indicates random segregation of DNA strands

15N-thymidine administered for 2 wks to adult mice (osmotic mini-pump: 20µg/h), then BrdU for 24h before sacrifice (See Supplemental Fig 8).

Dividing cells (BrdU+) dilute 15N-thymidine label (small arrows) relative to undivided cells (BrdU) (large arrows). Note 2 CBC cells with elongated nuclei at the crypt base. Scale bar=10µm.

Divided crypt cells (BrdU+), residing in CBC or +4–10 positions, demonstrated 15N-dilution consistent with 1 or 2 rounds of division during the chase (n = 3 mice per group).

Mitotic crypt cell. Segregating chromosomes are visible in 14N and 31P images. 15N-label and BrdU were measured in both segregating chromosomal complements consistent with symmetric chromosomal segregation. Scale bar=2µm.

Figure 4
Figure 4. Extending MIMS to Drosophila and human metabolism

Drosophila anterior midgut enterocytes (gut, left panels) accumulate more 13C from 24hr exposure to dietary 13C-palmitate than adipocytes (fat body cells; fb, right panels). Lipid droplets (arrows) have low nitrogen content and so appear dark in 14N images (top panels). The 13C/12C HSI images reveal 13C incorporation into lipid droplets. Scale bars=10µm.

Timecourse of 13C accumulation in Drosophila lipid droplets of anterior midgut enterocytes (Gut) and adipocytes (Fat body) over 24hr of larval exposure to dietary 13C-palmitate. Initial rates of 13C-incorporation in the lipid droplets were 9.7%/hr (Gut) and 1.16%/hr (Fat body) with the first timepoint after pulse taken at 1hr.

Rapid lipid turnover in Drosophila anterior midgut enterocytes (gut) after larval dietary 13C-palmitate pulse-chase. Upper panels show 14N images and lower panels show corresponding 13C/12C HSI images, indicating strong 13C incorporation at the end of the pulse (bottom left) and rapid elimination during a 4 hr chase (bottom right). Scale bar=5µm.

13C tracer dilution in the lipid droplets of anterior midgut enterocytes during a label-free chase fits an exponential decay curve (R2=0.90, half-life=9.1 h).

15N-thymidine administered for 48hrs: no labeled WBC were found. After a 4 wk chase we found few labeled lymphocytes. Top: unlabeled polymorphonuclear leukocyte (PMN) with multi-lobed nucleus. Bottom: 15N-labeled lymphocyte after chase. The round nucleus seen in the 31P image occupies the majority of the cell, as is typical for lymphocytes. Scale bars=3µm.

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