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1.
Figure 2

Figure 2. Lactate production and metabolic substrate requirements change with differentiation.. From: Endogenous Fluorescence Signatures in Living Pluripotent Stem Cells Change with Loss of Potency.

A. Lactate production as detected in the medium on days 3 through 8 of mEB development. Horizontal lines indicate statistical difference (P<0.05) (Holm-Sidak method pairwise multiple comparison following repeated measures ANOVA). B. Percentage of mEBs that exhibited beating areas when cultured in either standard medium (Glu & Pyr), medium with glucose only (Glu) or medium with pyruvate only (Pyr) as assessed on days 8, 9, 10 and 12 after EB formation. Horizontal lines indicate statistical difference (P<0.05) (Tukey test following ANOVA). C. Quantitation of cTnT antibody labeling using fluorescence intensity measures from EBs grown with glucose and pyruvate, glucose only or pyruvate only. Horizontal lines indicate statistical difference (P<0.05) (Tukey pairwise comparison test following ANOVA). D. Examples of cTnT antibody labeling in EBs cultured in the 3 media. Scale bar = 500 µm.

Jayne M. Squirrell, et al. PLoS One. 2012;7(8):e43708.
2.
Figure 5

Figure 5. Cellular level changes in endogenous fluorescence intensity and lifetime with loss of pluripotency of hESCs.. From: Endogenous Fluorescence Signatures in Living Pluripotent Stem Cells Change with Loss of Potency.

A. GFP intensity of MPLSM single optical section of live hESCs expressing pOct4:GFP: at 10 days post-EB formation using 890 nm excitation and 520/35 nm emission. Right image shows regions of interest (ROIs), roughly corresponding to single cells, with high intensity GFP (GFP(H)), outlined in red; low intensity GFP (GFP(L)), outlined in blue. B. Quantitative assessment of ROIs of GFP intensity. * indicates significant difference between GFP(H) ROIs and GFP(L) ROIs (P≤0.001). C. Intensity of the same MPLSM single optical section as (A) using 780 nm excitation and 457/50 nm emission corresponding primarily to NADH endogenous fluorescence. Right image shows same ROIs as (A). D. Quantitative assessment of ROIs of endogenous intensity. E. Color mapped mean lifetime (τm) images of endogenous fluorescence with quantitative assessment of ROIs (F). G. Color mapped long lifetime (τ2) images of endogenous fluorescence with quantitative assessment of ROIs (H). Scale bars = 20 µm. Color bar indicates lifetime values for τm (E) or τ2 (G). * indicates significant difference at P<0.01 (t-test).

Jayne M. Squirrell, et al. PLoS One. 2012;7(8):e43708.
3.
Figure 3

Figure 3. Cell level changes in endogenous fluorescence intensity and lifetime with loss of pluripotency of mESCs.. From: Endogenous Fluorescence Signatures in Living Pluripotent Stem Cells Change with Loss of Potency.

A. GFP intensity of MPLSM single optical section of live mESCs expressing the pOct4:GFP transgene at 5 days post-EB formation using 890 nm excitation and 520/35 nm emission filter. Right image shows regions of interest (ROI), roughly corresponding to single cells, with high intensity GFP (GFP(H)) outlined with red; medium intensity GFP (GFP(M)) outlined with orange; low intensity GFP (GFP(L)) outlined with blue. B. Quantitative assessment of ROIs of GFP intensity. C. Intensity images of the same MPLSM single optical section as (A) using 780 nm excitation and 457/50 nm emission, corresponding primarily to NADH endogenous fluorescence. Right images show same ROIs as (A). D. Quantitative assessment of ROIs of endogenous intensity. E. Color mapped mean lifetime (τm) images of endogenous fluorescence showing the same ROIs, with quantitative assessment in F. G. Color mapped long lifetime (τ2) images of endogenous fluorescence with quantitative assessment of ROIs (H). Scale bars = 20 µm. Color bar indicates lifetime values for τm (E) or τ2 (G). * indicates significant difference between GFP(H) ROIs and ROIs of medium or low GFP intensity (P≤0.001; ANOVA on ranks followed by Tukey multiple pairwise comparison test at P<0.05).

Jayne M. Squirrell, et al. PLoS One. 2012;7(8):e43708.
4.
Figure 4

Figure 4. Global endogenous fluorescence intensity and lifetime changes observed with differentiation of human EBs.. From: Endogenous Fluorescence Signatures in Living Pluripotent Stem Cells Change with Loss of Potency.

A. Background subtracted intensity of MPLSM single optical sections of the same live hEB at 5, 7, 10 and 16 days post-EB formation, using 780 nm excitation. B. Color mapped fluorescence lifetime (τm) of the same hEB as in upper row, over time. Color bar indicates lifetime values for τm. Scale bar = 50 µm. C. Quantitation shows changes in mean fluorescence intensity at the different time points (bars: for days 5, 7, 10, 16 n = 16, 17, 17, 16 EBs, respectively) while colored lines indicate the changes in intensity of individual EBs tracked over time. D. Quantitation of τm, with bar graph showing the mean τm at the given time points (for days 5, 7, 10, 16 n = 5, 6, 6, 5 EBs, respectively), while colored lines show the changes in lifetime of individual hEBs tracked over time. Horizontal bars indicate differences at P<0.05 (Holm-Sidak method for pairwise comparisons following repeated measures ANOVA). Quantitation of additional lifetime parameters and detailed statistics in Figure S6.

Jayne M. Squirrell, et al. PLoS One. 2012;7(8):e43708.
5.
Figure 6

Figure 6. Potential utilization of endogenous fluorescence parameters.. From: Endogenous Fluorescence Signatures in Living Pluripotent Stem Cells Change with Loss of Potency.

A. Scatter plot showing relationship between fluorescence lifetime (τm), endogenous fluorescence, and Oct4-GFP intensity category. Each circle represents one cell. Dotted lines indicate arbitrary threshold to illustrate the effect of segregating a population of cells based on two endogenous fluorescence parameters. B. Quantitation comparing the endogenous fluorescence intensity (background subtracted intensity per pixel of EB brightfield area) of hEBs on day 4 of EB formation, segregated by those that subsequently formed beating areas (BA) and those which did not (NBA). * indicates significant difference (P≤0.001, Mann-Whitney). Images above bars show MPLSM single optical sections of hEBs, using 780 nm excitation, on day 4 after hEB formation prior to attachment, to show background subtracted endogenous fluorescence intensity. hEB on right subsequently develops a beating area (BA) while the one on the left does not (NBA). Scale bar = 100 µm. C. Assessment of the putative enrichment for subsequent developmental of beating areas (day 24) based on fluorescence intensity on day 4. Graph shows the proportion of hEBs that developed beating area (filled red circles) when selected from the 30% of EBs that have the highest fluorescence intensity at day 4 (above horizontal dotted line), largest area (to right of vertical dotted line) or both (upper right quadrant) compared to those in the lower 70% (below or to left of dotted lines for intensity or area, respectively). Open circles represent EBs that did not develop beating areas. Red numbers indicate percentage of EBs with beating areas in that category (e.g. upper 30% of intensity or upper 30% of area) of the total number of EBs within that category while black numbers indicate the percentage of EBs with beating areas in that category of the total number of EBs examined (n = 106 EBs).

Jayne M. Squirrell, et al. PLoS One. 2012;7(8):e43708.
6.
Figure 7

Figure 7. Defining signatures for non-invasive enrichment of pluripotent stem cells and associated progeny.. From: Endogenous Fluorescence Signatures in Living Pluripotent Stem Cells Change with Loss of Potency.

This schematic outlines how the changes in endogenous fluorescence that occur with stem cell differentiation might be practically employed to enable both basic studies of the cues that dictate stem cell differentiation as well as clinical applications. A. Enrichment of pluripotent stem cell populations. It is difficult to maintain a pluripotent stem cell population in a culture dish as multiple stimuli can conspire to induce unwanted differentiation. Several approaches have been employed in an attempt to monitor and maintain pluripotent populations, but all are invasive in nature (i.e., exogenous labels or stably integrated antibiotic resistance genes). Instead, using a multiphoton-based flow cytometry system [5], threshold values (dashed lines) for intrinsic fluorescence lifetime elements might be identified and utilized to enrich populations. The cellular-scale analyses described here are particularly well suited to define threshold values. Stem cells enriched in this way could be used for a variety of applications, including the generation of EBs. B. Enrichment of EBs. EBs have long served as a developmentally-relevant format for inducing stem cell differentiation. The challenge is isolating EBs that give rise to particular differentiated cell types in an enhanced-throughput, noninvasive manner. Here we suggest use of parameters from our global-scale analyses, such as endogenous fluorescence intensity, to define thresholds that might be used to purify EBs at early time points that have a high probability of generating differentiated cell types of interest, such as cardiomyocytes. Differentiated cells enriched in this way would be quite valuable for basic studies of the cues that drive differentiation and perhaps, in the future, for regenerative therapies.

Jayne M. Squirrell, et al. PLoS One. 2012;7(8):e43708.
7.
Figure 1

Figure 1. Changes in global endogenous fluorescence intensity fluorescence lifetime with differentiation of mESCs.. From: Endogenous Fluorescence Signatures in Living Pluripotent Stem Cells Change with Loss of Potency.

A, B. MPLSM single optical sections of mouse ESCs at different stages of differentiation (ESC and days 1, 3, 5, and 8 of EB formation; ESC: n = 24, 27, 14, 23, 13, 4 colonies, respectively; EB: n = 7, 16, 6, 18, 8, 4 EBs, respectively) using 780 nm excitation. A. Endogenous fluorescence intensity images following application of a background subtraction FIJI plugin macro (see Methods) to remove 90% of the background of the region of interest prior to intensity analysis. Complete timeline of images, including raw image data, is in Figure S1. B. Color mapped lifetime values of MPLSM single optical sections of mouse ESCs at different stages of differentiation (mESC and days 1, 3, 5, and 8 of EB formation; ESC: n = 13, 20, 8, 18, 10, 18 colonies, respectively; EB: n = 7, 7, 6, 12, 7, 3 EBs respectively) using 780 nm excitation. Color bar indicates lifetime values for τm. Detailed statistics in text and Figure S2. Scale bars = 50µm. C. Background subtracted fluorescence levels were normalized by the mean background subtracted intensity per pixel of the mESCs for that day. For this, and all subsequent figures, bars are mean ± standard deviation. Horizontal lines indicate statistical difference (P<0.05) between normalized EB intensity values (Dunn’s method pairwise multiple comparison following ANOVA). D. Quantitation of lifetime values for τm of NADH (780 nm excitation). Values are normalized to corresponding mean mESC lifetime values. Horizontal lines indicate statistical difference (P<0.05) between normalized mEB lifetime values (Dunn’s method pairwise multiple comparison following ANOVA).

Jayne M. Squirrell, et al. PLoS One. 2012;7(8):e43708.

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