Research ArticleAPPLIED SCIENCES AND ENGINEERING

High-throughput intracellular biopsy of microRNAs for dissecting the temporal dynamics of cellular heterogeneity

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Science Advances  10 Jun 2020:
Vol. 6, no. 24, eaba4971
DOI: 10.1126/sciadv.aba4971
  • Fig. 1 Design of the inCell-Biopsy technique for miRNA profiling.

    (A) Schematic illustration of intracellular biopsy of miRNAs from live cells. (B) On-needle amplification of miRNA signals by hybridization chain reaction (HCR). (C) DNase-assisted multiple rounds of signal visualization. (D) Image processing and informatic approaches for miRNA transcriptome analysis.

  • Fig. 2 Technical characterization of inCell-biopsy.

    (A) SEM image of the diamond nanoneedles; scale bar, 50 μm. (B) Fluorescence images (top view) showing miRNA signals (red) on the nanoneedles (green); scale bar, 50 μm. For (A) and (B), the boxed region is enlarged below; scale bars, 10 μm. (C) Analysis of detection limit with violin plots showing the distribution of miRNA signals from all nanoneedles. *P < 0.005 by Kruskal-Wallis test. a.u., arbitrary units. (D) Relationship between miRNA concentration and fluorescence averaged from all nanoneedles. The red line indicates logarithmic fit (R2 = 0.99, P < 0.001 by F test). (E) Relationship between miRNA concentration and ratio of signal+ nanoneedles. The blue dashed line indicates a nonlinear fit by Langmuir isotherm model (R2 = 0.98, P < 0.001 in F test). (F) Analysis of detection specificity. NC, no target included. *P < 0.001 by ANOVA test. (G) Image of A549 cells after treatment; scale bar, 50 μm. (H) Fluorescence visualization of miRNAs (red, let-7a or miR-34a) on the nanoneedles (green); scale bars, 10 μm for three-dimensional and top view, 1 μm for enlarged view. (I) Comparison of miRNA (let-7a or miR-34a) signals from different controls. (J) Ratio of signal+ nanoneedles for experiments with or without HCR amplification. For (D), (E), (I), and (J), n = 3; the error bar indicates SEM; *P < 0.001 by ANOVA test.

  • Fig. 3 Temporal profiling of miRNA by inCell-biopsy.

    (A) Experimental design of monitoring miRNA dynamics over the differentiation of embryonic stem cells (ESCs). RA, retinoic acid; and SAG, smoothened agonist. (B) Phase (Ph)–contrast and fluorescence (green fluorescence indicates GFP) images showing morphological change of the cells along with differentiation. Scale bars, 50 μm. (C) Confocal fluorescence image (top view) of diamond nanoneedles after fishing and HCR amplification. Scale bar, 20 μm. The boxed region is enlarged below to show the expression of nine miRNAs from three rounds of amplification and visualization. Scale bars, 2 μm. (D) t-SNE clustering of the pooled multidimensional miRNA vectors that resulted from inCell-biopsy at all three stages, showing the overall evolution of miRNA expression along with ESC differentiation.

  • Fig. 4 The dynamic evolution of mESC heterogeneity revealed by inCell-biopsy.

    Self-diffusion–based spectral clustering and associated similarity network for the multidimensional miRNA measurements from thousands of nanoneedles at day 7 (A) or day 14 (B) of differentiation. (C) Separation of cellular subpopulations indicated by t-SNE analysis at day 7 or day 14. Heatmap showing distinct miRNA expression patterns between different clusters obtained from unsupervised classification at day 7 (D) or day 14 (E). (F) Sector graph showing the proportion of nanoneedles in each cluster out of the total number of nanoneedles on day 7 or day 14. Radar plots (left) and associated violin plots (right) show the averaged expression of the nine miRNAs for each of the identified clusters at day 7 (G) or day 14 (H).

  • Fig. 5 Mapping of miRNA transcriptome to cellular composition.

    (A) Hypergeometric tests for determining the closest pair of clusters from the two differentiation stages. Significant associations are labeled by red squares, colored in proportion to −log10(P value) (all P < 0.001). (B) The phylogenetic tree shows the evolutionary relationship among the clusters (cell subpopulations) as differentiation proceeds. The widths of the branches are proportionate to transformed P values [−log10(P values)] derived from hypergeometric tests. (C) Correlation between the cluster miRNA pattern (derived from inCell-biopsy) with the results acquired by miR-seq for sorted motor neurons/progenitors. *P < 0.001. (D) Quantitative analysis of the averaged expression of the nine miRNAs for motor neuron–like clusters at day 7 and day 14. Error bars indicate SEM from six independent experiments. (E) Density histograms and associated violin plots showing the variation and distribution of nine miRNAs expression for motor neuron–like clusters at day 7 and day 14.

Supplementary Materials

  • Supplementary Materials

    High-throughput intracellular biopsy of microRNAs for dissecting the temporal dynamics of cellular heterogeneity

    Zixun Wang, Lin Qi, Yang Yang, Mingxing Lu, Kai Xie, Xi Zhao, Elvis Hung Chi Cheung, Yuan Wang, Xuezhen Jiang, Wenjun Zhang, Linfeng Huang, Xin Wang, Peng Shi

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