Combination image flow cytometry for single-cell analysis reveals novel methods for isolating subsets of megakaryocyte progenitor populations

the progenitor (CMRP), the progenitor these on thrombopoietin (TPO)/c-Mpl the hematopoietic TPO/c-Mpl signaling essential self-renewal, survival, proliferation Commitment down the MK lineage characterized an increase in cell DNA upregulation of MK-specific

these limitations, enabling the categorical identification of rare cellular events within heterogeneous cell 23 populations.

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The principle goal of this study is to verify the phenotypic identity of rare cell populations initially

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HSCs and MKs are extremely rare cell types that constitute approximately 0.5% and 0.01% of the 20 mouse BM, respectively, presenting complications for large-scale studies 19,20 .

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Here, we quantified the effect of TPO on HSCs, MK progenitors, and mature MKs stained with two 23 different antibody panels (Supplementary Table 1

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indicating an MPP that dies or differentiates but cannot self-renew. R3 cells proliferate massively in 36 response to TPO and upregulate CD45, CD41, and CD42d expression. This demonstrates an initial 1 CMRP phenotype that can self-renew or directly gives rise to MKRPs after culture with TPO. R4 cells 2 emerge as a true subpopulation after TPO stimulation and are marked by low surface marker 3 expression and DNA content, indicating an MERPs that can self-renew in response to TPO but have 4 low MK production potential. R5 cells are CD45 -CD41 low CD42dand lack nuclei, which is typical of 5 maturing reticulocytes. R6 cells are CD45 -/low CD41 + CD42d + cells with low DNA content, exemplary of 6 an LT-HSC-derived MKRP that can self-renew but has not yet undergone endoreplication to increase 7 ploidy 9,10,12 . R7 cells are enriched after TPO stimulation, express the highest levels of all surface 8 markers and are highly ploidy, indicative of mature MKs produced through asymmetric cell division of 9 CMRPs, MERPs, or MKRPs 12 , respectively ( Supplementary Fig. 9).  Fig. 12).

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The methodology presented here has enabled the discovery of a previously unidentified MK progenitor 24 cell population using minimal antibody panels. This discovery can significantly accelerate the      Data from image flow cytometry experiments was acquired by gating for events that were in focus, did 3 not contain calibration focus beads, and excluded viability dyes (Supplemental Fig. 1,6). All 4 experiments used the ImageStream®X Mark II Imaging Flow Cytometer and all analysis was performed 5 using the IDEAS® Software.

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For the LSK stain, mean fluorescence intensity (MFI) values were calculated by plotting live cell 8 intensity of Lineage Cocktail, Sca1, and cKit staining, then taking the average value of the plot 9 (Supplemental Fig. 2). Further analysis of live cells by subgating for Lineage Negative cells resulted in 10 Sca1 vs. cKit intensity plots (Supplemental Fig. 1, Fig. 2). The shifts in the four subpopulations -R1, 11 R2, R3, and R4, respectively -were quantified by calculating the percentage of cells in each 12 subpopulation before and after exposure to TPO (Supplemental Fig. 3). Additional image files for each 13 of the four subpopulations were chosen based on surface marker expression and image quality 14 (Supplemental Fig. 4). The IDEAS® Software was used to calculate the circularity of brightfield images.

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Plots of Brightfield Circularity vs. Area were generated before and after TPO exposure for each of the 16 four subpopulations and overlaid (Supplemental Fig. 5a for each subpopulation were overlaid and averages were taken to produce MFI graphs (Supplemental 27 Fig. 9). The IDEAS® Software was used to calculate the circularity of brightfield images. Plots of 28 brightfield circularity vs. area were generated before and after TPO exposure for each of the four 29 subpopulations and overlaid (Supplemental Fig. 10a,b). Mean brightfield circularity vs. area scores