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It’s Now Easy to go with the Flow with Mouse Stem Cells - Cell-Surface Proteomics Identifies Lineage-Specific Markers of Embryo-Derived Stem Cells



From Developmental Cell
By Stuart P. Atkinson

Protocols for the reprogramming of somatic cells to induced pluripotent stem cells (iPSCs) and the differentiation of pluripotent cells such as iPSCs and embryonic stem cells (ESCs) have taken great strides forward in recent times, increasing the efficiency and the quality of the desired cell product. However, the isolation of cells after reprogramming or differentiation is a major problem, as pure populations of cells are required to better understand such cells and also, more importantly, if they are to be used in for regenerative medicine. Isolation of cells is such a problem as there is a lack of validated cell-surface markers for flow cytometric analysis. However, researchers from the groups of Janet Rossant and Thomas Kislinger have analysed recently the cell surface proteomes of multiple mouse stem cell populations and have uncovered cell-surface protein patterns which allow for the unambiguous separation of different stem cell types from in vitro  and in vivo sources (Rugg-Gunn, Cox and Lanner).

Treatment of mouse embryonic stem cells (ESC), trophoblast stem cells (TE), EpiSCs (epiblast stem cell) and extraembryonic endodermal cells (XEN) with sulfo-NHS-SS-biotin led to the biotinylation of surface proteins allowing the capture of cell-membrane-enriched protein samples. This led to the identification of 3,432 protein in total (1,758 for ES, 2,391 for TS, 2,442 for XEN and 2,169 for EpiSC) and using a stringent data mining model 551 of these proteins were identified as being localized at the cell surface (220 for ES, 222 for TS, 212 for XEN and 416 for EpiSC), with a strong enrichment for functional classes that are characteristic of cell-surface proteins, including signaling receptors, cell adhesion and cell migration molecules. While protein analysis correlated to RNA analysis for many factors, there also existed a larger set of proteins for which RNA and protein abundance correlation was poor; while RNA levels were similar between cell lines, protein abundance varied widely (e.g. discordance between RNA and protein for ES and TS cells occurred for 89/143 (62%) cell-surface proteins). Overall comparisons between RNA and protein for these cell surface markers demonstrated that only a minority (12%) of these factors would have been identified by RNA alone.

Cell-surface protein expression analysis also identified some unique markers for the cell lines utilised. Comparison between ES, TS and XEN cells revealed 71 cell-surface proteins unique to ES cells, 74 to TS cells and 66 to XEN cells while comparison between ES cells and EpiSC revealed 60 cell-surface proteins unique to ES cells and 256 to EpiSC. This knowledge has obvious applications in the unambiguous detection of specific cell types during ESC culturing, differentiation and iPSC reprogramming, and so this was further investigated.   Of 52 commercially available antibodies to the identified cell-surface proteins, 27 revealed the expected cell-specific cell-surface expression (while the other 25 gave no signal or multiple bands in western analysis). Interestingly, only 2 had been previously identified (Pecam1 in EPI/ES cells and Pdgfra in PE/XEN cells (Plusa et al, Robson et al, Vittet et al)). Of these 25, 9 gave excellent results in FACs based cell separation; Pecam1, Cd81 antigen, and Pvrl3 for ES cells; Cdcp1 and Cd40 antigen for TS cells; Pdgfra, Dpp4, and Robo2 for XEN cells; Cd40 antigen and Cd47 antigen for EpiSC and could be used to separate a mixed population of ES, TS and XEN cells.

This knowledge was then applied to understanding cell-surface changes during cell fate conversion. ESC to XEN cell conversion through the overexpression of the PE transcription factor Sox17 (Niakan et al, Qu et al and Shimoda et al) was analysed using Pecam1, Cd81, and Pvrl3 for ES cells; Dpp4, Pdgfra, and Robo2 for XEN cells, with complete conversion of cell phenotype observed within 8–12 days. Of further interest was the absence of all ESC/XEN markers at day 4 in one third of all the cells suggesting that an initial step in the differentiation process is the downregulation of ES cell proteins and this event precedes upregulation of XEN cell proteins. Next, the inter-conversion of ESC and EpiSC was studied, particularly interesting as no cell-surface markers have been previously shown to functionally isolate the two stem cell types from each other and instead previous reports have relied on transgene expression or cell morphology. Proteomic analysis and subsequent antibody-mediated validation identified nine cell-specific membrane proteins that are expressed by either ES cells or EpiSC: Pecam1, Pvrl3, and Cd81 antigen for ES cells; Notch3, Cd40 antigen, Cdh10, Sirpa, Cd47 antigen, and Cdh2 for EpiSC. This is of particular use in the inter-conversion of EpiSC to ESC which has a very low efficiency requiring accurate detection and isolation (Guo et al).

The next important step is to understand if these cell-surface markers are also expressed in a cell-specific manner in vivo and whether they can be subsequently used to isolate and properly characterise cells arising from the blastocyst. At embryonic day 4.5 (E.45), ESC markers (Pecam1, Cd81, Plxna4 and Pvrl3) were specifically localised to the epiblast, XEN markers (Pdgfra and Dpp4) were specifically localised to the primitive endoderm and TS markers (Cdcp1, Ggt1 and Scarb1) were specifically localised to the trophoectoderm. At E5.5, the XEN marker Robo2 was expressed in parietal endodermal cells, which are derived from the primitive endoderm and the TS marker Cd40 was expressed throughout the trophoblast. Cd40 also marks for EpiSCs, alongside Sirpa, Notch3, Cdh2 and Cd47, and are found localised to the epiblast at this stage, while ESC markers (Pecam1, Plxna4 and Pvrl3) were not detected, demonstrating that the markers discovered are able to distinguish between cells in vivo. This was further examined through the prospective isolation of lineages from E4.5 blastocysts by FACs after single cell dissociation. An unbiased computational analysis of the flow data demonstrated the existence of three distinct cell populations; epiblast, trophectoderm and primitive endoderm indicating that each cell lineage had been successfully isolated, thereby representing a significant advance in our ability to analyze specific cell types within the early embryo. This was also further confirmed by gene expression analysis using the BioMark Fluidigm System. Importantly, the sorted cells were deemed viable (after 96 hours in culture) allowing for functional assays, with each distinct population of sorted cells giving rise to the appropriate stem cell lineage (ESCs from the Pecam1 population, XEN cells from the Pdgfra population and TS cells from the Cdcp1 population).

Overall this research suggests that this direct proteomic approach for protein marker discovery is more reliable than RNA expression for providing predictions of cell-type. Through this approach specific cell-surface protein combinations have been identified which can separate populations of cell types into their individual lineages, allowing the accurate detection and isolation of specific cell populations without the need to use transgenic reporter cell lines. Further, the identification of these markers in the embryo and the ability to sort these cells after embryo dissociation further provide validation for the future use of these markers. However, detailed studies of human cells in such a way as delineated in this exciting research will also be required to be undertaken, providing a potentially important resource of information for all researchers working in stem cell biology.



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