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CD24 tracks divergent pluripotent states in mouse and human cells.

Shakiba N, White CA, Lipsitz YY, Yachie-Kinoshita A, Tonge PD, Hussein SM, Puri MC, Elbaz J, Morrissey-Scoot J, Li M, Munoz J, Benevento M, Rogers IM, Hanna JH, Heck AJ, Wollscheid B, Nagy A, Zandstra PW - Nat Commun (2015)

Bottom Line: Reprogramming is a dynamic process that can result in multiple pluripotent cell types emerging from divergent paths.Cell surface protein expression is a particularly desirable tool to categorize reprogramming and pluripotency as it enables robust quantification and enrichment of live cells.Thus, CD24 is a conserved marker for tracking divergent states in both reprogramming and standard pluripotent culture.

View Article: PubMed Central - PubMed

Affiliation: Institute of Biomaterials and Biomedical Engineering (IBBME), University of Toronto, Toronto, Ontario, Canada M5S 3E1.

ABSTRACT
Reprogramming is a dynamic process that can result in multiple pluripotent cell types emerging from divergent paths. Cell surface protein expression is a particularly desirable tool to categorize reprogramming and pluripotency as it enables robust quantification and enrichment of live cells. Here we use cell surface proteomics to interrogate mouse cell reprogramming dynamics and discover CD24 as a marker that tracks the emergence of reprogramming-responsive cells, while enabling the analysis and enrichment of transgene-dependent (F-class) and -independent (traditional) induced pluripotent stem cells (iPSCs) at later stages. Furthermore, CD24 can be used to delineate epiblast stem cells (EpiSCs) from embryonic stem cells (ESCs) in mouse pluripotent culture. Importantly, regulated CD24 expression is conserved in human pluripotent stem cells (PSCs), tracking the conversion of human ESCs to more naive-like PSC states. Thus, CD24 is a conserved marker for tracking divergent states in both reprogramming and standard pluripotent culture.

No MeSH data available.


Related in: MedlinePlus

Surface proteome analysis during reprogramming identifies CD24 as a differentially expressed surface marker.(a) Principal component analysis of the surface proteome (including only the subset of surface proteins that were also present in the global proteome screen to improve rigour of quantitative analysis) of reprogramming secondary MEF 1B cells derived from tetraploid complementation, showing divergent routes of F-class and ESC-like iPSCs. (b) Summary of sampling time course and DOX treatment protocols. (c) Representative flow cytometry plots of CD24 versus SSEA1 expression during reprogramming of secondary MEF 1B cells derived from tetraploid complementation, revealing emerging CD24high/SSEA1+ (CD24H) and CD24low/SSEA1+ (CD24L) subpopulations. ESC control is included for comparison. Flow plots are representative from three biological replicates.
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f1: Surface proteome analysis during reprogramming identifies CD24 as a differentially expressed surface marker.(a) Principal component analysis of the surface proteome (including only the subset of surface proteins that were also present in the global proteome screen to improve rigour of quantitative analysis) of reprogramming secondary MEF 1B cells derived from tetraploid complementation, showing divergent routes of F-class and ESC-like iPSCs. (b) Summary of sampling time course and DOX treatment protocols. (c) Representative flow cytometry plots of CD24 versus SSEA1 expression during reprogramming of secondary MEF 1B cells derived from tetraploid complementation, revealing emerging CD24high/SSEA1+ (CD24H) and CD24low/SSEA1+ (CD24L) subpopulations. ESC control is included for comparison. Flow plots are representative from three biological replicates.

Mentions: Consistent with the other ‘omic' analyses conducted as part of the Project Grandiose reprogramming analysis10, principal component analysis of surface proteins depicted the DOX-high time course diverging from an ESC-like state to a separate state, whereas the DOX-low time course converges on the ESC-like state (Fig. 1a). The divergence suggests the possibility of capturing an alternative reprogramming state, F-class9, by means of surface proteomics. We then conducted subsequent analysis to select surface proteins for additional scrutiny and validation (Supplementary Fig. 2). First, we conducted K-means clustering analysis to select genes that are differentially expressed in the F-class state, identifying two clusters of genes that are upregulated following DOX induction and maximally expressed at the F-class state (Supplementary Fig. 2). Of these genes, we selected those with the highest contribution to the principal components separating the F-class and ESC states as well as F-class and MEF states. This produced a list of genes, of which many were metabolic or neural markers. A handful of proteins were chosen based on this analysis as well as antibody availability (Supplementary Fig. 3) for validation along the reprogramming time course (Fig. 1b), which revealed CD24 as the best candidate based on its ability to differentiate between emerging F-class and ESC-like iPSCs (Fig. 1c). We next proceeded with a detailed characterization of CD24 expression during reprogramming.


CD24 tracks divergent pluripotent states in mouse and human cells.

Shakiba N, White CA, Lipsitz YY, Yachie-Kinoshita A, Tonge PD, Hussein SM, Puri MC, Elbaz J, Morrissey-Scoot J, Li M, Munoz J, Benevento M, Rogers IM, Hanna JH, Heck AJ, Wollscheid B, Nagy A, Zandstra PW - Nat Commun (2015)

Surface proteome analysis during reprogramming identifies CD24 as a differentially expressed surface marker.(a) Principal component analysis of the surface proteome (including only the subset of surface proteins that were also present in the global proteome screen to improve rigour of quantitative analysis) of reprogramming secondary MEF 1B cells derived from tetraploid complementation, showing divergent routes of F-class and ESC-like iPSCs. (b) Summary of sampling time course and DOX treatment protocols. (c) Representative flow cytometry plots of CD24 versus SSEA1 expression during reprogramming of secondary MEF 1B cells derived from tetraploid complementation, revealing emerging CD24high/SSEA1+ (CD24H) and CD24low/SSEA1+ (CD24L) subpopulations. ESC control is included for comparison. Flow plots are representative from three biological replicates.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4490408&req=5

f1: Surface proteome analysis during reprogramming identifies CD24 as a differentially expressed surface marker.(a) Principal component analysis of the surface proteome (including only the subset of surface proteins that were also present in the global proteome screen to improve rigour of quantitative analysis) of reprogramming secondary MEF 1B cells derived from tetraploid complementation, showing divergent routes of F-class and ESC-like iPSCs. (b) Summary of sampling time course and DOX treatment protocols. (c) Representative flow cytometry plots of CD24 versus SSEA1 expression during reprogramming of secondary MEF 1B cells derived from tetraploid complementation, revealing emerging CD24high/SSEA1+ (CD24H) and CD24low/SSEA1+ (CD24L) subpopulations. ESC control is included for comparison. Flow plots are representative from three biological replicates.
Mentions: Consistent with the other ‘omic' analyses conducted as part of the Project Grandiose reprogramming analysis10, principal component analysis of surface proteins depicted the DOX-high time course diverging from an ESC-like state to a separate state, whereas the DOX-low time course converges on the ESC-like state (Fig. 1a). The divergence suggests the possibility of capturing an alternative reprogramming state, F-class9, by means of surface proteomics. We then conducted subsequent analysis to select surface proteins for additional scrutiny and validation (Supplementary Fig. 2). First, we conducted K-means clustering analysis to select genes that are differentially expressed in the F-class state, identifying two clusters of genes that are upregulated following DOX induction and maximally expressed at the F-class state (Supplementary Fig. 2). Of these genes, we selected those with the highest contribution to the principal components separating the F-class and ESC states as well as F-class and MEF states. This produced a list of genes, of which many were metabolic or neural markers. A handful of proteins were chosen based on this analysis as well as antibody availability (Supplementary Fig. 3) for validation along the reprogramming time course (Fig. 1b), which revealed CD24 as the best candidate based on its ability to differentiate between emerging F-class and ESC-like iPSCs (Fig. 1c). We next proceeded with a detailed characterization of CD24 expression during reprogramming.

Bottom Line: Reprogramming is a dynamic process that can result in multiple pluripotent cell types emerging from divergent paths.Cell surface protein expression is a particularly desirable tool to categorize reprogramming and pluripotency as it enables robust quantification and enrichment of live cells.Thus, CD24 is a conserved marker for tracking divergent states in both reprogramming and standard pluripotent culture.

View Article: PubMed Central - PubMed

Affiliation: Institute of Biomaterials and Biomedical Engineering (IBBME), University of Toronto, Toronto, Ontario, Canada M5S 3E1.

ABSTRACT
Reprogramming is a dynamic process that can result in multiple pluripotent cell types emerging from divergent paths. Cell surface protein expression is a particularly desirable tool to categorize reprogramming and pluripotency as it enables robust quantification and enrichment of live cells. Here we use cell surface proteomics to interrogate mouse cell reprogramming dynamics and discover CD24 as a marker that tracks the emergence of reprogramming-responsive cells, while enabling the analysis and enrichment of transgene-dependent (F-class) and -independent (traditional) induced pluripotent stem cells (iPSCs) at later stages. Furthermore, CD24 can be used to delineate epiblast stem cells (EpiSCs) from embryonic stem cells (ESCs) in mouse pluripotent culture. Importantly, regulated CD24 expression is conserved in human pluripotent stem cells (PSCs), tracking the conversion of human ESCs to more naive-like PSC states. Thus, CD24 is a conserved marker for tracking divergent states in both reprogramming and standard pluripotent culture.

No MeSH data available.


Related in: MedlinePlus