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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

CD24H and CD24L subpopulations correspond to transgene-dependent (F-class) and ESC-like iPSCs, respectively.(a) Summary of approach to characterize CD24H and CD24L populations. (b) Percentage of CD24H and CD24L cells in DOXH, DOXL− and DOXH− culture time courses. Data bars show mean±s.d. (n=3 biological replicates). (c) Representative phase contrast images (n=3 biological replicates) of emerging colonies in the three DOX treatments, including ESC control. Scale bar, 125 μm. (d) Summary of sorting strategy used to separate CD24H and CD24L subpopulations from D30 culture. Representative phase contrast images (n=3 technical replicates) of sorted CD24H and CD24L cells in DOXH and DOX− conditions. Scale bar, 125 μm. (e) Effect of long-term passaging on levels of CD24H and CD24L cells in D30-sorted DOXH CD24H, DOXL− CD24L and DOXH− CD24L cells. ESCs are included as a control. Data bars show mean±s.d. (n=3 technical replicates). (f) EdU staining of DOXH CD24H, DOXL− CD24L and DOXH− CD24L cells in DOXH and DOX− conditions to assess DOX dependence of proliferation. Data bars show mean±s.d. (n=3 technical replicates). (g) Expression levels of various pluripotent and F-class-specific genes in CD24H and CD24L cells with unsupervised hierarchical clustering, including F-class and ESC cell controls, normalized to Gapdh and MEF cells.
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f2: CD24H and CD24L subpopulations correspond to transgene-dependent (F-class) and ESC-like iPSCs, respectively.(a) Summary of approach to characterize CD24H and CD24L populations. (b) Percentage of CD24H and CD24L cells in DOXH, DOXL− and DOXH− culture time courses. Data bars show mean±s.d. (n=3 biological replicates). (c) Representative phase contrast images (n=3 biological replicates) of emerging colonies in the three DOX treatments, including ESC control. Scale bar, 125 μm. (d) Summary of sorting strategy used to separate CD24H and CD24L subpopulations from D30 culture. Representative phase contrast images (n=3 technical replicates) of sorted CD24H and CD24L cells in DOXH and DOX− conditions. Scale bar, 125 μm. (e) Effect of long-term passaging on levels of CD24H and CD24L cells in D30-sorted DOXH CD24H, DOXL− CD24L and DOXH− CD24L cells. ESCs are included as a control. Data bars show mean±s.d. (n=3 technical replicates). (f) EdU staining of DOXH CD24H, DOXL− CD24L and DOXH− CD24L cells in DOXH and DOX− conditions to assess DOX dependence of proliferation. Data bars show mean±s.d. (n=3 technical replicates). (g) Expression levels of various pluripotent and F-class-specific genes in CD24H and CD24L cells with unsupervised hierarchical clustering, including F-class and ESC cell controls, normalized to Gapdh and MEF cells.

Mentions: In order to comprehensively compare CD24H and CD24L cells to F-class/ESC-like iPSCs, we chose the following criteria to characterize the newly defined cells (Fig. 2a): CD24/SSEA1 expression levels compared with ESC controls; morphology in native culture; morphology in response to DOX removal; stability following extensive passaging; dependence of proliferation on DOX; and gene expression profile.


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)

CD24H and CD24L subpopulations correspond to transgene-dependent (F-class) and ESC-like iPSCs, respectively.(a) Summary of approach to characterize CD24H and CD24L populations. (b) Percentage of CD24H and CD24L cells in DOXH, DOXL− and DOXH− culture time courses. Data bars show mean±s.d. (n=3 biological replicates). (c) Representative phase contrast images (n=3 biological replicates) of emerging colonies in the three DOX treatments, including ESC control. Scale bar, 125 μm. (d) Summary of sorting strategy used to separate CD24H and CD24L subpopulations from D30 culture. Representative phase contrast images (n=3 technical replicates) of sorted CD24H and CD24L cells in DOXH and DOX− conditions. Scale bar, 125 μm. (e) Effect of long-term passaging on levels of CD24H and CD24L cells in D30-sorted DOXH CD24H, DOXL− CD24L and DOXH− CD24L cells. ESCs are included as a control. Data bars show mean±s.d. (n=3 technical replicates). (f) EdU staining of DOXH CD24H, DOXL− CD24L and DOXH− CD24L cells in DOXH and DOX− conditions to assess DOX dependence of proliferation. Data bars show mean±s.d. (n=3 technical replicates). (g) Expression levels of various pluripotent and F-class-specific genes in CD24H and CD24L cells with unsupervised hierarchical clustering, including F-class and ESC cell controls, normalized to Gapdh and MEF cells.
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f2: CD24H and CD24L subpopulations correspond to transgene-dependent (F-class) and ESC-like iPSCs, respectively.(a) Summary of approach to characterize CD24H and CD24L populations. (b) Percentage of CD24H and CD24L cells in DOXH, DOXL− and DOXH− culture time courses. Data bars show mean±s.d. (n=3 biological replicates). (c) Representative phase contrast images (n=3 biological replicates) of emerging colonies in the three DOX treatments, including ESC control. Scale bar, 125 μm. (d) Summary of sorting strategy used to separate CD24H and CD24L subpopulations from D30 culture. Representative phase contrast images (n=3 technical replicates) of sorted CD24H and CD24L cells in DOXH and DOX− conditions. Scale bar, 125 μm. (e) Effect of long-term passaging on levels of CD24H and CD24L cells in D30-sorted DOXH CD24H, DOXL− CD24L and DOXH− CD24L cells. ESCs are included as a control. Data bars show mean±s.d. (n=3 technical replicates). (f) EdU staining of DOXH CD24H, DOXL− CD24L and DOXH− CD24L cells in DOXH and DOX− conditions to assess DOX dependence of proliferation. Data bars show mean±s.d. (n=3 technical replicates). (g) Expression levels of various pluripotent and F-class-specific genes in CD24H and CD24L cells with unsupervised hierarchical clustering, including F-class and ESC cell controls, normalized to Gapdh and MEF cells.
Mentions: In order to comprehensively compare CD24H and CD24L cells to F-class/ESC-like iPSCs, we chose the following criteria to characterize the newly defined cells (Fig. 2a): CD24/SSEA1 expression levels compared with ESC controls; morphology in native culture; morphology in response to DOX removal; stability following extensive passaging; dependence of proliferation on DOX; and gene expression profile.

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