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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

CD24 delineates 'primed' and 'naive' pluripotent states in human cells.(a) Flow cytometry analysis of CD24 expression in ‘primed' and ‘naive' human ESC lines following passages 6 and 10 in naive conditions. Flow plots are representative from three technical replicates. (b) CD24/Tra-1-60 staining of primed hES2 and naive-induced hES2 and H9 hESCs at indicated number of passages in naive conditions. Flow plots are representative from three technical replicates. (c) Gene expression analysis of CD24high/Tra-1-60+ and CD24low/Tra-1-60+ cells sorted from primed and naive culture at indicated passage number with unsupervised hierarchical clustering. Expression data are normalized to GAPDH and primed hES2 cells. (d) Gene expression analysis comparing unsorted, CD24H-sorted and CD24L-sorted hES2 cells. Expression data are normalized to GAPDH and primed hES2 cells. Statistical significance is assessed by a Student's t-test (heteroscedastic, two-sided). **P<0.05, *P<0.1. Data bars show mean±s.d. (n=3 technical replicates).
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f4: CD24 delineates 'primed' and 'naive' pluripotent states in human cells.(a) Flow cytometry analysis of CD24 expression in ‘primed' and ‘naive' human ESC lines following passages 6 and 10 in naive conditions. Flow plots are representative from three technical replicates. (b) CD24/Tra-1-60 staining of primed hES2 and naive-induced hES2 and H9 hESCs at indicated number of passages in naive conditions. Flow plots are representative from three technical replicates. (c) Gene expression analysis of CD24high/Tra-1-60+ and CD24low/Tra-1-60+ cells sorted from primed and naive culture at indicated passage number with unsupervised hierarchical clustering. Expression data are normalized to GAPDH and primed hES2 cells. (d) Gene expression analysis comparing unsorted, CD24H-sorted and CD24L-sorted hES2 cells. Expression data are normalized to GAPDH and primed hES2 cells. Statistical significance is assessed by a Student's t-test (heteroscedastic, two-sided). **P<0.05, *P<0.1. Data bars show mean±s.d. (n=3 technical replicates).

Mentions: In order to test this in vitro we cultured hESC lines in two conditions: standard hESC culture and culture supplemented with conditions reported to induce hESCs to an alternative naive-like state11. Flow cytometry analysis of these cells confirmed that CD24 expression is lowered in naive-like hESC states, achieving levels equivalent with mouse ESCs by passage 10 of culture in so-called naive conditions11 (Fig. 4a). Further development of our naive induction strategy led us to note a dependency of CD24 downregulation on levels of LDN in our naive induction protocol (Supplementary Fig. 16a). To ensure that CD24 was indeed a reliable marker for the primed-to-naive-like state transition, we removed LDN from subsequent experiments. hESCs were co-stained for CD24/Tra-1-60 (analogous to mouse CD24/SSEA1 staining) following changes in primed-to-naive associated culture conditions (Fig. 4b). As before, an observable (although reduced in size) CD24low/Tra-1-60+ fraction emerged. Analysis of Oct4/Sox2 expression in naive-like cells revealed maintenance of pluripotency (Supplementary Fig. 16b). Our observation was further supported by CD24 expression data extracted from a recent study by Theunissen et al.32, reporting generation of naive hESCs without LDN (Supplementary Fig. 15d). Characterization of our naive hESCs revealed that they exhibited gene expression levels consistent with previous reports113233. Furthermore, gene expression analysis revealed that sorted CD24high/Tra-1-60+ (hereafter called CD24H) cells and CD24low/Tra-1-60+ (hereafter called CD24L) cells clustered distinctly (Fig. 4c). Interestingly, CD24H cells derived from primed and naive-treated cultures clustered together, exhibiting especially low levels of Tbx3 expression (Fig. 4c,d) and expectedly higher levels of CD24a expression (Supplementary Fig. 16c). In addition, these CD24H cells clustered with the other primed cells. On the other hand, CD24L cells from naive culture clustered with unsorted naive-treated cells. Most notably, while unsorted naive hESCs derived here showed enrichment for naive markers Klf2, Tbx3, Otx2, Dnmt3a, LIF-R and Rex1, CD24L-sorted naive cells showed further enrichment for naive markers Stella, E-cadherin, Klf5 and Klf4 (Fig. 4d). This analysis may suggest heterogeneity in these naive hESC cultures, and support CD24 as a powerful tool to resolve this heterogeneity and enrich for hPSCs in different pluripotent states.


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)

CD24 delineates 'primed' and 'naive' pluripotent states in human cells.(a) Flow cytometry analysis of CD24 expression in ‘primed' and ‘naive' human ESC lines following passages 6 and 10 in naive conditions. Flow plots are representative from three technical replicates. (b) CD24/Tra-1-60 staining of primed hES2 and naive-induced hES2 and H9 hESCs at indicated number of passages in naive conditions. Flow plots are representative from three technical replicates. (c) Gene expression analysis of CD24high/Tra-1-60+ and CD24low/Tra-1-60+ cells sorted from primed and naive culture at indicated passage number with unsupervised hierarchical clustering. Expression data are normalized to GAPDH and primed hES2 cells. (d) Gene expression analysis comparing unsorted, CD24H-sorted and CD24L-sorted hES2 cells. Expression data are normalized to GAPDH and primed hES2 cells. Statistical significance is assessed by a Student's t-test (heteroscedastic, two-sided). **P<0.05, *P<0.1. Data bars show mean±s.d. (n=3 technical replicates).
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f4: CD24 delineates 'primed' and 'naive' pluripotent states in human cells.(a) Flow cytometry analysis of CD24 expression in ‘primed' and ‘naive' human ESC lines following passages 6 and 10 in naive conditions. Flow plots are representative from three technical replicates. (b) CD24/Tra-1-60 staining of primed hES2 and naive-induced hES2 and H9 hESCs at indicated number of passages in naive conditions. Flow plots are representative from three technical replicates. (c) Gene expression analysis of CD24high/Tra-1-60+ and CD24low/Tra-1-60+ cells sorted from primed and naive culture at indicated passage number with unsupervised hierarchical clustering. Expression data are normalized to GAPDH and primed hES2 cells. (d) Gene expression analysis comparing unsorted, CD24H-sorted and CD24L-sorted hES2 cells. Expression data are normalized to GAPDH and primed hES2 cells. Statistical significance is assessed by a Student's t-test (heteroscedastic, two-sided). **P<0.05, *P<0.1. Data bars show mean±s.d. (n=3 technical replicates).
Mentions: In order to test this in vitro we cultured hESC lines in two conditions: standard hESC culture and culture supplemented with conditions reported to induce hESCs to an alternative naive-like state11. Flow cytometry analysis of these cells confirmed that CD24 expression is lowered in naive-like hESC states, achieving levels equivalent with mouse ESCs by passage 10 of culture in so-called naive conditions11 (Fig. 4a). Further development of our naive induction strategy led us to note a dependency of CD24 downregulation on levels of LDN in our naive induction protocol (Supplementary Fig. 16a). To ensure that CD24 was indeed a reliable marker for the primed-to-naive-like state transition, we removed LDN from subsequent experiments. hESCs were co-stained for CD24/Tra-1-60 (analogous to mouse CD24/SSEA1 staining) following changes in primed-to-naive associated culture conditions (Fig. 4b). As before, an observable (although reduced in size) CD24low/Tra-1-60+ fraction emerged. Analysis of Oct4/Sox2 expression in naive-like cells revealed maintenance of pluripotency (Supplementary Fig. 16b). Our observation was further supported by CD24 expression data extracted from a recent study by Theunissen et al.32, reporting generation of naive hESCs without LDN (Supplementary Fig. 15d). Characterization of our naive hESCs revealed that they exhibited gene expression levels consistent with previous reports113233. Furthermore, gene expression analysis revealed that sorted CD24high/Tra-1-60+ (hereafter called CD24H) cells and CD24low/Tra-1-60+ (hereafter called CD24L) cells clustered distinctly (Fig. 4c). Interestingly, CD24H cells derived from primed and naive-treated cultures clustered together, exhibiting especially low levels of Tbx3 expression (Fig. 4c,d) and expectedly higher levels of CD24a expression (Supplementary Fig. 16c). In addition, these CD24H cells clustered with the other primed cells. On the other hand, CD24L cells from naive culture clustered with unsorted naive-treated cells. Most notably, while unsorted naive hESCs derived here showed enrichment for naive markers Klf2, Tbx3, Otx2, Dnmt3a, LIF-R and Rex1, CD24L-sorted naive cells showed further enrichment for naive markers Stella, E-cadherin, Klf5 and Klf4 (Fig. 4d). This analysis may suggest heterogeneity in these naive hESC cultures, and support CD24 as a powerful tool to resolve this heterogeneity and enrich for hPSCs in different pluripotent states.

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