Limits...
A resource for discovering specific and universal biomarkers for distributed stem cells.

Noh M, Smith JL, Huh YH, Sherley JL - PLoS ONE (2011)

Bottom Line: This delineation has several significant implications.These include: 1) providing experimental evidence that DSCs in vivo undergo asymmetric self-renewal as individual cells; 2) providing an explanation why earlier attempts to define a common gene expression signature for DSCs were unsuccessful; and 3) predicting that some ASRA proteins may be ideal biomarkers for DSCs.Indeed, two ASRA proteins, CXCR6 and BTG2, and two other related self-renewal pattern associated (SRPA) proteins identified in this gene resource, LGR5 and H2A.Z, display unique asymmetric patterns of expression that have a high potential for universal and specific DSC identification.

View Article: PubMed Central - PubMed

Affiliation: School of Pharmacy, Ajou University, Suwon, South Korea.

ABSTRACT
Specific and universal biomarkers for distributed stem cells (DSCs) have been elusive. A major barrier to discovery of such ideal DSC biomarkers is difficulty in obtaining DSCs in sufficient quantity and purity. To solve this problem, we used cell lines genetically engineered for conditional asymmetric self-renewal, the defining DSC property. In gene microarray analyses, we identified 85 genes whose expression is tightly asymmetric self-renewal associated (ASRA). The ASRA gene signature prescribed DSCs to undergo asymmetric self-renewal to a greater extent than committed progenitor cells, embryonic stem cells, or induced pluripotent stem cells. This delineation has several significant implications. These include: 1) providing experimental evidence that DSCs in vivo undergo asymmetric self-renewal as individual cells; 2) providing an explanation why earlier attempts to define a common gene expression signature for DSCs were unsuccessful; and 3) predicting that some ASRA proteins may be ideal biomarkers for DSCs. Indeed, two ASRA proteins, CXCR6 and BTG2, and two other related self-renewal pattern associated (SRPA) proteins identified in this gene resource, LGR5 and H2A.Z, display unique asymmetric patterns of expression that have a high potential for universal and specific DSC identification.

Show MeSH
Evaluation of ASRA protein biomarkers by sister pair (SPr) and cytochalasin D (CD) assay.Shown are examples of fluorescent photomicrographs from parallel SPr and CD analyses using dual indirect ISIF performed simultaneously with two different biomarker-specific antibodies of different species origin. Species-specific secondary antibodies conjugated to red and green fluorochromes were used, respectively, for biomarker-specific imaging. SYM, symmetric self-renewal by p53- Con-3 cells with ZnCl2. ASYM, asymmetric self-renewal by p53-inducible Ind-8 cells with ZnCl2 to induce p53 expression. DAPI, nuclear DNA fluorescence. CyA, indirect ISIF with specific antibodies for cyclin A, an indicator for cycling late G1, S, and G2 cells with greatest expression in G2 phase. A. Dual indirect ISIF with antibodies for cyclin A and cyclin E (CyE), an indicator for cycling late G1 and S phase cells, with highest expression in early S phase. B. Dual indirect ISIF with antibodies for cyclin A and cyclin D1 (CyD1), an indicator for cycling G1 or arrested late G1 and early S phase cells, with highest expression in late G1 phase. C. Dual indirect ISIF with antibodies for cyclin D1 and p53. D. Dual indirect ISIF with antibodies for cyclin A and Btg2. E. Dual indirect ISIF with antibodies for cyclin A and Lgr5. See Table 1 for quantitative analyses for the CyA∶CyD1 and CyA∶Btg2 SPr and CD assays. Only the CD analysis was quantified for the CyA∶Lgr5 assays (n = 61, 58; and % co-asymmetric CyA and Lgr5 expression = 20%, 4% for paired nuclei in the ASYM state versus the SYM state, respectively; p<0.006 by the two-tailed Fisher's exact test). Scale bars = 50 microns.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC3139609&req=5

pone-0022077-g006: Evaluation of ASRA protein biomarkers by sister pair (SPr) and cytochalasin D (CD) assay.Shown are examples of fluorescent photomicrographs from parallel SPr and CD analyses using dual indirect ISIF performed simultaneously with two different biomarker-specific antibodies of different species origin. Species-specific secondary antibodies conjugated to red and green fluorochromes were used, respectively, for biomarker-specific imaging. SYM, symmetric self-renewal by p53- Con-3 cells with ZnCl2. ASYM, asymmetric self-renewal by p53-inducible Ind-8 cells with ZnCl2 to induce p53 expression. DAPI, nuclear DNA fluorescence. CyA, indirect ISIF with specific antibodies for cyclin A, an indicator for cycling late G1, S, and G2 cells with greatest expression in G2 phase. A. Dual indirect ISIF with antibodies for cyclin A and cyclin E (CyE), an indicator for cycling late G1 and S phase cells, with highest expression in early S phase. B. Dual indirect ISIF with antibodies for cyclin A and cyclin D1 (CyD1), an indicator for cycling G1 or arrested late G1 and early S phase cells, with highest expression in late G1 phase. C. Dual indirect ISIF with antibodies for cyclin D1 and p53. D. Dual indirect ISIF with antibodies for cyclin A and Btg2. E. Dual indirect ISIF with antibodies for cyclin A and Lgr5. See Table 1 for quantitative analyses for the CyA∶CyD1 and CyA∶Btg2 SPr and CD assays. Only the CD analysis was quantified for the CyA∶Lgr5 assays (n = 61, 58; and % co-asymmetric CyA and Lgr5 expression = 20%, 4% for paired nuclei in the ASYM state versus the SYM state, respectively; p<0.006 by the two-tailed Fisher's exact test). Scale bars = 50 microns.

Mentions: To validate the SPr and CD assays, we used three well-described cell cycle-specific biomarkers: cyclin A (CyA) as a general biomarker for cycling cells, cyclin E (CyE) as a biomarker for cycling early S phase cells, and cyclin D1 (CyD1) as a biomarker for G1 cells and, more importantly, cells arrested at G1/S of the cell cycle [45]. Fig. 6 provides examples of the major types of SPr and CD expression patterns observed for these biomarkers under conditions of symmetric self-renewal versus asymmetric self-renewal with the engineered cell lines. In SPr analyses, all three cell cycle biomarkers showed primarily symmetric expression patterns when the engineered cell lines were cultured under conditions for symmetric self-renewal (Fig. 6A and 6B, SYM, SPr). In contrast, under conditions for asymmetric self-renewal, asymmetric expression patterns increased significantly (Fig. 6A and 6B, ASYM, SPr; see also Table 1, CyA∶CyD1, SPr assay). As predicted, CyD1, the biomarker for non-cycling non-stem sisters, was consistently reciprocally asymmetric with respect to the cycling cell biomarkers (Fig. 6B, ASYM, SPr). Similar relationships were observed in parallel CD assays (Fig. 6A and 6B, CD; see also Table 1, CyA∶CyD1, CD assay), validating this second assay for use with cyclins A, E, and D1. Simultaneous detection of CyA and CyD1 was particularly effective for detecting and quantifying asymmetric self-renewal and symmetric self-renewal (Fig. 6B, ASYM; Table 1, CyA∶CyD1). When p53 was induced, it was always expressed in both sister cells or sister nuclei, despite their clear phenotypic differences (Fig. 6C, ASYM; compare p53 and CyD1). Thus, although the shift to asymmetric self-renewal from symmetric self-renewal by the engineered cells is initiated by p53 expression, it is not due to asymmetric expression of p53 between sister cells.


A resource for discovering specific and universal biomarkers for distributed stem cells.

Noh M, Smith JL, Huh YH, Sherley JL - PLoS ONE (2011)

Evaluation of ASRA protein biomarkers by sister pair (SPr) and cytochalasin D (CD) assay.Shown are examples of fluorescent photomicrographs from parallel SPr and CD analyses using dual indirect ISIF performed simultaneously with two different biomarker-specific antibodies of different species origin. Species-specific secondary antibodies conjugated to red and green fluorochromes were used, respectively, for biomarker-specific imaging. SYM, symmetric self-renewal by p53- Con-3 cells with ZnCl2. ASYM, asymmetric self-renewal by p53-inducible Ind-8 cells with ZnCl2 to induce p53 expression. DAPI, nuclear DNA fluorescence. CyA, indirect ISIF with specific antibodies for cyclin A, an indicator for cycling late G1, S, and G2 cells with greatest expression in G2 phase. A. Dual indirect ISIF with antibodies for cyclin A and cyclin E (CyE), an indicator for cycling late G1 and S phase cells, with highest expression in early S phase. B. Dual indirect ISIF with antibodies for cyclin A and cyclin D1 (CyD1), an indicator for cycling G1 or arrested late G1 and early S phase cells, with highest expression in late G1 phase. C. Dual indirect ISIF with antibodies for cyclin D1 and p53. D. Dual indirect ISIF with antibodies for cyclin A and Btg2. E. Dual indirect ISIF with antibodies for cyclin A and Lgr5. See Table 1 for quantitative analyses for the CyA∶CyD1 and CyA∶Btg2 SPr and CD assays. Only the CD analysis was quantified for the CyA∶Lgr5 assays (n = 61, 58; and % co-asymmetric CyA and Lgr5 expression = 20%, 4% for paired nuclei in the ASYM state versus the SYM state, respectively; p<0.006 by the two-tailed Fisher's exact test). Scale bars = 50 microns.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3139609&req=5

pone-0022077-g006: Evaluation of ASRA protein biomarkers by sister pair (SPr) and cytochalasin D (CD) assay.Shown are examples of fluorescent photomicrographs from parallel SPr and CD analyses using dual indirect ISIF performed simultaneously with two different biomarker-specific antibodies of different species origin. Species-specific secondary antibodies conjugated to red and green fluorochromes were used, respectively, for biomarker-specific imaging. SYM, symmetric self-renewal by p53- Con-3 cells with ZnCl2. ASYM, asymmetric self-renewal by p53-inducible Ind-8 cells with ZnCl2 to induce p53 expression. DAPI, nuclear DNA fluorescence. CyA, indirect ISIF with specific antibodies for cyclin A, an indicator for cycling late G1, S, and G2 cells with greatest expression in G2 phase. A. Dual indirect ISIF with antibodies for cyclin A and cyclin E (CyE), an indicator for cycling late G1 and S phase cells, with highest expression in early S phase. B. Dual indirect ISIF with antibodies for cyclin A and cyclin D1 (CyD1), an indicator for cycling G1 or arrested late G1 and early S phase cells, with highest expression in late G1 phase. C. Dual indirect ISIF with antibodies for cyclin D1 and p53. D. Dual indirect ISIF with antibodies for cyclin A and Btg2. E. Dual indirect ISIF with antibodies for cyclin A and Lgr5. See Table 1 for quantitative analyses for the CyA∶CyD1 and CyA∶Btg2 SPr and CD assays. Only the CD analysis was quantified for the CyA∶Lgr5 assays (n = 61, 58; and % co-asymmetric CyA and Lgr5 expression = 20%, 4% for paired nuclei in the ASYM state versus the SYM state, respectively; p<0.006 by the two-tailed Fisher's exact test). Scale bars = 50 microns.
Mentions: To validate the SPr and CD assays, we used three well-described cell cycle-specific biomarkers: cyclin A (CyA) as a general biomarker for cycling cells, cyclin E (CyE) as a biomarker for cycling early S phase cells, and cyclin D1 (CyD1) as a biomarker for G1 cells and, more importantly, cells arrested at G1/S of the cell cycle [45]. Fig. 6 provides examples of the major types of SPr and CD expression patterns observed for these biomarkers under conditions of symmetric self-renewal versus asymmetric self-renewal with the engineered cell lines. In SPr analyses, all three cell cycle biomarkers showed primarily symmetric expression patterns when the engineered cell lines were cultured under conditions for symmetric self-renewal (Fig. 6A and 6B, SYM, SPr). In contrast, under conditions for asymmetric self-renewal, asymmetric expression patterns increased significantly (Fig. 6A and 6B, ASYM, SPr; see also Table 1, CyA∶CyD1, SPr assay). As predicted, CyD1, the biomarker for non-cycling non-stem sisters, was consistently reciprocally asymmetric with respect to the cycling cell biomarkers (Fig. 6B, ASYM, SPr). Similar relationships were observed in parallel CD assays (Fig. 6A and 6B, CD; see also Table 1, CyA∶CyD1, CD assay), validating this second assay for use with cyclins A, E, and D1. Simultaneous detection of CyA and CyD1 was particularly effective for detecting and quantifying asymmetric self-renewal and symmetric self-renewal (Fig. 6B, ASYM; Table 1, CyA∶CyD1). When p53 was induced, it was always expressed in both sister cells or sister nuclei, despite their clear phenotypic differences (Fig. 6C, ASYM; compare p53 and CyD1). Thus, although the shift to asymmetric self-renewal from symmetric self-renewal by the engineered cells is initiated by p53 expression, it is not due to asymmetric expression of p53 between sister cells.

Bottom Line: This delineation has several significant implications.These include: 1) providing experimental evidence that DSCs in vivo undergo asymmetric self-renewal as individual cells; 2) providing an explanation why earlier attempts to define a common gene expression signature for DSCs were unsuccessful; and 3) predicting that some ASRA proteins may be ideal biomarkers for DSCs.Indeed, two ASRA proteins, CXCR6 and BTG2, and two other related self-renewal pattern associated (SRPA) proteins identified in this gene resource, LGR5 and H2A.Z, display unique asymmetric patterns of expression that have a high potential for universal and specific DSC identification.

View Article: PubMed Central - PubMed

Affiliation: School of Pharmacy, Ajou University, Suwon, South Korea.

ABSTRACT
Specific and universal biomarkers for distributed stem cells (DSCs) have been elusive. A major barrier to discovery of such ideal DSC biomarkers is difficulty in obtaining DSCs in sufficient quantity and purity. To solve this problem, we used cell lines genetically engineered for conditional asymmetric self-renewal, the defining DSC property. In gene microarray analyses, we identified 85 genes whose expression is tightly asymmetric self-renewal associated (ASRA). The ASRA gene signature prescribed DSCs to undergo asymmetric self-renewal to a greater extent than committed progenitor cells, embryonic stem cells, or induced pluripotent stem cells. This delineation has several significant implications. These include: 1) providing experimental evidence that DSCs in vivo undergo asymmetric self-renewal as individual cells; 2) providing an explanation why earlier attempts to define a common gene expression signature for DSCs were unsuccessful; and 3) predicting that some ASRA proteins may be ideal biomarkers for DSCs. Indeed, two ASRA proteins, CXCR6 and BTG2, and two other related self-renewal pattern associated (SRPA) proteins identified in this gene resource, LGR5 and H2A.Z, display unique asymmetric patterns of expression that have a high potential for universal and specific DSC identification.

Show MeSH