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Circulating stem cell vary with NYHA stage in heart failure patients.

Fortini C, Toffoletto B, Fucili A, Puppato E, Olivares A, Beltrami AP, Fiorelli V, Bergamin N, Cesselli D, Morelli C, Francolini G, Ferrari R, Beltrami CA - J. Cell. Mol. Med. (2011)

Bottom Line: Level of CD45(-) CD34(-) CD90(+) CXCR4(+) cells progressively increased from class II to class IV (fold increases compared with controls: 8.5, 12 and 21.5, respectively).Both the entity and kinetic of this process varied in distinct cell subsets.Specifically, differently from HSCs and EPCs/CECs, MSCs and TCSCs significantly increased with the progression of the disease, suggesting a possible distinct role of these cells in the pathophysiology of HF.

View Article: PubMed Central - PubMed

Affiliation: University of Ferrara and Cardiovascular Research Center, Salvatore Maugeri Foundation, IRCCS, Lumezzane, Italy. cfortini@mtagroup.net

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Gating strategy. Orange box: MSC and HSPC enumeration. Pulse width versus FSC (A), FSC versus SSC (B), and CD45/CD34 (C) dot plots were utilized to exclude cell doublets, platelets, erythrocytes, dead cells, debris and neutrophilis, restricting the analysis to the lympho-monocyte fraction. On the basis of the level of expression of CD45 and CD34, we recognized, within the gated blood cells, a CD45+CD34+ population and a CD45−CD34− population (D). HSC were recognized within the CD45+CD34+ fraction, FL2-CD133+, FL2-CD90+ or FL2-CD105+ cells (HSC gate, D). MSC were studied within the CD45/CD34low/neg cells, as either FL2-CD90+ or FL2-CD105+ (MSC gate, D). Blue box: CD34lowSSC and EPC/EC enumeration. To count CD34+SSClow cells and EPC/EC, cells were first gated on pulse width versus FSC (E) and FSC versus SSC (F) dot plots. CD34 versus SSC dot plots were exploited to establish the gate identifying the CD34+ fraction characterized by low SSC (G). CD34 versus FL2-CD144 dot plots were utilized to recognize circulating CD144+ EPC/EC and to distinguish, within this class, the subpopulation of cells co-expressing CD144 and CD34 (H). White box: CXCR4+ cell enumeration and characterization. Regarding CXCR4+SCs, we first gated cells on the basis of pulse width versus FSC (I) and FSC versus SSC (J) dot plots. Samples stained with CD45 and isotype-matched antibodies were then plotted on CD45 versus CXCR4 charts to establish the gates identifying both the CXCR4+CD45+ and the CXCR4+CD45− populations (K). These two latter populations (L) were then analysed separately plotting CXCR4 versus either FL2-CD105, FL2-CD90 (M and N).
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fig01: Gating strategy. Orange box: MSC and HSPC enumeration. Pulse width versus FSC (A), FSC versus SSC (B), and CD45/CD34 (C) dot plots were utilized to exclude cell doublets, platelets, erythrocytes, dead cells, debris and neutrophilis, restricting the analysis to the lympho-monocyte fraction. On the basis of the level of expression of CD45 and CD34, we recognized, within the gated blood cells, a CD45+CD34+ population and a CD45−CD34− population (D). HSC were recognized within the CD45+CD34+ fraction, FL2-CD133+, FL2-CD90+ or FL2-CD105+ cells (HSC gate, D). MSC were studied within the CD45/CD34low/neg cells, as either FL2-CD90+ or FL2-CD105+ (MSC gate, D). Blue box: CD34lowSSC and EPC/EC enumeration. To count CD34+SSClow cells and EPC/EC, cells were first gated on pulse width versus FSC (E) and FSC versus SSC (F) dot plots. CD34 versus SSC dot plots were exploited to establish the gate identifying the CD34+ fraction characterized by low SSC (G). CD34 versus FL2-CD144 dot plots were utilized to recognize circulating CD144+ EPC/EC and to distinguish, within this class, the subpopulation of cells co-expressing CD144 and CD34 (H). White box: CXCR4+ cell enumeration and characterization. Regarding CXCR4+SCs, we first gated cells on the basis of pulse width versus FSC (I) and FSC versus SSC (J) dot plots. Samples stained with CD45 and isotype-matched antibodies were then plotted on CD45 versus CXCR4 charts to establish the gates identifying both the CXCR4+CD45+ and the CXCR4+CD45− populations (K). These two latter populations (L) were then analysed separately plotting CXCR4 versus either FL2-CD105, FL2-CD90 (M and N).

Mentions: Table 2 summarizes the Abs combination utilized to identify the different subpopulation of each class of BMSC. Figure 1 shows a typical example of analytical gates used to count total numbers and subsets of circulating stem cells. Putative MSCs were identified as CD45−CD34− cells, expressing either CD90 or CD105 [12]; HSCs as CD45+ and CD34+ cells co-expressing either CD90 or CD105 [13]; EPCs, a very heterogeneous group of cells, were characterized as CD45− with or without the surface markers CD133 and CD144 [14–16]. TCSCs were identified as CD34+CD45+ or CD45− cells co-expressing the CXCR4 receptor [17]. In view of the role of SDF-1α-CXCR4 interaction in homing, repopulation and recruitment of human stem cells [18], the expression of CXCR4 receptor was also evaluated in all groups except for the EPCs, because this was already evaluated in the CD45−CD34+ component of the TCSCs. Because of the role of PDGF-BB in MSC recruitment, we evaluated the axis PDGF-BB/PDGFR as well [19].


Circulating stem cell vary with NYHA stage in heart failure patients.

Fortini C, Toffoletto B, Fucili A, Puppato E, Olivares A, Beltrami AP, Fiorelli V, Bergamin N, Cesselli D, Morelli C, Francolini G, Ferrari R, Beltrami CA - J. Cell. Mol. Med. (2011)

Gating strategy. Orange box: MSC and HSPC enumeration. Pulse width versus FSC (A), FSC versus SSC (B), and CD45/CD34 (C) dot plots were utilized to exclude cell doublets, platelets, erythrocytes, dead cells, debris and neutrophilis, restricting the analysis to the lympho-monocyte fraction. On the basis of the level of expression of CD45 and CD34, we recognized, within the gated blood cells, a CD45+CD34+ population and a CD45−CD34− population (D). HSC were recognized within the CD45+CD34+ fraction, FL2-CD133+, FL2-CD90+ or FL2-CD105+ cells (HSC gate, D). MSC were studied within the CD45/CD34low/neg cells, as either FL2-CD90+ or FL2-CD105+ (MSC gate, D). Blue box: CD34lowSSC and EPC/EC enumeration. To count CD34+SSClow cells and EPC/EC, cells were first gated on pulse width versus FSC (E) and FSC versus SSC (F) dot plots. CD34 versus SSC dot plots were exploited to establish the gate identifying the CD34+ fraction characterized by low SSC (G). CD34 versus FL2-CD144 dot plots were utilized to recognize circulating CD144+ EPC/EC and to distinguish, within this class, the subpopulation of cells co-expressing CD144 and CD34 (H). White box: CXCR4+ cell enumeration and characterization. Regarding CXCR4+SCs, we first gated cells on the basis of pulse width versus FSC (I) and FSC versus SSC (J) dot plots. Samples stained with CD45 and isotype-matched antibodies were then plotted on CD45 versus CXCR4 charts to establish the gates identifying both the CXCR4+CD45+ and the CXCR4+CD45− populations (K). These two latter populations (L) were then analysed separately plotting CXCR4 versus either FL2-CD105, FL2-CD90 (M and N).
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Related In: Results  -  Collection

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fig01: Gating strategy. Orange box: MSC and HSPC enumeration. Pulse width versus FSC (A), FSC versus SSC (B), and CD45/CD34 (C) dot plots were utilized to exclude cell doublets, platelets, erythrocytes, dead cells, debris and neutrophilis, restricting the analysis to the lympho-monocyte fraction. On the basis of the level of expression of CD45 and CD34, we recognized, within the gated blood cells, a CD45+CD34+ population and a CD45−CD34− population (D). HSC were recognized within the CD45+CD34+ fraction, FL2-CD133+, FL2-CD90+ or FL2-CD105+ cells (HSC gate, D). MSC were studied within the CD45/CD34low/neg cells, as either FL2-CD90+ or FL2-CD105+ (MSC gate, D). Blue box: CD34lowSSC and EPC/EC enumeration. To count CD34+SSClow cells and EPC/EC, cells were first gated on pulse width versus FSC (E) and FSC versus SSC (F) dot plots. CD34 versus SSC dot plots were exploited to establish the gate identifying the CD34+ fraction characterized by low SSC (G). CD34 versus FL2-CD144 dot plots were utilized to recognize circulating CD144+ EPC/EC and to distinguish, within this class, the subpopulation of cells co-expressing CD144 and CD34 (H). White box: CXCR4+ cell enumeration and characterization. Regarding CXCR4+SCs, we first gated cells on the basis of pulse width versus FSC (I) and FSC versus SSC (J) dot plots. Samples stained with CD45 and isotype-matched antibodies were then plotted on CD45 versus CXCR4 charts to establish the gates identifying both the CXCR4+CD45+ and the CXCR4+CD45− populations (K). These two latter populations (L) were then analysed separately plotting CXCR4 versus either FL2-CD105, FL2-CD90 (M and N).
Mentions: Table 2 summarizes the Abs combination utilized to identify the different subpopulation of each class of BMSC. Figure 1 shows a typical example of analytical gates used to count total numbers and subsets of circulating stem cells. Putative MSCs were identified as CD45−CD34− cells, expressing either CD90 or CD105 [12]; HSCs as CD45+ and CD34+ cells co-expressing either CD90 or CD105 [13]; EPCs, a very heterogeneous group of cells, were characterized as CD45− with or without the surface markers CD133 and CD144 [14–16]. TCSCs were identified as CD34+CD45+ or CD45− cells co-expressing the CXCR4 receptor [17]. In view of the role of SDF-1α-CXCR4 interaction in homing, repopulation and recruitment of human stem cells [18], the expression of CXCR4 receptor was also evaluated in all groups except for the EPCs, because this was already evaluated in the CD45−CD34+ component of the TCSCs. Because of the role of PDGF-BB in MSC recruitment, we evaluated the axis PDGF-BB/PDGFR as well [19].

Bottom Line: Level of CD45(-) CD34(-) CD90(+) CXCR4(+) cells progressively increased from class II to class IV (fold increases compared with controls: 8.5, 12 and 21.5, respectively).Both the entity and kinetic of this process varied in distinct cell subsets.Specifically, differently from HSCs and EPCs/CECs, MSCs and TCSCs significantly increased with the progression of the disease, suggesting a possible distinct role of these cells in the pathophysiology of HF.

View Article: PubMed Central - PubMed

Affiliation: University of Ferrara and Cardiovascular Research Center, Salvatore Maugeri Foundation, IRCCS, Lumezzane, Italy. cfortini@mtagroup.net

Show MeSH
Related in: MedlinePlus