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Characterization of discrete subpopulations of progenitor cells in traumatic human extremity wounds.

Woodard GE, Ji Y, Christopherson GT, Wolcott KM, Hall DJ, Jackson WM, Nesti LJ - PLoS ONE (2014)

Bottom Line: The most abundant subpopulations were CD29+ and CD34+, which overlapped significantly.The CD29+ and CD34+ cells had the greatest proliferative and migratory capacity while the CD56+ subpopulation produced the highest amounts of TGFß1 and TGFß2.When cultured under endothelial differentiation conditions the CD29+ and CD34+ cells expressed VE-cadherin, Tie2 and CD31, all markers of endothelial cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Surgery, Uniformed Services University of Health Sciences, Bethesda, MD, United States of America.

ABSTRACT
Here we show that distinct subpopulations of cells exist within traumatic human extremity wounds, each having the ability to differentiate into multiple cells types in vitro. A crude cell suspension derived from traumatized muscle was positively sorted for CD29, CD31, CD34, CD56 or CD91. The cell suspension was also simultaneously negatively sorted for either CD45 or CD117 to exclude hematopoietic stem cells. These subpopulations varied in terms their total numbers and their abilities to grow, migrate, differentiate and secrete cytokines. While all five subpopulations demonstrated equal abilities to undergo osteogenesis, they were distinct in their ability to undergo adipogenesis and vascular endotheliogenesis. The most abundant subpopulations were CD29+ and CD34+, which overlapped significantly. The CD29+ and CD34+ cells had the greatest proliferative and migratory capacity while the CD56+ subpopulation produced the highest amounts of TGFß1 and TGFß2. When cultured under endothelial differentiation conditions the CD29+ and CD34+ cells expressed VE-cadherin, Tie2 and CD31, all markers of endothelial cells. These data indicate that while there are multiple cell types within traumatized muscle that have osteogenic differentiation capacity and may contribute to bone formation in post-traumatic heterotopic ossification (HO), the major contributory cell types are CD29+ and CD34+, which demonstrate endothelial progenitor cell characteristics.

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Inflammatory cytokine secretion of CD29, CD31, CD34, CD56 and CD91 positive cells.Flourescent-conjugated antibodies to CD29, CD31, CD34, CD56 and CD91 were used to separately sort and subculture cells form collagenase 2 digested human muscle tissue. The cells were cultured to confluence and the supernatants were removed and assessed for TGFß1, TGFß2 and TGFß3 levels by ELISA. The graph indicates the concentrations of the cytokines (mean ± SEM, n = 8). A significant difference in amounts (mean pg/ml) is noted (*) between CD34, CD56 and CD29 for TGFß1, CD56 for TGFß2 and CD34 for TGFß3 when compared to the other populations (Mann-Whitney U Test, p≤0.01).
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pone-0114318-g007: Inflammatory cytokine secretion of CD29, CD31, CD34, CD56 and CD91 positive cells.Flourescent-conjugated antibodies to CD29, CD31, CD34, CD56 and CD91 were used to separately sort and subculture cells form collagenase 2 digested human muscle tissue. The cells were cultured to confluence and the supernatants were removed and assessed for TGFß1, TGFß2 and TGFß3 levels by ELISA. The graph indicates the concentrations of the cytokines (mean ± SEM, n = 8). A significant difference in amounts (mean pg/ml) is noted (*) between CD34, CD56 and CD29 for TGFß1, CD56 for TGFß2 and CD34 for TGFß3 when compared to the other populations (Mann-Whitney U Test, p≤0.01).

Mentions: It has been previously demonstrated that members of the TGFß family are expressed to high levels in traumatized muscle [39], [40]. Since these inflammatory cytokines are thought to mediate much of the wound-healing and fibrotic response it was important to determine if a subpopulation of cells within the wound is responsible for expression of a critical TGFß family member. The five subpopulations were therefore assessed for production of TGFß1, TGFß2 and TGFß3 by Elisa assay and the results are shown in Fig. 7A, B and C respectively. From the data it is clear that there are significant differences in the protein expression profile between the different subpopulations with regard to secretion of TGFß1 and TGFß2. CD34+, CD56+ and CD29+ demonstrated high levels of TGFß1 expression while CD31+ had only moderate levels. CD91+ cells produced only low levels of TGFß1.


Characterization of discrete subpopulations of progenitor cells in traumatic human extremity wounds.

Woodard GE, Ji Y, Christopherson GT, Wolcott KM, Hall DJ, Jackson WM, Nesti LJ - PLoS ONE (2014)

Inflammatory cytokine secretion of CD29, CD31, CD34, CD56 and CD91 positive cells.Flourescent-conjugated antibodies to CD29, CD31, CD34, CD56 and CD91 were used to separately sort and subculture cells form collagenase 2 digested human muscle tissue. The cells were cultured to confluence and the supernatants were removed and assessed for TGFß1, TGFß2 and TGFß3 levels by ELISA. The graph indicates the concentrations of the cytokines (mean ± SEM, n = 8). A significant difference in amounts (mean pg/ml) is noted (*) between CD34, CD56 and CD29 for TGFß1, CD56 for TGFß2 and CD34 for TGFß3 when compared to the other populations (Mann-Whitney U Test, p≤0.01).
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC4260839&req=5

pone-0114318-g007: Inflammatory cytokine secretion of CD29, CD31, CD34, CD56 and CD91 positive cells.Flourescent-conjugated antibodies to CD29, CD31, CD34, CD56 and CD91 were used to separately sort and subculture cells form collagenase 2 digested human muscle tissue. The cells were cultured to confluence and the supernatants were removed and assessed for TGFß1, TGFß2 and TGFß3 levels by ELISA. The graph indicates the concentrations of the cytokines (mean ± SEM, n = 8). A significant difference in amounts (mean pg/ml) is noted (*) between CD34, CD56 and CD29 for TGFß1, CD56 for TGFß2 and CD34 for TGFß3 when compared to the other populations (Mann-Whitney U Test, p≤0.01).
Mentions: It has been previously demonstrated that members of the TGFß family are expressed to high levels in traumatized muscle [39], [40]. Since these inflammatory cytokines are thought to mediate much of the wound-healing and fibrotic response it was important to determine if a subpopulation of cells within the wound is responsible for expression of a critical TGFß family member. The five subpopulations were therefore assessed for production of TGFß1, TGFß2 and TGFß3 by Elisa assay and the results are shown in Fig. 7A, B and C respectively. From the data it is clear that there are significant differences in the protein expression profile between the different subpopulations with regard to secretion of TGFß1 and TGFß2. CD34+, CD56+ and CD29+ demonstrated high levels of TGFß1 expression while CD31+ had only moderate levels. CD91+ cells produced only low levels of TGFß1.

Bottom Line: The most abundant subpopulations were CD29+ and CD34+, which overlapped significantly.The CD29+ and CD34+ cells had the greatest proliferative and migratory capacity while the CD56+ subpopulation produced the highest amounts of TGFß1 and TGFß2.When cultured under endothelial differentiation conditions the CD29+ and CD34+ cells expressed VE-cadherin, Tie2 and CD31, all markers of endothelial cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Surgery, Uniformed Services University of Health Sciences, Bethesda, MD, United States of America.

ABSTRACT
Here we show that distinct subpopulations of cells exist within traumatic human extremity wounds, each having the ability to differentiate into multiple cells types in vitro. A crude cell suspension derived from traumatized muscle was positively sorted for CD29, CD31, CD34, CD56 or CD91. The cell suspension was also simultaneously negatively sorted for either CD45 or CD117 to exclude hematopoietic stem cells. These subpopulations varied in terms their total numbers and their abilities to grow, migrate, differentiate and secrete cytokines. While all five subpopulations demonstrated equal abilities to undergo osteogenesis, they were distinct in their ability to undergo adipogenesis and vascular endotheliogenesis. The most abundant subpopulations were CD29+ and CD34+, which overlapped significantly. The CD29+ and CD34+ cells had the greatest proliferative and migratory capacity while the CD56+ subpopulation produced the highest amounts of TGFß1 and TGFß2. When cultured under endothelial differentiation conditions the CD29+ and CD34+ cells expressed VE-cadherin, Tie2 and CD31, all markers of endothelial cells. These data indicate that while there are multiple cell types within traumatized muscle that have osteogenic differentiation capacity and may contribute to bone formation in post-traumatic heterotopic ossification (HO), the major contributory cell types are CD29+ and CD34+, which demonstrate endothelial progenitor cell characteristics.

Show MeSH
Related in: MedlinePlus