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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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Osteogenesis and adipogenesis of CD29+, CD31+, CD34+, CD56+ and CD91+ cells.A. Flourescent-conjugated antibodies to CD29, CD31, CD34, CD56 and CD91 were used to separately sort and subculture cells form collagenase 2 digested human muscle. The cells were cultured to confluence then the cells were cultured for an additional three weeks in either osteogenic media (OM), adipogenic media (AM) or regular growth media (GM). The cultures were fixed and processed for Alizarin Red or Oil Red O staining. Scale bar  = 100 µm. B. For each marker the bar graph reflects the number of optical density units (ODU) (mean ± SEM, n = 5), as measured with Image J, for Alizarin and Oil Red O staining of isolated cell types differentiated in osteogenic media (OM) and adipogenic media (AM). Significant difference is observed (*) in Oil Red O staining between CD34 and CD29 compared to CD31, CD56, and CD91 (Student's t-test, p≤0.05).
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pone-0114318-g008: Osteogenesis and adipogenesis of CD29+, CD31+, CD34+, CD56+ and CD91+ cells.A. Flourescent-conjugated antibodies to CD29, CD31, CD34, CD56 and CD91 were used to separately sort and subculture cells form collagenase 2 digested human muscle. The cells were cultured to confluence then the cells were cultured for an additional three weeks in either osteogenic media (OM), adipogenic media (AM) or regular growth media (GM). The cultures were fixed and processed for Alizarin Red or Oil Red O staining. Scale bar  = 100 µm. B. For each marker the bar graph reflects the number of optical density units (ODU) (mean ± SEM, n = 5), as measured with Image J, for Alizarin and Oil Red O staining of isolated cell types differentiated in osteogenic media (OM) and adipogenic media (AM). Significant difference is observed (*) in Oil Red O staining between CD34 and CD29 compared to CD31, CD56, and CD91 (Student's t-test, p≤0.05).

Mentions: From the data above it seems clear that these subpopulations differ in their abilities to proliferate, migrate and produce inflammatory cytokines; therefore, it may be expected that their abilities to differentiate may also be distinct. The subpopulations were monitored for their capacity to undergo osteogenesis and adipogenesis. As shown in Fig. 8, all five subpopulations displayed similar extents of mineralization after three weeks in osteogenic media, as shown by equal Alizarin Red staining. However, when adipogenesis was assessed, by Oil Red O staining, it appeared that the CD31+, CD56+ and CD91+ cells were approximately three to four fold better able to accumulate lipid than the CD34+ and CD29+ cells. These data indicate that while there were likely no differences in osteogenesis between the cell types, the subpopulations display significant differences in adipogenesis.


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)

Osteogenesis and adipogenesis of CD29+, CD31+, CD34+, CD56+ and CD91+ cells.A. Flourescent-conjugated antibodies to CD29, CD31, CD34, CD56 and CD91 were used to separately sort and subculture cells form collagenase 2 digested human muscle. The cells were cultured to confluence then the cells were cultured for an additional three weeks in either osteogenic media (OM), adipogenic media (AM) or regular growth media (GM). The cultures were fixed and processed for Alizarin Red or Oil Red O staining. Scale bar  = 100 µm. B. For each marker the bar graph reflects the number of optical density units (ODU) (mean ± SEM, n = 5), as measured with Image J, for Alizarin and Oil Red O staining of isolated cell types differentiated in osteogenic media (OM) and adipogenic media (AM). Significant difference is observed (*) in Oil Red O staining between CD34 and CD29 compared to CD31, CD56, and CD91 (Student's t-test, p≤0.05).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4260839&req=5

pone-0114318-g008: Osteogenesis and adipogenesis of CD29+, CD31+, CD34+, CD56+ and CD91+ cells.A. Flourescent-conjugated antibodies to CD29, CD31, CD34, CD56 and CD91 were used to separately sort and subculture cells form collagenase 2 digested human muscle. The cells were cultured to confluence then the cells were cultured for an additional three weeks in either osteogenic media (OM), adipogenic media (AM) or regular growth media (GM). The cultures were fixed and processed for Alizarin Red or Oil Red O staining. Scale bar  = 100 µm. B. For each marker the bar graph reflects the number of optical density units (ODU) (mean ± SEM, n = 5), as measured with Image J, for Alizarin and Oil Red O staining of isolated cell types differentiated in osteogenic media (OM) and adipogenic media (AM). Significant difference is observed (*) in Oil Red O staining between CD34 and CD29 compared to CD31, CD56, and CD91 (Student's t-test, p≤0.05).
Mentions: From the data above it seems clear that these subpopulations differ in their abilities to proliferate, migrate and produce inflammatory cytokines; therefore, it may be expected that their abilities to differentiate may also be distinct. The subpopulations were monitored for their capacity to undergo osteogenesis and adipogenesis. As shown in Fig. 8, all five subpopulations displayed similar extents of mineralization after three weeks in osteogenic media, as shown by equal Alizarin Red staining. However, when adipogenesis was assessed, by Oil Red O staining, it appeared that the CD31+, CD56+ and CD91+ cells were approximately three to four fold better able to accumulate lipid than the CD34+ and CD29+ cells. These data indicate that while there were likely no differences in osteogenesis between the cell types, the subpopulations display significant differences in adipogenesis.

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