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Bone marrow-derived progenitor cells in end-stage lung disease patients.

Gilpin SE, Lung K, de Couto GT, Cypel M, Sato M, Singer LG, Keshavjee S, Waddell TK - BMC Pulm Med (2013)

Bottom Line: An increase in CD45⁺Collagen-1⁺ fibrocytes was found in pulmonary fibrosis and bronchiolitis obliterans patients.Plasma cytokine levels differed between disease groups, with a significant correlation between SCGF-β and CCSP⁺ cells and between Monocyte Chemotactic Protein-1 and fibrocytes.An increase in the novel CCSP⁺ epithelial-like progenitors in cystic fibrosis patients was found.

View Article: PubMed Central - HTML - PubMed

Affiliation: Latner Thoracic Surgery Research Laboratories, Division of Thoracic Surgery, Toronto General Hospital, University Health Network, University of Toronto, North Wing, 9N - 949, 200 Elizabeth Street, Toronto, ON M5G 2C4, Canada.

ABSTRACT

Background: Chronic lung diseases are marked by progressive inflammation, tissue damage and remodelling. Bone marrow-derived progenitor cells may contribute to these processes. The objectives of this study were to (1) to quantify CD45⁺Collagen-1⁺ fibrocytes and a novel epithelial-like population of bone marrow-derived cells, which express Clara Cell Secretory Protein, in patients at the time of lung transplant and (2) to evaluate mediators that may act to recruit these cells during injury.

Methods: Using an observational design, progenitor cells were quantified by flow cytometry from both bone marrow (BM) and peripheral blood (PB). Migration was tested using in vitro transwell assays. Multiplex bead-based assays were used to quantify plasma cytokines.

Results: An increase in CD45⁺Collagen-1⁺ fibrocytes was found in pulmonary fibrosis and bronchiolitis obliterans patients. Cystic fibrosis patients had an increase in CCSP⁺ cells in both the BM and PB. The proportion of CCSP⁺ cells in the BM and PB was correlated. CCSP+ cells express the chemokine receptors CCR2, CCR4, CXCR3, and CXCR4, and significantly migrated in vitro toward Stromal Derived Factor-1 (SDF-1) and Stem Cell Growth Factor-β (SCGF-β). Plasma cytokine levels differed between disease groups, with a significant correlation between SCGF-β and CCSP⁺ cells and between Monocyte Chemotactic Protein-1 and fibrocytes.

Conclusions: Different bone marrow-derived cells are found in various lung diseases. Increased fibrocytes were associated with fibrotic lung diseases. An increase in the novel CCSP⁺ epithelial-like progenitors in cystic fibrosis patients was found. These differences may be mediated by alterations in plasma cytokines responsible for cell recruitment.

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Identification of progenitor populations. Taqman PCR measurement of (A) Clara Cell Secretory Protein (CCSP) gene expression in human bone marrow cells (BMCs), (B) peripheral blood mononuclear cells (PBMCs), and (C) PBMCs pre-sorted for CCSP by flow cytometry. No template and no reverse transcription negative controls are included. (D) Gel electrophoresis of PCR product after Taqman-based amplification. Positive control (bronchus) and negative no reverse transcription (NRT) controls are included. (E) Flow cytometry gating for measurement of CCSP+ PBMCs based on isotype control staining. (F) Flow cytometry gating for measurement of CD45+Collagen-1+ peripheral blood leukocytes (PBLs) based on isotype control staining.
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Figure 1: Identification of progenitor populations. Taqman PCR measurement of (A) Clara Cell Secretory Protein (CCSP) gene expression in human bone marrow cells (BMCs), (B) peripheral blood mononuclear cells (PBMCs), and (C) PBMCs pre-sorted for CCSP by flow cytometry. No template and no reverse transcription negative controls are included. (D) Gel electrophoresis of PCR product after Taqman-based amplification. Positive control (bronchus) and negative no reverse transcription (NRT) controls are included. (E) Flow cytometry gating for measurement of CCSP+ PBMCs based on isotype control staining. (F) Flow cytometry gating for measurement of CD45+Collagen-1+ peripheral blood leukocytes (PBLs) based on isotype control staining.

Mentions: Circulating bone marrow-derived cell populations were defined by (1) Clara Cell Secretory Protein (CCSP) expression for epithelial-like progenitors and by (2) dual expression of CD45 and intracellular collagen-1 expression for fibrocytes. To validate the expression of CCSP mRNA in the bone marrow and peripheral blood, Taqman PCR was utilized. CCSP mRNA was detected in human bone marrow cells (BMCs) and peripheral blood mononuclear cells (PBMCs) from 3 randomly selected lung transplant recipients, as well as in control lung bronchus tissue, but absent in experimental controls (no reverse transcription and no template controls) (Figure 1A-B). As a further proof-of-principle, peripheral blood cells from a healthy volunteer were isolated and sorted for CCSP by flow cytometery, representing less than 1% of total PBMCs, and then analyzed by PCR. CCSP mRNA was also detected in the pre-sorted population but not in CCSP-negative sorted cells (Figure 1C). The resulting amplification product was further analyzed by gel electrophoresis to confirm the correct amplicon size (Figure 1D), and compared to positive human bronchus tissue mRNA and a mixture of sample mRNAs not subjected to reverse transcription (NRT) to control for genomic DNA contamination. All subsequent quantification of progenitor cells populations was determined by flow cytometry. A representative plot identifying the positively gated populations based on initial isotype staining of 1% is shown for both CCSP+ cells and CD45+Collagen-1+ fibrocytes (Figure 1E-F). Further details of the gating strategy have been previously published [11].


Bone marrow-derived progenitor cells in end-stage lung disease patients.

Gilpin SE, Lung K, de Couto GT, Cypel M, Sato M, Singer LG, Keshavjee S, Waddell TK - BMC Pulm Med (2013)

Identification of progenitor populations. Taqman PCR measurement of (A) Clara Cell Secretory Protein (CCSP) gene expression in human bone marrow cells (BMCs), (B) peripheral blood mononuclear cells (PBMCs), and (C) PBMCs pre-sorted for CCSP by flow cytometry. No template and no reverse transcription negative controls are included. (D) Gel electrophoresis of PCR product after Taqman-based amplification. Positive control (bronchus) and negative no reverse transcription (NRT) controls are included. (E) Flow cytometry gating for measurement of CCSP+ PBMCs based on isotype control staining. (F) Flow cytometry gating for measurement of CD45+Collagen-1+ peripheral blood leukocytes (PBLs) based on isotype control staining.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3750607&req=5

Figure 1: Identification of progenitor populations. Taqman PCR measurement of (A) Clara Cell Secretory Protein (CCSP) gene expression in human bone marrow cells (BMCs), (B) peripheral blood mononuclear cells (PBMCs), and (C) PBMCs pre-sorted for CCSP by flow cytometry. No template and no reverse transcription negative controls are included. (D) Gel electrophoresis of PCR product after Taqman-based amplification. Positive control (bronchus) and negative no reverse transcription (NRT) controls are included. (E) Flow cytometry gating for measurement of CCSP+ PBMCs based on isotype control staining. (F) Flow cytometry gating for measurement of CD45+Collagen-1+ peripheral blood leukocytes (PBLs) based on isotype control staining.
Mentions: Circulating bone marrow-derived cell populations were defined by (1) Clara Cell Secretory Protein (CCSP) expression for epithelial-like progenitors and by (2) dual expression of CD45 and intracellular collagen-1 expression for fibrocytes. To validate the expression of CCSP mRNA in the bone marrow and peripheral blood, Taqman PCR was utilized. CCSP mRNA was detected in human bone marrow cells (BMCs) and peripheral blood mononuclear cells (PBMCs) from 3 randomly selected lung transplant recipients, as well as in control lung bronchus tissue, but absent in experimental controls (no reverse transcription and no template controls) (Figure 1A-B). As a further proof-of-principle, peripheral blood cells from a healthy volunteer were isolated and sorted for CCSP by flow cytometery, representing less than 1% of total PBMCs, and then analyzed by PCR. CCSP mRNA was also detected in the pre-sorted population but not in CCSP-negative sorted cells (Figure 1C). The resulting amplification product was further analyzed by gel electrophoresis to confirm the correct amplicon size (Figure 1D), and compared to positive human bronchus tissue mRNA and a mixture of sample mRNAs not subjected to reverse transcription (NRT) to control for genomic DNA contamination. All subsequent quantification of progenitor cells populations was determined by flow cytometry. A representative plot identifying the positively gated populations based on initial isotype staining of 1% is shown for both CCSP+ cells and CD45+Collagen-1+ fibrocytes (Figure 1E-F). Further details of the gating strategy have been previously published [11].

Bottom Line: An increase in CD45⁺Collagen-1⁺ fibrocytes was found in pulmonary fibrosis and bronchiolitis obliterans patients.Plasma cytokine levels differed between disease groups, with a significant correlation between SCGF-β and CCSP⁺ cells and between Monocyte Chemotactic Protein-1 and fibrocytes.An increase in the novel CCSP⁺ epithelial-like progenitors in cystic fibrosis patients was found.

View Article: PubMed Central - HTML - PubMed

Affiliation: Latner Thoracic Surgery Research Laboratories, Division of Thoracic Surgery, Toronto General Hospital, University Health Network, University of Toronto, North Wing, 9N - 949, 200 Elizabeth Street, Toronto, ON M5G 2C4, Canada.

ABSTRACT

Background: Chronic lung diseases are marked by progressive inflammation, tissue damage and remodelling. Bone marrow-derived progenitor cells may contribute to these processes. The objectives of this study were to (1) to quantify CD45⁺Collagen-1⁺ fibrocytes and a novel epithelial-like population of bone marrow-derived cells, which express Clara Cell Secretory Protein, in patients at the time of lung transplant and (2) to evaluate mediators that may act to recruit these cells during injury.

Methods: Using an observational design, progenitor cells were quantified by flow cytometry from both bone marrow (BM) and peripheral blood (PB). Migration was tested using in vitro transwell assays. Multiplex bead-based assays were used to quantify plasma cytokines.

Results: An increase in CD45⁺Collagen-1⁺ fibrocytes was found in pulmonary fibrosis and bronchiolitis obliterans patients. Cystic fibrosis patients had an increase in CCSP⁺ cells in both the BM and PB. The proportion of CCSP⁺ cells in the BM and PB was correlated. CCSP+ cells express the chemokine receptors CCR2, CCR4, CXCR3, and CXCR4, and significantly migrated in vitro toward Stromal Derived Factor-1 (SDF-1) and Stem Cell Growth Factor-β (SCGF-β). Plasma cytokine levels differed between disease groups, with a significant correlation between SCGF-β and CCSP⁺ cells and between Monocyte Chemotactic Protein-1 and fibrocytes.

Conclusions: Different bone marrow-derived cells are found in various lung diseases. Increased fibrocytes were associated with fibrotic lung diseases. An increase in the novel CCSP⁺ epithelial-like progenitors in cystic fibrosis patients was found. These differences may be mediated by alterations in plasma cytokines responsible for cell recruitment.

Show MeSH
Related in: MedlinePlus