Vasculogenic conditioning of peripheral blood mononuclear cells promotes endothelial progenitor cell expansion and phenotype transition of anti-inflammatory macrophage and T lymphocyte to cells with regenerative potential.
The resulting cells (QQMNCs) in EPC colony-forming assay generated significantly more definitive EPC colonies than PBMNCs.Using murine ischemic hindlimb models, the efficacy of QQMNC intramuscular transplantation (Tx) was compared to that of PBMNCTx, cultured "early EPC" Tx (eEPCTx), and granulocyte colony-stimulating factor mobilized CD34(+) cell Tx (GmCD34Tx).Laser Doppler imaging revealed the blood perfusion recovery in ischemic hindlimbs after QQMNCTx superior to after PBMNCTx and eEPCTx, but also earlier than after GmCD34Tx.
Affiliation: Department of Regenerative Medicine Science, Tokai University School of Medicine, Isehara, Japan (H.M., T.S., A.S., T.A.).
- Endothelial Progenitor Cells/cytology/drug effects/physiology*
- Leukocytes, Mononuclear/cytology/drug effects/physiology*
- Blood Vessels/physiology
- Cells, Cultured
- Flow Cytometry
- Lymphocyte Activation/physiology
- Membrane Proteins/pharmacology
- Mice, Inbred BALB C
- Neovascularization, Physiologic/physiology
- Stem Cell Factor/pharmacology
- T-Lymphocytes, Helper-Inducer/cytology/physiology
- Vascular Endothelial Growth Factor A/pharmacology
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fig02: Flow cytometry analysis of PBMNCs and QQMNCs. A, Representative features of PBMNCs at 3 hours after seeding and QQMNCs after 7 days. Scale bar=100 μm. B, Scatter diagrams of PBMNCs and QQMNCs in flow cytometry. The red lines indicate the cellular‐sized gates of lymphocyte (a), monocyte (b), or the larger cell (c). C, The bar graph shows the ratio of each percent (%) cell positivity in QQMNCs to that in PBMNCs. N=4 to 6 volunteers. The investigated cell surface markers were as follows: hematopoietic stem cell (CD34, CD133), endothelial cell (VEGFR‐2, CD31, vWF, CD105, and CD146), T cell (CD3, CD4, CD8, and CD3/CXCR4/CD31), NK cell (CD16 and CD56), B cell (CD19), monocyte (CD14), dendritic cell (CD11c), M1 macrophage (CCR2), M2 macrophage (CD206), and erythroid progenitor (CD235a). D, The bar graph indicates the ratio of each % helper T subset positivity in CD4+ cells of QQMNCs to that of PBMNCs. N=6 volunteers. *P<0.01; **P<0.01 in (C and D). The gray or white column represents a mean±SE in each increase or decrease. The number associated with each graph column shows the mean value. CCR2 indicates CC chemokine receptor 2; Foxp3, forkhead box P3; FSC‐A, forward scatter‐area; IL, interleukin; INF, interferon; NK, natural killer; PBMNCs, peripheral blood mononuclear cells; QQMNCs, quality and quantity control culture of mononuclear cells; SSC‐A, side scatter‐area; VEGFR, vascular endothelial growth factor receptor; vWF, von Willebrand factor.
Based on microscopy and fluorescent cell sorting, large cells were proportionally more common in QQMNC than in PBMNC samples (Figure 2A and 2B). In FCM, the proportion of each positive cell involved in the whole cells of (a), (b), and (c) gates separated with red lines was estimated (Figure 2B).