Serum-free Quality and Quantity Control Culture Improves the Angiogenic Potential of Peripheral Blood Mononuclear Cells Harvested from Patients with Connective Tissue Diseases

Abstract

Objectives: The number and quality of endothelial progenitor cells decrease in patients with connective tissue diseases. This limits the efficacy of mononuclear cell therapy for ischemic ulcers associated with connective tissue diseases. To overcome these problems, we developed a serum-free quality and quantity control culture method that potentially improves the function of endothelial progenitor cells and expands their numbers. Here, we show the effect of quality and quantity control culture on mononuclear cells from patients with connective tissue diseases.

Methods: Peripheral blood mononuclear cells were isolated from C57BL/6JJmsSlc-lpr/lpr mice with systemic lupus erythematosus, patients with connective tissue diseases, and healthy volunteers. Mononuclear cells were cultured using the quality and quantity control culture method, and the number of endothelial progenitor cells was analyzed using flow cytometry, an endothelial progenitor cell culture assay, and an endothelial progenitor cell colony-forming assay. Flow cytometry was also used to examine mononuclear cell subpopulations. A human umbilical vein endothelial cell tube-forming assay was used to examine the function of quality and quantity control cultured mononuclear cells.

Results: Mice with systemic lupus erythematosus showed a significantly lower number of endothelial progenitor cells, which increased to the same levels as those of the control mice after quality and quantity control culture. In humans, the numbers of endothelial progenitor cells and M2 macrophages were significantly increased and the number of proinflammatory cells was decreased after quality and quantity control culture in both healthy volunteers and patients with connective tissue diseases. The human umbilical vein endothelial cell tube formation assay showed higher angiogenic potential in quality and quantity control cultured mononuclear cells from patients with connective tissue diseases than that in quality and quantity control cultured mononuclear cells from healthy controls.

Conclusions: Our study suggests that the quality and quantity control culture method is effective in recovering the angiogenic ability of mononuclear cells from patients with connective tissue diseases.

Keywords: MNC-QQ cell; cell therapy; connective tissue disease; peripheral blood mononuclear cell; vasculogenesis.

Figure 1.

Depletion of EPCs in C57BL/6JJmsSlc-lpr/lpr (SLE) mice PBMNCs was recovered by QQ culture. (A) The number of PBMNCs in control mice and mice with SLE (n = 12). (B) Flow cytometry analysis detected EPCs that were double positive for Sca-1 and c-kit in control mice and mice with SLE pre- and post-QQc PBMNCs (pre-QQ, n = 6; post-QQ, n = 3). (C) The number of pEPC and dEPC colonies in control mice and mice with SLE pre- and post-QQc PBMNCs (pre-QQ, n = 11; post-QQ, n = 3). *p < 0.05, #p < 0.05 versus pre-MNCs. ns, not significant; EPCs, endothelial progenitor cells; pEPC, primitive EPC; dEPC, definitive EPC; SLE, systemic lupus erythematosus; PBMNCs, peripheral blood mononuclear cells

Figure 2.

Number of EPCs in PBMNCs was increased by QQ culture in patients with connective tissue disease. (A) The number of PBMNCs was counted in healthy controls and patients with CTD (healthy, n = 5; CTD, n = 12). (B) Flow cytometry analysis detected EPCs that were double positive for CD34 and CD133 in healthy and CTD pre- and post-QQc PBMNCs (healthy, n = 7; CTD, n = 12). (C) The EPCs stained with Ac-LDL and lectin were counted after the EPC culture was counted (n = 6). (D) The number of pEPC and dEPC colonies in pre- and post-QQc PBMNCs from healthy controls and patients with CTD (healthy, n = 6; CTD, n = 9). *p < 0.05, **p < 0.01. PBMNCs, peripheral blood mononuclear cells; QQc, serum-free quality and quantity control culture; H, healthy subjects; CTD, connective tissue disease; pEPC, primitive EPC; dEPC, definitive EPC; Ac-LDL, acetylated low-density lipoprotein

Figure 3

Flow cytometry analysis of the PBMNC subpopulation after QQc in healthy controls and patients with CTD. The percentages of CD14⁺, CCR2⁺, CD206⁺ macrophage markers, CD19⁺ B cell markers, CD56⁺ NK cell markers, and angiogenic T cell markers in PBMNCs and MNC-QQ cells were analyzed by flow cytometry (healthy, n = 7; CTD, n = 12). *p < 0.05, **p < 0.01, ***p < 0.001. H, healthy subjects; CTD, patients with connective tissue disease; PBMNCs, peripheral blood mononuclear cells; QQc, serum-free quality and quantity control culture; NK, natural killer

Figure 4.

Improvement in angiogenic ability after QQc in CTD PBMNCs was analyzed using the HUVEC tube formation assay. (A) The number of closed-circle formations on the Matrigel was counted. The graph shows the ratio of the combination of MNCs, MNC-QQ cells, and HUVECs to the HUVEC-only control group (n = 6). (B) Representative images of tube formation. *p < 0.05. H, healthy; CTD, connective tissue disease; PBMNCs, peripheral blood mononuclear cells; QQc, serum-free quality and quantity control culture; HUVEC, human umbilical vein endothelial cell