Characterization of Mesenchymal Stem Cells of “No-Options” Patients with Critical Limb Ischemia Treated by Autologous Bone Marrow Mononuclear Cells

Cestmir Altaner,Andrej Klepanec,Katarina Kaiserova,Ivan Vulev,Marina Cihova,Veronika Altanerova,Lubica Hunakova,Juraj Madaric,Xing-Ming Shi

doi:10.1371/journal.pone.0073722

Abstract

BackgroundApplication of autologous bone marrow mononuclear cells to “no option” patients with advanced critical limb ischemia (CLI) prevented major limb amputation in 73% patients during the 6-month follow-up. We examined which properties of bone marrow stromal cells also known as bone-marrow derived mesenchymal stem cells of responding and non-responding patients are important for amputation-free survival.Methods and FindingsMesenchymal stem cells of 41 patients with CLI unsuitable for revascularisation were isolated from mononuclear bone marrow concentrate used for their treatment. Based on the clinical outcome of the treatment, we divided patients into two groups: responders and non-responders. Biological properties of responders’ and non-responders’ mesenchymal stem cells were characterized according to their ability to multiply, to differentiate in vitro, quantitative expression of cell surface markers, secretion of 27 cytokines, chemokines and growth factors, and to the relative expression of 15 mesenchymal stem cells important genes. Secretome comparison between responders (n=27) and non-responders (n=14) revealed significantly higher secretion values of IL-4, IL-6 and MIP-1b in the group of responders. The expression of cell markers CD44 and CD90 in mesenchymal stem cells from responders was significantly higher compared to non-responders (p<0.01). The expression of mesenchymal stem cells surface markers that was analyzed in 22 patients did not differ between diabetic (n=13) and non-diabetic (n=9) patient groups. Statistically significant higher expression of E-cadherin and PDX-1/IPF1 genes was found in non-responders, while expression of Snail was higher in responders.ConclusionsThe quality of mesenchymal stem cells shown in the expression of cell surface markers, secreted factors and stem cell genes plays an important role in therapeutic outcome. Paracrine mechanisms are main drivers in the induction of reparatory processes in CLI patients. Differences in mesenchymal stem cells properties are discussed in relation to their involvement in the reparatory process.

Highlights

Despite the recent progress in medical and endovascular therapy of patients with critical limb ischemia (CLI), the prognosis of patients with no-option for revascularization remains poor
Human trials analyzing the effect of Vascular Endothelial Growth Factor (VEGF), Fibroblast Growth Factor (FGF-1), or combinations of growth factors (PDGF-BB and FGF-2) on prognosis of patients with CLI lead to an improvement of limb ischemia
Pilot human studies and randomized controlled trials have shown that autologous transplantation of bone marrow cells induced therapeutic angiogenesis in patients with limb ischemia

Summary

Introduction

Despite the recent progress in medical and endovascular therapy of patients with critical limb ischemia (CLI), the prognosis of patients with no-option for revascularization remains poor. Human trials analyzing the effect of Vascular Endothelial Growth Factor (VEGF), Fibroblast Growth Factor (FGF-1), or combinations of growth factors (PDGF-BB and FGF-2) on prognosis of patients with CLI lead to an improvement of limb ischemia They did not show significant effect on reduction of leg amputation [2,3]. We have demonstrated that application of autologous bone marrow mononuclear cells in forty-one patients with advanced CLI not eligible for revascularization prevented major limb amputation in 73% during the 6-month follow-up. Application of autologous bone marrow mononuclear cells to “no option” patients with advanced critical limb ischemia (CLI) prevented major limb amputation in 73% patients during the 6-month follow-up. Conclusions: The quality of mesenchymal stem cells shown in the expression of cell surface markers, secreted factors and stem cell genes plays an important role in therapeutic outcome. Differences in mesenchymal stem cells properties are discussed in relation to their involvement in the reparatory process

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Conclusion