5 research outputs found
Characterization of mesenchymal stem cells of "no-options" patients with critical limb ischemia treated by autologous bone marrow mononuclear cells.
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. 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.Mesenchymal 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.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. 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
Expression of cell surface markers characterizing MSCs.
<p>(A) Expression of MSCs markers on the surface of mesenchymal stem cells of group of responders versus non responders (** - statistic significance is ≤ 0.01; * - statistic significance is ≤ 0.02). (B) Expression of MSCs markers on the surface of mesenchymal stem cells of group of diabetic patients (n=13) versus non diabetic (n=9).</p
Doubling time of continually cultivated MSCs from CLI patients in comparison to healthy young donor.
<p>(<b>A</b>) Ability of MSCs of CLI patient to differentiate to osteogenic, adipogenic and chondrogenic lineages; (<b>B</b>) Doubling time of MSCs of CLI patients in first passage. The value is an average of two independent estimations; (<b>C</b>) Doubling time of continually cultivated MSCs from four CLI patients in comparison to healthy young donor. Lines represent the trend.</p
Secretion of factors from MSCs and gene expression.
<p>(<b>A</b>) Secretome comparison of several factors of responders (n=27) and non-responders (n=14) CLI patients; For detection of cytokines, chemokines and growth factors Bio-Plex Pro Human Cytokine 27-plex Assay from Bio-Rad was used. The concentration of each factor was calculated per mg of total proteins in 24 hour conditioned medium. The values of growth factors detected in control medium were deducted from values found in conditioned medium. (B) Relative expression of 15 genes typical for MSCs on the level of proteins. Cell extracts of MSCs in early passage of seven non-responders and eight responders were examined by Proteome Profiler Human Pluripotent Stem Cell Array.</p