Abstract: OBJECTIVE: Mesenchymal stromal cells (MSCs) from adult bone marrow (BM) are considered potential candidates for therapeutic neovascularization in cardiovascular disease. When implementing results from animal trials in clinical treatment, it is essential to isolate and expand the MSCs under conditions following good manufacturing practice (GMP). The aims of the study were first to establish culture conditions following GMP quality demands for human MSC expansion and differentiation for use in clinical trials, and second to compare these MSCs with MSCs derived from culture in four media commonly used for MSC cultivation in animal studies simulating clinical stem cell therapy. MATERIAL AND METHODS: Human mononuclear cells (MNCs) were isolated from BM aspirates by density gradient centrifugation and cultivated in a GMP-accepted medium (EMEA medium) or in one of four other media. RESULTS: FACS analysis showed that the plastic-adherent MSCs cultured in EMEA medium or in the other four media were identically negative for the haematopoietic surface markers CD45 and CD34 and positive for CD105, CD73, CD90, CD166 and CD13, which in combined expression is characteristic of MSCs. MSC stimulation with vascular endothelial growth factor (VEGF) increased expression of the characteristic endothelial genes KDR and von Willebrand factor; the von Willebrand factor and CD31 at protein level as well as the capacity to develop capillary-like structures. CONCLUSIONS: We established culture conditions with a GMP compliant medium for MSC cultivation, expansion and differentiation. The expanded and differentiated MSCs can be used in autologous mesenchymal stromal cell therapy in patients with ischaemic heart disease.
Abstract: BACKGROUND: YKL-40 is involved in remodelling and angiogenesis in non-cardiac inflammatory diseases. Aim was to quantitate plasma YKL-40 in patients with ST-elevation myocardial infarction (STEMI) or stable chronic coronary artery disease (CAD), and YKL-40 gene activation in human myocardium. METHODS AND RESULTS: We included 73 patients: I) 20 patients with STEMI; II) 28 patients with stable CAD; III) 15 CAD patients referred for coronary by-pass surgery. YKL-40 mRNA expression was measured in myocardium subtended by stenotic or occluded arteries and areas with no apparent disease; and IV) 10 age-matched healthy controls. Plasma YKL-40 was significantly increased in patients with STEMI (88 microg/l, median) and CAD (66 microg/l) compared to controls (16 microg/l, p<0.01 for both). Plasma YKL-40 correlated with CRP at baseline in STEMI (r=0.53, p=0.02) and CAD patients (r=0.41, p=0.031).YKL-40 gene expression was similar in ischemic and non-ischemic myocardium. CONCLUSIONS: Plasma YKL-40 was significantly increased in patients with STEMI and stable CAD. Further studies will define the role of YKL-40 as a clinically useful marker for myocardial ischemia, remodelling and maybe prognosis.
Abstract: Despite progress in percutaneous coronary intervention, bypass surgery and drug therapy, rates of mortality and morbidity after acute coronary syndrome are high due to ventricular remodeling and heart failure. Mesenchymal stromal cells (MSCs) from adult bone marrow or adipose tissue are considered potential candidates for therapeutic regenerative treatment in cardiovascular disease. Recent animal studies have demonstrated that MSCs can induce neovascularization and improve myocardial function in postinfarction myocardial ischemic hearts. This review will focus on the present preclinical and clinical knowledge about the use of mononuclear cells and MSCs for cardiac regenerative medicine, the source of MSCs for clinical use and problems to consider when conducting clinical MSC therapy.
Abstract: BACKGROUND: Studies have shown that stem cell therapy could be a novel option for improving neovascularization and cardiac function in patients with ischemic heart disease. Human mesenchymal stromal cells (MSC) have generated wide interest in the clinical setting because of their ability to regenerate tissue. The aim of the study was to test whether freezing and storage of human BM mononuclear cells (BM-MNC) and ex vivo-expanded MSC influenced their phenotypic and functional characteristics as well as proliferation capacity. METHODS: MNC were isolated from BM and divided into two portions: one part was immediately cultured (MSC P0) whereas the second part was frozen for a week before cultivation and analysis (F-MSC P1). Confluent MSC (P0) were harvested and divided: one was analyzed as MSC P1 and the other was frozen for a week before further cultivation and analysis as F-MSC P2. RESULTS: MSC P1, F-MSC P1 and F-MSC P2 had similar proliferation capacities and demonstrated almost identical expression levels of markers characteristic for MSC. The capacity to form endothelial vascular structures was independent of freezing. DISCUSSION: The proliferation and differentiation capacity as well as the cellular characteristics were identical in cultivated MSC derived from freshly isolated BM-MNC and MSC derived after freezing and storage of either freshly isolated BM-MNC or ex vivo-cultivated MSC. This highlights the potential clinical use of MSC in patients with cardiac and degenerative diseases, as it would be possible to inject MSC obtained from the same BM aspiration at different time points.
Abstract: BACKGROUND: Granulocyte-colony stimulating factor (G-CSF) after myocardial infarction does not affect systolic function when compared with placebo. In contrast, intracoronary infusion of bone marrow cells appears to improve ejection fraction. We aimed to evaluate the G-CSF mobilization of subsets of stem cells. METHODS AND RESULTS: We included 78 patients (62 men; 56+/-8 years) with ST-elevation myocardial infarction treated with primary percutaneous intervention <12 hours after symptom onset. Patients were randomized to double-blind G-CSF (10 microg/kg/d) or placebo. Over 7 days, the myocardium was exposed to 25x10(9) G-CSF mobilized CD34+ cells, compared with 3x10(9) cells in placebo patients (P<0.001); and to 4.9x10(11) mesenchymal stem cells, compared with 2.0x10(11) in the placebo group (P<0.001). The fraction of CD34+ cells/leukocyte increased during G-CSF treatment (from 0.3+/-0.2 to 1.1+/-0.9 x10(-3), P<0.001 when compared with placebo), whereas the fraction of putative mesenchymal stem cells/leukocyte decreased (from 22+/-17 to 14+/-11 x10(-3), P=0.01 when compared with placebo). An inverse association between number of circulating mesenchymal stem cells and change in ejection fraction was found (regression coefficient -6.8, P=0.004), however none of the mesenchymal cell subtypes analyzed, were independent predictors of systolic recovery. CONCLUSIONS: The dissociated pattern for circulating CD34+ and mesenchymal stem cells could be attributable to reduced mesenchymal stem cell mobilization from the bone marrow by G-CSF, or increased homing of mesenchymal stem cells to the infarcted myocardium. The inverse association between circulating mesenchymal stem cells and systolic recovery may be of clinical importance and should be explored further.
Abstract: OBJECTIVE: To investigate the spontaneous occurrence of circulating mesenchymal stem cells (MSC) and angiogenic factors in patients with ST elevation acute myocardial infarction (STEMI) treated with primary percutaneous coronary intervention (PCI). DESIGN: In 20 patients with STEMI, blood samples were obtained on days 1, 3, 7, 14, 21, and 28 after the acute PCI. Fifteen patients with a normal coronary angiography formed a control group. MSC (CD45-/CD34-), plasma stromal derived factor 1 (SDF-1), vascular endothelial growth factor A (VEGF-A), and fibroblast growth factor 2 (FGF-2) were measured by multiparametric flow cytometry and enzyme linked immunosorbent assay (ELISA). RESULTS: Circulating CD45-/CD34- cells were significantly decreased on day 7 compared with day 3. Cell counts normalised one month after the acute onset of STEMI. The changes were mainly seen in patients with a large infarction. Plasma SDF-1 increased significantly from day 3 to day 28, and VEGF-A and FGF-2 increased significantly from day 7 to day 28. CONCLUSIONS: Spontaneous sequential fluctuations in MSC and the increase in vascular growth factor concentrations after STEMI suggest that the optimal time for additional stem cell therapy is three weeks after a myocardial infarction to obtain the maximum effects by stimulating endogenous growth factors on the delivered stem cells.
Abstract: Aging is associated with decreased osteoblast-mediated bone formation leading to bone loss and increased risk for osteoporotic fractures. However, the cellular mechanisms responsible for impaired osteoblast functions are poorly understood. In the present study, we hypothesized that changes in bone microenvironment composition with aging are responsible for impaired osteoprogenitor cell recruitment and differentiation. As a model for bone microenvironment, we examined the effects of sera obtained from young (age 20-30 year old [yo], n=20) and old (70-84 yo, n=19) healthy female donors on cell proliferation and differentiation capacity into osteoblasts and adipocytes of human mesenchymal stem cells (hMSC). Cell proliferation rate determined by counting cell number was similar when the cells were cultured in the presence of media containing 5% sera from old or from young donors. Similarly, the number of adipocytes and levels of adipocytic gene expression was similar in cultures incubated with sera from young or old donors. We observed decreased osteoblastic gene expression in hMSC cultured either in pooled or individual sera of old donors compared to sera from young donors: core binding factor/runt-related binding factor 2 (Cbfa1/Runx2) 46%+/-2% (P<0.05), alkaline phosphatase (ALP) 45%+/-2% (P<0.05), collagen type I (Col-I) 50%+/-1% (P<0.05), and osteocalcin 65%+/-3% (P<0.05). This down-regulation of the mRNA was accompanied by reduced ALP enzyme activity by 25%+/-1% (P<0.01), immunocytochemical staining for osteoblastic markers: ALP, Col-I, and bone sialoprotein (BSP) as well as reduced in vitro mineralization as determined by Alizarin red staining. In conclusion, age-related changes in the serum composition and possibly hMSC microenvironment may contribute to the impaired osteoblast functions with aging. The factors mediating these changes remain to be determined.
Abstract: PURPOSE: Stem cell therapy seems to be a new treatment option within cardiac diseases to improve myocardial perfusion and function. However, the delivery and traceability of the cells represent a problem. Radioactive labelling with 111In could be a method for tracking mesenchymal stem cells (MSCs). However, 111In could influence the viability and differentiation capacity of MSCs, which would limit its use. Therefore, the aim of this study was to evaluate the influence of 111In labelling in doses relevant for SPECT imaging in humans on the viability and differentiation capacity of human MSCs. METHODS AND RESULTS: Human MSCs isolated from bone marrow were incubated with 111In-tropolone (15-800 Bq/cell). The labelling efficiency was approximately 25% with 30 Bq/cell 111In. The MSC doubling time was 1.04+/-0.1 days and was not influenced by 111In within the range 15-260 Bq/cell. Using 30 Bq 111In/cell it was possible to label MSCs to a level relevant for clinical scintigraphic use. With this dose, 111In had no effect on characteristic surface and intracellular markers of cultured MSCs analysed both by flow cytometry and by real-time polymerase chain reaction. Further, the labelled MSCs differentiated towards endothelial cells and formed vascular structures. CONCLUSION: It is possible to label human MSCs with 111In for scintigraphic tracking of stem cells delivered to the heart in clinical trials without affecting the viability and differentiation capacity of the MSCs. This creates an important tool for the control of stem cell delivery and dose response in clinical cardiovascular trials.
Abstract: Human bone marrow mesenchymal stem cells (hMSC) represent a population of stem cells that are capable of differentiation into multiple lineages. However, these cells exhibit senescence-associated growth arrest and phenotypic changes during long-term in vitro culture. We have recently demonstrated that overexpression of human telomerase reverse transcriptase (hTERT) in hMSC reconstitutes telomerase activity and extends life span of the cells [Nat. Biotechnol. 20 (2002) 592]. In the present study, we have performed extensive characterization of three independent cell lines derived from the parental hMSC-TERT cell line based on different plating densities during expansion in culture: 1:2 (hMSC-TERT2), 1:4 (hMSC-TERT4), and 1:20 (hMSC-TERT20). The 3 cell lines exhibited differences in morphology and growth rates but they all maintained the characteristics of self-renewing stem cells and the ability to differentiate into multiple mesoderm-type cell lineages: osteoblasts, adipocytes, chondrocytes, and endothelial-like cells over a 3-year period in culture. Also, surface marker studies using flow cytometry showed a pattern similar to that known from normal hMSC. Thus, telomerization of hMSC by hTERT overexpression maintains the stem cell phenotype of hMSC and it may be a useful tool for obtaining enough number of cells with a stable phenotype for mechanistic studies of cell differentiation and for tissue engineering protocols.
Abstract: Closely related signals often lead to very different cellular outcomes. We found that the differentiation of human mesenchymal stem cells into bone-forming cells is stimulated by epidermal growth factor (EGF) but not platelet-derived growth factor (PDGF). We used mass spectrometry-based proteomics to comprehensively compare proteins that were tyrosine phosphorylated in response to EGF and PDGF and their associated partners. More than 90% of these signaling proteins were used by both ligands, whereas the phosphatidylinositol 3-kinase (PI3K) pathway was exclusively activated by PDGF, implicating it as a possible control point. Indeed, chemical inhibition of PI3K in PDGF-stimulated cells removed the differential effect of the two growth factors, bestowing full differentiation effect onto PDGF. Thus, quantitative proteomics can directly compare entire signaling networks and discover critical differences capable of changing cell fate.
