Edyta Paczkowska

Mobilization of bone marrow-derived Oct-4+ SSEA-4+ very small embryonic-like stem cells in patients with acute myocardial infarction.

OBJECTIVES This study sought to assess of the mobilization of nonhematopoietic very small embryonic-like stem cells (VSELs) in acute myocardial infarction (MI). BACKGROUND Acute MI induces mobilization of bone marrow stem cells. Recently, a rare population of VSELs, expressing markers of embryonic pluripotent stem cells (PSCs), was identified in adult murine bone marrow and human umbilical cord blood. METHODS Thirty-one patients with acute MI and 30 healthy subjects were enrolled. Blood was sampled on admission, after 24 h, and 5 days later. Erythrocytes were lysed and lin(-)CD45(-) VSELs were isolated using a live cell sorting system (FACSAria, Beckton Dickinson, San Jose, California). RESULTS In healthy subjects the median number of circulating VSELs was very low (median 0.8 [range 0 to 1.3]) cells/microl. In acute MI, VSELs were mobilized early (median 2.7 [range 0.2 to 3.9] cells/microl; p < 0.001) and remained elevated after 24 h and 5 days (median 4.7 [range 0.2 to 6.4] cells/microl; p < 0.003, and median 2.6 [range 0.3 to 3.6] cells/microl; p < 0.03, respectively). The mobilization of VSEL was significantly reduced in patients older than 50 years and with diabetes in comparison with younger and nondiabetic patients. Circulating VSELs were small (7 to 8 microm) and enriched in the messenger ribonucleic acid of PSC markers (Oct-4, Nanog), cardiac lineage (GATA-4, Nkx2.5/Csx, MEF2C), and endothelial (VE-cadherin) markers. The presence of PSC markers (Oct-4, SSEA-4) and the chemokine receptor CXCR4 in circulating VSELs was confirmed at the protein level by immunofluorescent staining and ImageStream system (Amnis Corporation, Seattle, Washington) analysis. CONCLUSIONS Acute MI induced mobilization of VSELs expressing pluripotent markers, early cardiac and endothelial markers, and chemokine receptor CXCR4.

Clinical Evidence That Very Small Embryonic-Like Stem Cells Are Mobilized Into Peripheral Blood in Patients After Stroke

Background and Purpose— In a murine model of stroke, we identified a population of very small embryonic-like (VSEL) stem cells (SCs) in adult murine bone marrow that could be mobilized into peripheral blood (PB). This raised the question of whether a similar population of cells is mobilized in human stroke patients. Methods— We evaluated a number of cells that corresponded to VSEL SCs in the PB of 44 stroke patients and 22 age-matched controls. After each patient’s stroke, PB samples were harvested during the first 24 hours, on day +3, and on day +7 and then compared with normal controls. The circulating human cells with the phenotype of VSEL SCs were evaluated in PB by real-time quantitative polymerase chain reaction, fluorescence-activated cell sorting analysis, and direct immunofluorescence staining. In parallel, we also measured the serum concentration of stromal derived factor-1 by ELISA. Results— In stroke patients, we found an increase in the number of circulating cells expressing SC-associated antigens, such as CD133, CD34, and CXCR4. More important, we found an increase in the number of circulating primitive cells expressing the VSEL phenotype (CXCR4+lin-CD45- small cells), mRNA for Octamer-4 and Nanog, and Octamer-4 protein. All changes were accompanied by an increased serum concentration of stromal derived factor-1. Additionally, we found a positive correlation between stroke extensiveness, stromal derived factor-1 concentration in serum, and the number of CXCR4+ VSEL SCs circulating in the PB. Conclusions— We conclude that stroke triggers the mobilization of CXCR4+ VSEL SCs that have potential prognostic value in stroke patients. However, the potential role of these mobilized cells in brain regeneration requires further study.

Increased circulating endothelial progenitor cells in patients with haemorrhagic and ischaemic stroke: The role of Endothelin-1

Ischaemic stroke induces endothelial progenitor cell (EPC) mobilisation from bone marrow into peripheral blood. Circulating EPCs play an important role in post-injury regeneration of vasculature, whereas endothelial cells (ECs) have been shown to reflect endothelial damage and may be responsible for increased Endothelin-1 (ET-1) expression. We investigated herein the association between numbers of circulating ECs and EPCs, the levels of soluble factors regulating their migration and function, and the clinical outcome in patients with haemorrhagic (HS) or ischaemic stroke (IS). Sixteen patients with HS and eighteen with IS were assessed during the first 24h, day 3, and day 7 after stroke and compared them with twenty-three control subjects. We found elevated EPC and EC concentrations using flow cytometry and increase in VEGF, SDF-1, HGF, and ET-1 plasma levels by ELISA in the HS patients, while ET-1 mRNA expression in peripheral blood cells was elevated in the IS patients. Significant correlations were observed between EPCs or ECs and Big ET-1 protein or mRNA levels in HS but not in the IS patients. We suggest that ET-1 may play a role in pathophysiology of stroke and subsequent EPC mobilisation; however, further studies aimed at the precise elucidation of this issue are required.

Different populations of circulating endothelial cells in patients with age-related macular degeneration: a novel insight into pathogenesis.

PURPOSE Circulating endothelial cells (CECs) and endothelial progenitor cells (EPCs) may serve as novel markers of endothelial dysfunction. The presence and clinical implications of CECs and the expression of endothelin (ET)-1, one of the most potent vasoconstrictors, have not been evaluated in patients with the neovascular form of age-related macular degeneration (AMD). This study was conducted to determine the different populations of endothelial cells (ECs) in the peripheral blood of AMD patients and to correlate these findings with the expression of ET-1 and the cytokines and growth factors responsible for EC migration and function. METHODS Peripheral blood samples were collected from 29 patients with diagnosed neovascular AMD and from 38 healthy control subjects. CD133(-)CD144(+) CECs and CD34(+)CD133(+)CD144(+) EPCs were counted and analyzed by flow cytometry. The intracellular expression of ET-1 in peripheral blood nuclear cells (PBNCs) was studied by using qRT-PCR, Western blot, and immunocytofluorescence assays, and ET-1, IGF-1, VEGF, SDF-1, and HGF plasma concentrations were measured in enzyme-linked immunosorbent assays. RESULTS Increased CECs and EPCs were found in the AMD patients compared with the counts in healthy individuals. The expression of intracellular ET-1 was significantly elevated in PBNCs from the AMD patients compared with the control subjects. In addition a significantly higher plasma concentration of IGF-1 was observed, but a lower SDF-1 level in the group of AMD patients. CONCLUSIONS These findings suggest that circulating endothelial cells, together with high ET-1 content, may contribute to the development of AMD. Further prospective investigations on the mechanism involved may be relevant to the potential treatment of this disease.

The influence of 3,3′,5-triiodo-l-thyronine on human haematopoiesis

Abstract.  Objectives: Thyroid hormones mediate many physiological and developmental functions in humans. The role of the 3,3′,5‐triiodo‐l‐thyronine (T3) in normal human haematopoiesis at the cellular and molecular levels has not been determined. In this study, it was revealed that the human haematopoietic system might be directly depended on T3 influence. Materials and methods: We detected the TRα1 and TRβ1 gene expression at the mRNA level in human cord blood, peripheral blood and bone marrow CD34+‐enriched progenitor cells, using the RT‐PCR method. Furthermore, we performed Western blotting to prove TRα1 and TRβ1 expression occurs at the protein level in human cord blood, peripheral blood and bone marrow CD34+ cells. In addition, the examined populations of cells were exposed in serum‐free conditions to increasing doses of T3 and were subsequently investigated for clonogenic growth of granulocyte‐macrophage colony‐forming unit and erythrocyte burst‐forming unit in methylcellulose cultures, and for the level of apoptosis, by employing annexin V staining and the terminal deoxynucleotidyltransferase‐mediated dUTP nick‐end labelling method. We investigated expression levels of apoptosis‐related Bax and antiapoptotic Bcl‐2 and Bcl‐xL genes in the examined cells. Results: We found that exposure to higher and lower than normal concentration of thyroid hormone significantly influenced clonogenecity and induced apoptosis in human haematopoietic progenitor cells. Conclusions: This study expands the understanding of the role of thyroid disorders in normal human haematopoiesis and indicates a direct influence of T3 on this process.

The impact of IL18 gene polymorphisms on mRNA levels and interleukin-18 release by peripheral blood mononuclear cells.

INTRODUCTION Interleukin-18 (IL-18) is a pleiotropic cytokine playing an important role as a modulator of immune responses, found to play a role in pathogenesis of numerous inflammatory-associated disorders. In the present study a potential association between 7 common single-nucleotide polymorphisms (SNPs) spanning the whole IL18 gene, gene expression and the release of IL-18 from the stimulated peripheral blood mononuclear cells (PBMCs) was investigated. MATERIALS/METHODS PBMCs were isolated from peripheral blood of 29 healthy volunteers, genotyped for the presence of IL18 SNPs: rs1946518: A>C, rs187238: G>C, rs360718: A>C, rs360722: C>T, rs360721: C>G, rs549908: T>G, and rs5744292: A>G. IL-18 concentration and IL18 mRNA levels were investigated after incubation of cells for 48 h with different stimulants (PHA, LPS, and anti-CD3/CD28 antibodies). RESULTS After treatment with LPS and antibodies IL-18 concentrations were significantly lower in rs1946518AA homozygotes than in C allele carriers. When differences in IL18 mRNA levels between non-stimulated and stimulated cells were analyzed, significantly decreased gene expression was noted in rs1946518 AA homozygotes (as compared with C allele carriers) in samples treated with PHA and LPS. Similar trends were observed in the case of rs187238 SNP; however, the differences reached statistical significance only after PHA treatment. CONCLUSIONS Our study supports the role of rs1946518 (-607A>C) and rs187238 (-137G>C) SNPs as genetic determinants of the observed variability in IL18 expression.

Isolation of hematopoietic stem cells from heparinized cadaveric multiple organ donors: potential clinical implications

THE HEPARINIZED cadaveric multiple organ donors (HCMOD) are an important source of organs and tissues for transplant purposes. Unfortunately, the HCMODs hematopoietic stem cells (HSC) are wasted at this moment. Since HCMOD are heparinized before an organ donation, blood in their bone marrow cavities remains liquid and can be easily aspirated. The fact that before the donation HCMOD are always phenotyped for HLA antigens and evaluated for the presence of any potential blood transmitted diseases (HIV, HBV, CMV etc.) makes this source of cells safe and has an important economic aspect. Thinking in advance about a worldwide initiative to create a bank of HSC isolated from organ donors, we are working on an efficient, economic, and rapid method for isolating and banking cadaveric HSC. The aim of our study was to: (1) establish an efficient and rapid method for extracting hematopoietic stem cells from HCMODs, (2) evaluate if bone marrow in HCMODs remains not infected after removal of other organs, before the marrow cells are harvested, (3) elaborate an efficient protocol for sending extracted marrow cells from place of donation to the central bank, which will collect, process, and long-term store these cells.

Humoral Activity of Cord Blood-Derived Stem/Progenitor Cells: Implications for Stem Cell-Based Adjuvant Therapy of Neurodegenerative Disorders

Background Stem/progenitor cells (SPCs) demonstrate neuro-regenerative potential that is dependent upon their humoral activity by producing various trophic factors regulating cell migration, growth, and differentiation. Herein, we compared the expression of neurotrophins (NTs) and their receptors in specific umbilical cord blood (UCB) SPC populations, including lineage-negative, CD34+, and CD133+ cells, with that in unsorted, nucleated cells (NCs). Methods and Results The expression of NTs and their receptors was detected by QRT-PCR, western blotting, and immunofluorescent staining in UCB-derived SPC populations (i.e., NCs vs. lineage-negative, CD34+, and CD133+ cells). To better characterize, global gene expression profiles of SPCs were determined using genome-wide RNA microarray technology. Furthermore, the intracellular production of crucial neuro-regenerative NTs (i.e., BDNF and NT-3) was assessed in NCs and lineage-negative cells after incubation for 24, 48, and 72 h in both serum and serum-free conditions. We discovered significantly higher expression of NTs and NT receptors at both the mRNA and protein level in lineage-negative, CD34+, and CD133+ cells than in NCs. Global gene expression analysis revealed considerably higher expression of genes associated with the production and secretion of proteins, migration, proliferation, and differentiation in lineage-negative cells than in CD34+ or CD133+ cell populations. Notably, after short-term incubation under serum-free conditions, lineage-negative cells and NCs produced significantly higher amounts of BDNF and NT-3 than under steady-state conditions. Finally, conditioned medium (CM) from lineage-negative SPCs exerted a beneficial impact on neural cell survival and proliferation. Conclusions Collectively, our findings demonstrate that UCB-derived SPCs highly express NTs and their relevant receptors under steady-state conditions, NT expression is greater under stress-related conditions and that CM from SPCs favorable influence neural cell proliferation and survival. Understanding the mechanisms governing the characterization and humoral activity of subsets of SPCs may yield new therapeutic strategies that might be more effective in treating neurodegenerative disorders.

Influence of Ranibizumab on Vascular Endothelial Growth Factor Plasma Level and Endothelial Progenitor Cell Mobilization in Age-Related Macular Degeneration Patients: Safety of Intravitreal Treatment for Vascular Homeostasis

PURPOSE Intravitreal ranibizumab, which neutralizes vascular endothelial growth factor (VEGF), nowadays constitutes the first-line treatment in neovascular age-related macular degeneration (AMD). However, its potential systemic effect on vascular homeostasis as the consequence of such therapy has not been extensively investigated. METHODS Peripheral blood (PB) samples from 12 patients with newly diagnosed neovascular AMD were analyzed before as well as 1 and 4 weeks after intravitreal treatment with ranibizumab. VEGF plasma levels, the number of circulating endothelial progenitor cells (EPCs), and the intracellular expression of hypoxia-inducible factor (HIF) in PB cells were determined by enzyme-linked immunosorbent assay, flow cytometry, and real-time quantitative reverse transcriptase-polymerase chain reaction assays, respectively. RESULTS No significant changes within the analyzed parameters were found in the first or fourth weeks after ranibizumab injection compared with the primary, basic values before treatment. Based on our findings, intravitreal ranibizumab does not induce significant systemic effects or vascular impairment. CONCLUSIONS Evaluation of the VEGF plasma level, the PB EPC concentration, and intracellular HIF expression may be supportive indicators of drug safety for ranibizumab.

Neural stem/progenitor cells circulating in peripheral blood of patients with neovascular form of AMD: a novel view on pathophysiology

BackgroundThe neovascular form of age-related macular degeneration (AMD) manifested with choroidal neovascularization (CNV) is one of the leading causes of rapid and irreversible visual loss. Recent reports suggest that bone marrow-derived stem/progenitor cells (SPCs) play a crucial role in the development and progression of the disease. The purpose of this study was to investigate whether or not undifferentiated non-haematopoietic stem cells, including those capable of differentiating into neural phenotypes, play a role in the pathological state of CNV formation.MethodsPeripheral blood samples were collected from 46 patients diagnosed with CNV and from 46 controls. The CXCR4+Lin-CD45- stem cells were counted and analysed by flow cytometry. Using qRT-PCR and immunocytofluorescence, the expression of early neural and glial cell markers (β-III-tubulin, nestin, and glial fibrillary acidic protein) in the sorted cells was analysed, and correlated with plasma concentrations of stromal cell-derived factor 1 (SDF-1) (enzyme-linked immunosorbent assay), which is a pivotal chemokine that regulates the trafficking of SPCs.ResultsWe found that the number of circulating CXCR4+Lin-CD45- cells did not differ in patients with active CNV as compared to the controls. However, we noticed significant intracellular overexpression of β-III-tubulin in the cells derived from AMD patients. Moreover, we observed significantly lower SDF-1 plasma levels in neovascular AMD patients compared to healthy individuals.ConclusionsOur findings suggest that neural progenitor cells, together with low SDF-1 concentrations, may play a considerable role in the process of AMD progression. Further investigations aimed at the precise elucidation of these issues may help with the future development of effective prevention against, and the treatment of, this disease.