Epigenetic dysregulation has been shown to limit functional capacity of aging hematopoietic stem cells, which may contribute to impaired outcome of hematopoietic stem cell-based therapies. The aim of our study was to gain better insight into the epigenetic profile of CD34+-enriched cell products intended for autologous CD34+ cell transplantation in patients with cardiomyopathy. We found global DNA methylation content significantly higher in immunoselected CD34+ cells compared to leukocytes in leukapheresis products (2.33 +- 1.03% vs. 1.84 +- 0.86%, p=0.04). Global DNA hydroxymethylation content did not differ between CD34+ cells and leukocytes (p=0.30). By measuring methylation levels of 94 stem cell transcription factors on a ready-to-use array, we identified 15 factors in which average promoter methylation was significantly different between leukocytes and CD34+ cells. The difference was highest for HOXC12 (58.18 +- 6.47% vs. 13.34 +- 24.18%, p=0.0009) and NR2F2 (51.65 +-25.89% vs. 7.66 +- 21.43%, p=0.0045) genes. Our findings suggest that global DNA methylation and hydroxymethylation patterns as well as target methylation profile of selected genes in CD34+-enriched cell products do not differ significantly compared to leukapheresis products and, thus, can tell us little about the functional capacity and regenerative properties of CD34+ cells. Future studies should examine other CD34+ cell graft characteristics, which may serve as prognostic tools for autologous CD34+ cell transplantation.
COBISS.SI-ID: 33302489
Age-related telomere attrition in stem/progenitor cells may diminish their functional capacity and thereby impair the outcome of cell-based therapies. The aim of the present study was to investigate the effect of CD34+ cell telomere length and hTERT expression on the clinical outcome of autologous CD34+ cell transplantation. We studied 43 patients with cardiomyopathy. Their peripheral blood CD34+ cells were mobilized with granulocyte colony-stimulating factor, enriched by immunoselection and delivered transendocardially. Relative telomere length and expression levels of hTERT were measured using a real-time PCR assay. Immunoselected CD34+ cells had longer telomere length compared to leukocytes in leukapheresis products (p=0.001). In multivariate analysis, CD34+ cell telomere length was not associated with the clinical outcome (b=3.306, p=0.540). While hTERT expression was undetectable in all leukapheresis products, 94.4% of the CD34+ enriched cell products expressed hTERT. Higher CD34+hTERT expression was associated with a better clinical outcome on univariate analysis (b=87.911, p=0.047). Our findings demonstrate that CD34+ cell telomere length may not influence the clinical outcome in cardiomyopathy patients treated with autologous CD34+ cell transplantation. Larger studies are needed to validate the impact of the CD34+hTERT expression on the clinical outcome of autologous CD34+ cell transplantation.
COBISS.SI-ID: 33303001
Background aims. Clinical protocols for dendritic cell (DC) generation from monocytes require the use of animal serum- free supplements. Serum-free media can also require up to 1% of serum supplementation. In addition, recommendations based on the 3Rs (Refinement, Reduction, Replacement) principle also recommend the use of non-animal sera in in vitro studies. The aim of this study was to explore the potential use of platelet lysate (PL) for generation of optimally differen- tiated DCs from monocytes. Methods. Cells were isolated from buffy coats from healthy volunteers using immunomagnetic selection. DCs were differentiated in RPMI1640 supplemented with either 10% fetal bovine serum (FBS), 10% AB serum or 10% PL with the addition of granulocyte monocyte colony stimulating factor and interleukin-4. Generated DCs were assessed for their morphology, viability, endocytotic capacity, surface phenotype (immature, mature and tolerogenic DCs) and activation of important signaling pathways. DC function was evaluated on the basis of their allostimulatory capacity, cytokine profile and ability to induce different T-helper subsets. Results. DCs generated with PL displayed normal viability, morphology and endocytotic capacity. Their differentiation and maturation phenotype was comparable to FBS-cultured DCs. They showed functional plasticity and up-regulated tolerogenic markers in response to their environment. PL-cultured mature DCs displayed unhindered allostimulatory potential and the capacity to induce Th1 responses. The use of PL allowed for activation of crucial signaling proteins associated with DC differentiation and maturation. Discussion. This study demon- strates for the first time that human PL represents a successful alternative to FBS in differentiation of DCs from monocytes. DCs display the major phenotypic and functional characteristics compared with existing culture protocols.
COBISS.SI-ID: 33118169
Durable engraftment of transplanted CD34+ cells largely depends on the quality of the cell product. Limited data are currently available about extended storage of immunoselected CD34+ cells. The aim of our study was to assess the stability of CD34+ cell product with the cells stored in high concentration (80 × 106 in 6 mL) in small bags intended for cell implantation. Cell products were prepared by leukapheresis and immunoselection (Clinimacsplus procedure) from 13 patients with chronic dilated cardiomyopathy. CD34+ cell products were stored at 2–8 °C and analyzed at time 0 (fresh products), 24, 48 h, 4 and 6 days. Product viability, absolute number of viable CD34+ cells and apoptosis were determined by flow cytometry. Microbiological contamination of the cell products was tested by BACTEC system. The mean viability of CD34+ cells decreased by 2.7% within 24 h, by 13.4% within 48 h and by 37.5% within 6 days. The mean recovery of viable CD34+ cells was 91.1%, 74.8%, 66.3% and 56.2% at 24, 48 h, 4 and 6 days, respectively. The mean fraction of early apoptotic cells in fresh and stored products was 4.9 ± 3.5% at 0 h, 5.9 ± 3.8% at 24 h, 4.2 ± 3.1% at 48 h, 6.3 ± 2.6% at 4 days and 9.3 ± 4.6% at 6 days. All products were negative for microbial contamination.
COBISS.SI-ID: 33342937
Cystatin F is a cysteine peptidase inhibitor which, unlike other cystatin family members, is targeted to endosomal/lysosomal compartments. It is synthesized as an inactive disulfide-linked dimer which is then converted to an active monomer by proteolytic cleavage of 15 N-terminal residues. Cystatin F has been suggested to regulate the cytotoxicity of natural killer (NK) cells by inhibiting the major granzyme convertases, cathepsins C and H. To test this hypothesis, we prepared variants of cystatin F and analyzed their uptake, subcellular trafficking, and peptidase inhibition, as well as their impact on the cytotoxicity of NK-92 cells and primary NK cells. The N-glycosylation pattern is responsible for the secretion, uptake, and subcellular sorting of cystatin F in HeLa and Hek293 cells, whereas the legumain binding site had no effect on these processes. Active, N-terminally truncated, monomeric cystatin F can also be internalized by recipient cells and targeted to endo/lysosomes, affecting also cells lacking the activating peptidase. Cystatin F mutants capable of cell internalization and trafficking through the endo/lysosomal pathway significantly decreased cathepsin C and H activities, both in situ, following transfection and in trans, using conditioned media. Further, incubation of IL-2 stimulated NK-92 and primary NK cells with full-length and N-terminally truncated cystatin F mutants led to suppression of their granule-mediated cytotoxicity. This effect was most significant with the N-terminally truncated mutants. These results suggest that cystatin F can be an important mediator within tumor microenvironment affecting the cytotoxicity of NK cells and consequently antitumor immune response.
COBISS.SI-ID: 30930471