COVID-19 is caused by the SARS-CoV-2 virus and has spread globally in 2020. Cellular immunity may serve as an important functional marker of the disease, especially in the asymptomatic cases. Blood samples were collected from 46 convalescent donors with a history of COVID-19 and 38 control donors. Quantification of the T-cell response upon contact with SARS-CoV-2 proteins in vitro was based on IFN-γ. Significantly higher numbers of activated cells were measured in patients who underwent COVID-19. Anti-SARS-CoV-2 T cells were detected weeks after the active virus disappeared from the organism. Repeated sample collection after five months proved that the T-cell activation was weaker in time in 79 % of the patients. In the majority of cases, the CD4+ helper T-cell subpopulation was responsible for the immune reaction. Moreover, different viral proteins triggered activation in CD4+ helper and in CD8+ cytotoxic T cells. Together, these findings suggest that the T-cell activation level identifies the individuals who underwent COVID-19 and may become a diagnostic tool for the disease.
- MeSH
- aktivace lymfocytů MeSH
- COVID-19 * MeSH
- lidé MeSH
- protilátky virové MeSH
- SARS-CoV-2 MeSH
- T-lymfocyty MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- Publikační typ
- abstrakt z konference MeSH
The generation of haematopoietic progenitors from human pluripotent stem cells (hPSCs) presents great promise for cell-replacement therapies. However, current protocols for haematopoietic differentiation of hPSCs suffer from low efficiency and functional defects in the derived cells. The technology is also limited by variable ability of hPSC lines to generate blood cells in vitro. To address this issue, methodologies for haematopoietic differentiation in feeder-free conditions were applied to available human embryonic stem cell (hESC) and human induced pluripotent stem cell (hiPSC) lines in this study. It was found that these cell lines did not generate haematopoietic progenitors to such an extent as did H1 and H9 hESC lines that were used for this purpose in the vast majority of relevant studies. These results suggest that for clinical application of blood cells derived from hPSCs, possibly from autologous hiPSCs, it is necessary to overcome the variability in the haematopoietic developmental potential of individual hPSC lines.
Preclinical studies have demonstrated the promising potential of human induced pluripotent stem cells (hiPSCs) for clinical application. To fulfil this goal, efficient and safe methods to generate them must be established. Various reprogramming techniques were presented during seven years of hiPSCs research. Genome non-integrating and completely xeno-free protocols from the first biopsy to stable hiPSC clones are highly preferable in terms of future clinical application. In this short communication, we summarize the reprogramming experiments performed in our laboratories. We successfully generated hiPSCs using STEMCCA lentivirus, Sendai virus or episomal vectors. Human neonatal fibroblasts and CD34(+) blood progenitors were used as cell sources and were maintained either on mouse embryonic feeder cells or in feeder-free conditions. The reprogramming efficiency was comparable for all three methods and both cell types, while the best results were obtained in feeder-free conditions.
- MeSH
- antigeny CD34 metabolismus MeSH
- biologické markery metabolismus MeSH
- buněčné kultury metody MeSH
- buněčné linie MeSH
- genom lidský genetika MeSH
- imunohistochemie MeSH
- indukované pluripotentní kmenové buňky cytologie metabolismus MeSH
- lidé MeSH
- myši MeSH
- pluripotentní kmenové buňky cytologie metabolismus MeSH
- přeprogramování buněk genetika MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- myši MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
CD34 is the most frequently used marker for the selection of cells for bone marrow (BM) transplantation. The use of CD133 as an alternative marker is an open research topic. The goal of this study was to evaluate the proliferation and differentiation potential for hematopoiesis (short and long term) of CD133+ and CD34+ populations from bone marrow and mobilized peripheral blood. Eight cell populations were compared: CD34+ and CD133+ cells from both the BM (CML Ph-, CML Ph+, and healthy volunteers) and mobilized peripheral blood cells. Multicolor flow cytometry and cultivation experiments were used to measure expression and differentiation of the individual populations. It was observed that the CD133+ BM population showed higher cell expansion. Another finding is that during a 6-day cultivation with 5(6)-carboxyfluorescein diacetate N-succinimidyl ester (CFSE), more cells remained in division D0 (non-dividing cells). There was a higher percentage of CD38- cells observed on the CD133+ BM population. It was also observed that the studied populations contained very similar but not the same pools of progenitors: erythroid, lymphoid, and myeloid. This was confirmed by CFU-GM and CFU-E experiments. The VEGFR antigen was used to monitor subpopulations of endothelial sinusoidal progenitors. The CD133+ BM population contained significantly more VEGFR+ cells. Our findings suggest that the CD133+ population from the BM shows better proliferation activity and a higher distribution of primitive progenitors than any other studied population.
- MeSH
- antigeny CD34 krev imunologie MeSH
- biologické markery metabolismus MeSH
- buněčná diferenciace imunologie MeSH
- buněčný rodokmen MeSH
- buňky kostní dřeně cytologie fyziologie MeSH
- CD antigeny krev imunologie MeSH
- glykoproteiny krev imunologie MeSH
- hematopoetické kmenové buňky cytologie fyziologie MeSH
- krevní buňky cytologie fyziologie MeSH
- kultivované buňky MeSH
- lidé MeSH
- peptidy krev imunologie MeSH
- proliferace buněk MeSH
- separace buněk MeSH
- vaskulární endoteliální růstový faktor A metabolismus MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
This study combines mRNA and protein analysis using cDNA and antibody microarray techniques, respectively. These create a novel, integrated perspective into cellular molecular profiles. The aims of this study were to establish a reliable way of integrating these two approaches in order to obtain complex molecular profiles of the cell and to find suitable methods to normalize the data obtained using these approaches.
Antibody microarray and cDNA microarray techniques were used to study expression alterations in HL-60 cells that were differentiated into granulocytes using all-trans retinoic acid (ATRA). We selected this model to evaluate this combined profiling technique because the expression levels of most of the mRNA and protein species in these cells are not altered; therefore it is easier to track and define those species that are changed. The proteins whose levels were altered included c-myc, c-jun, Pyk2, FAK, PKC, TRF1, NF-kappaB and certain caspase types. These proteins are involved in apoptosis and hematopoietic differentiation pathways, and some have also been reported to have oncogenic potential. We compared the results obtained using the two methods, verified them by immunoblotting analysis, and devised normalization approaches.
This is one of the first demonstrations that a combination of antibody microarray and cDNA microarray techniques is required for complex molecular profiling of cells based on multiple parameters. This approach allows a more detailed molecular phenotype of the given sample to be obtained. The results obtained using a combination of the two profiling methods are consistent with those from previous studies that used more traditional methods.
Keywords: microarray, cell profiling, protein expression, mRNA expression, HL-60.- MeSH
- čipová analýza proteinů MeSH
- financování organizované MeSH
- fokální adhezní kinasa 2 analýza MeSH
- geny myc MeSH
- HL-60 buňky MeSH
- lidé MeSH
- messenger RNA analýza MeSH
- protein TRF1 analýza MeSH
- sekvenční analýza hybridizací s uspořádaným souborem oligonukleotidů MeSH
- tretinoin farmakologie MeSH
- Check Tag
- lidé MeSH
