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
cDNA microarray technology is widely used in various biological and medical disciplines to determine gene expression profiles. Unfortunately, this technology requires a large quantity of input RNA. Since there is an increasing need for more precise analyses of defined cell subpopulations with low cell counts, working protocols using a minimal number of input cells are required. Optimal RNA isolation and its accurate amplification are crucial to the success of these protocols. The HL-60 cell line was used in the search for a suitable protocol that can be used for clinical samples of CD34+ haematopoietic cells obtained from bone marrow. The goal was to discover the best method for isolating and amplifying RNA from a small number of cells. Our evaluation of various methods and kits available in the market revealed that the combination of RNAqueous™ Kit for RNA isolation and the SenseAmp Plus Kit for one-round RNA amplification produced the best results. This article presents a verified protocol describing a reliable and reproducible method for obtaining enough input RNA for microarray experiments when the number of cells is limited.
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
