Cord blood (CB) is a valid alternative to mobilized peripheral blood (mPB) as a source of hematopoietic stem cell for clinical transplantation. The qualitative differences among CD133+ cells from these sources have been demonstrated by in vitro and in vivo studies but the molecular phenotype of these cells is only partially understood. In view of better exploiting the peculiarities and advantages of both the stem cell sources, we performed a comparative analysis of gene expression of CD133+ from CB and mPB using Affymetrix HG-U133A arrays. The CD133+ cells were selected using the MiniMacs immunomagnetic separation system (Miltenyi Biotec) from 6 CB units and 6 mPB samples. Since microarray studies are strictly dependent on the purity of the cell population and on the high RNA quality, it was crucial to apply rigorous quality controls. The purity of CD133+ cells was consistently more than 93% in all samples. In addition, after extraction, the RNA was purified by the RNeasy system (Qiagen) and the RNA quality was evaluated using the RNA 6000 Nano LabChip kit (Agilent Technologies). Given the low amount of RNA due to the limited numbers of CB CD133+ cells, we used a double-cycle amplification protocol for both sources to obtain biotin-labeled target for hybridization starting from as low as 100 ng of total RNA. Probe level data have been converted to expression values using Affymetrix MAS 5.0 algorithm. Differentially expressed genes have been identified using Significance Analysis of Microarrays (SAM). The cutoff for significance has been determined tuning the  parameter on the false positive rate and controlling the q-value for the gene list. We found that mPB CD133+ cells expressed cell cycle driving, transcription factor and DNA synthesis genes at distinctly higher levels than CB CD133+ cells, indicating a higher cycling activity in mPB CD133+ cells. Furthermore apoptosis driving genes were up-regulated in mPB CD133+ cells, probably due to G-CSF mobilization. Conversely in CB CD133+ cells we found an overexpression of genes involved in immunoresponse and immunomodulation. Since all the CB units were collected after vaginal delivery, this finding could be related to the stress status during labor. Finally, many genes related to different metabolic pathways such as alcohol, sterol, nucleotide, lipid and sugar biosynthesis, were found up-regulated in CB CD133+ cells, reflecting a neonatal wider potentiality of these cells.In conclusion, mPB CD133+ cells include a more committed and cycling population while CB CD133+ represent a cell subset with higher potentialities probably due to their immaturity. This suggest that even if CB has a lower number of stem cells, CB CD133+ cells are more prone/open/susceptible to different stimuli and potential fates.

Different gene expression profiles of CD133+cells from cord blood and mobilized peripheral blood / Giorgetti, A; Agnelli, L; Mattioli, M; Lombardi, L; Neri, A; Bicciato, Silvio; Giordano, R; Rebulla, P; Lazzari, L.. - In: BLOOD. - ISSN 0006-4971. - STAMPA. - 106:(2005), pp. 4210-4210.

Different gene expression profiles of CD133+cells from cord blood and mobilized peripheral blood

BICCIATO, Silvio;
2005

Abstract

Cord blood (CB) is a valid alternative to mobilized peripheral blood (mPB) as a source of hematopoietic stem cell for clinical transplantation. The qualitative differences among CD133+ cells from these sources have been demonstrated by in vitro and in vivo studies but the molecular phenotype of these cells is only partially understood. In view of better exploiting the peculiarities and advantages of both the stem cell sources, we performed a comparative analysis of gene expression of CD133+ from CB and mPB using Affymetrix HG-U133A arrays. The CD133+ cells were selected using the MiniMacs immunomagnetic separation system (Miltenyi Biotec) from 6 CB units and 6 mPB samples. Since microarray studies are strictly dependent on the purity of the cell population and on the high RNA quality, it was crucial to apply rigorous quality controls. The purity of CD133+ cells was consistently more than 93% in all samples. In addition, after extraction, the RNA was purified by the RNeasy system (Qiagen) and the RNA quality was evaluated using the RNA 6000 Nano LabChip kit (Agilent Technologies). Given the low amount of RNA due to the limited numbers of CB CD133+ cells, we used a double-cycle amplification protocol for both sources to obtain biotin-labeled target for hybridization starting from as low as 100 ng of total RNA. Probe level data have been converted to expression values using Affymetrix MAS 5.0 algorithm. Differentially expressed genes have been identified using Significance Analysis of Microarrays (SAM). The cutoff for significance has been determined tuning the  parameter on the false positive rate and controlling the q-value for the gene list. We found that mPB CD133+ cells expressed cell cycle driving, transcription factor and DNA synthesis genes at distinctly higher levels than CB CD133+ cells, indicating a higher cycling activity in mPB CD133+ cells. Furthermore apoptosis driving genes were up-regulated in mPB CD133+ cells, probably due to G-CSF mobilization. Conversely in CB CD133+ cells we found an overexpression of genes involved in immunoresponse and immunomodulation. Since all the CB units were collected after vaginal delivery, this finding could be related to the stress status during labor. Finally, many genes related to different metabolic pathways such as alcohol, sterol, nucleotide, lipid and sugar biosynthesis, were found up-regulated in CB CD133+ cells, reflecting a neonatal wider potentiality of these cells.In conclusion, mPB CD133+ cells include a more committed and cycling population while CB CD133+ represent a cell subset with higher potentialities probably due to their immaturity. This suggest that even if CB has a lower number of stem cells, CB CD133+ cells are more prone/open/susceptible to different stimuli and potential fates.
106
4210
4210
Different gene expression profiles of CD133+cells from cord blood and mobilized peripheral blood / Giorgetti, A; Agnelli, L; Mattioli, M; Lombardi, L; Neri, A; Bicciato, Silvio; Giordano, R; Rebulla, P; Lazzari, L.. - In: BLOOD. - ISSN 0006-4971. - STAMPA. - 106:(2005), pp. 4210-4210.
Giorgetti, A; Agnelli, L; Mattioli, M; Lombardi, L; Neri, A; Bicciato, Silvio; Giordano, R; Rebulla, P; Lazzari, L.
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Utilizza questo identificativo per citare o creare un link a questo documento: http://hdl.handle.net/11380/421444
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