Physiological and proteomic aspects of functional cord blood cd34+cells maintenance at 4 degrees c under hypoxia and hypercapnia
Аутори
Vlaški, MarijaNegroni, Luc
Kovačević-Filipović, Milica
Berets, S.
Le Reverend, G.
Hammoud, M.
Chevaleyre, Jean
Duchez, Pascale
de la Grange, P. Brunet
Lafarge, Xavier
Boiron, Jean-Michel
Ivanović, Z.
Конференцијски прилог (Објављена верзија)
Метаподаци
Приказ свих података о документуАпстракт
Background. The short-term conservation of hematopoietic stem and progenitors cells is actually performed in hypothermia (+4°C). In nature, long-term
survival of some animals in hypothermia is enabled by regulated metabolic
depression which can be induced by their exposure to low O2 (hypoxia) and
increased CO2 (hypercapnia) environment. Similarly, in physiological conditions, the primitive hematopoietic cells are maintained in a hypometabolic state
in the poorly oxygenated bone marrow niche. Recently, we demonstrated that
the same protective mechanism is operational in mobilized peripheral blood
primitive hematopoietic progenitors and stem cells during their storage at4°C
(Jeanne et al, Transfusion 2009). Aims. The aim of this study was to test if
exposure to hypoxic/hypercapnic gas mixture could be also beneficial for the
preservation of functional cord blood CD34+ hematopoetic progenitors in
hypothermia and, if so, to analyse physiological and proteomic aspects of this
phe...nomenon. Methods. Cord blood CD34+ cells were incubated in conservation medium Stem-α S3 for 10 days at +4°C under hypoxic (1 and 5% O2) and
hypercapnic (2,5 and 9% CO2) concentration or air (20% O2 and 0,05% CO2).
The functional maintenance of committed hematopoietic progenitors (CFC
assay) and stem cells (Scid-Repopulating Cells (SRCs) assay estimated by the
presence of human markers [CD45, CD19, CD33] and human CFC in
NOG/Scid mice femur 6 weeks after transplantation) in parallel with a flowcytometry analysis of phenotype and vital functions (Aldefluor, AnnexinV/PI,
ATP bioluminescent assay, DiBAC4, TMRM, H2DCFDA staining). Proteomic
analysis was performed using iTRAQ labelling followed by peptide fractionation (SXC chromatography) and mass spectrometry (LC/MS/MS). Results.
Incubation in hypoxia and hypercapnia doubled the survival CD34+ cells (60%)
comparing to air (30%). Similar ratio is obtained for CFC (34±9% vs 18±6% in
air). 5%O2/9%CO2 ratio appeared to be the optimal hypoxic/hypercapnic combination. Better cell maintenance in this condition was associated with a higher frequency of primitive aldehyde dehydrogenase (ALDH) expressing cells
and SRCs. These cell-protective effects seem to concern a better preservation
of the plasma and mitochondrial membrane potential. Hypoxia/hypercapnia
also ensures maintenance of viable cells and induces less apoptosis, in spite
of a production of deleterious ROS and a drop of ATP amount/per viable cell
which were equivalent to that obtained in air. These effects seem to be principally mediated via hypercapnia. Proteomic study revealed that overall protein’s
content was better preserved in hypoxia/hypercapnia. In addition, this analysis enabled to identify and to distinguish proteins sensitive and insensitive to
hypothermia irrespective to the gas phase, as well as the proteins contributing
specifically to the hypoxia/hypercapnia cell protective effect. Among them are
some protein families known to be implicated in the long time survival of hibernating animals in hypothermia. Conclusions. Present work demonstrates the
critical physiological effects and indicates protein candidates implicated in
hypoxia/hypercapnia-mediated functional maintenance of hematopoietic progenitors in hypothermia. These results suggest a way to optimise cell conservation without freezing and to design a new generation of conservation media.
Извор:
Haematologica, 2012, 97, 288-288Издавач:
- Ferrata Storti Foundation, Pavia
Колекције
Институција/група
Fakultet veterinarske medicineTY - CONF AU - Vlaški, Marija AU - Negroni, Luc AU - Kovačević-Filipović, Milica AU - Berets, S. AU - Le Reverend, G. AU - Hammoud, M. AU - Chevaleyre, Jean AU - Duchez, Pascale AU - de la Grange, P. Brunet AU - Lafarge, Xavier AU - Boiron, Jean-Michel AU - Ivanović, Z. PY - 2012 UR - https://vet-erinar.vet.bg.ac.rs/handle/123456789/927 AB - Background. The short-term conservation of hematopoietic stem and progenitors cells is actually performed in hypothermia (+4°C). In nature, long-term survival of some animals in hypothermia is enabled by regulated metabolic depression which can be induced by their exposure to low O2 (hypoxia) and increased CO2 (hypercapnia) environment. Similarly, in physiological conditions, the primitive hematopoietic cells are maintained in a hypometabolic state in the poorly oxygenated bone marrow niche. Recently, we demonstrated that the same protective mechanism is operational in mobilized peripheral blood primitive hematopoietic progenitors and stem cells during their storage at4°C (Jeanne et al, Transfusion 2009). Aims. The aim of this study was to test if exposure to hypoxic/hypercapnic gas mixture could be also beneficial for the preservation of functional cord blood CD34+ hematopoetic progenitors in hypothermia and, if so, to analyse physiological and proteomic aspects of this phenomenon. Methods. Cord blood CD34+ cells were incubated in conservation medium Stem-α S3 for 10 days at +4°C under hypoxic (1 and 5% O2) and hypercapnic (2,5 and 9% CO2) concentration or air (20% O2 and 0,05% CO2). The functional maintenance of committed hematopoietic progenitors (CFC assay) and stem cells (Scid-Repopulating Cells (SRCs) assay estimated by the presence of human markers [CD45, CD19, CD33] and human CFC in NOG/Scid mice femur 6 weeks after transplantation) in parallel with a flowcytometry analysis of phenotype and vital functions (Aldefluor, AnnexinV/PI, ATP bioluminescent assay, DiBAC4, TMRM, H2DCFDA staining). Proteomic analysis was performed using iTRAQ labelling followed by peptide fractionation (SXC chromatography) and mass spectrometry (LC/MS/MS). Results. Incubation in hypoxia and hypercapnia doubled the survival CD34+ cells (60%) comparing to air (30%). Similar ratio is obtained for CFC (34±9% vs 18±6% in air). 5%O2/9%CO2 ratio appeared to be the optimal hypoxic/hypercapnic combination. Better cell maintenance in this condition was associated with a higher frequency of primitive aldehyde dehydrogenase (ALDH) expressing cells and SRCs. These cell-protective effects seem to concern a better preservation of the plasma and mitochondrial membrane potential. Hypoxia/hypercapnia also ensures maintenance of viable cells and induces less apoptosis, in spite of a production of deleterious ROS and a drop of ATP amount/per viable cell which were equivalent to that obtained in air. These effects seem to be principally mediated via hypercapnia. Proteomic study revealed that overall protein’s content was better preserved in hypoxia/hypercapnia. In addition, this analysis enabled to identify and to distinguish proteins sensitive and insensitive to hypothermia irrespective to the gas phase, as well as the proteins contributing specifically to the hypoxia/hypercapnia cell protective effect. Among them are some protein families known to be implicated in the long time survival of hibernating animals in hypothermia. Conclusions. Present work demonstrates the critical physiological effects and indicates protein candidates implicated in hypoxia/hypercapnia-mediated functional maintenance of hematopoietic progenitors in hypothermia. These results suggest a way to optimise cell conservation without freezing and to design a new generation of conservation media. PB - Ferrata Storti Foundation, Pavia C3 - Haematologica T1 - Physiological and proteomic aspects of functional cord blood cd34+cells maintenance at 4 degrees c under hypoxia and hypercapnia VL - 97 SP - 288 EP - 288 UR - https://hdl.handle.net/21.15107/rcub_veterinar_927 ER -
@conference{ author = "Vlaški, Marija and Negroni, Luc and Kovačević-Filipović, Milica and Berets, S. and Le Reverend, G. and Hammoud, M. and Chevaleyre, Jean and Duchez, Pascale and de la Grange, P. Brunet and Lafarge, Xavier and Boiron, Jean-Michel and Ivanović, Z.", year = "2012", abstract = "Background. The short-term conservation of hematopoietic stem and progenitors cells is actually performed in hypothermia (+4°C). In nature, long-term survival of some animals in hypothermia is enabled by regulated metabolic depression which can be induced by their exposure to low O2 (hypoxia) and increased CO2 (hypercapnia) environment. Similarly, in physiological conditions, the primitive hematopoietic cells are maintained in a hypometabolic state in the poorly oxygenated bone marrow niche. Recently, we demonstrated that the same protective mechanism is operational in mobilized peripheral blood primitive hematopoietic progenitors and stem cells during their storage at4°C (Jeanne et al, Transfusion 2009). Aims. The aim of this study was to test if exposure to hypoxic/hypercapnic gas mixture could be also beneficial for the preservation of functional cord blood CD34+ hematopoetic progenitors in hypothermia and, if so, to analyse physiological and proteomic aspects of this phenomenon. Methods. Cord blood CD34+ cells were incubated in conservation medium Stem-α S3 for 10 days at +4°C under hypoxic (1 and 5% O2) and hypercapnic (2,5 and 9% CO2) concentration or air (20% O2 and 0,05% CO2). The functional maintenance of committed hematopoietic progenitors (CFC assay) and stem cells (Scid-Repopulating Cells (SRCs) assay estimated by the presence of human markers [CD45, CD19, CD33] and human CFC in NOG/Scid mice femur 6 weeks after transplantation) in parallel with a flowcytometry analysis of phenotype and vital functions (Aldefluor, AnnexinV/PI, ATP bioluminescent assay, DiBAC4, TMRM, H2DCFDA staining). Proteomic analysis was performed using iTRAQ labelling followed by peptide fractionation (SXC chromatography) and mass spectrometry (LC/MS/MS). Results. Incubation in hypoxia and hypercapnia doubled the survival CD34+ cells (60%) comparing to air (30%). Similar ratio is obtained for CFC (34±9% vs 18±6% in air). 5%O2/9%CO2 ratio appeared to be the optimal hypoxic/hypercapnic combination. Better cell maintenance in this condition was associated with a higher frequency of primitive aldehyde dehydrogenase (ALDH) expressing cells and SRCs. These cell-protective effects seem to concern a better preservation of the plasma and mitochondrial membrane potential. Hypoxia/hypercapnia also ensures maintenance of viable cells and induces less apoptosis, in spite of a production of deleterious ROS and a drop of ATP amount/per viable cell which were equivalent to that obtained in air. These effects seem to be principally mediated via hypercapnia. Proteomic study revealed that overall protein’s content was better preserved in hypoxia/hypercapnia. In addition, this analysis enabled to identify and to distinguish proteins sensitive and insensitive to hypothermia irrespective to the gas phase, as well as the proteins contributing specifically to the hypoxia/hypercapnia cell protective effect. Among them are some protein families known to be implicated in the long time survival of hibernating animals in hypothermia. Conclusions. Present work demonstrates the critical physiological effects and indicates protein candidates implicated in hypoxia/hypercapnia-mediated functional maintenance of hematopoietic progenitors in hypothermia. These results suggest a way to optimise cell conservation without freezing and to design a new generation of conservation media.", publisher = "Ferrata Storti Foundation, Pavia", journal = "Haematologica", title = "Physiological and proteomic aspects of functional cord blood cd34+cells maintenance at 4 degrees c under hypoxia and hypercapnia", volume = "97", pages = "288-288", url = "https://hdl.handle.net/21.15107/rcub_veterinar_927" }
Vlaški, M., Negroni, L., Kovačević-Filipović, M., Berets, S., Le Reverend, G., Hammoud, M., Chevaleyre, J., Duchez, P., de la Grange, P. B., Lafarge, X., Boiron, J.,& Ivanović, Z.. (2012). Physiological and proteomic aspects of functional cord blood cd34+cells maintenance at 4 degrees c under hypoxia and hypercapnia. in Haematologica Ferrata Storti Foundation, Pavia., 97, 288-288. https://hdl.handle.net/21.15107/rcub_veterinar_927
Vlaški M, Negroni L, Kovačević-Filipović M, Berets S, Le Reverend G, Hammoud M, Chevaleyre J, Duchez P, de la Grange PB, Lafarge X, Boiron J, Ivanović Z. Physiological and proteomic aspects of functional cord blood cd34+cells maintenance at 4 degrees c under hypoxia and hypercapnia. in Haematologica. 2012;97:288-288. https://hdl.handle.net/21.15107/rcub_veterinar_927 .
Vlaški, Marija, Negroni, Luc, Kovačević-Filipović, Milica, Berets, S., Le Reverend, G., Hammoud, M., Chevaleyre, Jean, Duchez, Pascale, de la Grange, P. Brunet, Lafarge, Xavier, Boiron, Jean-Michel, Ivanović, Z., "Physiological and proteomic aspects of functional cord blood cd34+cells maintenance at 4 degrees c under hypoxia and hypercapnia" in Haematologica, 97 (2012):288-288, https://hdl.handle.net/21.15107/rcub_veterinar_927 .