Extensive amplification of single cells from CD34+ subpopulations in umbilical cord blood and identification of long-term culture-initiating cells present in two subsets.

2.50
Hdl Handle:
http://hdl.handle.net/10541/95821
Title:
Extensive amplification of single cells from CD34+ subpopulations in umbilical cord blood and identification of long-term culture-initiating cells present in two subsets.
Authors:
De Wynter, Erika A; Nadali, Gianpaolo; Coutinho, Lucia H; Testa, Nydia G
Abstract:
CD34+ cord blood cells were isolated with immunomagnetic beads and fractionated by fluorescence-activated cell sorting (FACS) into three subpopulations: CD34+38+DR+, CD34+38-DR+ and CD34+38-DR-, using antibodies specific for these cell surface markers. Cells from each of the three subsets were plated as single cells in serum-free medium supplemented with a combination of growth factor and individual cells were monitored for proliferation and the capacity to form colony-forming cells. Single cells from the CD34+38+DR+ subset showed the lowest expansion capacity, generating up to 1.1 x 10(6) cells at five weeks, while individual cells from both the CD34+38-DR+ and CD34+38-DR- subsets could be expanded up to 1.8 x 10(6) and 9.2 x 10(6) cells, respectively, over a period of six weeks. The different subpopulations also generated colony-forming cells which gave rise to erythroid, myeloid and erythroid/myeloid colonies. CD34+38-DR+ cells generated large numbers of colonies within two weeks in liquid culture, but this rapidly declined. Generation of lineage-committed colony-forming cells was better sustained in the CD34+38-DR- population and continued for up to six weeks in culture. Overall, the generation of colony-forming cells declined with time in culture, although the cell numbers continued to expand. However, when the same populations were plated on irradiated bone marrow stroma, both the CD34+38-DR+ and the CD34+38-DR- cells were capable of producing granulocytemacrophage colony-forming cells (GM-CFCs) for 10 to 12 weeks. As hemopoiesis was sustained for almost three months, it appears that these populations were significantly enriched in long-term culture-initiating cells (LTC-ICs). Although both populations generated GM-CFCs, the CD34+38-DR- cells sustained production of higher numbers of colony-forming cells than the CD34+38-DR+ population. These results demonstrate that cells from cord blood can be efficiently monitored at the single-cell level for proliferation, expansion and colony-forming capacity. Furthermore, at least two populations of LTC-ICs can be distinguished in cord blood CD34+38- cells by the differential expression of the HLA-DR antigen.
Affiliation:
CRC Department of Experimental Haematology, Paterson Institute for Cancer Research, Withington, Manchester, United Kingdom.
Citation:
Extensive amplification of single cells from CD34+ subpopulations in umbilical cord blood and identification of long-term culture-initiating cells present in two subsets. 1996, 14 (5):566-76 Stem Cells
Journal:
Stem Cells
Issue Date:
Sep-1996
URI:
http://hdl.handle.net/10541/95821
DOI:
10.1002/stem.140566
PubMed ID:
8888497
Type:
Article
Language:
en
ISSN:
1066-5099
Appears in Collections:
All Paterson Institute for Cancer Research

Full metadata record

DC FieldValue Language
dc.contributor.authorDe Wynter, Erika Aen
dc.contributor.authorNadali, Gianpaoloen
dc.contributor.authorCoutinho, Lucia Hen
dc.contributor.authorTesta, Nydia Gen
dc.date.accessioned2010-04-07T09:09:04Z-
dc.date.available2010-04-07T09:09:04Z-
dc.date.issued1996-09-
dc.identifier.citationExtensive amplification of single cells from CD34+ subpopulations in umbilical cord blood and identification of long-term culture-initiating cells present in two subsets. 1996, 14 (5):566-76 Stem Cellsen
dc.identifier.issn1066-5099-
dc.identifier.pmid8888497-
dc.identifier.doi10.1002/stem.140566-
dc.identifier.urihttp://hdl.handle.net/10541/95821-
dc.description.abstractCD34+ cord blood cells were isolated with immunomagnetic beads and fractionated by fluorescence-activated cell sorting (FACS) into three subpopulations: CD34+38+DR+, CD34+38-DR+ and CD34+38-DR-, using antibodies specific for these cell surface markers. Cells from each of the three subsets were plated as single cells in serum-free medium supplemented with a combination of growth factor and individual cells were monitored for proliferation and the capacity to form colony-forming cells. Single cells from the CD34+38+DR+ subset showed the lowest expansion capacity, generating up to 1.1 x 10(6) cells at five weeks, while individual cells from both the CD34+38-DR+ and CD34+38-DR- subsets could be expanded up to 1.8 x 10(6) and 9.2 x 10(6) cells, respectively, over a period of six weeks. The different subpopulations also generated colony-forming cells which gave rise to erythroid, myeloid and erythroid/myeloid colonies. CD34+38-DR+ cells generated large numbers of colonies within two weeks in liquid culture, but this rapidly declined. Generation of lineage-committed colony-forming cells was better sustained in the CD34+38-DR- population and continued for up to six weeks in culture. Overall, the generation of colony-forming cells declined with time in culture, although the cell numbers continued to expand. However, when the same populations were plated on irradiated bone marrow stroma, both the CD34+38-DR+ and the CD34+38-DR- cells were capable of producing granulocytemacrophage colony-forming cells (GM-CFCs) for 10 to 12 weeks. As hemopoiesis was sustained for almost three months, it appears that these populations were significantly enriched in long-term culture-initiating cells (LTC-ICs). Although both populations generated GM-CFCs, the CD34+38-DR- cells sustained production of higher numbers of colony-forming cells than the CD34+38-DR+ population. These results demonstrate that cells from cord blood can be efficiently monitored at the single-cell level for proliferation, expansion and colony-forming capacity. Furthermore, at least two populations of LTC-ICs can be distinguished in cord blood CD34+38- cells by the differential expression of the HLA-DR antigen.en
dc.language.isoenen
dc.subjectFoetal Blooden
dc.subjectHaematopoiesisen
dc.subjectHaematopoietic Stem Cellsen
dc.subject.meshADP-ribosyl Cyclase-
dc.subject.meshAntigens, CD-
dc.subject.meshAntigens, CD34-
dc.subject.meshAntigens, CD38-
dc.subject.meshAntigens, Differentiation-
dc.subject.meshBone Marrow-
dc.subject.meshBone Marrow Cells-
dc.subject.meshCell Division-
dc.subject.meshCells, Cultured-
dc.subject.meshCulture Media, Serum-Free-
dc.subject.meshFemale-
dc.subject.meshFetal Blood-
dc.subject.meshFlow Cytometry-
dc.subject.meshFluorescent Antibody Technique-
dc.subject.meshGrowth Substances-
dc.subject.meshHLA-DR Antigens-
dc.subject.meshHematopoiesis-
dc.subject.meshHematopoietic Stem Cells-
dc.subject.meshHumans-
dc.subject.meshImmunophenotyping-
dc.subject.meshLymphocyte Subsets-
dc.subject.meshMembrane Glycoproteins-
dc.subject.meshN-Glycosyl Hydrolases-
dc.subject.meshTime Factors-
dc.titleExtensive amplification of single cells from CD34+ subpopulations in umbilical cord blood and identification of long-term culture-initiating cells present in two subsets.en
dc.typeArticleen
dc.contributor.departmentCRC Department of Experimental Haematology, Paterson Institute for Cancer Research, Withington, Manchester, United Kingdom.en
dc.identifier.journalStem Cellsen
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