Intrinsic potential of phenotypically defined human hemopoietic stem cells to self-renew in short-term in vitro cultures

S Knaän-Shanzer, S F Verlinden, V W van Beusechem, D W Van Bekkum, D Valerio

Research output: Contribution to journalArticleAcademicpeer-review

Abstract

In search for culture conditions that will facilitate hemopoietic stem cell (HSC) replication while preserving their primitive properties, we have made use of a multi-parameter FACS assay to define HSCs on basis of their phenotypic characteristics, i.e., CD34++CD33,38,71(-). Bone marrow and umbilical cord blood samples of CD34(+) cells from 31 donors were loaded with the membrane dye PKH26 and each exposed to various culture conditions for 6 days. The cells that retained the primitive CD34(++)CD33,38,71(-) phenotype were analysed for the number of cell replications they underwent, by measuring loss of PKH26 fluorescence after 6 days. A most striking observation was the large inter-sample variation in the proliferative response of cells that retained the CD34(++)CD33,38,71(-) phenotype. In general, samples could be characterised as either good- or poorly-replicating, according to the proliferation property of their CD34(++)CD33,38,71(-) subset. In comparison to this 'intrinsic' potential, the effects of the applied growth stimuli on CD34(++)CD33,38,71(-) cell replication were negligible. In contrast, the overall recovery of the CD34(++)CD33,38,71(-) cells was clearly dependent on the culture stimuli. Of the various conditions tested, serum-free cultures with pre-established stroma maintained the cells with this primitive phenotype most effectively. In cultures supplemented with various combinations of recombinant HGFs, HSC differentiation prevailed. These findings with phenotypically defined HSCs should assist in the design of systems for expansion and ex vivo gene therapy of early hemopoietic cells.

Original languageEnglish
Pages (from-to)1440-50
Number of pages11
JournalExperimental Hematology
Volume27
Issue number9
Publication statusPublished - Sep 1999

Cite this

Knaän-Shanzer, S ; Verlinden, S F ; van Beusechem, V W ; Van Bekkum, D W ; Valerio, D. / Intrinsic potential of phenotypically defined human hemopoietic stem cells to self-renew in short-term in vitro cultures. In: Experimental Hematology. 1999 ; Vol. 27, No. 9. pp. 1440-50.
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abstract = "In search for culture conditions that will facilitate hemopoietic stem cell (HSC) replication while preserving their primitive properties, we have made use of a multi-parameter FACS assay to define HSCs on basis of their phenotypic characteristics, i.e., CD34++CD33,38,71(-). Bone marrow and umbilical cord blood samples of CD34(+) cells from 31 donors were loaded with the membrane dye PKH26 and each exposed to various culture conditions for 6 days. The cells that retained the primitive CD34(++)CD33,38,71(-) phenotype were analysed for the number of cell replications they underwent, by measuring loss of PKH26 fluorescence after 6 days. A most striking observation was the large inter-sample variation in the proliferative response of cells that retained the CD34(++)CD33,38,71(-) phenotype. In general, samples could be characterised as either good- or poorly-replicating, according to the proliferation property of their CD34(++)CD33,38,71(-) subset. In comparison to this 'intrinsic' potential, the effects of the applied growth stimuli on CD34(++)CD33,38,71(-) cell replication were negligible. In contrast, the overall recovery of the CD34(++)CD33,38,71(-) cells was clearly dependent on the culture stimuli. Of the various conditions tested, serum-free cultures with pre-established stroma maintained the cells with this primitive phenotype most effectively. In cultures supplemented with various combinations of recombinant HGFs, HSC differentiation prevailed. These findings with phenotypically defined HSCs should assist in the design of systems for expansion and ex vivo gene therapy of early hemopoietic cells.",
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Intrinsic potential of phenotypically defined human hemopoietic stem cells to self-renew in short-term in vitro cultures. / Knaän-Shanzer, S; Verlinden, S F; van Beusechem, V W; Van Bekkum, D W; Valerio, D.

In: Experimental Hematology, Vol. 27, No. 9, 09.1999, p. 1440-50.

Research output: Contribution to journalArticleAcademicpeer-review

TY - JOUR

T1 - Intrinsic potential of phenotypically defined human hemopoietic stem cells to self-renew in short-term in vitro cultures

AU - Knaän-Shanzer, S

AU - Verlinden, S F

AU - van Beusechem, V W

AU - Van Bekkum, D W

AU - Valerio, D

PY - 1999/9

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N2 - In search for culture conditions that will facilitate hemopoietic stem cell (HSC) replication while preserving their primitive properties, we have made use of a multi-parameter FACS assay to define HSCs on basis of their phenotypic characteristics, i.e., CD34++CD33,38,71(-). Bone marrow and umbilical cord blood samples of CD34(+) cells from 31 donors were loaded with the membrane dye PKH26 and each exposed to various culture conditions for 6 days. The cells that retained the primitive CD34(++)CD33,38,71(-) phenotype were analysed for the number of cell replications they underwent, by measuring loss of PKH26 fluorescence after 6 days. A most striking observation was the large inter-sample variation in the proliferative response of cells that retained the CD34(++)CD33,38,71(-) phenotype. In general, samples could be characterised as either good- or poorly-replicating, according to the proliferation property of their CD34(++)CD33,38,71(-) subset. In comparison to this 'intrinsic' potential, the effects of the applied growth stimuli on CD34(++)CD33,38,71(-) cell replication were negligible. In contrast, the overall recovery of the CD34(++)CD33,38,71(-) cells was clearly dependent on the culture stimuli. Of the various conditions tested, serum-free cultures with pre-established stroma maintained the cells with this primitive phenotype most effectively. In cultures supplemented with various combinations of recombinant HGFs, HSC differentiation prevailed. These findings with phenotypically defined HSCs should assist in the design of systems for expansion and ex vivo gene therapy of early hemopoietic cells.

AB - In search for culture conditions that will facilitate hemopoietic stem cell (HSC) replication while preserving their primitive properties, we have made use of a multi-parameter FACS assay to define HSCs on basis of their phenotypic characteristics, i.e., CD34++CD33,38,71(-). Bone marrow and umbilical cord blood samples of CD34(+) cells from 31 donors were loaded with the membrane dye PKH26 and each exposed to various culture conditions for 6 days. The cells that retained the primitive CD34(++)CD33,38,71(-) phenotype were analysed for the number of cell replications they underwent, by measuring loss of PKH26 fluorescence after 6 days. A most striking observation was the large inter-sample variation in the proliferative response of cells that retained the CD34(++)CD33,38,71(-) phenotype. In general, samples could be characterised as either good- or poorly-replicating, according to the proliferation property of their CD34(++)CD33,38,71(-) subset. In comparison to this 'intrinsic' potential, the effects of the applied growth stimuli on CD34(++)CD33,38,71(-) cell replication were negligible. In contrast, the overall recovery of the CD34(++)CD33,38,71(-) cells was clearly dependent on the culture stimuli. Of the various conditions tested, serum-free cultures with pre-established stroma maintained the cells with this primitive phenotype most effectively. In cultures supplemented with various combinations of recombinant HGFs, HSC differentiation prevailed. These findings with phenotypically defined HSCs should assist in the design of systems for expansion and ex vivo gene therapy of early hemopoietic cells.

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KW - Animals

KW - Antigens, CD/analysis

KW - Blood Cells/cytology

KW - Bone Marrow Cells/cytology

KW - Cattle

KW - Cell Culture Techniques/methods

KW - Cell Division/drug effects

KW - Cells, Cultured/drug effects

KW - Coculture Techniques

KW - Colony-Forming Units Assay

KW - Culture Media/pharmacology

KW - Culture Media, Serum-Free/pharmacology

KW - Fetal Blood/cytology

KW - Flow Cytometry

KW - Hematopoietic Stem Cells/cytology

KW - Humans

KW - Infant, Newborn

KW - Organ Specificity

KW - Phenotype

KW - Stromal Cells/cytology

M3 - Article

VL - 27

SP - 1440

EP - 1450

JO - Experimental Hematology

JF - Experimental Hematology

SN - 0301-472X

IS - 9

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