Long-term generation and expansion of human primitive hematopoietic progenitor cells in vitro

Edward Srour, John E. Brandt, Robert A. Briddell, Susan Grigsby, Tom Leemhuis, Ronald Hoffman

Research output: Contribution to journalArticle

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Abstract

Although sustained production of committed human hematopoietic progenitor cells in long-term bone marrow cultures (LTBMC) is well documented, evidence for the generation and expansion of human primitive hematopoietic progenitor cells (PHPC) in such cultures is lacking. For that purpose, we attempted to determine if the human high proliferative potential colony-forming cell (HPP-CFC), a primitive hematopoietic marrow progenitor cell, is capable of generation and expansion in vitro. To that effect, stromal cell-free LTBMC were initiated with CD34+ HLA-DR- CD15- rhodamine 123dull bone marrow cells and were maintained with repeated addition of c-kit ligand and a synthetic interleukin-3/granulocyte-macrophage colony-stimulating factor fusion protein. By day 21 of LTBMC, a greater than twofold increase in the number of assayable HPP-CFC was detected. Furthermore, the production of HPP-CFC in LTBMC continued for up to 4 weeks, resulting in a 5.5-fold increase in HPP-CFC numbers. Weekly phenotypic analyses of cells harvested from LTBMC showed that the number of CD34+ HLA-DR- cells increased from 104 on day O to 56 × 104 by day 21. To examine further the nature of the in vitro HPP-CFC expansion, individual HPP-CFC colonies were serially cloned. Secondary cloning of individual, day 28 primary HPP-CFC indicated that 46% of these colonies formed an average of nine secondary colony-forming unitgranulocyte-macrophage (CFU-GM)- derived colonies, whereas 43% of primary HPP-CFC gave rise to between one and six secondary HPP-CFC colonies and 6 to 26 CFU-GM. These data show that CD34+ HLA-DR- CD15- rhodamine 123dull cells represent a fraction of human bone marrow highly enriched for HPP-CFC and that based on their regeneration and proliferative capacities, a hierarchy of HPP-CFC exists. Furthermore, these studies indicate that in the presence of appropriate cytokine stimulation, it is possible to expand the number of PHPC in vitro.

Original languageEnglish
Pages (from-to)661-669
Number of pages9
JournalBlood
Volume81
Issue number3
StatePublished - Feb 1 1993

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Hematopoietic Stem Cells
Bone
HLA-DR Antigens
Bone Marrow
Rhodamines
Macrophages
Stem Cell Factor
Interleukin-3
Cloning
Granulocyte-Macrophage Colony-Stimulating Factor
In Vitro Techniques
Fusion reactions
Cells
Cytokines
Stromal Cells
Bone Marrow Cells
Organism Cloning
Regeneration
Cell Count
Proteins

ASJC Scopus subject areas

  • Hematology

Cite this

Srour, E., Brandt, J. E., Briddell, R. A., Grigsby, S., Leemhuis, T., & Hoffman, R. (1993). Long-term generation and expansion of human primitive hematopoietic progenitor cells in vitro. Blood, 81(3), 661-669.

Long-term generation and expansion of human primitive hematopoietic progenitor cells in vitro. / Srour, Edward; Brandt, John E.; Briddell, Robert A.; Grigsby, Susan; Leemhuis, Tom; Hoffman, Ronald.

In: Blood, Vol. 81, No. 3, 01.02.1993, p. 661-669.

Research output: Contribution to journalArticle

Srour, E, Brandt, JE, Briddell, RA, Grigsby, S, Leemhuis, T & Hoffman, R 1993, 'Long-term generation and expansion of human primitive hematopoietic progenitor cells in vitro', Blood, vol. 81, no. 3, pp. 661-669.
Srour E, Brandt JE, Briddell RA, Grigsby S, Leemhuis T, Hoffman R. Long-term generation and expansion of human primitive hematopoietic progenitor cells in vitro. Blood. 1993 Feb 1;81(3):661-669.
Srour, Edward ; Brandt, John E. ; Briddell, Robert A. ; Grigsby, Susan ; Leemhuis, Tom ; Hoffman, Ronald. / Long-term generation and expansion of human primitive hematopoietic progenitor cells in vitro. In: Blood. 1993 ; Vol. 81, No. 3. pp. 661-669.
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abstract = "Although sustained production of committed human hematopoietic progenitor cells in long-term bone marrow cultures (LTBMC) is well documented, evidence for the generation and expansion of human primitive hematopoietic progenitor cells (PHPC) in such cultures is lacking. For that purpose, we attempted to determine if the human high proliferative potential colony-forming cell (HPP-CFC), a primitive hematopoietic marrow progenitor cell, is capable of generation and expansion in vitro. To that effect, stromal cell-free LTBMC were initiated with CD34+ HLA-DR- CD15- rhodamine 123dull bone marrow cells and were maintained with repeated addition of c-kit ligand and a synthetic interleukin-3/granulocyte-macrophage colony-stimulating factor fusion protein. By day 21 of LTBMC, a greater than twofold increase in the number of assayable HPP-CFC was detected. Furthermore, the production of HPP-CFC in LTBMC continued for up to 4 weeks, resulting in a 5.5-fold increase in HPP-CFC numbers. Weekly phenotypic analyses of cells harvested from LTBMC showed that the number of CD34+ HLA-DR- cells increased from 104 on day O to 56 × 104 by day 21. To examine further the nature of the in vitro HPP-CFC expansion, individual HPP-CFC colonies were serially cloned. Secondary cloning of individual, day 28 primary HPP-CFC indicated that 46{\%} of these colonies formed an average of nine secondary colony-forming unitgranulocyte-macrophage (CFU-GM)- derived colonies, whereas 43{\%} of primary HPP-CFC gave rise to between one and six secondary HPP-CFC colonies and 6 to 26 CFU-GM. These data show that CD34+ HLA-DR- CD15- rhodamine 123dull cells represent a fraction of human bone marrow highly enriched for HPP-CFC and that based on their regeneration and proliferative capacities, a hierarchy of HPP-CFC exists. Furthermore, these studies indicate that in the presence of appropriate cytokine stimulation, it is possible to expand the number of PHPC in vitro.",
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AB - Although sustained production of committed human hematopoietic progenitor cells in long-term bone marrow cultures (LTBMC) is well documented, evidence for the generation and expansion of human primitive hematopoietic progenitor cells (PHPC) in such cultures is lacking. For that purpose, we attempted to determine if the human high proliferative potential colony-forming cell (HPP-CFC), a primitive hematopoietic marrow progenitor cell, is capable of generation and expansion in vitro. To that effect, stromal cell-free LTBMC were initiated with CD34+ HLA-DR- CD15- rhodamine 123dull bone marrow cells and were maintained with repeated addition of c-kit ligand and a synthetic interleukin-3/granulocyte-macrophage colony-stimulating factor fusion protein. By day 21 of LTBMC, a greater than twofold increase in the number of assayable HPP-CFC was detected. Furthermore, the production of HPP-CFC in LTBMC continued for up to 4 weeks, resulting in a 5.5-fold increase in HPP-CFC numbers. Weekly phenotypic analyses of cells harvested from LTBMC showed that the number of CD34+ HLA-DR- cells increased from 104 on day O to 56 × 104 by day 21. To examine further the nature of the in vitro HPP-CFC expansion, individual HPP-CFC colonies were serially cloned. Secondary cloning of individual, day 28 primary HPP-CFC indicated that 46% of these colonies formed an average of nine secondary colony-forming unitgranulocyte-macrophage (CFU-GM)- derived colonies, whereas 43% of primary HPP-CFC gave rise to between one and six secondary HPP-CFC colonies and 6 to 26 CFU-GM. These data show that CD34+ HLA-DR- CD15- rhodamine 123dull cells represent a fraction of human bone marrow highly enriched for HPP-CFC and that based on their regeneration and proliferative capacities, a hierarchy of HPP-CFC exists. Furthermore, these studies indicate that in the presence of appropriate cytokine stimulation, it is possible to expand the number of PHPC in vitro.

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