Ex vivo expansion of stem and progenitor cells in co-culture of mobilized peripheral blood CD34+ cells on human endothelium transfected with adenovectors expressing thrombopoietin, c-kit ligand, and Flt-3 ligand

Pierre Feugier, Yeon Jo Deog, Hung Shieh Jae, Karen L. Mackenzie, Shahin Rafii, Ronald Crystal, Malcolm A S Moore

Research output: Contribution to journalArticle

23 Citations (Scopus)

Abstract

To optimize conditions for ex vivo expansion of adult hematopoietic stem cells, we evaluated the co-culture of G-CSF mobilized human peripheral blood (PB) CD34+ cells with endothelial cells engineered to overexpress various hematopoietic growth factors. Immortalized human bone marrow endothelial cells (BMEC) transfected with an expression vector carrying cDNA encoding the human telomerase reverse transcriptase (hTERT) and human umbilical vein endothelial cells (HUVEC) were transfected with combinations of adenovectors expressing murine c-kit ligand (KL), human thrombopoietin (TPO), human Flt3 ligand (FL), and human granulocyte-macrophage colony-stimulating factor (GM-CSF). Ex vivo expansion of PB CD34+ cells from normal donors and non-Hodgkin lymphoma (NHL) patients in endothelial co-culture was evaluated weekly for total cell production, progenitor (CFU-GM, BFU-E) cell production, and stem cell production as measured by Week-5 Cobblestone Area Forming Cell assay (Wk-5 CAFC). HUVEC transfected with adenovectors expressing TPO, KL, and FL provided the best co-culture system for expanding CD34+ cells. Maximal total nuclear cell, CFU-GM, and Wk-5 CAFC production occurred between weeks 2 and 3 with 113-fold, 25-fold, and 2.2-5.5-fold expansions, respectively. We did not detect significant differences when GM-CSF was added to the co-culture system. Expansion was also obtained using recombinant human cytokines, but was not maintained beyond 3 weeks. We demonstrated that continuous generation of high levels of TPO, FL, and KL as well as other factors secreted by endothelium provided a clinically relevant co-culture method for ex vivo expansion of stem and progenitor cells from cryopreserved CD34+ populations.

Original languageEnglish
Pages (from-to)127-138
Number of pages12
JournalJournal of Hematotherapy and Stem Cell Research
Volume11
Issue number1
DOIs
Publication statusPublished - 4 Mar 2002
Externally publishedYes

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Thrombopoietin
Stem Cell Factor
Coculture Techniques
Endothelium
Blood Cells
Stem Cells
Granulocyte-Macrophage Progenitor Cells
Human Umbilical Vein Endothelial Cells
Granulocyte-Macrophage Colony-Stimulating Factor
Endothelial Cells
Erythroid Precursor Cells
Adult Stem Cells
Granulocyte Colony-Stimulating Factor
Hematopoietic Stem Cells
Bone Marrow Cells
Non-Hodgkin's Lymphoma
flt3 ligand protein
Intercellular Signaling Peptides and Proteins
Complementary DNA
Tissue Donors

ASJC Scopus subject areas

  • Hematology
  • Immunology

Cite this

Ex vivo expansion of stem and progenitor cells in co-culture of mobilized peripheral blood CD34+ cells on human endothelium transfected with adenovectors expressing thrombopoietin, c-kit ligand, and Flt-3 ligand. / Feugier, Pierre; Deog, Yeon Jo; Jae, Hung Shieh; Mackenzie, Karen L.; Rafii, Shahin; Crystal, Ronald; Moore, Malcolm A S.

In: Journal of Hematotherapy and Stem Cell Research, Vol. 11, No. 1, 04.03.2002, p. 127-138.

Research output: Contribution to journalArticle

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