Cord blood cd34+ cells co-cultured with huvec transfected with adenovirus expressing kl, fl and tpo leads to expansion of progenitors, week-5 cafc and nod-scid repopulating cells

Pierre Feugier, Jae Hung Shieh, D. Y. Jo, Karen L. MacKenzie, Shahin Rafii, Ronald G. Crystal, Malcolm A S Moore

Research output: Contribution to journalArticle

2 Citations (Scopus)

Abstract

The major limitation to more widespread use of human cord blood (CB) transplantation is the low number of cells obtained, which restricts its use in adult patients due to delayed engraftment of platelets and neutrophils. Ex vivo expansion has been proposed to overcome these drawbacks.In this study, we evaluated the expansion capability of CB CD34+ cells co-cultured on human umbilical vascular endothelial cells (HUVEC) transfected with adenoviruses expressing Flk2Flt3 ligand (FL), c-kit ligand (KL) and thrombopoietin (Tpo). The co-cultures resulted in 67±6- and 311+25-fold increase of total nucleated cells after one and two weeks respectively; progenitor cells, including CFU-GM, BFU-E and CFUGEMM, increased 13.5 ±2- and 70 ±6-fold; and week-5 cobble-stone area forming cells increased 23 ±2- and 75±7-fold, respectively. In addition, this co-culture system also markedly increased NOD/SCID populating cells. Positive engraftment of human cells in 350 rad irradiated-NOD/SCID mice was defined as 0.1% of human CD45+ cells detected in murine bone marrow cells after 8 to 10 weeks transplantation. In a control cohort, no human cells were detected in mice receiving IxlO4 non-cultured CD34+ cells (n=3), while mice receiving 105 non-cultured CD34+ cells engrafted with human cells (6.5%). In a cohort of mice receiving the equivalent of IxlO4 CD34+ cells after one week co-culture, all animals (n=3) had human cells (2.61%). One out of three mice receiving the equivalent of 2xl03 CD34+ cells cultured for 1 week had human cell engraftment. In a cohort of animals (n=3) receiving the equivalent of IxlO4 CD34+ following two weeks of co-culture, all mice were positive for human cells (average 0.8%). Our data demonstrates that FL, KL and Tpo adenovector-transfected HUVEC provides an optimal microenvironment for expansion of hematopoietic stem cells detected by in vitro and in vivo assay. Since autologous HUVEC can be used, this culture system provides a clinically relevant method for optimizing human CB ex vivo expansion.

Original languageEnglish
JournalBlood
Volume96
Issue number11 PART I
Publication statusPublished - 1 Dec 2000
Externally publishedYes

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Fetal Blood
Adenoviridae
Blood Cells
Blood
Cells
Endothelial cells
Cell culture
Thrombopoietin
Stem Cell Factor
Coculture Techniques
Umbilicus
Animals
Ligands
Endothelial Cells
Platelets
Stem cells
Assays
Transplantation
Bone
Granulocyte-Macrophage Progenitor Cells

ASJC Scopus subject areas

  • Hematology

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Cord blood cd34+ cells co-cultured with huvec transfected with adenovirus expressing kl, fl and tpo leads to expansion of progenitors, week-5 cafc and nod-scid repopulating cells. / Feugier, Pierre; Shieh, Jae Hung; Jo, D. Y.; MacKenzie, Karen L.; Rafii, Shahin; Crystal, Ronald G.; Moore, Malcolm A S.

In: Blood, Vol. 96, No. 11 PART I, 01.12.2000.

Research output: Contribution to journalArticle

Feugier, Pierre ; Shieh, Jae Hung ; Jo, D. Y. ; MacKenzie, Karen L. ; Rafii, Shahin ; Crystal, Ronald G. ; Moore, Malcolm A S. / Cord blood cd34+ cells co-cultured with huvec transfected with adenovirus expressing kl, fl and tpo leads to expansion of progenitors, week-5 cafc and nod-scid repopulating cells. In: Blood. 2000 ; Vol. 96, No. 11 PART I.
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abstract = "The major limitation to more widespread use of human cord blood (CB) transplantation is the low number of cells obtained, which restricts its use in adult patients due to delayed engraftment of platelets and neutrophils. Ex vivo expansion has been proposed to overcome these drawbacks.In this study, we evaluated the expansion capability of CB CD34+ cells co-cultured on human umbilical vascular endothelial cells (HUVEC) transfected with adenoviruses expressing Flk2Flt3 ligand (FL), c-kit ligand (KL) and thrombopoietin (Tpo). The co-cultures resulted in 67±6- and 311+25-fold increase of total nucleated cells after one and two weeks respectively; progenitor cells, including CFU-GM, BFU-E and CFUGEMM, increased 13.5 ±2- and 70 ±6-fold; and week-5 cobble-stone area forming cells increased 23 ±2- and 75±7-fold, respectively. In addition, this co-culture system also markedly increased NOD/SCID populating cells. Positive engraftment of human cells in 350 rad irradiated-NOD/SCID mice was defined as 0.1{\%} of human CD45+ cells detected in murine bone marrow cells after 8 to 10 weeks transplantation. In a control cohort, no human cells were detected in mice receiving IxlO4 non-cultured CD34+ cells (n=3), while mice receiving 105 non-cultured CD34+ cells engrafted with human cells (6.5{\%}). In a cohort of mice receiving the equivalent of IxlO4 CD34+ cells after one week co-culture, all animals (n=3) had human cells (2.61{\%}). One out of three mice receiving the equivalent of 2xl03 CD34+ cells cultured for 1 week had human cell engraftment. In a cohort of animals (n=3) receiving the equivalent of IxlO4 CD34+ following two weeks of co-culture, all mice were positive for human cells (average 0.8{\%}). Our data demonstrates that FL, KL and Tpo adenovector-transfected HUVEC provides an optimal microenvironment for expansion of hematopoietic stem cells detected by in vitro and in vivo assay. Since autologous HUVEC can be used, this culture system provides a clinically relevant method for optimizing human CB ex vivo expansion.",
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AU - Shieh, Jae Hung

AU - Jo, D. Y.

AU - MacKenzie, Karen L.

AU - Rafii, Shahin

AU - Crystal, Ronald G.

AU - Moore, Malcolm A S

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N2 - The major limitation to more widespread use of human cord blood (CB) transplantation is the low number of cells obtained, which restricts its use in adult patients due to delayed engraftment of platelets and neutrophils. Ex vivo expansion has been proposed to overcome these drawbacks.In this study, we evaluated the expansion capability of CB CD34+ cells co-cultured on human umbilical vascular endothelial cells (HUVEC) transfected with adenoviruses expressing Flk2Flt3 ligand (FL), c-kit ligand (KL) and thrombopoietin (Tpo). The co-cultures resulted in 67±6- and 311+25-fold increase of total nucleated cells after one and two weeks respectively; progenitor cells, including CFU-GM, BFU-E and CFUGEMM, increased 13.5 ±2- and 70 ±6-fold; and week-5 cobble-stone area forming cells increased 23 ±2- and 75±7-fold, respectively. In addition, this co-culture system also markedly increased NOD/SCID populating cells. Positive engraftment of human cells in 350 rad irradiated-NOD/SCID mice was defined as 0.1% of human CD45+ cells detected in murine bone marrow cells after 8 to 10 weeks transplantation. In a control cohort, no human cells were detected in mice receiving IxlO4 non-cultured CD34+ cells (n=3), while mice receiving 105 non-cultured CD34+ cells engrafted with human cells (6.5%). In a cohort of mice receiving the equivalent of IxlO4 CD34+ cells after one week co-culture, all animals (n=3) had human cells (2.61%). One out of three mice receiving the equivalent of 2xl03 CD34+ cells cultured for 1 week had human cell engraftment. In a cohort of animals (n=3) receiving the equivalent of IxlO4 CD34+ following two weeks of co-culture, all mice were positive for human cells (average 0.8%). Our data demonstrates that FL, KL and Tpo adenovector-transfected HUVEC provides an optimal microenvironment for expansion of hematopoietic stem cells detected by in vitro and in vivo assay. Since autologous HUVEC can be used, this culture system provides a clinically relevant method for optimizing human CB ex vivo expansion.

AB - The major limitation to more widespread use of human cord blood (CB) transplantation is the low number of cells obtained, which restricts its use in adult patients due to delayed engraftment of platelets and neutrophils. Ex vivo expansion has been proposed to overcome these drawbacks.In this study, we evaluated the expansion capability of CB CD34+ cells co-cultured on human umbilical vascular endothelial cells (HUVEC) transfected with adenoviruses expressing Flk2Flt3 ligand (FL), c-kit ligand (KL) and thrombopoietin (Tpo). The co-cultures resulted in 67±6- and 311+25-fold increase of total nucleated cells after one and two weeks respectively; progenitor cells, including CFU-GM, BFU-E and CFUGEMM, increased 13.5 ±2- and 70 ±6-fold; and week-5 cobble-stone area forming cells increased 23 ±2- and 75±7-fold, respectively. In addition, this co-culture system also markedly increased NOD/SCID populating cells. Positive engraftment of human cells in 350 rad irradiated-NOD/SCID mice was defined as 0.1% of human CD45+ cells detected in murine bone marrow cells after 8 to 10 weeks transplantation. In a control cohort, no human cells were detected in mice receiving IxlO4 non-cultured CD34+ cells (n=3), while mice receiving 105 non-cultured CD34+ cells engrafted with human cells (6.5%). In a cohort of mice receiving the equivalent of IxlO4 CD34+ cells after one week co-culture, all animals (n=3) had human cells (2.61%). One out of three mice receiving the equivalent of 2xl03 CD34+ cells cultured for 1 week had human cell engraftment. In a cohort of animals (n=3) receiving the equivalent of IxlO4 CD34+ following two weeks of co-culture, all mice were positive for human cells (average 0.8%). Our data demonstrates that FL, KL and Tpo adenovector-transfected HUVEC provides an optimal microenvironment for expansion of hematopoietic stem cells detected by in vitro and in vivo assay. Since autologous HUVEC can be used, this culture system provides a clinically relevant method for optimizing human CB ex vivo expansion.

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