Adherent cells generated during long-term culture of human umbilical cord blood CD34+ cells have characteristics of endothelial cells and beneficial effect on cord blood ex vivo expansion.
Hematopoiesis depends on the association of hematopoietic stem cells with stromal cells that constitute the hematopoietic microenvironment. The in vitro development of the endothelial cell from umbilical cord blood (UCB) is not well established and has met very limited success. In this study, UCB CD34(+) cells were cultured for 5 weeks in a stroma-free liquid culture system using thrombopoietin, flt3 ligand, and granulocyte-colony stimulating factor. By week 4-5, we found that firmly adherent fibroblast-like cells were established. These cells showed characteristics of endothelial cells expressing von Willebrand factor, human vascular cell adhesion molecule-1, human intracellular adhesion molecule-1, human CD31, E-selectin, and human macrophage. Furthermore, when comparing an ex vivo system without an established endothelial monolayer to an ex vivo system with an established endothelial monolayer, better expansion of total nucleated cells, CD34(+) cells, and colony-forming units (CFUs)-granulocyte-macrophage and CFUs-granulocyte-erythroid-megakaryocyte-macrophage were found during culture. This phenomenon was in part due to the fact that a significant reduction of apoptotic fractions was found in the CD34(+) cells, which were cultured on the adherent monolayer for up to 5 weeks. To gather quantitative data on the number of endothelial cells derived from a given number of CD34 cells, we performed limiting dilution assay by using Poisson distribution: the number of tested cells (linear scale) producing a 37% negative culture (logarithmic scale) is the number of cells containing one endothelial cell. By this method, one endothelial cell may be found from 314 CD34(+) cells after 5 weeks of culture. These results suggest that the UCB CD34(+) cell fraction contains endothelial cell precursors, establishing the hematopoietic microenvironment and providing the beneficial effects through downregulating apoptosis on UCB expansion protocols. These observations may provide insight for future cellular therapy or graft engineering.
Yoo
Eun-Sun
ES
Department of Pediatrics, College of Medicine, Ewha Women's University, Seoul, Korea.
Lee
Kyung-Eun
KE
Seo
Jeong-Wan
JW
Yoo
Eun-Hee
EH
Lee
Mi-Ae
MA
Im
Seock-Ah
SA
Mun
Yeung-Chul
YC
Lee
Soon-Nam
SN
Huh
Jung-Won
JW
Kim
Mi-Jung
MJ
Jo
Duck-Yeon
DY
Ahn
Jee-Young
JY
Lee
Sun-Mee
SM
Chung
Wha-Soon
WS
Kim
Jae-Hong
JH
Seong
Chu-Myong
CM
D000906:Antibodies; D018952:Antigens, CD34 / Q000032:analysis* / Q000276:immunology; D017209:Apoptosis; D002469:Cell Separation; D002478:Cells, Cultured; D003609:Dactinomycin / Q000031:analogs & derivatives*; D015536:Down-Regulation; D004730:Endothelium, Vascular / Q000166:cytology*; D005312:Fetal Blood / Q000166:cytology*; D005347:Fibroblasts / Q000166:cytology; D005456:Fluorescent Dyes; D006412:Hematopoietic Stem Cells / Q000737:chemistry* / Q000166:cytology*; D006801:Humans; D018799:Intercellular Adhesion Molecule-1 / Q000032:analysis / Q000276:immunology; D019010:Vascular Cell Adhesion Molecule-1 / Q000032:analysis / Q000276:immunology; D014841:von Willebrand Factor / Q000032:analysis / Q000276:immunology
D000906:Antibodies; D018952:Antigens, CD34; D005456:Fluorescent Dyes; D019010:Vascular Cell Adhesion Molecule-1; D014841:von Willebrand Factor; D018799:Intercellular Adhesion Molecule-1; D003609:Dactinomycin; C025942:7-aminoactinomycin D