Ultrastructural and phenotypic analysis of in vitro erythropoiesis from human cord blood CD34+ cells

J. H. Kie; Y. J. Jung; S. Y. Woo; K. H. Ryu; H. Y. Park; W. S. Chung; J. Y. Seoh

doi:10.1007/s00277-003-0629-x

Ultrastructural and phenotypic analysis of in vitro erythropoiesis from human cord blood CD34⁺ cells

J. H. Kie, Y. J. Jung, S. Y. Woo, K. H. Ryu, H. Y. Park, W. S. Chung, J. Y. Seoh

Research output: Contribution to journal › Article › peer-review

8 Scopus citations

Abstract

Erythropoietin (EPO) induces erythropoiesis in vitro as well as in vivo, and the process of erythroid differentiation has been explored phenotypically and morphologically. However, morphological analysis of in vitro erythropoiesis of human hematopoietic progenitor cells at the ultrastructural level has not been reported before. In the present study, we have traced the ultrastructural changes of erythroid differentiation during ex vivo expansion of human cord blood (CB) CD34⁺ cells in the presence of EPO by electron microscopy (EM), along with concurrent phenotypic analysis. CD34⁺ cells purified from ten CBs by immunomagnetic selection were cultured in serum-free essential media in the presence of a combination of the several cytokines including EPO, thrombopoietin, flt3-ligand (FL), stem cell factor (SCF), granulocyte colony-stimulating factor, interleukin (IL)-3 and/or IL-11. Phenotypic analysis was performed by flow cytometric analysis for erythroid markers, including glycophorin C (GPC), Kell-related, glycophorin A (GPA , band 3, Lu^b , and RhD. Ultrastructural analysis was performed by electron-microscopic examination of the cultured cells stained with uranyl acetate and lead citrate. Phenotypic analysis revealed that in the absence of EPO, genuine erythroid fraction expressing the typical pattern of erythroid markers did not appear. The order of the above markers expressed in the cultured cells in the presence of EPO was GPC, Kell-related, GPA, band 3, Lu^b, and RhD, irrespective of the type of cytokine added. Of the cytokines used in combination with EPO, FL + IL-3 was the most efficient in inducing erythroid differentiation, which was followed by SCF + IL-3. EM examination demonstrated complete process of erythroid development from pronormoblasts to reticulocytes with nuclei having been extruded and mature erythrocytes. These results suggest that morphologically intact erythrocytes could be produced by ex vivo expansion of CB CD34⁺ cells using EPO.

Original language	English
Pages (from-to)	278-283
Number of pages	6
Journal	Annals of Hematology
Volume	82
Issue number	5
DOIs	https://doi.org/10.1007/s00277-003-0629-x
State	Published - 1 May 2003

Bibliographical note

Funding Information:
Acknowledgements This study was supported by a grant of the Korea Health 21 R&D Project, Ministry of Health and Welfare, Republic of Korea (HMP-00-CH-04–0004).

Keywords

Cord blood
Erythropoiesis
Ex vivo expansion
Phenotype
Ultrastructure

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10.1007/s00277-003-0629-x

Cite this

@article{95bb44f5cd4143e88a52365452be388c,

title = "Ultrastructural and phenotypic analysis of in vitro erythropoiesis from human cord blood CD34+ cells",

abstract = "Erythropoietin (EPO) induces erythropoiesis in vitro as well as in vivo, and the process of erythroid differentiation has been explored phenotypically and morphologically. However, morphological analysis of in vitro erythropoiesis of human hematopoietic progenitor cells at the ultrastructural level has not been reported before. In the present study, we have traced the ultrastructural changes of erythroid differentiation during ex vivo expansion of human cord blood (CB) CD34+ cells in the presence of EPO by electron microscopy (EM), along with concurrent phenotypic analysis. CD34+ cells purified from ten CBs by immunomagnetic selection were cultured in serum-free essential media in the presence of a combination of the several cytokines including EPO, thrombopoietin, flt3-ligand (FL), stem cell factor (SCF), granulocyte colony-stimulating factor, interleukin (IL)-3 and/or IL-11. Phenotypic analysis was performed by flow cytometric analysis for erythroid markers, including glycophorin C (GPC), Kell-related, glycophorin A (GPA , band 3, Lub , and RhD. Ultrastructural analysis was performed by electron-microscopic examination of the cultured cells stained with uranyl acetate and lead citrate. Phenotypic analysis revealed that in the absence of EPO, genuine erythroid fraction expressing the typical pattern of erythroid markers did not appear. The order of the above markers expressed in the cultured cells in the presence of EPO was GPC, Kell-related, GPA, band 3, Lub, and RhD, irrespective of the type of cytokine added. Of the cytokines used in combination with EPO, FL + IL-3 was the most efficient in inducing erythroid differentiation, which was followed by SCF + IL-3. EM examination demonstrated complete process of erythroid development from pronormoblasts to reticulocytes with nuclei having been extruded and mature erythrocytes. These results suggest that morphologically intact erythrocytes could be produced by ex vivo expansion of CB CD34+ cells using EPO.",

keywords = "Cord blood, Erythropoiesis, Ex vivo expansion, Phenotype, Ultrastructure",

author = "Kie, {J. H.} and Jung, {Y. J.} and Woo, {S. Y.} and Ryu, {K. H.} and Park, {H. Y.} and Chung, {W. S.} and Seoh, {J. Y.}",

note = "Funding Information: Acknowledgements This study was supported by a grant of the Korea Health 21 R&D Project, Ministry of Health and Welfare, Republic of Korea (HMP-00-CH-04–0004).",

year = "2003",

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doi = "10.1007/s00277-003-0629-x",

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T1 - Ultrastructural and phenotypic analysis of in vitro erythropoiesis from human cord blood CD34+ cells

AU - Kie, J. H.

AU - Jung, Y. J.

AU - Woo, S. Y.

AU - Ryu, K. H.

AU - Park, H. Y.

AU - Chung, W. S.

AU - Seoh, J. Y.

N1 - Funding Information: Acknowledgements This study was supported by a grant of the Korea Health 21 R&D Project, Ministry of Health and Welfare, Republic of Korea (HMP-00-CH-04–0004).

PY - 2003/5/1

Y1 - 2003/5/1

N2 - Erythropoietin (EPO) induces erythropoiesis in vitro as well as in vivo, and the process of erythroid differentiation has been explored phenotypically and morphologically. However, morphological analysis of in vitro erythropoiesis of human hematopoietic progenitor cells at the ultrastructural level has not been reported before. In the present study, we have traced the ultrastructural changes of erythroid differentiation during ex vivo expansion of human cord blood (CB) CD34+ cells in the presence of EPO by electron microscopy (EM), along with concurrent phenotypic analysis. CD34+ cells purified from ten CBs by immunomagnetic selection were cultured in serum-free essential media in the presence of a combination of the several cytokines including EPO, thrombopoietin, flt3-ligand (FL), stem cell factor (SCF), granulocyte colony-stimulating factor, interleukin (IL)-3 and/or IL-11. Phenotypic analysis was performed by flow cytometric analysis for erythroid markers, including glycophorin C (GPC), Kell-related, glycophorin A (GPA , band 3, Lub , and RhD. Ultrastructural analysis was performed by electron-microscopic examination of the cultured cells stained with uranyl acetate and lead citrate. Phenotypic analysis revealed that in the absence of EPO, genuine erythroid fraction expressing the typical pattern of erythroid markers did not appear. The order of the above markers expressed in the cultured cells in the presence of EPO was GPC, Kell-related, GPA, band 3, Lub, and RhD, irrespective of the type of cytokine added. Of the cytokines used in combination with EPO, FL + IL-3 was the most efficient in inducing erythroid differentiation, which was followed by SCF + IL-3. EM examination demonstrated complete process of erythroid development from pronormoblasts to reticulocytes with nuclei having been extruded and mature erythrocytes. These results suggest that morphologically intact erythrocytes could be produced by ex vivo expansion of CB CD34+ cells using EPO.

AB - Erythropoietin (EPO) induces erythropoiesis in vitro as well as in vivo, and the process of erythroid differentiation has been explored phenotypically and morphologically. However, morphological analysis of in vitro erythropoiesis of human hematopoietic progenitor cells at the ultrastructural level has not been reported before. In the present study, we have traced the ultrastructural changes of erythroid differentiation during ex vivo expansion of human cord blood (CB) CD34+ cells in the presence of EPO by electron microscopy (EM), along with concurrent phenotypic analysis. CD34+ cells purified from ten CBs by immunomagnetic selection were cultured in serum-free essential media in the presence of a combination of the several cytokines including EPO, thrombopoietin, flt3-ligand (FL), stem cell factor (SCF), granulocyte colony-stimulating factor, interleukin (IL)-3 and/or IL-11. Phenotypic analysis was performed by flow cytometric analysis for erythroid markers, including glycophorin C (GPC), Kell-related, glycophorin A (GPA , band 3, Lub , and RhD. Ultrastructural analysis was performed by electron-microscopic examination of the cultured cells stained with uranyl acetate and lead citrate. Phenotypic analysis revealed that in the absence of EPO, genuine erythroid fraction expressing the typical pattern of erythroid markers did not appear. The order of the above markers expressed in the cultured cells in the presence of EPO was GPC, Kell-related, GPA, band 3, Lub, and RhD, irrespective of the type of cytokine added. Of the cytokines used in combination with EPO, FL + IL-3 was the most efficient in inducing erythroid differentiation, which was followed by SCF + IL-3. EM examination demonstrated complete process of erythroid development from pronormoblasts to reticulocytes with nuclei having been extruded and mature erythrocytes. These results suggest that morphologically intact erythrocytes could be produced by ex vivo expansion of CB CD34+ cells using EPO.

KW - Cord blood

KW - Erythropoiesis

KW - Ex vivo expansion

KW - Phenotype

KW - Ultrastructure

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