TY - JOUR
T1 - Erythroid differentiation and maturation from peripheral CD34+ cells in liquid culture
T2 - Cellular and molecular characterization
AU - Ronzoni, Luisa
AU - Bonara, Paola
AU - Rusconi, Daniela
AU - Frugoni, Cecilia
AU - Libani, Ilaria
AU - Cappellini, Maria Domenica
PY - 2008/3
Y1 - 2008/3
N2 - In vitro models of human erythropoiesis are useful in studying the mechanisms of erythroid differentiation from BFU-E to mature erythrocytes both in normal and pathological conditions. Most of the available in vitro liquid cultures are from cell lines or are limited by the production of few erythroid cells mixed with myeloid cells. Here we describe an erythroid liquid culture system starting from CD34+-enriched cells obtained from peripheral blood. CD34+ cells were cultured for 21 days in different conditions. Precisely stem cell factor (SCF, 20 ng/mL) and IL-3 (10 ng/mL) were added at starting point plus Epo (3 U/mL) at day 0 or 7 of culture with or without cyclosporine A (Cy; 1 mg/mL). In all the conditions, the highest recovery was obtained at day 14 of culture. Epo and Cy added at day 0 produced the highest cell expansion (170-fold mean amplification of the initial cell input by day 14) and recovery of erythroid cell. Sixty seven percent of the cells were GP+ at day 7 and 97% by day 14 respectively. Most of the cells were proerythroblasts at day 7 and mature erythroblasts at day 14 (> 90% were benzidinepos). The presence of Cy favoured erythroid differentiation and maturation and reduced the percentage of non-erythroid CD45+ cells (2% with Cy versus 5% without Cy). Cells cultured with Epo and Cy reproduced erythropoiesis also at the molecular level. The results suggest that in 14 days different steps of human erythropoiesis from peripheral CD34+ cells could be reproduced, with high recovery of highly purified erythroid cells. The high number and purity of erythroid cells produced from a small amount of peripheral blood make this method useful for studying either normal or pathological erythropoiesis.
AB - In vitro models of human erythropoiesis are useful in studying the mechanisms of erythroid differentiation from BFU-E to mature erythrocytes both in normal and pathological conditions. Most of the available in vitro liquid cultures are from cell lines or are limited by the production of few erythroid cells mixed with myeloid cells. Here we describe an erythroid liquid culture system starting from CD34+-enriched cells obtained from peripheral blood. CD34+ cells were cultured for 21 days in different conditions. Precisely stem cell factor (SCF, 20 ng/mL) and IL-3 (10 ng/mL) were added at starting point plus Epo (3 U/mL) at day 0 or 7 of culture with or without cyclosporine A (Cy; 1 mg/mL). In all the conditions, the highest recovery was obtained at day 14 of culture. Epo and Cy added at day 0 produced the highest cell expansion (170-fold mean amplification of the initial cell input by day 14) and recovery of erythroid cell. Sixty seven percent of the cells were GP+ at day 7 and 97% by day 14 respectively. Most of the cells were proerythroblasts at day 7 and mature erythroblasts at day 14 (> 90% were benzidinepos). The presence of Cy favoured erythroid differentiation and maturation and reduced the percentage of non-erythroid CD45+ cells (2% with Cy versus 5% without Cy). Cells cultured with Epo and Cy reproduced erythropoiesis also at the molecular level. The results suggest that in 14 days different steps of human erythropoiesis from peripheral CD34+ cells could be reproduced, with high recovery of highly purified erythroid cells. The high number and purity of erythroid cells produced from a small amount of peripheral blood make this method useful for studying either normal or pathological erythropoiesis.
KW - CD34 peripheral blood cells
KW - Erythroid differentiation
KW - Erythroid gene expression
KW - Erythropoietin
KW - Flow cytometric analysis
UR - http://www.scopus.com/inward/record.url?scp=38949133445&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=38949133445&partnerID=8YFLogxK
U2 - 10.1016/j.bcmd.2007.07.006
DO - 10.1016/j.bcmd.2007.07.006
M3 - Article
C2 - 17889571
AN - SCOPUS:38949133445
VL - 40
SP - 148
EP - 155
JO - Blood Cells, Molecules, and Diseases
JF - Blood Cells, Molecules, and Diseases
SN - 1079-9796
IS - 2
ER -