In vitro expansion of human cord blood CD36+ erythroid progenitors: temporal changes in gene and protein expression.
OBJECTIVE
Erythropoiesis involves proliferation and differentiation of committed erythroid progenitors to mature red blood cells. The objective of this study was to characterize growth characteristics of human CD36+ erythroid progenitors and to profile temporal expression of lineage-specific transcription factors, structural proteins, and growth factor receptors involved in erythropoiesis.


METHODS
Erythropoietin-induced differentiation of human cord blood CD36+ erythroid progenitors was profiled for GATA-1, GATA-2, NFE2, EKLF, SCL, PU.1, Id1, Evi-1, c-myb, Hox2.2, c-kit, EpoR, glycophorin A (GPA), CD71, beta- and gamma-globin, and protein 4.2 gene and/or protein expression and DNA content analysis on days 4, 7, and 15 of culture.


RESULTS
Real-time RT-PCR analysis revealed upregulation of GATA-1, Id1, glycophorin A, and protein 4.2 mRNA expression on day 7 when compared to day 4 and decreased expression on day 15. EKLF, GATA-2, Hox2.2, c-myb, Evi-1, c-kit, and PU.1 mRNA expression decreased on days 7 and 15. NFE2, CD71, SCL, and EPO-R mRNA expression remained similar on days 4 and 7 but decreased on day 15. Expression of globin genes beta- and gamma-globin increased on both day 7 and day 15 compared to day 4. Values from flow cytometric quantitation of glycophorin A, transferrin receptor (CD71), and hemoglobin A proteins correlated with gene expression results. DNA analysis demonstrated that most cells lacked DNA content by day 15, a finding consistent with enucleation and terminal erythroid differentiation.


CONCLUSIONS
These data indicate that in vitro liquid cultures of committed CD36+ erythroid progenitor cells retain, in part, many features of erythropoiesis at the cellular and molecular level and may provide a useful model for assessment of disease-related or drug-induced erythropoietic abnormalities.
Scicchitano
Marshall S
MS
GlaxoSmithKline, Safety Assessment Department, King of Prussia, Pa. 19406, USA. Marshall.2.Scicchitano@gsk.com
McFarland
David C
DC
Tierney
Lauren A
LA
Narayanan
Padma K
PK
Schwartz
Lester W
LW
D018955:CD36 Antigens; D002454:Cell Differentiation; D002455:Cell Division; D019070:Cell Lineage / Q000235:genetics; D002478:Cells, Cultured; D015672:Erythroid Precursor Cells / Q000166:cytology* / Q000502:physiology*; D004920:Erythropoiesis / Q000235:genetics; D005434:Flow Cytometry; D018507:Gene Expression Regulation, Developmental; D006801:Humans; D014176:Protein Biosynthesis; D011506:Proteins / Q000235:genetics
D018955:CD36 Antigens; D011506:Proteins