Unicellular-unilineage erythropoietic cultures: molecular analysis of regulatory gene expression at sibling cell level.
In vitro studies on hematopoietic control mechanisms have been hampered by the heterogeneity of the analyzed cell populations, ie, lack of lineage specificity and developmental stage homogeneity of progenitor/precursor cells growing in culture. We developed unicellular culture systems for unilineage differentiation of purified hematopoietic progenitor cells followed by daughter cell analysis at cellular and molecular level. In the culture system reported here, (1) the growth factor (GF) stimulus induces cord blood (CB) progenitor cells to proliferate and differentiate/mature exclusively along the erythroid lineage; (2) this erythropoietic wave is characterized by less than 4% apoptotic cells; (3) asymmetric divisions are virtually absent, ie, nonresponsive hematopoietic progenitors with no erythropoietic potential are forced into apoptosis; (4) the system is cell division controlled (cdc), ie, the number of divisions performed by each cell is monitored. Single-cell reverse transcriptase-polymerase chain reaction (RT-PCR) analysis was applied to this culture system to investigate gene expression of diverse receptors, markers of differentiation, and transcription factors (EKLF, GATA-1, GATA-2, p45 NF-E2, PU.1, and SCL/Tal1) at discrete stages of erythropoietic development. Freshly isolated CD34(+) cells expressed CD34, c-kit, PU.1, and GATA-2 but did not express CD36, erythropoietin receptor (EpoR), SCL/Tal1, EKLF, NF-E2, GATA-1, or glyocophorin A (GPA). In early to intermediate stages of erythroid differentiation we monitored the induction of CD36, Tal1, EKLF, NF-E2, and GATA-1 that preceeded expression of EpoR. In late stages of erythroid maturation, GPA was upregulated, whereas CD34, c-kit, PU.1, and GATA-2 were barely or not detected. In addition, competitive single-cell RT-PCR was used to assay CD34 mRNA transcripts in sibling CD34(+)CD38(-) cells differentiating in unilineage erythroid cultures: this analysis allowed us to semiquantitate the gradual downmodulation of CD34 mRNA from progenitor cells through their differentiating erythroid progeny. It is concluded that this novel culture system, coupled with single-cell RT-PCR analysis, may eliminate the ambiguities intrinsic to molecular studies on heterogeneous populations of hematopoietic progenitors/precursors growing in culture, particularly in the initial stages of development.
Ziegler
B L
BL
Department of Medicine, Division of Hematology and Oncology, Eberhard-Karls-University Tübingen, Tübingen, Germany. benedikt.ziegler@uni-tuebingen.de
Müller
R
R
Valtieri
M
M
Lamping
C P
CP
Thomas
C A
CA
Gabbianelli
M
M
Giesert
C
C
Bühring
H J
HJ
Kanz
L
L
Peschle
C
C
D018952:Antigens, CD34 / Q000235:genetics; D017209:Apoptosis; D001483:Base Sequence; D018929:Cell Culture Techniques / Q000379:methods; D002454:Cell Differentiation; D002455:Cell Division; D002478:Cells, Cultured; D017931:DNA Primers; D004268:DNA-Binding Proteins / Q000235:genetics; D015672:Erythroid Precursor Cells / Q000166:cytology* / Q000502:physiology*; D004920:Erythropoiesis / Q000235:genetics / Q000502:physiology; D005312:Fetal Blood / Q000166:cytology; D005434:Flow Cytometry; D050989:GATA2 Transcription Factor; D005786:Gene Expression Regulation; D005809:Genes, Regulator; D006801:Humans; D007231:Infant, Newborn; D008969:Molecular Sequence Data; D011518:Proto-Oncogene Proteins / Q000235:genetics; D019009:Proto-Oncogene Proteins c-kit / Q000235:genetics; D012333:RNA, Messenger / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction; D015534:Trans-Activators / Q000235:genetics; D014157:Transcription Factors / Q000235:genetics; D014158:Transcription, Genetic; D000099232:Proto-Oncogene Protein Spi-1
D018952:Antigens, CD34; D017931:DNA Primers; D004268:DNA-Binding Proteins; D050989:GATA2 Transcription Factor; D011518:Proto-Oncogene Proteins; D019009:Proto-Oncogene Proteins c-kit; D012333:RNA, Messenger; D015534:Trans-Activators; D014157:Transcription Factors; D000099232:Proto-Oncogene Protein Spi-1; C494711:GATA2 protein, human