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Blood, Vol. 93 No. 10 (May 15), 1999:
pp. 3355-3368
Unicellular-Unilineage Erythropoietic Cultures: Molecular Analysis
of Regulatory Gene Expression at Sibling Cell Level
Benedikt L. Ziegler,
Robert Müller,
Mauro Valtieri,
Christa
P. Lamping,
Christian A. Thomas,
Marco Gabbianelli,
Christina Giesert,
Hans-Jörg Bühring,
Lothar Kanz, and
Cesare Peschle
From the Department of Medicine, Division of Hematology and Oncology,
Eberhard-Karls-University Tübingen, Tübingen, Germany; the
Thomas Jefferson Cancer Center, Philadelphia, PA; the Department of
Hematology-Oncology, Istituto Superiore di Sanitá, Rome, Italy;
Jackson Laboratory, Bar Harbor, ME; and the Department of Physiology
and Cellular Biophysics, College of Physicians and Surgeons of Columbia
University, New York, NY.
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.
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