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 Previous Article | Table of Contents | Next Article 
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
By
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.
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ABSTRACT |
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.
© 1999 by The American Society of Hematology.
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INTRODUCTION |
DIFFERENTIATION OF hematopoietic stem
cells is a regulated developmental cascade that generates
lineage(s)-committed progenitor cells that feed into maturing
precursors and then terminal elements circulating in peripheral blood
(PB). Hematopoietic stem cells possess three important properties: (1)
extensive self-renewal capacity, (2) broad differentiation potential,
and (3) prolonged maintenance in a noncycling state. These features
gradually taper off in differentiating progenitors.1-3 Upon
induction to cycling, stem cells and primitive progenitors may undergo
symmetric divisions (ie, either self-replication or differentiation)
and/or asymmetric divisions (ie, generation of a self-replicated and a
differentiated cell).4,5
Hematopoietic stem/progenitor cell differentiation has been interpreted
in terms of stochastic,6 inductive,7 or hybrid models.8 According to the stochastic hypothesis, random
intrinsic molecular events are responsible for committment, whereas
exogenous hematopoietic growth factors allow survival and proliferation of preprogrammed cells.9,10 According to the inductive
model, hematopoietic growth factors and cell-mediated regulatory
mechanisms trigger stem/progenitor cells to differentiate along a
particular lineage(s).11 Alternatively, a hybrid model
proposes that stochastic events prevail at early developmental stages,
whereas growth factor-mediated inductive events operate
thereafter.12 Indeed, purified hematopoietic progenitors
express the receptors for multilineage growth factors (ie,
interleukin-3 [IL-3] and granulocyte-macrophage colony-stimulating factor [GM-CSF]), but barely express or do not express the receptors for late-acting unilineage cytokines (ie, erythropoietin [Epo], and
macrophage colony-stimulating factor [M-CSF]) as well as mainly late-acting unilineage cytokines (ie, thrombopoietin [TPO] and granulocyte colony-stimulating factor [G-CSF]). The latter receptors accumulate through differentiation/maturation in their specific lineage, whereas they are downmodulated in the other series.
Furthermore, interaction of early hematopoietic growth factors with
their receptors upmodulates distal growth factor
receptors.13 Regardless of the proposed model, it is
generally accepted that lineage choice is mediated via activation of
differentiation gene program(s) and specifically via a network of
transcription factors, which orchestrate these gene programs at the
transcriptional level.14-16 Leukemia cell lines and murine
hematopoietic progenitor cell lines (32D and FDCP-Mix)17,18
have been extensively used for transcription factor studies; however,
they reflect selected stages and lineages of normal hematopoiesis and
are partially or totally independent of growth factors and other
physiological control mechanisms. Studies on knock-out mice have
provided basic insight into molecular mechanisms underlying
hematopoiesis,16 but results may not always apply to human
adult hematopoiesis due to several limitations such as gene redundancy,
position effects of the disrupted gene on adjacent genes, lethal
effects in early ontogenesis, lack of tissue specificity, and species
differences.14,19 In vitro studies on hematopoietic control
mechanisms have been hampered by (1) the extreme rarity of early
hematopoietic progenitors and stem cells, (2) limitations of current
protocols for purification of homogeneous stem/progenitor cell subsets,
and (3) unavailability of pure progenitors and precursors at discrete
developmental stages through a selected lineage. Analysis of the
molecular basis of human adult hematopoiesis has been facilitated by
development of unilineage progenitor cell differentiation in bulk
culture.20-27 In these systems, purified early
progenitor/stem cells are induced into a wave of gradual
differentiation/maturation along a specific lineage(s), thus providing
a tool to evaluate expression and function of developmentally regulated
genes. However, unilineage bulk cultures bear two limitations: (1)
hematopoietic progenitors programmed for differentiation along other
lineage(s) undergo apoptosis and (2) cells analyzed at a given culture
time reflect a narrow but still overlapping range of different
developmental stages.
We have developed a novel strategy based on hematopoietic progenitor
cell unicellular, unilineage differentiation culture, followed by
daughter cell analysis at cellular and molecular levels. This requires
well-defined culture conditions20-27 as well as stringently controlled reverse transcriptase-polymerase chain reaction
(RT-PCR) technology, ie, the unambiguous detection of
multiple genes in single progenitor/precursors undergoing unilineage
differentiation/maturation. This approach was applied here to evaluate
the pattern of gene expression at discrete stages of cord blood (CB)
progenitor cell differentiation and maturation along the erythroid
lineage by RT-PCR analysis at the single-cell level. Single-cell
competitive RT-PCR was also performed in selected experiments. We show
that discrete developmental stages of erythroid differentiation
(defined by the number of cell divisions performed by each progenitor
cell) are characterized by a specific expression pattern of genes
coding for transcription factors, growth factor receptors, and
differentiation markers.
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MATERIALS AND METHODS |
Cell Preparation
CB was obtained from healthy, full-term neonates according to
institutional guidelines in 50 mL polypropylene tubes containing 20 U/mL preservative-free heparin, 1 mmol/L adenosine, and 2 mmol/L theophylline.28 Low-density cells (<1.077 g/mL) were
isolated using first Ficoll (Biochrom KG, Berlin, Germany) and then a
discontinuous Percoll gradient (LD/P cells) as described
previously.28 The CD34+ cells were isolated by
using the MiniMACS CD34 isolation system (Miltenyi, Bergisch Gladbach,
Germany) following the manufacturer`s instructions and then sorted by
flow cytometry. The CD34+Lin cells were
purified by a positive/negative selection strategy using the MiniMACS
CD34 Multisort kit (Miltenyi). Briefly, 2 to 3 × 108
LD/P cells were incubated for 5 minutes at 12°C in 200 µL FcR blocking reagent (Miltenyi). Cells were then incubated without washing
with 200 µL CD34 Multisort microbeads and 25 µL of each of the
following biotinylated Lin-Moabs in a total volume of 1 mL in
phosphate-buffered saline (PBS)-EDTA/0.5% bovine serum albumin (BSA):
anti-CD2 (Immunotech, IM, Hamburg, Germany), anti-CD5, anti-CD10,
anti-CD13, anti-CD19, anti-CD56 (Leinco, Ballwin, MO), anti-CD7
(Serotec, Oxford, UK), anti-CD11b, anti-CD11c, anti-CD14, anti-CD15
(Sigma, München, Germany), anti-CD16 (Pharmingen, Hamburg, Germany), anti-CD33, anti-CD45RA (Becton Dickinson, Heidelberg, Germany), anti-CD20, and anti-CD61 (Southern Biotechnology Associates, Birmingham, AL). Cells were incubated for 45 minutes at 4°C,
centrifuged for 10 minutes at 300g, and resuspended in
PBS-EDTA. Rinsed MS separation columns (Miltenyi) were placed into a
magnetic support and 1 × 108 cells in 500 µL were
loaded onto the column. The eluate comprising the
CD34 fraction was disregarded and the column was
washed 3 times with 500 µL of buffer. The CD34+ cells
were isolated after removal of the column from the magnet and eluted
with a total volume of 1.5 mL of buffer. This fraction containing
CD34+ cells was loaded onto a second freshly prepared
column and CD34+ cells isolated as described above. The
microbead-labeled CD34+ cell suspension was incubated with
20 µL of Multisort Release Reagent per milliliter for 10 minutes at
12°C to release beads from cells. The cell suspension was then
loaded onto a third freshly prepared MS column placed in a magnet. The
eluate containing bead-free CD34+ cells was centrifuged
through a cushion of PBS/10% BSA for 10 minutes at 600g. The
pellet was resuspended after adding 30 µL of stop reagent and 10 µL
of streptavidin-conjugated microbeads (Miltenyi) and then incubated for
30 minutes at 6°C. Cells were resuspended in 500 µL of buffer and
loaded onto a freshly prepared MS column placed in a magnet. The eluate
representing CD34+Lin
cells was further processed for fluorescence-activated cell sorting (FACS) analysis and cell culture.
Immunofluorescence Staining and Flow Cytometry
For purity control, purified CD34+Lin
cells were incubated for 10 minutes at 4°C in PBS/1% BSA
containing 10 µg/100 µL of purified mouse IgG (Sigma). Cells were
incubated without washing for 30 minutes at 4°C with phycoerythrin
(PE)-conjugated anti-CD34 (8G12; Becton Dickinson) and
streptavidin-fluorescein isothiocyanate (FITC; DAKO, Hamburg, Germany),
or a cocktail of FITC-labeled Lin-MoAbs was used. After 3 washes with
PBS/1% BSA, cells were analyzed using a Calibur flow cytometer (Becton
Dickinson). CD34+Lin cells induced to
unilineage differentiation (bulk culture) were stained accordingly
using PE-conjugated anti-CD34 and FITC-labeled anti-CD36, anti-c-kit,
anti-GPA (Immunotech), or anti-CD71 (Becton Dickinson). For cell
sorting, column-purified CD34+ cells (0.5 to 1 × 106/100 µL) were stained with anti-CD34-FITC (8G12) and
anti-CD38-PE (Becton Dickinson) and then sorted with a FACS Vantage
(Becton Dickinson) equipped with an automatic cell deposition unit
(ACDU) essentially as described previously.29 Single sorted
cells were processed for RT-PCR or unicellular, unilineage culture as
described below.
Serum-Free Liquid Suspension Culture
Single ACDU-sorted CD34+CD38 cells or
CD34+Lin cells were grown in individual
wells of a round-bottom 96-well plate (Falcon, Heidelberg, Germany) in
serum-free medium26 containing 0.1 U/mL IL-3, 0.05 U
GM-CSF, and 3 U/mL Epo.22 Cells were incubated in a fully
humidified atmosphere of 5% CO2/5% O2 in air.
Each well was examined daily for evaluation of sibling cells and
processed as shown in Fig 1. Briefly, when
wells contained 4 cells (round I, 2 cell divisions), 1 cell was
transferred in liquid culture and allowed to terminally
differentiate/proliferate for morphologic evaluation, 1 cell was
replated for serum-free liquid suspension culture, and each of the
remaining 2 cells was processed separately for RT-PCR. A second, third,
and fourth round of culture (4, 6, and 8 cell divisions, respectively)
and RT-PCR analysis was performed for sibling cells. This culture
system was designated cell division controlled (cdc) culture. For
comparative experiments to examine the homogeneity of gene expression
among daughter cells, 3 of 4 cells of round I, II, III, or IV from cdc
cultures (Fig 1) were each subjected to RT-PCR as described
below. In addition, single cells were grown in unilineage erythroid
cultures until wells contained 4, 8 to 12, or 16 to 24 cells, ie,
non-cell division number controlled (non-cdc) cultures, followed by
single-cell RT-PCR analysis. Individual cells were isolated by using a
siliconized Pasteur pipet attached to a micromanipulator. The frequency
of apoptosis at the single-cell level was determined at initiation of
single cell cultures, after 1 and 2 cell divisions, as shown in
Fig 2, as well as after 6 cell divisions.
Only wells containing a single cell at initiation of each culture as
evaluated by light microscopy were monitored for 10 days. Cells were
scored apoptotic if no cell division or no cell was detected during the
evaluation period. For evaluation of symmetric cell divisions, 3 cells
from each 4-cell stage (rounds I through III) were transferred into separate wells containing the unilineage erythroid growth factor cocktail and allowed to terminally differentiate/proliferate for morphologic analysis. The remaining fourth cell was used to initiate the subsequent culture round. For flow cytometry analysis (bulk culture
experiments), 2.5 × 104
CD34+Lin cells were grown in 500 µL of
the same medium used for unicellular culture and analyzed by FACS at
days 0, 6, 10, and 14.
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| Fig 1.
Unilineage, single-cell culture of purified
CD34+Lin cells or ACDU-sorted
CD34+CD38 cells from CB: sibling analysis
(clonogenic capacity and gene expression, as evaluated by single cell
RT-PCR) at sequential rounds of cell division.
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| Fig 2.
Evaluation of apoptosis in unicellular, unilineage
erythroid cultures. Single cells were scored apoptotic if no cell
division(s) was detectable or if no cell(s) was detectable after 10 days of culture. At inititation of single-cell culture, each well was
monitored by an inverted microscope. Only wells containing 1 cell were
monitored. The proportion of single apoptotic cells was evaluated for
freshly sorted CD34+CD38 cells or purified
CD34+Lin cells (A), siblings thereof, ie,
after the first cell division (B), and single cells after 2 cell
divisions (C).
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Apoptosis in Unilineage Erythroid Bulk Culture
CB LD/P cells were labeled with PKH 26 (Sigma) essentially as described
by the manufacturer. Briefly, 2 × 108 LD/P cells were
incubated for 3 minutes at room temperature in 10 mL of diluent C
(Sigma) containing 2 × 106 mol/L PKH26. The
labeling reaction was stopped by adding 10 mL of heat-inactivated fetal
calf serum (FCS; CCPro, Neustadt, Germany). Cells were washed with
PBS/0.5% BSA and then processed for isolation of
CD34+Lin cells as described above. For
detection of apoptotic cells, freshly purified PKH26 labeled
CD34+Lin cells and the same cells after
2 and 6 days of unilineage erythroid bulk culture were stained with 10 µL of FITC-conjugated Annexin V in 100 µL binding buffer (Apoptosis
staining kit; R&D Systems, Minneapolis, MN). Cells were analyzed by
using a FACSCalibur (Becton Dickinson).
Isolation of mRNA From Single Cells/Small Numbers of Cells and
Sequence Specific (SSP) RT-PCR
The poly(A)+ mRNA was isolated by using the PolyATract
system 1000 (Promega, Heidelberg, Germany). For single cells or small numbers of cells, ie, 100 cells, the system was scaled down to allow
isolation of mRNAs.30,31 The mRNA isolation was performed with slight modifications of the manufacturer's instructions. Briefly,
cells were transferred to 25 µL guanidine thiocyanate (GuSCN) lysis
buffer containing 2%  -mercaptoethanol (-ME) in a 1.5 mL
polypropylene tube and immediately vortexed thoroughly. Fifty
microliters of dilution buffer preheated to 70°C containing 1%
-ME and 5 pmol biotinylated oligo(dT) were added. The homogenate was
incubated at 70°C for 5 minutes and then centrifuged at
12.000g for 15 minutes at room temperature. The supernatant
containing poly(A)+ mRNA hybridized to biotinylated
oligo(dT) was transferred to a fresh tube containing washed
streptavidin-conjugated paramagnetic particles (SA-PMP) and incubated
for 5 minutes at room temperature. The SA-PMP-mRNA conjugates were then
isolated by magnetic separation. To dissociate bound mRNAs from
SA-PMPs, washed SA-PMP-mRNA hybrids were resuspended in RNase-free
dH2O for 2 minutes. After magnetic separation, the
supernatant containing dissociated mRNAs was transferred to a fresh
tube, ethanol precipitated, and further processed for cDNA synthesis.
The mRNA was dissolved in 10 µL reverse transcriptase buffer (50 mmol/L Tris-HCl, pH 8.3, 75 mmol/L KCl, 3 mmol/L MgCl2) containing 100 U Moloney murine leukemia virus (MMLV) reverse transcriptase (Superscript; BRL/LTI, Eggenstein, Germany), 0.2 µg
oligo(dT), 15 U Rnasin (Promega), 10 mmol/L dithiothreitol (DTT), and
0.5 mmol/L of each dNTP and was reverse transcribed for 1 hour at
42°C. For RT-PCR analysis, the cDNA from individual cells was
divided in up to 4 aliquots. Genbank locus and primer sequence were
reported previously28,29 or were CD34
(Table 1), EKLF (HSU65404) positions
726-745 and 1051-1032; GATA-1 (HSERYF1), 1081-1100 and 1401-1381;
GATA-2 (HUMGATA2A), 1007-1026 and 1493-1474; p45 NF-E2 (HUMNFE2A),
781-801 and 1214-1194; SCL/Tal1 (HUMSCLA), 2817-2838 and 3136-3115;
PU.1 (HSSPI1), 213-232 and 758-739; 2-microglobulin (HUMB2M),
118-137 and 341-321; -globin (HSBGL1), 377-396 and 531-510; EpoR
(HUMERYTH), 887-897 and 1204-1185; CD13 (HUMAMIPEP), 2691-2710 and
2995-2976.
Each 50 µL of PCR reaction contained 1.25 U AmpliTaq DNA polymerase
(Perkin Elmer Cetus, Überlingen, Germany), 200 µmol/L of
each dNTP, and 0.5 µmol/L of each oligonucleotide primer in PCR
buffer (10 mmol/L Tris-HCl, pH 8.3 to 9.0, 50 mmol/L KCl, 1.5 to 3.5 mmol/L MgCl2, and 0.001% gelatin). Reactions were
amplified in a DNA thermal cycler (PCR 9600 system; Perkin Elmer Cetus) for 40 to 60 cycles using predetermined optimal cycling parameters for
each primer pair. The sensitivity of the RT-PCR was determined by
serial dilution of cDNA obtained from sorted cells that were CD34+, CD34+CD36+,
CD34+c-kit+, GPA+CD36+,
CD7+, CD13+, CD14+,
CD16+, CD19+ cells or from single erythroblasts
isolated from erythroid colonies (burst-forming unit-erythroid
[BFU-E]), as described previously.28 Cell
lines K562 or HL-60 were used for sensitivity testing for GATA-1,
GATA-2, p45 NF-E2, EKLF, SCL/Tal1, and PU.1. The RT-PCR products were
electrophoresed through a 2% SEAKEM (FMC BioProducts, Biozym, Hess.
Oldendorf, Germany) agarose gel, stained, transferred onto Genescreen
Plus membranes (DuPont, Bad Homburg, Germany), and hybridized with
corresponding 32P-labeled 30-mer oligonucleotide probes
using standard techniques.
Sequence-Independent (SIP) RT-PCR
SIP-RT-PCR was performed on mRNA, which was reverse transcribed with
slight modifications as described for SSP-RT-PCR.32 Briefly, the mRNA was dissolved in 4 µL reverse transcriptase buffer
(see above) containing 100 U MMLV reverse transcriptase (BRL/LTI) and 2 U of AMV RTase (Promega), 0.1 µg oligo(dT)24, 15 U
RNasin, 10 mmol/L DTT, and 2 µmol/L of each dNTP and reverse transcribed for 15 minutes at 37°C. Samples were heat-inactivated at 65°C for 10 minutes and then stored on ice. The cDNA was
polyadenylate-tailed at 37°C for 30 minutes in an 8 µL reaction
containing 200 µmol/L dATP and 10 U terminal transferase by using a
terminal transferase kit (Boehringer Mannheim, Mannheim, Germany). PCR
was performed in a volume of 50 µL using 5 µmol/L
oligo(dT)24X primer.32 Ten microliters of
SIP-RT-PCR product was electrophoresed through 2% agarose and
hybridized using 32P-labeled 35-mer probes (sequences not
shown) for the detection of the CD34, CD36, glycophorin A, epoR, c-kit,
GATA-1, GATA-2, NF-E2, SCL/Tal1, EKLF, PU.1, or 2-microglobulin PCR
product. In addition, 2.5 to 5 µL of SIP-RT-PCR product was
reamplified using sequence-specific primers as well as intron spanning
primers for detection of the 2-microglobulin gene.
Competitive RT-PCR
The cRNA deletion construct (dc) for the semiquantitation of CD34 mRNA
was synthesized by a non-plasmid-based technique. Briefly, the RT-PCR
product corresponding to wild-type (wt) CD34 was isolated from the
agarose gel and reamplified with upstream primer DelCD34 (Table 1) and
the downstream primer used for amplification of wt CD34. The resulting
construct was separated by agarose gel electrophoresis, isolated, and
then reamplified with wt upstream primer T7DelCD34 containing the T7
RNA polymerase promoter sequence at the 5'-end (Table 1) and the
wt downstream primer pTCD34 containing a 5'-poly(T)24
tail. The resulting PCR product was purified from the agarose gel and 1 µg was processed for in vitro mRNA transcription with the
T7-Megascript kit following the manufacturer's instructions (Ambion,
Austin, TX). The DNase I-treated cRNA CD34 deletion construct was
phenol/chloroform extracted, precipitated, and then quantitated by UV
spectrophotometry. RT-PCR of the cRNA dc results in a 156-bp amplification product, whereas a 229-bp product is detected after RT-PCR of wt CD34 mRNA. To evaluate expression levels of CD34 mRNA,
sorted CD34+CD38 cells were used for the
competitive RT-PCR. A dilution series containing known amounts of cRNA
dc, ie, 10, 100, 200, 400, 800, 1,600, or 3,200 transcripts, was added
to each cell lysate derived from 10 CD34+CD38 cells and coisolated with the
unknown amounts of wt CD34 mRNA by using the PolyATract System 1000 (data not shown). After reverse transcription of both dc cRNA and wt
mRNA in a one-tube reaction, 40 cycles of competitive PCR in the
presence of 50 µCi/mL  -32P-dATP was performed.
Quantitation of mRNA was performed essentially as described
previously.33 Briefly, RT-PCR products corresponding to wt
CD34 and dc CD34 were excised from the agarose gel and the radioactive
counts in each determined. To correct for lower
-32P-dATP incorporation into the smaller dc PCR product,
cpm from dc bands were multiplied by 229/156. To determine the point of equivalence,33 values were plotted as log of input dc
concentration (X-axis) versus the ratio of the corrected dc cpm to the
wt cpm (Y-axis). Based on two experiments, 10 CD34+CD38 cells contained approximately
100 CD34 mRNA transcripts.
A simplified competitive RT-PCR protocol was performed for the
semiquantitation of CD34 mRNA transcripts in individual cells. Thus,
titration was omitted and the 8/10 of the mRNA isolated from a single
cell was processed for RT-PCR in the presence of 10 CD34 cRNA dc
transcripts. Based on prior titrations (see above), the number of cRNA
dc transcripts corresponded to the approximate number of wt CD34
transcripts per CD34+CD38 cell, ie,
approximate point of equivalence.
Immunohistochemistry
Immunohistochemistry was performed for
CD34+Lin cells induced to unilineage
erythroid differentiation/proliferation (bulk culture) by immunogold
staining with silver enhancement. Freshly isolated CD34+Lin cells as well as cells after 6 and 12 days of culture were cytospun onto poly-L-lysine-coated slides,
fixed for 20 minutes at 4°C with cold PBS/10% formaldehyde, and
washed 2 times with PBS. Cytospins were treated for 3.5 minutes with
PBS/0.125% Triton-X 100, washed with PBS, and then incubated for 30 minutes at room temperature with PBS/5% BSA/5% goat serum/0.5% cold
water fish gelatin (blocking buffer). Slides were washed twice for 5 minutes with PBS and once with PBS/0.1% Aurion BSA-c/20 mmol/L
NaN3 (incubation buffer; Biotrend, Köln, Germany) for
5 minutes and then incubated with rat anti-GATA-1 MoAb, mouse
anti-GATA-2 MoAb, or rabbit anti-NF-E2 antibody (Santa Cruz
Biotechnology, Heidelberg, Germany) for 45 minutes at room temperature.
Slides were washed thoroughly twice for 10 minutes each with PBS and
for 10 minutes with incubation buffer and then incubated with the
appropriate secondary gold-conjugated antibody (ultrasmall
gold-conjugated goat-antirat IgG, goat-antimouse IgG, goat-antirabbit
IgG; Biotrend) for 2 hours at room temperature. Slides were washed
thoroughly, postfixed for 5 minutes with PBS/2% glutaraldehyde, and
then washed with PBS and 4 times for 5 minutes with dH2O.
Slides were then incubated for 20 to 25 minutes at room temperature
with silverenhancement reagent (Biotrend) and monitored by light
microscopy for color development. After three 5-minute washes with
dH2O, cells were counterstained with
May-Grünwald-Giemsa.
Clonogenic Progenitor Cell Assay
One hundred CD34+Lin cells/mL were
plated in 35-mm plastic tissue culture dishes containing a 1 mL mixture
of IMDM (Sigma), 1% methylcellulose (Sigma), 40% of pretested FCS
(CCPro), 1% BSA (Sigma), 2 × 104 mol/L
-thioglycerol (Sigma), and 400 µg/mL fully iron-saturated human
transferrin (Sigma). Cultures were supplemented with 500 U/mL IL-3
(Genzyme, Rüsselsheim, Germany), 100 U/mL GM-CSF (Genzyme), 100 ng/mL human stem cell factor (hSCF; kindly provided by
Amgen, Thousand Oaks, CA), 80 ng flt-3 ligand (CCPro), 50 ng G-CSF (R&D Systems), 50 ng M-CSF (R&D Systems), and 3 U/mL Epo (kindly provided by
Boehringer Mannheim). Cultures were incubated at 37°C in a 100%
humidified atmosphere of 5% CO2/5% O2 in air.
BFU-E, colony-forming units-granulocyte-macrophage (CFU-GM), and
CFU-Mix comprising granulocytes and/or monocytes and erythroid cells
were scored on day 14 using an inverted microscope.
| |
RESULTS |
Column Purified CD34+Lin Cells
The CD34+Lin cells isolated by
positive/negative selection comprised 99.1% ± 0.8% (mean ± SD; n = 8) pure CD34+ cells by flow cytometry analysis
(Fig 3, day 0). Less than 2% of purified
CD34+Lin cells expressed lineage markers
(not shown). The CD34+Lin population
comprised 87.5% ± 4% (n = 6) clonogenic progenitors (55% ± 9% BFU-E, 35% ± 7% CFU-GM, and 10% ± 2%
CFU-Mix), from which the majority (>70%) gave rise to macroscopic
visible colonies after 14 days of culture.
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| Fig 3.
Flow cytometry analysis of purified CB
CD34+Lin cells induced to unilineage
erythroid differentiation in bulk culture.
CD34+Lin cells were seeded at 2.5 × 104 cells/500 µL in serum-free medium containing 0.1 U/mL
IL-3, 0.05 U/mL GM-CSF, and 3 U/mL Epo.
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Expression of Progenitor Cell Surface Antigens and Transcription
Factors in Unilineage Erythroid Bulk Culture
Freshly purified CD34+Lin cells were
forced to unilineage erythroid differentiation in serum-free liquid
culture as previously described.20 Surface antigens linked
to erythroid differentiation were analyzed by FACS until day 14. Figure
3 shows that, at day 0, CD34+Lin cells
are GPA, predominantly c-kit+/low,
CD71low/, and largely CD36. The
proportion of GPA+ cells gradually increased in culture,
and at day 14 virtually all cells expressed GPA. In contrast,
expression of the CD34 antigen gradually decreased from day 0 to 14. A
distinct pattern of expression was noted for CD36, CD71, and c-kit. The
c-kit antigen initially expressed at low levels was first upregulated
at day 6 and then progressively decreased until day 14. The majority of
cells became strongly positive for CD71 at day 10 and were weakly to
strongly CD71+ at day 14. Similarly, the vast majority of
cells expressed CD36 at day 10 and then became weakly to strongly
positive for CD36 after 14 days of culture. After 12 to 14 days of
culture, almost all cells were
CD34c-kitCD36+CD71+GPA+
and were erythroblasts by morphology analysis
(Fig 4, day 12).
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| Fig 4.
Expression of transcription factors GATA-1, GATA-2, and
p45 NF-E2 in CB CD34+Lin cells induced to
unilineage erythroid differentiation. Transcription factor expression
was evaluated by immunohistochemistry (immunogold staining with
silverenhancement method) using specific MoAbs (original magnification × 400 and × 1,000). Arrows indicate nuclear staining of selected
cells. Control staining was performed with irrelevant, isotype-matched
rat or mouse MoAbs as well as with irrelevant rabit antibody.
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Immunohistochemistry was applied to investigate expression of
transcription factors in CD34+Lin cells
induced to unilineage erythroid differentiation in bulk culture.
Virtually all of the freshly purified
CD34+Lin cells expressed GATA-2 at day 0 (Fig 4, upper panel). Day 0 cells did not stain with anti-GATA-1 or
anti-p45 NF-E2 (Fig 4, upper panel). At days 6 and 12 of culture,
virtually all cells stained positive with anti-GATA-1, anti-GATA-2,
and anti-p45 NF-E2. All transcription factor antibodies intensely
immunolabeled cells and immunoreactivity was predominantly confined to
the nuclei (Fig 4).
Apoptosis in Unilineage Erythroid Culture
Apoptosis in erythroid bulk culture.
Apoptosis was evaluated in unilineage erythroid bulk culture of
PKH26-labeled CD34+Lin cells using
Annexin-V-FITC. We used the red fluorescent membrane dye PKH26
(half-life of elution >100 days),34 which partitions in
daughter cells after cell division. Thus, the PKH26/Annexin V staining
allowed determination of the proportion of apoptotic cells in
nondividing and proliferating populations.
Figure 5A demonstrates FSC/SSC properties
of freshly purified and cultured cells. Figure 5B shows that virtually
all day 0 CD34+Lin cells were brightly
stained with PKH26, and approximately 4.5% of those scored apoptotic
as determined by Annexin V staining. Backgating on Annexin
V+ cells showed that almost all apoptotic day 0 cells were
found in the low FSC gate (gate R1; Fig 5A). At day 2 of culture,
almost all cells were still PKH26+ (Fig 5B; day 2).
However, approximately one half of the PKH26+ cells were
positive for Annexin V. Again, backgating of Annexin V+
cells showed that virtually all apoptotic day-2 cells were
characterized by low FSC (gate R2; Fig 5A) but stained bright for
PKH26. After 6 days of culture, the vast majority of cells had
proliferated and showed low to barely detectable PKH26 fluorescence
(Fig 5B). Notably, less than 6% of day 6 PKH26/low
cells were apoptotic. Again, the vast majority of day-6 Annexin V+ cells were characterized by low FSC as determined by
backgating (R3; Fig 5A). Interestingly, after 6 days of culture (Fig
5B), a minority of cells remained PKH26+ and were not
stained with Annexin V (Fig 5B, day 6, upper left quadrant).
However, this population was not further characterized.
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| Fig 5.
Analysis of apoptosis and cell division in unilineage
erythroid bulk culture. Freshly purified PKH26 labeled
CD34+Lin cells were stained with
Annexin V-FITC (day 0) or transferred to unilineage erythroid culture
and stained with Annexin V at days 2 and 6. (A) FSC/SSC of day-0, -2, and -6 cells. (B) Ungated cells stained with PKH26 and Annexin V-FITC.
Day 0: note that only a minority of freshly isolated
CD34+Lin cells scored apoptotic when
stained with Annexin V. Day 2: virtually all cells are still
PKH26+ (nondividing cells), but more than 50% stain with
Annexin V. Day 6: the majority of cells are proliferating because they
stain barely or do not stain with PKH26. Some of these cells are
Annexin V+, ie, they are apoptotic. Gates R1, R2, and R3:
dead, apoptotic cells with low FSC.
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|
Unicellular cultures.
In the described unicellular, unilineage culture system (Fig 1),
apoptosis of individual cells may occur. In this case, the surviving
cell(s) must perfom one or more cell divisions to generate 4 siblings.
Because proliferation/cell division is usually associated with
differentiation, apoptotic events in the described culture system would
hinder the accurate analysis of developmentally regulated genes. We
therefore investigated the frequency of apoptotic cell death in
unicellular, unilineage erythroid cultures of
CD34+Lin cells and
CD34+CD38 cells as outlined in Fig 2.
Table 2 summarizes the proportion of
apoptotic cells from both populations that had evolved after 1, 2, or 6 cell divisions, ie, cycling cells. Fifty-four percent ± 16% (mean ± SD; n = 4 independent experiments) and 97.5% ± 1.5% (n = 4 experiments) of freshly purified
CD34+Lin cells and sorted
CD34+CD38 cells, respectively, underwent
apoptosis in unilineage erythroid culture. The proportion of apoptotic
cells among cycling cells from both CD34+ populations
ranged from 0.7% to 4% (Table 2). The proportion of apoptotic cells
among cycling cells from both CD34+ popluations was not
different between either tested group (P for all >.3;
Student's t-test).
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|
Table 2.
Frequency of Apoptotic Cell Death Among Single Fresh
CD34+LIN Cells, Single Fresh
CD34+CD38 Cells, and the Same Cells After
1, 2, or 6 Cell Divisions in Unicellular-Unilineage Erythroid
Culture
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Unicellular-Unilineage Erythroid Cultures and Single-Cell RT-PCR
Figure 1 outlines processing of single cells grown in the unilineage,
unicellular culture system. To investigate whether symmetric divisions
occur in unilineage culture, individual cells from cultures containing
4 siblings at culture rounds I, II, and III were transferred to a
separate culture and allowed to generate terminally differentiated progeny for morphology analysis. Single cells from round IV usually did
not generate a sufficient number of progeny for cytospin preparations. By morphology analysis, all cells (>150 cells were processed) from
round I to III forced to erythroid differentiation in hematopoietic progenitor cell liquid phase culture pertained to the erythroid lineage. Figure 6A shows a freshly seeded
single CD34+Lin cell that generated 4 siblings (round I; Fig 6B). Figure 6C shows a representative
May-Grünwald-Giemsa-stained cytospin preparation of
erythroblasts generated from an individual round I cell. SSP-RT-PCR allowed the analysis of up to 4 genes per single cell in separate reactions and was reproducible in approximately 90% of the experiments when mRNA was divided into 2 or 3 aliquots and processed for SSP-RT-PCR in separate reactions. Notably, using intron-spanning primers for
-actin (not shown) or 2-microglobulin for SSP-RT-PCR on mRNA
isolated from single cells (eg, diverse subsets of CD34+
cells as well as colony-derived macrophages, erythroblasts, or megakaryocytes), coamplification of genomic DNA sequences was not
detected in more than 400 SSP-RT-PCR reactions. Therefore, genomic DNA
is not copurified along with mRNA using the described mRNA isolation
methodology.
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| Fig 6.
Morphology of CD34+Lin cells
from unicellular, unilineage erythroid cultures. (A) A freshly seeded
single cell is shown that had generated 4 siblings after approximately
50 hours of culture (B) (original magnification for [A] and [B] was
100-fold). One of the siblings was transferred to a separate well
containing unilineage erythroid culture medium and allowed to further
differentiate/proliferate (C) (a representative
May-Grünwald-Giemsa-stained cytospin preparation is shown;
original magnification × 1,000).
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Individual CD34+Lin cells processed for
RT-PCR expressed GATA-2, PU.1, and c-kit but lacked expression of
GATA-1, p45 NF-E2, SCL/Tal-1, EKLF, EpoR, CD36, and GPA (round 0;
Fig 7). At round I of sibling analysis,
individual cells became additionally positive at the transcriptional
level for GATA-1, NF-E2, SCL/Tal1, EKLF, and CD36, whereas mRNA
transcripts for EpoR and GPA were still undetectable (Fig 7). Round II
cells were characterized by additional expression of EpoR and barely
detectable expression of GPA. The mRNA phenotype of round III cells was
similar to cells analyzed from round II (Fig 7). Late stages of
erythroid differentiation (round IV) were characterized by the absence
of or barely detectable levels of PU.1, GATA-2, and c-kit expression
(Fig 7).
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| Fig 7.
Nonquantitative single-cell RT-PCR of mRNA derived from
individual cells. Data from representative experiments are shown.
Single CD34+Lin cells were induced to
unilineage erythroid differentiation as described in Materials and
Methods. When the 4-cell stage was reached (at round I, II, III, and
IV), 2 individual daughter cells were removed from culture and
processed separately for RT-PCR. mRNA was isolated from each cell and
reverse transcribed into cDNA as described in Materials and Methods.
The cDNA was divided into 3 aliquots that were processed in separate
PCR reactions. Lane 0: Gene expression pattern of freshly isolated
single CD34+Lin cells. Lane I: single
daughter cell (first round). Lane II: single daughter cell (second
round). Lane III: single daughter cell (third round). Lane IV: single
daughter cell (fourth round). Note that only GATA-2, PU.1, and c-kit
are expressed in freshly isolated highly purified
CD34+Lin cells. Copurification of genomic
DNA with mRNA from single cells was not detected by RT-PCR using
intron-spanning primers for -2 microglobulin (not shown).
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Because the number of genes that can be detected in single cells by
SSP-RT-PCR is limited, we applied SIP-RT-PCR methodology to generate
abundant quantities of representative cDNA species from a single cell
for further analysis. Genomic DNA is not necessarily removed by whole
cell lysate RNA extraction protocols32,35 and may bias the
RT-PCR reactions. In our hands, single cells processed for SIP-RT-PCR
by using the whole cell lysate (w.c.l.) method32,35
resulted in coamplification of genomic DNA in approximately 30% of the
experiments (Fig 8B; outermost right lane).
Therefore, we used mRNA for SIP-RT-PCR. In addition to hybridizing
SIP-RT-PCR amplification products with radiolabeled target
gene-specific probes, we reamplified SIP-RT-PCR products by PCR using
target gene-specific primers and limited our experiments to single
cells isolated from round III (Fig1) of unicellular, unilineage
erythroid culture. Figure 8 shows a representative experiment using an
individual cell. SIP-RT-PCR (Fig 8) provided the same gene expression
pattern as detected by SSP-RT-PCR (Fig 7). Moreover, single-cell RT-PCR data are supported by analysis at protein level of CD34, CD36, GPA, and
c-kit as well as GATA-1, GATA-2, and NF-E2, using flow cytometry (Fig
3) and immunohistochemistry (Fig 4), respectively. Notably, in some
experiments, sequence-specific reamplification of SIP-RT-PCR product
resulted in only barely detectable PCR signals, although strong signals
were observed after direct hybridization of corresponding SIP-RT-PCR
products.
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| Fig 8.
Nonquantitative, sequence-independent RT-PCR (SIP-RT-PCR)
of single daughter cells induced to unilineage erythroid
differentiation. A representative experiment is shown. (Upper panel)
The mRNA isolated from a single round III cell was processed for
SIP-RT-PCR or SIP-RT-PCR was performed on whole cell lysate (w.c.l.;
upper panel right lane). PCR products were then hybridized with
32P-labeled probes (upper panel). Autoradiographs are
shown. (Lower panel) Autoradiographs of corresponding sequence-specific
PCR products from SIP-RT-PCR products shown in the upper panel.
SIP-RT-PCR product (2.5 µL) was reamplified by PCR using
sequence-specific pairs of primers (SSP-RT-PCR) recognizing the
respective target gene. Note that the whole cell lysate (w.c.l.)
SIP-RT-PCR results in coamplification of genomic DNA (outermost right
lane) by using intron-spanning primers for -microglobulin. No PCR
products corresponding to genomic 2-microglobulin DNA sequences were
detected when SIP-RT-PCR product from mRNA was reamplified by the same
2-microglobulin specific primers (lower panel, second lane from the
right).
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To investigate whether heterogeneity existed among single cells from
each culture round in cdc cultures, 3 individual cells from round I,
II, III, or IV were separately processed for SSP-RT-PCR and analyzed
for the expression of stage-specific genes CD34, CD36, GPA, EpoR, and
-globin. In addition, heterogeneity analysis was performed by
analysis of single cells from non-cdc cultures that contained 4, 8 to
12, or 16 to 24 cells. In cdc cultures, each culture round comprised
cells that were either all positive or all negative for a specific
transcript (Fig 9). Moreover, no differences in culture round specific gene expression patterns between
independent experiments were observed. Thus, single cells from specific
rounds of cdc cultures displayed homogeneity with respect to the
pattern of gene expression. In sharp contrast, cells derived from
non-cdc cultures were heterogeneous at mRNA level in cultures
containing 8 to 12 and 16 to 24 cells
(Table 3).
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| Fig 9.
Heterogeneity analysis by SSP-RT-PCR in cdc unicellular
erythroid cultures of CD34+Lin cells. At
each culture round, 3 cells were processed separately for RT-PCR as
described in Materials and Methods and 1 cell was transferred to the
next culture round, and so forth. Note the absence of heterogeneity of
individual cells from each culture round. An ethidium bromide-stained
gel is shown.
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Expression of Nonerythroid Genes in Unilineage Erythroid Bulk
Culture
Freshly purified CD34+Lin cells and the
same cells isolated from unilineage erythroid bulk culture at day 6 or
12 were analyzed by RT-PCR using primers recognizing nonerythroid
markers CD7, CD13, CD14, CD16, and CD19 as well as erythroid marker
GPA. Freshly purified CD34+Lin cells did
not express CD13, CD14, CD16, or GPA, whereas CD7 and CD19 were barely
detectable (Fig 10, day 0). After 6 or 12 days of unilineage erythroid culture, cells were RT-PCR-negative for CD7, CD13, CD14, CD16, and CD19 but did express GPA (Fig 10).
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| Fig 10.
RT-PCR products from
CD34+Lin cells, unilineage erythroid
differentiating cells and single sorted cells using primers recognizing
lineage-specific markers linked to differentiation. Autoradiographs are
shown. At days 0, 6, and 12, the mRNA from 100 cells was isolated and
processed as described in Materials and Methods. The cDNA derived from
100 cells was divided into 7 aliquots and each aliquot was processed
for RT-PCR using primers recognizing CD7 (pre-T/T cells), CD13
(myelomonocytic cells), CD14 (monocytic cells), CD16 (NK cells), CD19
(pre-B/B cells), GPA (erythroid cells), or 2-microglobulin (2m).
Day 0: freshly purified CD34+Lin cells
isolated by positive/negative selection (MiniMACS). Day 6:
CD34+Lin cells from unilineage erythroid
cultures as described in Materials and Methods. Day 12:
CD34+Lin cells induced to unilineage
erythroid differentiation. Control: RT-PCR sensitivity testing. Each
hybridization signal represents RT-PCR products from mRNA corresponding
to 0.5 cell equivalents of flow cytometry sorted cells that expressed
CD7, CD13, CD14, CD16, CD19, or GPA. The outermost right band
represents the 2-microglobulin RT-PCR product of a single sorted
CD34+CD38 cell. Note the absence of
nonerythroid CD7, CD13, CD14, CD16, and CD19 transcripts in erythroid
differentiating cells at days 6 and 12 of unilineage erythroid
culture.
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Competitive RT-PCR of Single Sibling Cells
A polyadenylated CD34 cRNA deletion construct was used for the
semiquantitation of CD34 mRNA expression in single
CD34+CD38 cells by competitive RT-PCR
that allowed control of each variable RT-PCR step.
Figure 11 shows a representative
competitive RT-PCR experiment from which all daughter cells (Fig 1)
transferred to unilineage erythroid culture medium generated pure
erythroblasts and illustrates gradual downregulation of CD34 mRNA in
single sibling cells from culture round I to IV.
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| Fig 11.
Competitive single-cell RT-PCR of sibling cells induced
to unilineage erythroid differentiation. A representative experiment is
shown. Single CD34+CD38 cells were induced
to unilineage erythroid differentiation as described in Materials and
Methods. When the 4-cell stage was reached (at round I, II, III, and
IV), a single daughter cell was isolated and the RNA corresponding to
0.8 cell equivalents processed for competitive RT-PCR to semiquantitate
the CD34 mRNA. Polyadenylated cRNA CD34 deletion construct
corresponding to 10 transcripts (based on prior titration) was added to
each single-cell lysate and coprocessed with the wt mRNA.
Ethidiumbromide-stained RT-PCR products were separated over a 2.5%
agarose gel (Metaphor; Biozym) and photographed. Each band was excised
from the gel and the radioactive counts in each were determined. Based
on Cerenkov counts of excised bands, round I, II, III, and IV daughter
cells contain 60%, 20%, 3%, and 0.01% of the CD34-mRNA transcripts
present in a freshly sorted CD34+CD38
cell, respectively. Lane M.W.: size marker. Lane 0: single sorted
CD34+CD38 cell before culture. Lane I:
single daughter cell (first round). Lane II: single daughter cell
(second round). Lane III: single daughter cell (third round). wt CD34
RT-PCR product is only barely detectable. Lane IV: single daughter cell
(fourth round). Note that the wt CD34 mRNA message is gradually
downregulated from round I to IV. Products corresponding to
2m-mRNA-transcripts but not to genomic sequences were detected when
the RNA corresponding to 0.2 cell equivalents from each daughter cell
was processed for RT-PCR using 2m primers (not shown).
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| |
DISCUSSION |
To investigate expression of multiple genes at discrete stages of
erythropoietic development, we applied a novel strategy consisting of
hematopoietic progenitor/stem cell unicellular-unilineage culture
followed by daughter cell analysis at cellular and molecular levels.
The cultures were seeded with greater than 98% purified CD34+Lin cells or sorted
CD34+CD38 CB cells. Both cell
populations are heterogeneous, in that they comprise putative
hematopoietic stem cells (eg, long-term culture-initiating cells
[LTC-ICs]) and more committed progenitors (eg, CFUs).
To offset the limitations of this heterogeneity, we have developed single progenitor cell unilineage cultures, combined with single-cell RT-PCR analysis.
In the standard protocol, (1) a single progenitor cell generates 4 siblings in unilineage erythroid culture; (2) two siblings are analyzed
by RT-PCR, whereas another sibling is grown to generate control
unilineage progenies for morphologic evaluation; (3) the fourth sibling
generates a second round of 4 sibling cells; and so forth. This
approach may be successfully applied if the following experimental
conditions are met: (1) asymmetric, (2) asynchronous cell divisions,
and (3) apoptotic cell death are largely absent, whereas (4) the
single-cell RT-PCR technology is well controlled; furthermore, (5)
competitive RT-PCR analysis may add to this approach.
These experimental conditions were met in the present study. Thus, (1)
asymmetric divisions did not occur; (2) in these cdc cultures, the
impact of asynchronous cell divisions was negligible, ie, in each
culture round the initiating cell performs 2 cell divisions generating
4 siblings; (3) the frequency of apoptotic cells was  4% in cycling
progenitors and their progeny; finally, (4/5) RT-PCR methodology
allowed unambiguous detection of multiple mRNA transcripts as well as
mRNA semiquantitation at the single-cell level.
(1) The absence of asymmetric divisions is indicated by morphological
analysis of mature progeny cells generated by individual siblings from
each culture round. At the transcriptional level, single cells derived
from culture round I to IV consistently displayed an erythroid-specific
pattern of gene expression (eg, GPA, EpoR, and CD36), whereas
transcripts of nonerythroid genes, ie, CD7, CD13, CD14, CD16, and CD19,
were not detected by RT-PCR in erythroid differentiating cells.
(2) In these cdc cultures, the individual cells from each culture round
(4 cells/round) displayed an identical mRNA expression profile: these
data strongly suggest that in each round all cells pertained to an
identical stage of erythroid development. In non-cdc unilineage
cultures, a single progenitor cell is allowed to terminally differentiate/proliferate without repetetive replating of individual cells for initiation of subsequent culture rounds: in these culture conditions, we observed heterogeneity of the mRNA profile of the progeny after 3 cell divisions, ie, greater than 8 to 12 cells/well. This observation suggests that cells generated after 3 or more mitoses
are increasingly heterogeneous in terms of developmental stage, due to
asynchronuous divisions, ie, wells containing greater than 8 to 12 cells comprise cells generated by a different number of mitoses, due to
different cycling times.
Conversely, asynchronuous cell divisions do not affect the
unicellular-unilineage cdc culture approach. The number of mitoses is
under strict control, in that after every second division a single cell
is replated to initiate the next culture round.
(3) Under the described unilineage erythroid culture conditions, cells
may undergo apoptosis due to growth factor starvation, as reported for
hematopoietic progenitor cell lines (FDCP and 32D)36 and
human erythroleukemia cells37 when deprived of IL-3 and
Epo, respectively. Evaluation of apoptosis in unilineage-unicellular culture systems represented a crucial issue. Indeed, genes involved in
apoptosis may interact with other ones (eg, transcription factors) and
thus possibly interfere with the developmentally regulated program of
gene expression. In addition, one or more sibling(s) may undergo
apoptosis, whereas nonapoptotic cell(s) further proliferate. In this
case, a nonapoptotic cell(s) must perform more cell division(s) to feed
into the next 4-cell stage and thereby reflect more advanced stages of
differentiation. Approximately 54% of individually seeded CD34+Lin cells did not clone at the
initiation of culture and were scored apoptotic. However, upon cell
cycling, ie, after the first and subsequent cell divisions, apoptosis
affected only 0.7% to 4% of progeny cells. These data demonstrate
that apoptosis occurs frequently at the start of unilineage-unicellular
cultures but represents a rare event among cycling cells. Notably, the
initial 54% apoptosis frequency in unicellular cultures is in line
with the results from unilineage erythroid bulk culture of
CD34+Lin cells (the ratio of
apoptotic/viable cells both comprising predominantly nonproliferating
PKH26+ cells peaked to ~50% at day 2 of culture).
Similar to the observed low apoptosis frequencies among cycling cells
in unicellular cultures, proliferating, ie,
PKH26/low, cells at day 6 of bulk culture
comprised only a low proportion, ie, 6%, of Annexin V+
cells. In the bulk culture system, the initial apoptosis frequency could be considerably reduced by the addition of saturating doses of
multilineage (IL-3/GM-CSF) or early acting (c-kit and flt-3 ligand)
hematopoietic growth factors; however, this resulted in loss of
unilineage erythroid growth (data not shown).
A high proportion, ie, 97.5%, of
CD34+CD38 cells initially underwent
apoptosis in unicellular-unilineage culture. This finding suggests that
this subset predominantly comprises cells that are more primitive than
those CD34+Lin. Indeed, primitive
CD34+CD38 cells express c-kit, FLT-3,
and only barely detectable transcripts for IL3 receptor -chain but
no Epo receptor, whereas addition of saturating doses of IL-3 and early
acting growth factors (c-kit and flt-3 ligands) considerably reduced
apoptosis frequency in unicellular culture to approximately 35%; this
resulted in loss of unilineage growth (data not shown).
Altogether, our observations suggest that the unilineage erythroid
culture conditions select for progenitor cells potentially responsive
to the Epo stimulus. In line with the model of cascade transactivation
of hematopoietic growth factor receptors,13 the purified
quiescent progenitor cells express IL-3R and GM-CSFR,12 and
in unilineage erythroid culture are triggered into cycling by
IL-3/GM-CSF. These growth factors also induce upmodulation of
unilineage hematopoietic growth factor receptors, particularly EpoR on
progenitor cells with erythroid differentiation
potential.13 In the presence of saturating levels of Epo
(and the absence of other unilineage growth factors),
these progenitor cells are induced to increasing expression of EpoR and
hence differentiation and then maturation along the erythroid
lineage.12 On the other hand, progenitor cells with no
erythroid differentiation potential do not express EpoR and undergo apoptosis.
(4) The symmetry and synchrony of cell divisions, coupled with low
frequency of apoptosis for dividing cells, enabled us to investigate
the pattern of gene expression at discrete stages of erythroid
differentiation by single-cell RT-PCR. RT-PCR analysis of RNA from
single cells32,35,38 is easily obscured by the copurified
genomic DNA. To address this issue, we isolated mRNA from individual
cells and then performed RT-PCR for detection of multiple mRNA species.
Furthermore, we developed single-cell competitive RT-PCR
methodology33 and applied it to semiquantitate CD34 mRNA
transcripts in single progenitor cells induced to erythroid maturation.
The number of CD34 transcripts gradually declined from freshly sorted
CD34+CD38 erythroid-induced cells from
round I through IV, thus indicating that single-cell RT-PCR can be
rendered semiquantitative in the unicellular-unilineage culture system.
Our results demonstrate that discrete stages of erythoid development
are characterized by a specific pattern of gene expression. In
unicellular erythroid culture, expression of the early transcription
factors GATA-2 and PU.1 is monitored in the initial hematopoietic
progenitor cells and gradually tapers off in erythropoietic
differentiation; inversely, expression of the late transcription
factors GATA-1, p45 NF-E2, and EKLF is absent in freshly purified
progenitors, whereas it is gradually induced and sustainedly expressed
in erythroid development. A similar expression pattern is observed for
the receptors of early acting hematopoietic growth factors (eg, c-kit) as compared with lineage-specific late growth factor receptor EpoR, ie,
they are expressed as early and late hematopoietic growth factor
receptors, respectively. Although GATA-2 and PU.1 were detected in
individual freshly purified CB CD34+Lin
cells or CB CD34+CD38 cells, the
expression/function of these transcription factors may not be strictly
limited to early hematopoietic development.16,22,39-41
In line with the present and previous bulk culture studies using
progenitor cells undergoing unilineage erythroid or granulopoietic differentiation (previous reports20,22,25,42 and this
report), the single-cell RT-PCR data on GATA-1 and p45 NF-E2
demonstrate that both transcription factors are not expressed in
freshly purified CD34+Lin cells but are
detectable during erythroid differentiation. Accordingly, GATA-1 mRNA
is expressed in more differentiated CD34+CD38++
but not in early PB CD34++CD38
cells.43 No data about expression of EKLF at discrete
stages of adult erythroid differentiation have been reported. Our
results show that expression of EKLF during erythroid development
parallels expression of GATA-1 and NF-E2. Thus, EKLF not expressed in
early CD34+Lin cells was found
upregulated before its late expressed target gene
-globin.44 These expression results are in line with
functional studies indicating that (1) GATA-1
embryonic stem cells fail to contribute to mature erythroid
cells,45 whereas other hematopoietic lineages are not
affected46; (2) targeted disruption of NF-E2 or EKLF does
not affect the early steps of hematopoiesis47-49; and (3)
treatment of adult hematopoietic progenitor cells with antisense
oligomers targeting GATA-1 or NF-E2 mRNA selectively inhibits erythroid
but not myeloid colony formation.22
SCL/Tal1 is barely or not expressed in adult24 and CB
(present finding) early progenitors, but is induced upon erythroid unilineage differentiation through terminal maturation, as observed for
late erythroid transcription factors. Similar observations were
previously reported.43 The lack of expression of SCL/Tal1 in early progenitor cells is possibly due to the biphasic
expression/function profile of this transcription factor,50
which may peak in early ontogeny at hematopoietic stem cell level and
in perinatal/postnatal life during hematopoietic progenitor cell
erythroid differentiation.
A few studies on transcription factor expression in hematopoiesis are
apparently in contrast with our results. Expression of GATA-1 and
SCL/Tal1 was demonstrated in murine multipotential FDCPmix
cells.51 Furthermore, GATA-1, NF-E2, and SCL/Tal1
transcripts were monitored in human BM
CD34+CD38 or quiescent G0
phase cells by a modified SIP-RT-PCR method, which also allowed
semiquantitation of gene expression at the single-cell
level.38 These findings may be reconciled with the present
results22,24,43 in view of (1) differences between primary
cells and cell lines as well as species specificity; (2) differences of
analyzed progenitor/stem cell populations and purification methods; and
(3) possible lack of SIP-RT-PCR specificity (eg, coamplification of
genomic DNA and/or hybridization of specific probes to coamplified
sequences related to the target gene), as indicated by our own
observations (see Results).
Our study shows that RT-PCR analysis can be applied to assay multiple
RNA species in single hematopoietic progenitor cells and daughter cells
at discrete, sequential stages of unilineage erythroid
differentiation/maturation. The described unilineage-unicellular culture system together with single-cell RT-PCR, preferably of competitive type, has the potential to functionally investigate relevant genes (eg, cell cycle genes, transcription factors, and growth
factor receptor genes) through gene inhibition or overexpression strategies (eg, oligomer antisense or retroviral sense/antisense treatment) of purified progenitors to shed light on the cross-talk between the downmodulated gene(s) and other genes.
In conclusion, the described novel approach may eliminate ambiguities
deriving from molecular analysis of heterogeneous populations of
hematopoietic progenitors/ precursors growing in culture, particularly in the initial stages of development. This approach will enable studies
on hematopoietic stem/progenitor cells aimed to analyze both markers
linked to differentiation and expression of genes regulating their
proliferation and differentiation in normal or malignant hematopoiesis;
particularly, the single-cell RT-PCR approach will be used for gene
expression studies in highly purified progenitors undergoing unilineage
erythroid,25 granulocytic,23 monocytic,27 or megakaryocytic26
differentiation in liquid suspension culture.
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FOOTNOTES |
Submitted July 13, 1998; accepted January 12, 1999.
Supported by Grant No. E/B 41G/T0347/T5920 from the BMVg, Grant No.
FI4P-CT95-0029 from the European Communities, the Arthur N. Saydman
Trust Fund, and a Research Fellowship from the Lucille P. Markey
Charitable Trust.
The publication costs of this
article were defrayed in part by
page charge payment. This article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. section
1734 solely to indicate this fact.
Address reprint requests to Benedikt L. Ziegler, MD, University of
Tübingen, Department of Medicine, Division of Hematology & Oncology, Otfried-Müller-Str. 10, D-72076 Tübingen,
Germany; e-mail: benedikt.ziegler{at}uni-tuebingen.de.
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