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Blood, Vol. 93 No. 10 (May 15), 1999:
pp. 3355-3368
By
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
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.
Cell Preparation
Immunofluorescence Staining and Flow Cytometry
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.
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.
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, or2-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.
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.
Column Purified 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.
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).
Apoptosis in Unilineage Erythroid Culture Apoptosis in erythroid bulk culture.
Apoptosis was evaluated in unilineage erythroid bulk culture of
PKH26-labeled CD34+Lin
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
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.
Expression of Nonerythroid Genes in Unilineage Erythroid Bulk
Culture
Competitive RT-PCR of Single Sibling Cells
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ABSTRACT
INTRODUCTION
