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=20
Cell Cycle-Related Changes in Repopulating = Capacity of=20 Human Mobilized Peripheral Blood CD34+ Cells in Non-Obese=20 Diabetic/Severe Combined Immune-Deficient Mice
Abstract
Most primitive hematopoietic progenitor cells reside in vivo = within=20 the G0/G1 phase of the cell cycle. By simultaneous = DNA/RNA=20 staining it is possible to distinguish G0 and G1 = states=20 and to isolate cells in defined phases of the cell cycle. We report here = the use=20 of cell cycle fractionation to separate human mobilized peripheral blood = (MPB)=20 CD34+ cells capable of repopulating the bone marrow (BM) of = non-obese=20 diabetic/severe combined immune-deficient (NOD/SCID) mice. In freshly = isolated=20 MPB, repopulating cells were predominant within the G0 phase, = because=20 transplantation of CD34+cells residing in G0=20 (G0CD34+) resulted on average in a 16.6- =C2=B1 = 3.2-fold higher=20 BM chimerism than infusion of equal numbers of CD34+ cells = isolated=20 in G1. We then investigated the effect of ex vivo cell cycle=20 progression, in the absence of cell division, on engraftment capacity. = Freshly=20 isolated G0CD34+ cells were activated by = interleukin-3=20 (IL-3), stem cell factor (SCF), and flt3-ligand (FL) for a 36-hour = incubation=20 period during which a fraction of cells progressed from G0 = into=20 G1 but did not complete a cell cycle. The repopulating = capacity of=20 stimulated cells was markedly diminished compared with that of = unmanipulated=20 G0CD34+ cells. Cells that remained in = G0 during=20 the 36-hour incubation period and those that traversed into = G1 were=20 sorted and assayed separately in NOD/SCID recipients. The repopulating = ability=20 of cells remaining in G0 was insignificantly reduced compared = with=20 that of unstimulated G0CD34+ cells. On the = contrary,=20 CD34+ cells traversing from G0 into G1 = were=20 largely depleted of repopulating capacity. Similar results were obtained = when=20 G0CD34+ cells were activated by the combination of = thrombopoietin-SCF-FL. These studies provide direct evidence of the = quiescent=20 nature of cells capable of repopulating the BM of NOD/SCID mice. = Furthermore,=20 these data also demonstrate that G0-G1 progression = in=20 vitro is associated with a decrease in engraftment capacity.
=C2=A9 1998 by The American Society of Hematology.
IN VITRO MANIPULATION of hematopoietic stem cells (HSC) has = been the=20 focus of numerous studies. Two main objectives have been under = evaluation: HSC=20 expansion and somatic gene therapy. The use of expanded grafts would in = theory=20 speed up the hematological recovery after HSC transplantation and/or = allow for=20 the use of small HSC collections, such as cord blood samples.1=20 Information collected from ex vivo expansion studies is also critical to = design=20 gene transfer protocols, because retroviral transduction requires in = vitro=20 cycling of the target cells.2=20 3=20
In defining strategies for HSC expansion or gene therapy, a =
decisive=20
factor to consider is the responsiveness of the most primitive cells to =
cytokine=20
stimulation in vitro. This is largely dependent on the cell cycle status =
of the=20
target cells, as cycling cells will be able to respond quickly to =
mitogenic=20
factors, whereas more quiescent cells will not.4-7=20
In addition, cell cycle activation may have a negative effect on the =
engraftment=20
capability. Studies in murine models have documented an engraftment =
defect of=20
stimulated HSC: in vitro HSC activation by various combinations of =
hematopoietic=20
growth factors resulted in a decrease of in vivo repopulating ability, =
compared=20
with unmanipulated marrow.8-10=20
Consistent with these results were data acquired in our laboratory by =
PKH=20
membrane cell tracking11=20
that demonstrated the superiority of quiescent cells over proliferating =
cells in=20
rescuing lethally irradiated recipients. In another report, evidence was =
presented that not only in vitro stimulation, but also in vivo HSC =
cycling, as=20
observed after 5-fluorouracil (5-FU) treatment, resulted in transient=20
alterations in the functional state of HSC, leading to an engraftment =
defect.
In humans, experimental studies examining the impact of cell = cycle=20 status on engraftment capacity have been unattainable due to the absence = of an=20 in vivo model of human HSC. Xenotransplant models have been recently = developed=20 which can address this question, most notably the in vivo severe = combined=20 immune-deficient (SCID) mouse repopulating cell (SRC) assay.13=20 In this model, human HSC intravenously injected into immunodeficient = recipients=20 home to the bone marrow and are able to proliferate to a large extent = and=20 differentiate into various lineages.14=20 15=20 High levels of chimerism are especially achieved with the non-obese=20 diabetic/SCID (NOD/SCID) strain, which was used in the present study. =
To investigate the relationship of cell cycle status with =
the=20
engraftment capability of human hematopoietic cells, we took advantage =
of a new=20
method of cell cycle analysis and fractionation recently developed in =
our=20
laboratory.16-18=20
Conventional cell cycle analysis using DNA dyes classifies cells in the=20
G0/G1 phase as noncycling cells and cells in the=20
S/G2+M phase as cycling cells. This approach is of limited =
utility=20
when applied to the characterization of the cell cycle status of human=20
hematopoietic cells, because more than 85% of bone marrow (BM) =
CD34+=20
cells and almost all of mobilized peripheral blood (MPB) =
CD34+ cells=20
are in the G0/G1 phase. Simultaneous DNA/RNA =
staining with=20
Hoechst 33342 (Hst) and Pyronin Y (PY) allows for further fractionation =
of the=20
G0/G1 phase. Using this procedure, we have been =
able to=20
isolate viable CD34+ cells in the G0 or =
G1=20
phase of the cell cycle and examine their properties in vitro.
MATERIALS AND METHODS
Mice.
NOD/LtSz-scid/scid (NOD/SCID)19=20 animals were kindly provided by Dr Leonard Shultz (Jackson Laboratory, = Bar=20 Harbor, ME). Mice were housed in microisolators under pathogen-free = conditions=20 and received autoclaved food and acidified water ad libitum. Animal = experiments=20 were performed in accordance with institutional guidelines approved by = the=20 Animal Care Committee of the Indiana University School of Medicine. =
Human cells.
BM and MPB samples were obtained from healthy adult = volunteers=20 according to the guidelines established by the Human Investigation = Committee of=20 the Indiana University School of Medicine. Mobilization was achieved by = daily=20 granulocyte colony-stimulating factor (G-CSF) administration at 5 = =CE=BCg/kg=20 (maximum, 480 =CE=BCg/d) for 4 consecutive days. MPB cells were = collected by=20 apheresis on day 5. MPB CD34+ cells were isolated by = immunomagnetic=20 selection using a large scale Isolex 300i system (Baxter Healthcare, = Irvine, CA)=20 as per the manufacturer=E2=80=99s instructions. CD34+ cell = purity in the=20 selected product always exceeded 95%. In one experiment, BM cells = instead of MPB=20 cells were used. BM cells were pooled from 5 normal donors and selected = with the=20 same procedure. CD34+ cell purity of this BM collection was = 86%.=20
Cell cycle fractionation with Hst and Pyronin Y.
Fresh or cultured CD34+ cells were resuspended at =
5 =C3=97=20
106 cells/mL in a 1 =CE=BCg/mL solution of Hst (Molecular =
Probes, Eugene,=20
OR) in Hst buffer. Hst buffer consisted of Hanks Balanced Salt Solution =
(HBSS;=20
Biowhittaker, Walkersville, MD), 20 mmol/L HEPES (Biowhittaker), 1 g/L =
glucose,=20
and 10% fetal calf serum (FCS; Hyclone, Logan, UT). After incubation at =
37=C2=B0C for=20
45 minutes, PY (Sigma, St Louis, MO), prepared in Hst Buffer, was added =
at a=20
final concentration of 1 =CE=BCg/mL and cells were further incubated for =
another 45=20
minutes at 37=C2=B0C. Cells were washed once, resuspended in Hst buffer, =
and sorted=20
on a FACStar Plus equipped with an argon laser providing the 488 nm =
excitation=20
for PY and a krypton laser providing the 350 nm excitation for Hst. PY =
signal=20
was selected with a 575 =C2=B1 13 nm bandpass filter and Hst was =
detected with a 424=20
=C2=B1 22 nm bandpass filter. Sorting windows were constructed as =
depicted in Fig
Single- and dual-parameter cell cycle = analysis of=20 BM and MPB CD34+ cells. DNA histogram of BM (A) and MPB (B)=20 CD34+ cells, respectively, showing the relative absence of=20 replicating progenitor cells in MPB. Simultaneous DNA/RNA staining with = Hst and=20 PY, identifying G0 and G1 regions in similar = samples of BM=20 (C) and MPB (D) CD34+ cells.
High resolution cell cycle analysis.
Cells were analyzed for Ki-67 expression and DNA content as = recently=20 described by Jordan et al,20=20 with minor modification. Cells were washed and resuspended in 1 mL=20 phosphate-buffered saline (PBS) + 0.4% formaldehyde. After 30 minutes at = 4=C2=B0C, 1=20 mL of PBS + 0.2% Triton X-100 was added and cells were left overnight at = 4=C2=B0C.=20 Cells were then washed twice in PBS + 1% bovine serum albumin (BSA) and = stained=20 with fluorescein isothiocyanate (FITC)-conjugated anti-Ki-67 (clone = MIB-1;=20 Immunotech, Westbrook, ME) for 60 minutes at 4=C2=B0C. Isotype controls = were stained=20 in parallel. Finally, cells were washed and resuspended in PBS + 1% BSA=20 containing 5 =CE=BCg/mL 7-aminoactinomycin-D (7-AAD; Sigma). After 3 = hours of=20 incubation on ice, samples were run on a FACScan flow cytometer (BDIS, = San Jose,=20 CA) using FL-1 and FL-3 channels for Ki-67 and 7-AAD, respectively. =
Human progenitor cell assay.
Human CD34+ cells were assayed in 1.3% = methylcellulose,=20 30% FCS, 100 ng/mL stem cell factor (SCF), 10 ng/mL interleukin-3 = (IL-3), 10=20 ng/mL IL-6, 5 ng/mL granulocyte-macrophage colony-stimulating factor = (GM-CSF),=20 and 2 U/mL erythropoietin (EPO) suspended in Iscove=E2=80=99s modified = Dulbecco=E2=80=99s medium=20 (IMDM). When unseparated BM cells from NOD/SCID recipient mice were = used, IL-6=20 was omitted to avoid the growth of murine progenitors.14=20 21The=20 selectivity of the assay for human progenitors was confirmed by = polymerase chain=20 reaction (PCR) detection of human =CE=B2-globin gene in individual = colonies (see=20 below). Hematopoietic colonies were scored after 2 weeks according to = standard=20 criteria.
LTC-IC assay by limiting dilution analysis.
M2-10B4 cells,22=20 obtained from the American Type Culture Collection (Rockville, MD), were = used to=20 establish stromal layers. M2-10B4 cells harvested from large-scale = cultures in=20 RPMI 1640 with 10% FCS were irradiated at 8,000 cGy and plated in = 96-well plates=20 at 15,000 cells/well in 100 =CE=BCL long-term culture medium (LTCM). = LTCM consisted=20 of Myelocult (Stem Cell Technologies, Vancouver, British Columbia, = Canada)=20 containing 10=E2=88=926 mmol/L hydrocortisone (Sigma). Within = 1 week, human=20 test cells were plated in limiting dilution at 24 wells/cell dose in = another 100=20 =CE=BCL LTCM and maintained at 37=C2=B0C in 100% humidified atmosphere = containing 5%=20 CO2, with weekly half-medium change. After 5 to 6 weeks, 120 = =CE=BCL=20 medium were removed from each well followed by the addition of 150 = =CE=BCL of a=20 mixture consisting of 3 parts FCS and 4 parts 3.3% methylcellulose and=20 containing at final concentration 5 =C3=97 10=E2=88=922 = mmol/L 2-mercaptoethanol,=20 100 ng/mL SCF, 25 ng/mL IL-3, 25 ng/mL IL-6, 25 ng/mL GM-CSF, and 2 U/mL = EPO.=20 After an additional 2 weeks, wells were scored for the presence or = absence of=20 hematopoietic colonies and the frequency of LTC-IC was calculated using = the=20 maximum likelihood estimator.23=20
Short-term culture.
CD34+ cells initially isolated in G0 = were=20 plated in a serum-free medium consisting of IMDM supplemented with 25 = mmol/L=20 HEPES (Biowhittaker), 10 =CE=BCg/mL BSA, 10 =CE=BCg/mL bovine insulin, = 200 =CE=BCg/mL=20 transferrin (all from Stem Cell Technologies), 2 mmol/L = alanyl-glutamine, 1%=20 (vol/vol) lipids cholesterol-rich, 1 mmol/L sodium pyruvate (all from = Sigma),=20 100 U/mL penicillin, 100 =CE=BCg/mL streptomycin, and 5 =C3=97 = 10=E2=88=922 mmol/L=20 2-mercaptoethanol. Cells were stimulated during 36 hours by a = combination of 50=20 ng/mL IL-3, 100 ng/mL SCF, and 100 ng/mL flt-3 ligand (FL) or a = combination of=20 50 ng/mL thrombopoietin (TPO), 100 ng/mL SCF, and 100 ng/mL FL. = Viability of=20 cultured cells was always greater than 98% as judged by trypan blue = exclusion.=20
Transplantation of human cells into NOD/SCID mice.
Twelve- to 14-week-old NOD/SCID mice were sublethally = irradiated with=20 300 cGy from a 137Cs source (GammaCell 40; Nordion = International,=20 Kanata, Ontario, Canada). Mice received 10 =C3=97 106 = nonadherent=20 CD34=E2=88=92cells irradiated with 2,000 cGy, as accessory = cells, by=20 intravenous tail injection. Two hours later, mice were transplanted with = fresh=20 or cultured CD34+ cells. Animals were not treated with = cytokines=20 during the experiments. After 6 to 7 weeks,14=20 24mice=20 were killed by cervical dislocation and bone marrow cells were harvested = from=20 femurs and tibias by flushing the bones with HBSS-5% FCS. Nucleated = cells were=20 isolated by centrifugation over Ficoll-Paque (Pharmacia Fine Chemicals,=20 Piscataway, NJ), washed, and resuspended in IMDM with 10% FCS for = further=20 analyses.
Flow cytometric analysis of engraftment.
Between 1.0 =C3=97 105 and 2.5 =C3=97 = 105NOD/SCID BM=20 cells were pelletted, resuspended in 50 =CE=BCL mouse serum (Sigma), and = incubated=20 with various mouse antihuman monoclonals for 20 minutes at 4=C2=B0C. = FITC-conjugated=20 antibodies included anti-CD45, CD33, CD19, CD15 (BDIS), and CD38 = (Immunotech).=20 Phycoerythrin (PE)-conjugated anti-CD34 was obtained from BDIS and = anti-CD45-PE=20 was obtained from Pharmingen (San Diego, CA). Samples were analyzed on a = FACScan=20 (BDIS). Positive cells were identified by comparison with isotypic = controls and=20 with cells harvested from control (not transplanted) NOD/SCID mice = stained with=20 the same antibodies.
In the case of highly engrafted mice (>10% = CD45+=20 cells), CD45+CD34+ cells were sorted on a FACStar = Plus=20 (BDIS) and assayed for their progenitor and LTC-IC frequencies. =
PCR detection of human =CE=B2-globin gene.
Briefly, individual colonies were plucked and transferred = into=20 Eppendorf tubes, lyzed with 500 =CE=BCL water, and pelletted. DNA was = extracted with=20 InstaGene matrix (Bio-Rad, Hercules, CA) as per the = manufacturer=E2=80=99s instructions.=20 Positive controls consisted of individual colonies grown from purified = human=20 CD34+ cells and negative controls were samples of = 103 and=20 10 =C3=97 103 murine BM cells harvested from a control = animal. PCR was=20 performed using a 20-bp 5=E2=80=B2 primer GAA TCC AGA TGC TCA AGG CC and = a 20-bp 3=E2=80=B2=20 primer CAA TCC AGC TAC CAT TCT GCamplifying a 345-bp fragment of human = =CE=B2-globin=20 gene. After an initial denaturation step of 5 minutes at 94=C2=B0C, a = hot start at=20 85=C2=B0C was used, followed by 40 cycles consisting of 4 minutes at = 94=C2=B0C, 2 minutes=20 at 56=C2=B0C, and 1.5 minutes at 72=C2=B0C. The final cycle had a = 10-minute extension=20 step. PCR products were analyzed on a 1.5% agarose gel after staining = with=20 ethidium bromide.
Growth factors.
Human recombinant IL-3, IL-6, SCF, TPO, and GM-CSF were = kind gifts=20 from Amgen (Thousand Oaks, CA). Human recombinant EPO was obtained from = Amgen.=20 Human recombinant FL was a kind gift from Immunex (Seattle, WA). =
Statistical analysis.
Comparisons were made using the Student=E2=80=99s = t-test.=20 AllP values are two-sided.
RESULTS
Cell cycle fractionation of MPB CD34+ cells by staining = with Hst=20 and PY.
We17=20 25=20 and others26=20 27=20 have already reported the homogeneous G0/G1 cell = cycle=20 status of MPB CD34+ cells compared with their BM = counterparts. When=20 analyzed with a standard DNA histogram, up to 15% of BM CD34+ = cells=20 are in the S/G2+M phase, whereas less than 1% of MPB = CD34+=20 cells are in the same actively cycling compartment (Fig 1A=20 and B). Thus, the vast majority of MPB CD34+ cells cannot be = ascribed=20 to a precisely defined position in the cell cycle, because cells in=20 G0 and in G1 have the same DNA content. = Simultaneous=20 DNA/RNA staining with Hst and PY, respectively, was used in the present = study to=20 discriminate the G0 phase from the G1 phase of the = cell=20 cycle (Fig 1C=20 and D). Cells in G0 were identified by minimal RNA content, = whereas=20 cells traversing into G1were defined as cells with maximal = RNA=20 staining. The validity of this approach has been demonstrated in = previous=20 reports by kinetic and phenotypic data from this laboratory.16-18=20 Most notably, expression of Ki-67, a nuclear antigen specifically = expressed by=20 cycling cells,20=20 was restricted to PYhighcells, whereas PYlow cells = were=20 Ki-67 negative.
Differential cell cycle status of colony-forming cells (CFC) and = LTC-IC in=20 MPB CD34+cells.
The frequency of late and primitive progenitor cells was = determined=20 in CD34+ cells isolated in G0 or in = G1by=20 standard CFC and LTC-IC assays, respectively. The overall clonogenic = activity=20 was very similar, approximately 30%, in G0 and=20 G1CD34+ cells (P > .05; Fig 2A).=20 However, as already reported for BM CD34+ cells,17=20 consistent differences were observed in the distribution of the = different types=20 of progenitors: the G0 compartment was relatively enriched in = multipotential progenitors (colony-forming unit-mixed [CFU-Mix]; 15% in=20 G0 v 4% in G1; P < .05), = whereas=20 there were more committed myeloid progenitors in = G1(colony-forming=20 unit=E2=80=93granulocyte-macrophage [CFU-GM]; 16% in G0 = v 28% in=20 G1; P < .05).
In vitro functional assays of MPB=20 CD34+cells isolated in G0 or in G1 = phase of the=20 cell cycle. (A) Progenitor cell assay. Numbers of hematopoietic colonies = are=20 given per 100 cells plated. Data shown are the mean =C2=B1 SEM of four = experiments=20 performed in duplicate. Total indicates total number of hematopoietic = colonies;=20 BFU-E, burst-forming unit-erythroid; CFU-GM, colony-forming=20 unit-granulocyte/macrophage; CFU-MIX, CFU-Mixed. (B) Frequencies of = LTC-IC among=20 G0 and G1CD34+ cells. Frequencies per = 100 cells=20 are reported as the mean =C2=B1 SEM, n =3D 4. *P < .05 = versus=20 G1CD34+ cells, paired t-tests.
As for more primitive cells (Fig 2B),=20 the G0 compartment was significantly enriched in LTC-ICs = compared=20 with the G1compartment (3.1% for = G0CD34+ cells=20 v0.8% for G1CD34+ cells; P < = .05).=20
NOD/SCID repopulating ability of MPB CD34+cells isolated = in=20 G0 or in G1.
Comparison of the repopulating ability of=20 G0CD34+ and G1CD34+cell = populations=20 was assessed by transplanting equal numbers of either cell fraction = sorted from=20 single-donor MPB samples. After 6 to 7 weeks,14=20 24=20 the extent of human cell engraftment was evaluated by flow cytometric=20 determination of human CD45+and/or CD34+ cells in=20 suspensions of BM cells harvested from recipient mice. High numbers of = human=20 CD45+cells (up to 75% after injection of 2 =C3=97 = 106 cells)=20 were detected in the BM of animals transplanted with=20 G0CD34+ cells. Of interest is the fact that a = large=20 fraction of chimeric human CD45+ cells was also = CD34+ (Fig=20 3B).=20 In sharp contrast, low numbers of CD45+ cells were = demonstrated after=20 transplantation of G1CD34+ cells and chimeric=20 CD34+ cells were mostly undetectable (Fig 3D).=20 In four separate experiments in which MPB CD34+ cells sorted = in=20 either G0 or G1 were transplanted at cell doses = ranging=20 from 2 =C3=97 106 to 0.7 =C3=97 106, the = repopulating activity of=20 G0CD34+ cells was consistently severalfold higher = than=20 that of G1CD34+ cells. The percentage of human=20 CD45+ cells in mice receiving G0CD34+ = cells was=20 on average 16-fold higher (range, 12.3- to 19.1-fold; P < = .05,=20 pairedt-test) than in mice injected with = G1CD34+=20 cells (Fig4A).=20 In one additional experiment in which fractions of BM CD34+ = cells=20 instead of MPB CD34+ cells were used as grafts, similar = results were=20 obtained: at a cell dose of 0.8 =C3=97 106 cells = transplanted, the average=20 percentage of chimeric human CD45+ cells was 3.6% in mice=20 transplanted with G0CD34+ cells (n =3D 2 mice) and = only 0.2%=20 in animals receiving G1CD34+ cells (n =3D 3 mice). =
Comparison of FACScan analysis of BM = cells=20 harvested from NOD/SCID mice transplanted 6 weeks previously with 1.0 = =C3=97=20 106 MPB CD34+ cells isolated in G0 or = in=20 G1 phase of the cell cycle. Cells were stained either with = isotypic=20 control antibodies (upper panels) or with antihuman CD45-FITC and = antihuman=20 CD34-PE (lower panels). (A and B) Mouse transplanted with=20 G0CD34+ cells; (C and D) mouse transplanted with=20 G1CD34+ cells.
Comparison of engraftment capacity of = MPB=20 CD34+ cells isolated in G0 or in G1 = phase of=20 the cell cycle. Data from four experiments are reported as the mean = =C2=B1 SEM. Each=20 experiment was performed with MPB CD34+ cells isolated from a = different donor (n =3D 2 to 4 animals per experiment). (A) Percentages = of chimeric=20 human CD45+ cells in the BM of mice transplanted 6 weeks = previously=20 with indicated numbers of G0 or G1CD34+ = cells.=20 Average chimerism was statistically higher after transplantation of=20 G0CD34+ cells than after transplantation of=20 G1CD34+ cells (P < .05,=20 pairedt-test). (B) Numbers of human progenitor cells per 50 = =C3=97=20 103 BM cells 6 weeks posttransplantation in the same = recipient mice.=20 Average human progenitor output was statistically higher in = G0-=20 versus G1-transplanted mice (P < .05, paired=20 t-test).
The presence of human clonogenic cells in BM low-density = cells=20 harvested from recipient mice was also determined by a progenitor cell = assay=20 selective for human cells.14=20 21=20 The numbers of engrafted human progenitors were in good correlation with = those=20 of CD45+ cells (Fig 4B),=20 indicating that functional human hematopoietic cells were present in = chimeric=20 mice. Human progenitors in the marrow were on average 22-fold more = numerous in=20 mice transplanted with G0CD34+ cells than in those = receiving G1CD34+ cells (P < .05,=20 pairedt-test). The selectivity of the assay for human = clonogenic cells=20 was confirmed by PCR amplification of the human =CE=B2-globin gene in = DNA extracts=20 prepared from individual colonies. In two experiments in which DNA from=20 individual colonies derived from the BM of 2 to 4 mice was analyzed by = PCR,=20 human sequences were detected in 23 of 24 progenitors.
Differentiative capacity of repopulating cells present in MPB=20 G0CD34+ cells.
The phenotypic profile of engrafted CD45+ cells =
in=20
G0-transplanted mice was determined by two-color staining =
(Table
- In=20 a new window
Phenotype of Chimeric Human Cells = Harvested From=20 NOD/SCID Mice Transplanted With G0CD34+Cells
CD34+ cells were reisolated from the BM of = highly positive=20 animals and assayed in vitro. Both granulo-monocytic (CFU-GM) and = erythroid=20 progenitors (burst-forming unit-erythroid [BFU-E]) were demonstrated in=20 progenitor cell assays at frequencies of 1.36% =C2=B1 0.1% and 0.1% = =C2=B1 0.02%,=20 respectively. Primitive hematopoietic progenitor cells assayed as = LTC-ICs were=20 also present, although at a lower frequency than in the original graft = (0.1% =C2=B1=20 0.02% v 3.1% =C2=B1 0.8%).
Effect of G0-G1 progression in vitro on the=20 repopulating ability of MPB CD34+ cells.
Given the marked difference of repopulating activity =
between MPB=20
CD34+ cells residing in vivo (ie, in a fresh MPB sample) in=20
G0 or in G1, we were interested in examining =
whether the=20
same relationship existed for cells progressing from G0 to=20
G1 under in vitro cytokine stimulation. The experimental =
design is=20
shown in Fig 5.=20
A first Hst/PY fractionation was used to isolate CD34+cells =
in=20
G0 or in G1 (Fig 5A),=20
and defined cell doses were used for mice transplantation on the =
original day of=20
cell isolation. The remaining cells isolated in G0 were =
plated in=20
serum-free conditions and stimulated with a combination of 100 ng/mL FL, =
100=20
ng/mL SCF, and 50 ng/mL IL-3, a cytokine mixture previously shown to =
promote the=20
expansion of primitive cells detected as LTC-ICs.28=20
After 36 hours in culture, a second cell cycle fractionation step was =
used to=20
separate cells remaining in G0 from those traversing into=20
G1 under these conditions (Fig 5B).=20
A short culture period was chosen to minimize cell proliferation such =
that only=20
the effect of progression to an activated state was investigated and not =
the=20
result of maturational cell divisions potentially leading to stem cell=20
exhaustion. At this time point, the mean increase in total cells was =
1.18- =C2=B1=20
0.17-fold (n =3D 3). Viability of cultured cells always exceeded 98%, as =
judged by=20
trypan blue exclusion. To assess the fidelity of the cell cycle =
fractionation at=20
36 hours, aliquots of sorted cells were reanalyzed with 7-AAD and Ki-67 =
(Fig
Cell sorting strategy to isolate=20 CD34+cells during ex vivo G0-G1 = progression.=20 (A) MPB CD34+ cells in G0 were isolated in a first = Hst/PY=20 sort and either directly injected into recipient animals or plated in ex = vivo=20 conditions. (B) After 36 hours, a second Hst/PY sort was used to = separate=20 cultured cells transiting to G1 from those remaining in=20 G0. Both cell subsets were then assayed in NOD/SCID mice. = Cell-cycle=20 status of cells isolated in G1(C) or G0 (D) after = culture=20 was confirmed by restaining samples of sorted cells with 7-AAD and = Ki-67.=20 Percentages of cells detected in each quadrant are indicated.
- In=20 a new window
Effect of Cell Cycle Progression Under = IL-3-SCF-FL=20 Stimulation on the Repopulating Activity of CD34+ Cells = Initially=20 Isolated in G0
As already shown, freshly isolated CD34+ cells =
residing in=20
G0 were able to produce a high level of engraftment. As for =
cells=20
remaining in G0 after 36 hours of cytokine stimulation, the =
level of=20
repopulating activity was comparable or slightly reduced compared with =
that=20
observed in noncultured G0 cells (experiments 1 and 2, Table =
This differential engraftment potential of cultured cells = could be=20 explained by the presence of two different subsets of=20 G0CD34+ cells: a subset enriched in repopulating = cells,=20 which responded poorly to mitogenic stimulation in vitro and remained=20 sequestrated in the G0 compartment after 36 hours in culture, = and a=20 highly responsive subset, transiting quickly into G1, but = initially=20 devoid of repopulating activity. Alternatively, these observations could = be the=20 result of a loss of repopulating capacity resulting directly from the=20 progression to an active state in the cell cycle. To distinguish between = these=20 two options, we performed two experiments in which fresh=20 G0CD34+ cells were cultured for 36 hours under the = same=20 conditions as those described above, then directly transplanted into = NOD/SCID=20 recipients without restaining with Hst/PY and separation into quiescent = and=20 activated fractions (experiments 2 and 3, Table 2).=20 In both experiments, the engraftment efficiency of unfractionated = cultured cells=20 was massively reduced compared with that of fresh = G0CD34+=20 cells indicating that repopulating activity was compromised after ex = vivo=20 culture, most likely by progression into G1. Because no = adjustment to=20 the number of cells remaining in G0 among total cells used as = a graft=20 was made, it is difficult to estimate the magnitude of chimerism lost = due to=20 decreased numbers of G0 cells in the graft.
A negative effect of IL-3 on the in vitro maintenance of =
primitive=20
hematopoietic cells has now been highly suspected,29=20
30whereas=20
TPO has emerged as a potent stimulator of putative stem cells.
- In=20 a new window
Effect of Cell Cycle Progression Under = TPO-SCF-FL=20 Stimulation on the Repopulating Activity of CD34+ Cells = Initially=20 Isolated in G0
DISCUSSION
The present report describes for the first time a strong = relationship=20 between cell cycle status and BM repopulating potential, based on a = direct cell=20 cycle fractionation method originally described by our group.16-18=20 Using simultaneous DNA/RNA staining and various in vitro assays, we = previously=20 demonstrated that primitive hematopoietic cells were mainly found in the = G0 phase of the cell cycle. We show herein that this = relationship=20 also applies to NOD/SCID repopulating cells contained within freshly = isolated=20 human MPB CD34+ cells. Although we did not use limiting = dilution=20 analysis to determine the frequency of SRC in mitotically quiescent and=20 activated subsets of MPB CD34+ cells,13=20 we show a significantly large difference in the readout of these two = cell=20 populations in the NOD/SCID mouse model. This suggests that SRC are = highly=20 enriched in the G0 compartment, that SRC present in the = G1=20 phase have a reduced proliferative capacity in the murine = microenvironment, or a=20 combination of both phenomena. This is further evidence of the ability = of our=20 cell cycle fractionation method to isolate functionally defined = populations of=20 human hematopoietic progenitor/stem cells based on their cycling status. = Although the endpoint analysis of engraftment at 6 to 7 weeks=20 posttransplantation is in agreement with other investigators,14=20 24=20 the exact nature and position of these cells in the hierarchy of the = pool of=20 engrafting hematopoietic progenitor cells remain to be fully elucidated. =
Few studies have examined the outcome of ex vivo culture of = putative=20 stem cells in the NOD/SCID model. Differences were pointed out in the = behavior=20 of SRC compared with that of in vitro assayable human hematopoietic = cells, ie,=20 CFC and LTC-IC. Gan et al32=20 showed that incubation of hematopoietic cells on stromal cells = maintained or=20 even expanded CFC and LTC-IC, whereas SRC declined over the same period = of time.=20 In gene marking experiments, after prestimulation over the C-terminal = fragment=20 of fibronectin,33=20 high levels of transduction were observed in CFC and LTC-IC, but no = transduced=20 cells were detected in the BM of recipient mice. Finally, using CB=20 CD34+CD38=E2=88=92 cells, Bhatia et al34=20 were able to demonstrate a twofold increase of SRC in short-term = culture,=20 although in a similar study, Conneally et al24=20 showed that ex vivo expanded SRC had a reduced proliferative capacity = compared=20 with that of freshly isolated SRC. It appears therefore that expansion = of SRC is=20 at best limited and achieved at the expense of a decrease in the = generative=20 capacity of individual repopulating cells.
The ability of the DNA/RNA staining method to sort cells in =
precise=20
phases of the cell cycle was used in the present study to determine the =
effect=20
of in vitro cell cycle activation on the repopulating ability of =
putative stem=20
cells, in the absence of cell division. A short cytokine stimulation =
period of=20
36 hours was used to induce a fraction of CD34+ cells =
initially=20
isolated in G0 to progress into G1 without =
executing a=20
complete cell cycle, while allowing almost an equal number of cells to =
remain in=20
G0. Thus, the only variable tested in these experiments was =
the=20
movement along the G0/G1 phase of the cell cycle =
and not=20
the effect of cellular divisions. This was confirmed by the absence of =
an=20
increase in cell numbers during ex vivo culture. Our data show that =
engagement=20
in G1 after cytokine stimulation is associated with a =
decrease in=20
repopulating capacity. Two mutually nonexclusive mechanisms may underlie =
these=20
observations. It is possible that SRC are sequestrated in the =
G0=20
compartment during culture, whereas more differentiated cells quickly =
traverse=20
into G1. This is most likely, because we previously =
demonstrated that=20
CD34+ cells isolated in G0 by the Hst/PY method =
were still=20
heterogeneous in their ability to respond to cytokine stimulation,
This study provides evidence that ex vivo culture can =
modify=20
primitive hematopoietic functions of putative stem cells before any =
cellular=20
division actually occurs. This is compatible with at least two =
hypotheses.=20
First, commitment and/or differentiation may take place during the=20
G0-G1 transition. Second, homing of primitive =
progenitor=20
cells to the BM microenvironment is possibly impaired after cell cycle=20
activation. The latter hypothesis is also suggested by studies that have =
now=20
established a direct relationship between cytokine stimulation and =
affinity=20
and/or expression of integrins at the surface of human hematopoietic=20
progenitors35-38=20
or between cell cycle status, expression of adhesion molecules, and =
engraftment=20
of murine progenitor cells.39=20
Because the BM microenvironment is thought to comprise multiple loci =
supporting=20
hematopoietic progenitors at different levels of differentiation,
ACKNOWLEDGMENT
The authors thank Dr David A. Williams (Herman B Wells = Center for=20 Pediatric Research, Indiana University School of Medicine) for the use = of the=20 NOD/SCID mice facility, Susan Rice and Jon McMahel for technical = assistance in=20 flow cytometric cell sorting, and Ryan Cooper for help in immunomagnetic = CD34+ cell selection. We are also grateful to Amgen and = Immunex for=20 generous gifts of recombinant human cytokines.
Footnotes
-
Supported by National Institutes of Health Grant No. R01 = HL55716=20 and a research award from the Phi Beta Psi Sorority to E.F.S. Herman B = Wells=20 Center for Pediatric Research is a Center of Excellence in Molecular=20 Hematology (NIDDK P50 DK 49218). A.G. is supported by a fellowship = from the=20 Fonds National de la Recherche Scientifique (FNRS, Brussels, Belgium) = and by a=20 travel grant from the Centre Anticanc=C3=A9reux pr=C3=A8s = l=E2=80=99ULg (University of Li=C3=A8ge,=20 Li=C3=A8ge, Belgium).
-
Address reprint requests to Edward F. Srour, PhD, Indiana = University School of Medicine, 1044 W Walnut St, R4-202, Indianapolis, = IN=20 46202.
-
The publication costs of this article were defrayed in = part by page=20 charge payment. This article must therefore be hereby marked = "advertisement"=20 is accordance with 18 U.S.C. section 1734 solely to indicate this = fact.=20
- Copyright =C2=A9 1998 = American Society of=20 Hematology
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