Original Paper
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file on Synergy OPEN |
Acta Biochim Biophys
Sin 2008, 40: 711-720
doi:10.1111/j.1745-7270.2008.00448.x
Simultaneous knockdown of p18INK4C, p27Kip1 and
MAD1 via RNA interference results in the expansion of long-term
culture-initiating cells of murine bone marrow cells in vitro
Yan-Yi Wang1*, Yong Yang2, Qingyong Chen3, Jianping Yu1, Yongzhong Hou4, Lizhen Han1, Jun He1, Demin Jiao1, and Huihui Yu1
1
Department of
Pharmaceutical Engineering, College of Life Science, Guizhou University,
Guiyang 550025, China
2
Department of Biomedical
Engineering, Hangzhou Dianzi University, Hangzhou 310018, China
3
Department of Pulmonary
Diseases, the 117th Hospital, Hangzhou 310013, China
4
Department of
Microbiology and Infectious Diseases, University of Calgary, Calgary, Alberta
T2N 4N1, Canada
Received: March 27, 2008
Accepted: May 16,
2008
This work was
supported by the grants from the Science and Research Project of Guizhou
University for the Recruit Talent [(2007)033] and the Natural Science
Foundation of Guizhou Province [(2008)2204]
*Corresponding
author: Tel, 86-13765813178; E-mail, [email protected]
A combination of extrinsic hematopoietic
growth regulators, such as stem cell factor (SCF), interleukin (IL)-3 and IL-6,
can induce division of quiescent hematopoietic stem cells (HSCs), but it
usually impairs HSCs self-renewal ability. However, intrinsic negative cell
cycle regulators, such as p18INK4C (p18), p27Kip1 (p27)
and MAD1, can regulate the self-renewal of HSCs. It is unknown whether the
removal of some extrinsic regulators and the knockdown of intrinsic negative
cell cycle regulators via RNA interference (RNAi) induce ex vivo
expansion of the HSCs. To address this question, a lentiviral vector-based RNAi
tool was developed to produce two copies of small RNA that target multiple
genes to knockdown the intrinsic negative cell cycle regulators p18, p27 and
MAD1. Colony-forming cells, long-term culture-initiating cells (LTC-IC) and
engraftment assays were used to evaluate the effects of extrinsic and intrinsic
regulators. Results showed that the medium with only SCF, but without IL-3 and
IL-6, could maintain the sca-1+c-kit+ bone marrow cells with high LTC-IC
frequency and low cell division. However, when the sca-1+c-kit+ bone
marrow cells were cultured in a medium with only SCF and simultaneously knocked
down the expression of p18, p27 and MAD1 via the lentiviral vector-based RNAi,
the cells exhibited both high LTC-IC frequency and high cell division, though
engraftment failed. Thus, the simultaneous knockdown of p18, p27 and MAD1 with
a medium of only SCF can induce LTC-IC expansion despite the loss of
engraftment ability.
Keywords p18INK4C; p27Kip1; MAD1; hematopoietic
stem cell
A variety of extrinsic and intrinsic regulators influence
hematopoietic stem cell (HSC) self-renewal and differentiation. Although the
extrinsic hematopoietic growth regulators, such as stem cell factor (SCF),
interleukin (IL)-3 and IL-6, were commonly used to induce murine HSC division,
the self-renewal ability of HSC was usually impaired [1–3]. SCF was reported to be
required for the maintenance of ex vivo HSC culture in the absence of
cell division [4,5], while the interrupted expression of intrinsic
negative cell cycle regulators, such as p18, p21, p27 and MAD1, was reported to
increase stem cell division and maintain the ability of cells to renew
themselves [6–10]. Therefore, we hypothesized that the only addition of SCF to the
medium and the knockdown of the intrinsic negative cell cycle regulators via
RNA interference (RNAi) would favor the expansion of HSC in vitro. The short interfering RNA (siRNA) technique has been widely used in
the study of gene functions. siRNA are short double-stranded RNA molecules that
can target and degrade complementary messenger RNA via a cellular process
termed RNAi [11]. Alternate methods for generating siRNA are: (1) vector-based in
vivo expression; (2) chemical synthesis; (3) in vitro transcription
(IVT); (4) ribonuclease III-mediated hydrolysis; and (5) polymerase chain
reaction (PCR)-based siRNA expression cassettes. Among them, vector-based,
especially lentiviral vector-based, in vivo expression offers a durable
and effective gene knockdown. Knockdown of multiple genes can be accomplished
by delivering either multiple separate lentiviral vectors bearing single siRNA
or a single lentiviral vector bearing multiple siRNA that target multiple genes
[12,13]. For knockdown of multiple genes in the primary HSC, applying a single
lentiviral vector bearing multiple siRNA should be advantageous as it avoids
repeated infections. Therefore, in this study, we used a double polymerase III promoter
(H1/U6) lentiviral vector to develop a highly efficient RNAi tool to produce
two copies of small RNA that target multiple genes to knockdown intrinsic
negative cell cycle regulators. We demonstrated that only the addition of the
extrinsic regulator SCF in the medium and the simultaneous knockdown of the
three intrinsic negative cell cycle regulators, p18, p27 and MAD1, could induce
expansion of long-term culture-initiating cells (LTC-IC).
Materials and Methods
Lentiviral constructs and lentivirus preparation The pFIV-H1/U6-CopGFP (copepod green fluorescent protein) lentiviral
vector (System Biosciences, Mountain View, USA) containing double H1 and U6 RNA
polymerase III promoters was used for lentiviral vector-based gene knockdown.
To enhance the CopGFP expression in HSC, the cytomegalovirus (CMV) promoter
driving CopGFP expression was substituted between SpeI and XbaI
sites with the murine stem cell virus (MSCV) promoter, which was derived from
MigR1 plasmid [Fig. 1(A)]. In this study, we tested only the effects of
p18, p27 and MAD1, as p21 reportedly leads to premature exhaustion of stem
cells under conditions of stress [6,14]. p18, p27 and MAD1 gene target
sequences TAATGTAAACGTCAACGCT, GTGGAATTTCGACTTTCAG and CAAGCCCAAGAAGAACAGC,
respectively, were identified as templates for producing siRNA as determined
using an Ambion siRNA Target Finder (http://www.ambion.com/techlib/misc/siRNA_finder.html)
and the mouse p18, p27 and MAD1 cDNA sequences. A
sequence (GCCGAAACTATTTAGACAT ) was designed as the template for producing the
control siRNA. Blast search was carried out to ensure that the p18, p27 and
MAD1 siRNA was targeting only mouse p18, p27 and MAD1 and that control siRNA
was not targeting any mouse genes. For single gene knockdown, two complementary
DNA oligonucleotides were chemically synthesized, annealed, and inserted
immediately into the pFIV-H1/U6-CopGFP vector between the H1 and U6 promoters
according to the manual (http://www.systembio.com/)
[Fig. 1(A)]. The single control siRNA was used for all experiments. For
simultaneous knockdown of two genes, the p18, p27 and MAD1-specific hairpin
siRNA inserts (sense-loop-antisense) were determined using a
computerized insert design tool based on a target sequence from
the instructions on the Ambion website (http://www.ambion.com). The
oligonucleotides encoding the p18-, p27- and MAD1-specific hairpin siRNA
inserts were designed to contain a unique restriction enzyme site (HindIII)
and a sticky end for ligation of both of the p18-, p27- and MAD1-hairpin siRNA
inserts but avoidance of ligation of the same hairpin siRNA inserts [Fig.
1(A)]. Then, the two ligated hairpin siRNA inserts were ligated into the
pFIV-H1/U6-CopGFP vector to build the double gene knockdown construct [Fig.
1(A)]. For simultaneous knockdown of three genes, H1-siRNA cassettes were
obtained by PCR using the double gene knockdown constructs as the template; the
PCR products for the H1-siRNA cassettes were ligated into the pFIV-H1/U6-CopGFP
vector that had been built for simultaneous knockdown of two genes in the
unique restriction enzyme site (HindIII) [Fig. 1(A)]. The
pFIV-H1/U6-CopGFP plasmid bearing the siRNA inserts and the Packaging Plasmids
(System Biosciences, Mountain View, USA) were used to produce the lentivirus
using the packaging cell line 293T/17 (ATCC, Manassas, USA) according to the
manufacturers instructions. The virus titer was determined by
infection of NIH 3T3 cells using following formula:
Eq.
Stable clones expressing CopGPF were sorted by
fluorescence-activated cell sorting (FACS). The levels of p18, p27 and MAD1
knockdown, respectively, were determined by Western blot analysis from the cell
lysate of cell lines known to express p18, p27 or MAD1. These cell lines were
transduced with the lentivirus and the CopGFP+ cells
were sorted (Fig. 1).
Isolation, lentiviral transduction and culture of hematopoietic stem
cells
To test the effect of the extrinsic hematopoietic growth regulators
(SCF, IL-3 and IL-6) on cell self-renewal, bone marrow (BM) cells were obtained
by flushing the tibias and femurs of male C57BL/6J mice with phosphate-buffered
saline (Gibco, Gaithersburg, USA). sca-1+ BM cells
were collected using MACS (Miltenyi Biotech, Bergisch Gladbach, Germany)
according to the manufacturers instructions. The sca-1+ BM cells
were cultured for 7 d in a medium [high glucose Dulbeccos modified Eagles medium
(Gibco), 15% fetal bovine serum (embryonic stem specific; Gibco), 2 mM L-glutamine
(Gibco), 0.1 mM non-essential amino acid (Stemcell Technologies, Vancouver,
Canada), 1% Pen/Strep (100?; Gibco #15070-014), 0.1 mM b-mercaptoethanol
(Sigma, St. Louis, USA)] either supplemented with only SCF (50 ng/ml) or with a
combination of 50 ng/ml SCF, 20 ng/ml IL-3, and 50 ng/ml IL-6. To test the effect of the intrinsic negative cell cycle regulators
(p18, p27 and MAD1) on cell self-renewal, BM cells were obtained by flushing
the tibias and femurs of male C57BL/6J mice with PBS (Gibco). The sca-1+ BM cells were collected using MACS and transduced with the
lentivirus bearing p18, p27 or MAD1 siRNA, combined p18, p27 and MAD1 siRNA, or
control siRNA by spinoculation. The sca-1+ BM cells
were briefly suspended in 2 ml lentiviral supernatants supplemented with 4 mg/ml polybrene
in a 6-well plate; the plate was then spinoculated at 900 g for 50 min
at room temperature. After spinoculation, the lentiviral supernatants were
replaced with the medium supplemented with 50 ng/ml SCF. After 24 h, the
lentiviral infection procedure was repeated. The lentiviral infected sca-1+ BM cells were cultured in the medium with only 50 ng/ml SCF for 1
week. Then, the infected sca-1+ BM cells were traced by their
expression of CopGFP and sorted for CopGFP+sca-1+c-kit+ cells using FACS. The test cells were cultured
on a 15 Gy irradiated primary mouse stromal monolayer in a medium supplemented
with only 50 ng/ml SCF; the number of days the experiment lasted depended on
the method of testing. The medium was changed with 2/3 fresh medium every 5 d.
Cell numbers were counted using a hemocytometer.
To test the effect of the intrinsic negative cell cycle regulators
(p18, p27 and MAD1) on cell self-renewal, BM cells were obtained by flushing
the tibias and femurs of male C57BL/6J mice with PBS (Gibco). The sca-1+ BM cells were collected using MACS and transduced with the
lentivirus bearing p18, p27 or MAD1 siRNA, combined p18, p27 and MAD1 siRNA, or
control siRNA by spinoculation. The sca-1+ BM cells
were briefly suspended in 2 ml lentiviral supernatants supplemented with 4 mg/ml polybrene
in a 6-well plate; the plate was then spinoculated at 900 g for 50 min
at room temperature. After spinoculation, the lentiviral supernatants were
replaced with the medium supplemented with 50 ng/ml SCF. After 24 h, the
lentiviral infection procedure was repeated. The lentiviral infected sca-1+ BM cells were cultured in the medium with only 50 ng/ml SCF for 1
week. Then, the infected sca-1+ BM cells were traced by their
expression of CopGFP and sorted for CopGFP+sca-1+c-kit+ cells using FACS. The test cells were cultured
on a 15 Gy irradiated primary mouse stromal monolayer in a medium supplemented
with only 50 ng/ml SCF; the number of days the experiment lasted depended on
the method of testing. The medium was changed with 2/3 fresh medium every 5 d.
Cell numbers were counted using a hemocytometer.
Colony-forming cells (CFC) assay
Test cells were cultured in Complete M3434 (Stemcell Technologies).
Cells were plated at 1000 cells/ml into low adherence 35 mm dishes (Stemcell
Technologies). Along with an open 35 mm dish containing sterile water for
humidification, the cultures were placed in a covered Petri dish and incubated
at 37 ?C, 5% CO2. At 10 d, colonies (>30 cells) were scored
by phase microscopy and reported as CFC.
LTC-IC assayLTC-IC assay was performed as described [6,15–17] with minor
modifications. The test cells were briefly plated with 2-fold diluted single-cell
suspensions on a 15 Gy irradiated primary mouse stromal monolayer in 96-well
plates containing 150 ml M5300 medium (Stemcell Technologies) supplemented with 10–6 M hydrocortisone. The medium was changed with half fresh medium
weekly. After four weeks, the Complete M3434 (Stemcell Technologies) was
overlaid into the wells. The colonies (>30 cells) were counted on 38 d.
Limiting dilution analysis software (Stemcell Technologies) was used to
calculate the frequency of LTC-IC in the cell population.
Cell cycle analysis
The test cells were fixed in 90% methanol for 60 min at 4 ?C and
stained with 50 mg/ml propidium iodide (Sigma) to determine cell cycle distribution
by FACS.
Engraftment assay
To evaluate test cells engraftment ability, they were transplanted
by retro orbital injection into lethally irradiated 8-week-old female mice.
Peripheral blood and BM cells were obtained from each recipient mouse to
determine chimerism or detect CopGFP using FACS and PCR. The sense and
antisense PCR primers for CopGFP are 5‘-AGGACAGCGTGATCTTCACC-3‘
and 5‘-CTTGAAGTGCATGTGGCTGT-3‘ respectively.
To verify that CopGFP is an indicator of siRNA expression, freshly
isolated sca-1+ BM cells were infected twice by spinoculation
with the lentivirus bearing siRNA. They were then directly transplanted into
the lethally irradiated 8-week-old female mice. After the blood was
reconstituted, the CopGFP+ cells were isolated from the
mouse spleen cells by FACS, and the expression of p18, p27 and MAD1, respectively,
in the CopGFP+ cells was detected by Western blot analysis.
Results
Knockdown of negative cell cycle regulator genes via double-copy
RNAi
In the lentiviral vector construct, the siRNA cassettes were
embedded in the 3‘–DLTR [Fig. 1(A)]. During RT, the U3 region of the 5‘-LTR
was synthesized using its 3‘ homolog as a template, which resulted in a
duplication of the siRNA cassette in the provirus integrated into the
transduced cells genome [Fig. 1(B)]. Therefore, the siRNA were generated
in double-copy manner. The titers of lentiviruses bearing single, double or
triple siRNA cassettes were estimated to be at least 1?106 cfu/ml by infection of NIH-3T3 cells. Because
the expression of negative cell cycle regulators in primary HSC might not occur
simultaneously and there were too few primary stem cells to assess the
knockdown effect of RNAi by Western blot analysis [18,19], we chose the cell
lines known to express p18, p27 or MAD1 to assess the knockdown effect of the
RNAi. The NIH-3T3 cell line is known to express p18 and MAD1, while the 10 d
hematopoietic differentiating murine embryonic stem cell line is known to
express p27. The cell lysates used for Western blot analysis were from the
CopGFP+ cells that stably expressed p18, p27 or MAD1 siRNA or combined p18,
p27 and MAD1 siRNA. Results showed that both the individual regulators and the
combination of p18, p27 and MAD1 siRNA worked well [Fig. 2(A)]. To verify if CopGFP expression is indicative of siRNA expression
after long-term culture, the freshly isolated sca-1+ BM cells
were infected twice by spinoculation and then directly transplanted into
lethally irradiated mice. The CopGFP+ cells were isolated from
the spleen cells by FACS after the blood was reconstituted. The expression of
p18, p27 and MAD1 in the CopGFP+ spleen cells was detected by
Western blotting. Fig. 2(B) shows the expression of p18, p27 and MAD1 in
the CopGFP+ cells was markedly knocked down, indicating the CopGFP expression
is indicative of siRNA expression.
Effect of extrinsic hematopoietic growth regulators on in vitro
self-renewal of HSC
SCF is required for maintenance of ex vivo hematopoietic stem
cell culture [4,5]. The results of our experiments showed that sca-1+ BM cells undergo apoptosis when cultured in medium without any
hematopoietic growth factor; however, they can be maintained for a relatively
long time (ie over 4 weeks) if cultured in medium with SCF (data not
shown). The commonly used hematopoietic growth regulators in mouse BM cell
cultures are IL-3, IL-6 and SCF. The combination of IL-3, IL-6 and SCF can
dramatically drive division of HSC, but it impairs the cells engraftment
ability [1–3]. Therefore, we considered whether SCF alone would impair
engraftment ability of HSC. Isolated sca-1+ BM cells were cultured in
medium supplemented with 50 ng/ml SCF or with the combination of 50 ng/ml SCF,
20 ng/ml IL-3, and 50 ng/ml IL-6. After 7 d, CFC assay and engraftment assay
were performed on these cells. Results showed that the total number of colonies
dramatically decreased in cells cultured in medium with the combination of
regulators compared to those cultured in medium with only 50 ng/ml SCF in CFC
assay [Fig. 3(A)]. This indicated that IL-3 and IL-6 impair the
colony-forming ability of the stem or progenitor cells in the sca-1+ BM cells. In engraftment assay, 1?106 cultured or freshly isolated sca-1+ BM cells from male C57BL/6J mice were transplanted by retro orbital
injection into the lethally 10 Gy irradiated 8-week-old female C57BL/6J mice.
Of the six mice that received cells cultured for 7 d in medium with the
combination of 50 ng/ml SCF, 20 ng/ml IL-3, and 50 ng/ml IL-6, none survived
for more than 6 weeks after transplantation. Of the six mice that received
cells cultured for 7 d in medium with only 50 ng/ml SCF, all survived for more
than 1 year, as did the six control mice that received freshly isolated cells.
This suggests that medium with SCF alone does not impair the engraftment
ability of stem cells. Nevertheless, the increase in the number of sca-1+ BM cells cultured in medium with only 50 ng/ml SCF was
significantly lower than the increase in those cultured in medium with the
combination of 50 ng/ml SCF, 20 ng/ml IL-3, and 50 ng/ml IL-6 [Fig. 3(B)].
Furthermore, cells cultured in medium with the combination of 50 ng/ml SCF, 20
ng/ml IL-3, and 50 ng/ml IL-6 appeared more attached to the bottom of plate
than those cultured in medium with 50 ng/ml SCF [Fig. 3(C)].
Effect of intrinsic negative cell cycle regulators (p18, p27 and
MAD1) on in vitro expansion of HSCs
Effect of negative cell cycle regulators on cell division Although
medium with only 50 ng/ml SCF does not impair the engraftment ability of stem
cells, the increase in cell number is very low [Fig. 3(B)]. To overcome
this drawback, we knocked down the individual and combined expression of
negative cell cycle regulators p18, p27 and MAD1 using lentiviral vector-based
siRNA strategy to induce proliferation of the HSC or hematopoietic progenitor
cells. The effect of p21 was not tested in this study as it was reported to
lead to premature exhaustion of stem cells under conditions of stress [6,14].
We infected sca-1+ BM cells with lentivirus supernatants. Then we
isolated CopGFP+sca-1+c-kit+ BM cells and compared the effects of individual knockdown of p18,
p27, and MAD1 and simultaneous knockdown of p18 and p27 (p18+p27), p18 and MAD1
(p18+MAD1), p27 and MAD1 (p27+MAD1), and p18, p27 and MAD1 (p18+p27+MAD1) on
HSC division in medium with only 50 ng/ml SCF. After the cells were cultured
for 35 d and 92 d, all seven knockdown samples exhibited significant cell
division when compared with the control [Fig. 4(A)]. To test whether the
negative cell cycle regulators affected the cell cycle status, we used FACS to
analyze the cell cycles of the transduced cells that were cultured for 7 d.
Knockdown of p18+p27 siRNA led more cells to enter the cell cycle than
knockdown of only MAD1, knockdown of p27+MAD1 siRNA or the control did.
Likewise, knockdown of p18+MAD1 siRNA and p18+p27+MAD1 siRNA led more cells to
enter the cell cycle than the control [Fig. 4(B)]. Although the cells
entering cell cycle did not appear proportional to the fold increase in cell
numbers for each sample, there was still a trend suggesting that
down-regulation of the negative cell cycle regulators results in cell division.
To test whether the siRNA expressions were sustained, we checked CopGFP
expression in these cultured cells under fluorescent microscope because the
CopGFP expression had previously indicated siRNA expression. The cells were all
positive for CopGFP after being cultured for 92 d [Fig. 4(C)]. Effect of negative cell cycle regulators on maintenance of LTC-IC The results showed that
knockdown of negative cell cycle regulators can dramatically increase cell
division. Next, we assessed whether these divided cells retain LTC-IC. CFC
assay and LTC-IC assay were performed on these cells to quantify the functional
populations of progenitor cells and more primitive cells [7]. Except
simultaneous knockdown of p18+p27, single knockdown of negative cell cycle
regulators led to less functional populations of progenitor cells (CFC) and
primitive cells (LTC-IC) than simultaneous knockdown of two or three negative
cell cycle regulators after the cells were cultured for 35 or 92 days [Fig.
5(A–D)]. Furthermore, simultaneous knockdown
of p18+p27+MAD1 resulted in the highest CFC and LTC-IC frequency [Fig. 5(A–D)]. The control also exhibited high CFC and LTC-IC frequency [Fig.
5(A–D)], though cell division rate was low [Fig.
4(A)], indicating medium with only SCF can maintain LTC-IC. Moreover, the
colonies of p18+p27+MAD1 siRNA sample were the largest among the seven
knockdown samples and the control, but were smaller than the colonies of fresh
BM cells in the CFC assay [Fig. 5(E)]. Nevertheless, after the cells
were cultured for 35 d and 92 d, the colonies formed in CFC assay all were
colony-forming unit granulocyte-macrophage (CFU-GM) (data not shown). The
typical colonial morphologies are shown in Fig. 5(E). However, when
these cells were cultured for 35 d and 92 d in medium with a combination of 50
ng/ml SCF, 20 ng/ml IL-3, and 50 ng/ml IL-6, they hardly formed colonies in CFC
and LTC-IC assays (data not shown).Assess the engraftment ability of these expanded HSC Although our results
showed that the long-term cultured GFP+sca-1+c-kit+ cells had a high rate of cell division rate
and a high LTC-IC frequency, it was still unknown whether these cells had the
engraftment ability, so an engraftment assay was performed. About 5?104 freshly isolated sca-1+c-kit+ BM cells or 5?104 GFP+sca-1+c-kit+ cells cultured for 6 d, 35 d or 92 d were transplanted by retro
orbital injection into the lethally 9.5 Gy irradiated 8-week-old female
C57BL/6J mice. The recipient mice (seven knockdown samples and one control)
transplanted with GFP+sca-1+c-kit+ cells cultured for 35 d or 92 d died within 3 weeks after
transplantation, whereas the mice transplanted with the same number of freshly
isolated sca-1+c-kit+ BM cells or GFP+sca-1+c-kit+ cells cultured for 6 d
all survived more than 15 weeks. This failure of engraftment may have resulted
from the lack of short-term HSC responsible for rapid reconstitution. To verify
this possibility, we transplanted 5?104 freshly isolated sca-1+c-kit+ BM cells in which there were short-term HSC and 5?104 GFP+sca-1+c-kit+ cells cultured for 35 d or 92 d together into
the lethally 9.5 Gy irradiated recipient mice. We detected GFP in the
peripheral blood and BM cells of the recipient mice using FACS and PCR 3 months
after transplantation, there was no GFP detected by FACS or PCR (data not
shown), indicating the GFP+sca-1+c-kit+ cells did not contribute to the reconstitution. Therefore, the
failure of engraftment did not result from the lack of short-term HSC in GFP+sca-1+c-kit+ cells.
Discussion
This study shows that the culture medium with 50 ng/ml SCF, but
without IL-3 and IL-6, maintains the LTC-IC of murine BM cells for a quite long
period, but fails to enhance cell division. However, both cell division and
high LTC-IC frequency could be achieved when RNAi simultaneously knocked down
the negative cell cycle regulators p18, p27 and MAD1. These results demonstrate
that the extrinsic hematopoietic growth regulators IL-3 and IL-6 are the
factors that impair the self-renewal ability of HSC. Moreover, these results
show that removing IL-3 and IL-6 from the medium and simultaneous knockdown of
the intrinsic negative cell cycle regulators p18, p27 and MAD1 favors the
expansion of LTC-IC in vitro. Nevertheless, the ability to favor the
expansion of LTC-IC in vitro is different among p18, p27 and MAD1.
Simultaneous knockdown of p18+p27 did not favor LTC-IC expansion more than the
individual knockdowns, whereas simultaneous knockdown of MAD1+p18, MAD1+p27 or
MAD1+p18+p27 dramatically increased the expansion ability of LTC-IC, suggesting
that the negative cell cycle regulators have their different functions. The
simultaneous knockdown of p18+p27 increased only cell division, not CFC and
LTC-IC frequency, while simultaneous knockdown of other negative cell cycle
regulators exhibited a synergistic effect both in cell division and CFC and
LTC-IC frequency. Some investigations have demonstrated that p18–/– or both p27–/– and MAD1–/– enhance
self-renewal in vivo [8,10]. Therefore, this study further confirmed
that these negative cell cycle regulators function not only in vivo but
also in vitro. However, long-term culture in vitro seems to alter
the properties of expanded cells so that the colonies formed in CFC assay all
became CFU-GM. When these in vitro expanded cells were transplanted into
the lethal irradiated mice, the engraftment failed. The failed engraftment did
not seem to result from the knockdown of negative cell cycle regulators, as it
also failed in transplantation of control samples. Rather, it might result from
long-term culture in vitro, as the engraftment was successful in
transplantation of short-term (6 d) cultured cells with knockdown of negative
cell cycle regulators. Possible reasons for this failure may relate to the
following: (1) homing failure; (2) long-term culture in vitro altered
some HSC properties, which can be elucidated by the colony type (CFU-GM) and
colony morphologies that differ from the colonies from fresh BM cells [Fig.
5(E)]; (3) transplantation of insufficient numbers of cells as short- and
long-term repopulating cells may be depleted under these conditions; and (4)
the cells we have detected are not HSC.Other investigators have reported the simultaneous knockdown of
multiple genes by single lentiviral vector [12,13]. The lentivirus is known to
be able to deliver genetic material to most cell types, including non-dividing
and hard-to-transfect cells (primary, blood and stem cells) in vitro. In
this study, we used the pFIV-H1/U6-CopGFP lentiviral vector, specifically
designed by System Biosciences for expression of natural double-stranded siRNA
constructs rather than hairpin-type siRNA constructs. We modified it to express
multiple hairpin-type siRNA constructs to knockdown multiple genes (Fig. 1).
Since the siRNA cassettes were embedded in the 3‘–DLTR, the siRNA
would theoretically be expressed in double copies so that this lentiviral
vector would exhibit more knockdown efficiency than the lentiviral vector
expressing a single copy of siRNA. The results in this study indicate that
modified pFIV-H1/U6-CopGFP lentiviral vector worked well [Fig. 2(A)].
Therefore, it would be suitable for application in primary HSC. It could also
be a useful tool to study the cooperative effects of multiple genes on cell
division and differentiation of HSC, as its one-step infection for simultaneous
knockdown of multiple genes has advantages. In summary, we have shown that knockdown of negative cell cycle
regulators will induce expansion of LTC-IC despite the loss of engraftment
ability after long-term culture in medium with only SCF in vitro.
Further studies should be carried out to overcome the engraftment failure.
Acknowledgement
The authors wish to thank Xiao Yuan for assisting with the
manuscript preparation.
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