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Original Paper
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Acta Biochim Biophys
Sin 2008, 40: 711-720 |
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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, GTG�GAA�TTTCGACTTTCAG and CAAGCCCAAG�A�A�G�AACAGC, 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 (GCCGAAACTATTT�AGACAT ) 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 manufacturer�s 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 manufacturer�s instructions. The sca-1+ BM cells were cultured for 7 d in a medium [high glucose Dulbecco�s modified Eagle�s 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.
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 assay
LTC-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'-AGGA�C�A�G�CG�TGATCTTCACC-3'
and 5'-CTTGAAGTG�CATG�T�G�G�CTGT-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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