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RAPID COMMUNICATION |
a Department of Surgery, University of Cambridge, Addenbrooke’s Hospital, Cambridge, United Kingdom;
b School of Animal and Microbial Sciences, University of Reading, Reading, United Kingdom
Key Words. Human embryonic stem cells • Fluorescent reporter genes • Green fluorescent protein • Red fluorescent protein • Transfection • Small interfering RNA • Gain-of-function • Loss-of-function • Stem cell therapy • Regenerative medicine
Correspondence:
Ludovic Vallier, Ph.D., Box 202, Level E9, Addenbrooke’s Hospital, Hills Road, Cambridge, CB2 2QQ, United Kingdom. Telephone: 44-1223-762025; Fax: 44-1223-410772; e-mail: lv225{at}cam.ac.uk
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ABSTRACT |
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 Top Abstract IntroductionMaterials and MethodsResults and DiscussionReferences |
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INTRODUCTION |
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TopAbstractIntroductionMaterials and MethodsResults and DiscussionReferences |
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MATERIALS AND METHODS |
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TopAbstractIntroductionMaterials and MethodsResults and DiscussionReferences |
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The cells were incubated for 24 hours and then rinsed twice with phosphate-buffered saline (PBS). For transitory transfection, the cells were harvested 48 hours after transfection. For stable expression, the cells were passed 3 days after transfection onto 60-mm gelatin-coated tissue culture plates containing puromycin-resistant mouse fetal fibroblasts as feeders. After 3 days, puromycin (1 µg/ml final concentration) was added. Puromycin-resistant colonies that appeared by 12 days of selection were picked, dissociated, and plated onto 24-well gelatin-coated feeder-containing plates and expanded as described above for further analysis. The same procedure was used for stable cotransfection of the pSuper-HPRT vector with the puro-TK selection gene [7]. The DNA ratio was 3.5 µg of siRNA expression vector for 0.5 µg of selection gene. For pSuper-HPRT single transfection, hES cells were transfected on six-well matrigel-coated plates (Becton Dickinson; Franklin Lakes, NJ; http://www.bd.com) and then grown in feeder-free conditions as described [8] in order to avoid the use of 6-thioguanine (6-TG)-resistant feeder cells. After 4 days, 6-TG (3 µg/ml final concentration) was added to conditioned media. Selection was stopped 10 days later, and 6-TG-resistant colonies were picked after 2 additional days. Colonies were then dissociated, plated onto 24-well gelatin-coated feeder-containing plates, and expanded for further analyses as described above.
siRNA Design, Synthesis, and Transfection
siRNAs corresponding to hr-green fluorescent protein (hrGFP) or DsRed2 fluorescent reporter genes were designed as recommended [5] and synthesized using the Silencer siRNA Construction Kit (Ambion; Austin, TX; http://www.ambion.com). The following sequences were used: si-hrGFP6 sense 5'-CAACCACGUGUUCACCAUGUU-3' and antisense 5'-CAU GGUGAACACGUGGUUGUU-3'; si-hrGFP14 sense 5'-CCU GAUCGAGGAGAUGUUCUU-3' and antisense 5'-GAACA UCUCCUCGAUCAGGUU-3'; si-hrGFP22 sense 5'-GUUCU ACAGCUGCCACAUGUU-3' and antisense 5'-CAUGUGG CAGCUGUAGAACUU-3'; and si-DsRed2 sense 5'-CACCG UGAAGCUGAAGGUGUU-3' and antisense 5'-CACCUUC AGCUUCACGGUGUU-3'. Transfection of siRNA was performed with Oligofectamine (Invitrogen) in 12-well plates, following manufacturer instructions. After 24 hours, the cells were retransfected as before. The fluorescence-activated cell sorting (FACS), real-time reverse transcription-polymerase chain reaction (RT-PCR), and the fluorescence microscopy analysis were done 48 hours after the second transfection.
For stable transfection, siRNA primers corresponding to hypoxanthine guanine phosphoribosyl transferase (HPRT) were designed using OligoEngine workstation software (Seattle, WA; http://www.oligoengine.com). The primers were subcloned downstream from the H1 promoter in the pSuper vector [9]. The resulting vector, pSuper-HPRT, was cotransfected into hES cells with the puro-TK vector for selection using puromycin [7], or alternatively with the hrGFP-pTP6 vector for selection using 6-TG. Preliminary observations revealed that 6-TG-treated hES cells tended to undergo differentiation, so only the hardiest colonies persisted under these conditions.
Real-Time and Semiquantitative RT-PCR
For real-time RT-PCR, total RNA was extracted from fluorescent green hES cells transfected with siRNA Anti-green 22 using the RNeasy Mini Kit (Qiagen; Valencia, CA; http://www.qiagen.com). Each sample was treated with RNase-Free DNase (Qiagen) in order to avoid DNA contamination. One-half microgram of total RNA was reverse transcribed using Superscript II Reverse Transcriptase (Invitrogen). Real-time Taqman PCR was performed using an ABI 7700, with 1 x Mastermix (Eurogentec; Liège, Belgium; http://www.eurogentec.be), 500 nM of each primer, 200 nM Taqman probe, and 100 ng cDNA. Cycle conditions were as recommended by Eurogentec. Sequences were: porphobilinogen deaminase (PBGD)-FP: 5'-GGAGCCATGTCTGGTAACGG-3'; PBGD-BP: 5'-CCACGCGAATCACTCTCATCT-3'; PBGD Probe: 5'-TTTCTTCCGCCGTTGCAGCCG-3'; hrGFP-FP: 5'-CTTC GACATCCTGAGCCCC-3'; hrGFP-BP: 5'-GAAGTCGCTG ATGTCCTCGG-3'; and hrGFP Probe: 5'-TTCCAGTACGG CAACCGCACCTTC-3'.
For semiquantitative RT-PCR, total RNA was extracted from hES cells stably transfected with pSuper-HPRT vector and wild-type hES cells as described above. Total RNA was reverse transcribed using Superscript II Reverse Transcriptase. PCR reaction mixtures were prepared as described (Promega protocol for Taq polymerase) then were denatured at 94°C for 2 minutes and cycled at 94°C for 30 seconds, 60°C for 30 seconds (unless otherwise stated), and 72°C for 30 seconds. A final extension at 72°C for 10 minutes was performed after cycling. PCR primers were optimized for annealing temperature and a time course of cycle number was done, allowing semiquantitative comparisons within the log phase of amplification. Primers sequences were: HPRT-FP 5'-ATGCTGAGG ATTTGGAAAGG-3'; HPRT-RP 5'-TACTGGCGATGTCA ATAGG-3'; FGF-4-FP 5'-ACCTTGGTGCACTTTCTTCG-3'; FGF-4-RP 5'-CTCCACTGTTGCACCAGAAA-3' (55°C); Cyclin D1-FP 5'-ATGAACTACCTGGACCGCTTCC-3'; Cyclin D1-RP 5'-ACAAGAGGCAACGAAGGTCTGC-3'; ß-2 microglobulin (ß2M)-FP 5'-ACTGAAAAAGATGAGTA TGCCTGCCGTGTGAACC-3'; ß2M-RP 5'-CCTGCTCAGA TACATCAAACATGGAGACAGCACT-3' (55°C).
Mouse ES Cell Culture and Transfection
The mouse ES cell lines R1 and CGR8 were cultured as described [10]. For transitory transfection, 1 x 107 cells were trypsinized and washed once in medium containing fetal calf serum (FCS; PAA Laboratories; Pasching, Austria; http://www.paa.at) and twice in DMEM without serum. The cells were then electroporated with a mix of 40 µg of circular DNA at 300 volts and 960 µF using the Gene Pulser II System (Bio-Rad Laboratories; Hercules, CA; http://www.bio-rad.com). The mix of DNA was made of 35 µg pTP6 vector plus 5 µg of a control vector containing a ß-galactosidase reporter gene regulated by the CAGG promoter. The expression of the fluorescent reporter genes was analyzed 24 hours after electroporation. To assess experimental variations due to electroporation efficiency, a ß-galactosidase essay was done on the same amount of protein for each sample. The fraction of cells was then normalized in function to the ß-galactosidase activity. Stable transfection was performed as described [11]. Colonies selected for brightness using a Zeiss Axiovert 200 fluorescent microscope (Oberkochen, Germany; http://www.zeiss.com) were picked and expanded for further analysis.
Immunostaining
Human ES cells or their differentiated derivatives were fixed for 20 minutes in 4% paraformaldehyde and washed three times in PBS. Cells were incubated for 20 minutes at room temperature in PBS containing 10% goat serum (Serotec; Oxford, UK; http://www.serotec.co.uk) and subsequently incubated for 2 hours at room temperature with primary antibody diluted in 1% goat serum in PBS as follows: stage-specific embryonic antigen (SSEA)-1 (clone MC480, 1:50; Developmental Studies Hybridoma Bank [DSHB]; University of Iowa; http://www.uiowa.edu), SSEA-4 (clone MC813, 1:50; DSHB), Tra-1-60 (a gift from Dr. P.W. Andrews, 1:20), and ß-tubulin (a gift from Dr. S. Chandran). Cells were then washed three times in PBS and incubated with fluorescein-isothiocyanate-conjugated anti-mouse IgG or IgM (Sigma; 1:200 in 1% goat serum in PBS) for 2 hours at room temperature. Unbound secondary antibodies were removed by three washes in PBS. Hoechst staining was added to the first wash (Sigma, 1:10,000).
Flow Cytometry
Mouse and human ES cells were dissociated with trypsin (0.25%) plus EDTA (1 mM; GIBCO), washed once in medium containing FCS, and washed twice in PBS containing 0.1% serum (hES). The cells were then immediately analyzed on a FACSCalibur flow cytometer (Becton Dickinson) using CellQuest acquisition and analysis software (Becton Dickinson).
Statistical Analysis
All results are presented as mean ± standard error. Comparisons between experimental and control data were made using Student’s t-test (Excel, two-tailed). Responses were considered as different if p < 0.05.
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RESULTS AND DISCUSSION |
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TopAbstractIntroductionMaterials and MethodsResults and DiscussionReferences |
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We tested five different fluorescent proteins (destabilized d2GFP [Clontech; Franklin Lakes, NJ; http://www.cambrex.com], eGFP [Clontech], TauGFP [14], hrGFP [Stratagene; La Jolla, CA; http://www.stratagene.com], and DsRed2 [Clontech]) in mouse ES cells to identify those with maximal brightness and minimal toxicity. Each fluorescent reporter gene was subcloned downstream of the CAGG promoter into the pTp6 expression vector [14] (Fig. 1A
) in order to obtain equivalent levels of expression (data not shown). The CAGG promoter confers strong and ubiquitous expression in mouse (m)ES cells and in mice derived from them [12, 14, 15]. Moreover, by linking the fluorescent reporter gene to the puromycin resistance gene through an internal ribosome entry site (IRES) [16], strong selective pressure could be applied on transgene expression during isolation and subsequent passaging of transfected colonies. Of the fluorescent reporter genes tested, hrGFP was the brightest and one of the least toxic for mES cells (Fig. 1B, 1C). Furthermore, silencing of this gene was observed only at low levels in mES cells (Fig. 1D), and the green protein was resistant to paraformaldehyde fixation, allowing its use for immunochemistry (data not shown). As a complementary fluorescent reporter gene, DsRed2 was chosen for its brightness and protein stability, although it showed a tendency towards silencing when mES cells were passaged repeatedly without drug selection (Fig. 1D). While recent results suggest lentiviral vectors have a high transfection efficiency when used in hES cells [17], they have the disadvantage of being complex to generate and accommodate a limited size of transgene. However, there is no clear consensus on relative efficiencies of the more conventional approaches to transfection of hES cells [6, 18]. We thus examined the efficiency of several transfection reagents in preliminary experiments (data not shown). On this basis, we then compared Lipofectamine 2000 and Exgen 500 [6] for stable transgene expression in hES cells using hrGFP and DsRed2 (Fig 2A, 2C). Colonies generated using the hrGFP vector with Lipofectamine transfection (n = 40), as well as those generated using Exgen 500 (n = 10) retained green fluorescence after five passages without puromycin selection. However, only 6 of 48 DsRed2-expressing clones retained red fluorescence after five passages without selection. This reduction in fluorescence was lessened and sometimes reversed by resuming puromycin selection. Evidently, DsRed2 is sensitive to epigenetic silencing in hES cells, confirming the results obtained with mouse ES cells (Fig. 1D), thus requiring their continuous maintenance under drug selection. After 12 passages (2 months of culture), FACS analysis revealed that 99% of cells were fluorescent in several green- and red-fluorescing hES cell lines selected for optimal expression by fluorescent microscopy (Fig. 2B).
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, 2E). Also, RT-PCR was used to detect the expression of 
fetoprotein, albumin, and somatostatin, which are specific markers of endoderm [19] (data not shown). These results show that green and red fluorescent hES cells can be differentiated into all three germ layers. However, further studies will be needed to assess the functionality of differentiated cells derived from fluorescent hES cells. Finally, the pluripotent stem cell markers, Oct-4 and FGF-4, as well as the embryonic stem cell surface markers, SSEA-4, Tra-160, and Tra-181, were all expressed in fluorescent reporter transgene-expressing hES cells, whereas the differentiation marker SSEA-1 remained absent (data not shown). Furthermore, the karyotype and morphology of these cells were normal (data not shown), so despite the transfection and the selection process, the red- and green-fluorescing hES cells retained molecular markers of their undifferentiated state as well as the ability to differentiate into derivatives of each of the three germ layers. These findings provide additional compelling evidence that such genetic alteration of hES cells is compatible with maintenance of their pluripotency [6, 17, 18]. This expands the use of fluorescent reporter-expressing hES cell lines as powerful tools for cell tracing during tissue differentiation in in vitro and in animal models. The principal approach that has been used to study gene function in mammals is gene targeting by homologous recombination in mES cells [20]. Such an approach recently has been used with hES cells to generate a mutation in a single allele of the HPRT and Oct-4 genes [21]. However, generating a homozygous null mutation would require targeting of the second allele in the case of autosomal genes, or might be accomplished less reproducibly by gene conversion [22]. To circumvent this obstacle to perturbing gene function in hES cells, we evaluated the use of short-interfering double-stranded RNA, which has been shown to be efficient in mouse ES cells [23–26].
We first sought to define the efficacy of siRNA in hES cells by targeting the hrGFP and DsRed2 genes in the fluorescent cell lines generated above using transitory transfection of siRNAs. Fluorescent reporter genes provide distinct advantages as siRNA targets, in that they are capable of single cell assessment, can sustain multiple observations in living cells without perturbing viability and development, and they encode readily detectable proteins, whose absence does not alter cellular phenotype or provide a proliferative disadvantage. We designed three siRNA oligonucleotides targeted against the mRNA of each gene and transfected them into stably expressing green or red hES cell lines using Oligofectamine. After 48 hours, we observed the disappearance of fluorescence in a fraction (5%–10%) of the cells. To increase this effect, cells were retransfected 24 hours after the initial transfection. The siRNA effect was analyzed 48 hours later by counting the number of colonies containing negative cells using fluorescence microscopy (data not shown), by counting the negative cells using FACS (Fig. 3A), or by direct measurement of fluorescent gene expression using real time RT-PCR (Fig. 3C). Two of three anti-green siRNAs (14 and 22) reduced the expression of the green protein in 10% and 20% of the cells, respectively (Fig. 3A) (t-test, p = 0.007), and negative cells were detected in all the colonies treated with them (Fig. 3B).
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and data not shown). The effect of the anti-red siRNA was more difficult to assay by FACS because of the stability of the red protein (data not shown). At the RNA level, anti-green siRNA 22 induced a 40% decrease in the green fluorescent gene transcript (Fig. 3C), suggesting that siRNA had a greater impact at the RNA level than at the protein level (Fig. 3A and 3C). Thus, transient transfection of siRNA led to a decrease of gene products in a substantial fraction of cells; however, the knock down apparently did not reach a null level.
The siRNA-induced knock down of gene function was specific to the targeted gene. The transfection of anti-red siRNA into green hES cells or anti-green siRNA into red hES cells had no effect, respectively, on hrGFP or DsRed2 expression as compared with the controls. Nonspecific effects were similarly absent when we examined the expression of endogenous genes using real-time PCR (Fig 3D). Likewise, siRNA transfection into hES cells did not alter expression of pluripotent markers such as Oct-4 (data not shown), SSEA-4, or Tra-1-60 (Fig. 3E). Therefore, siRNA does not appear to activate the interferon response or the PKR kinase pathway, which would lead to a generalized suppression of transcription, as with long double-stranded RNAs, nor does it affect the expression of markers indicative of the pluripotent state. Therefore, these findings provided proof of principle that siRNA knocks down gene expression in hES cells, although the fluorescent reporter genes were challenging targets for detecting such effects. First, the sensitivity of detecting the fluorescent proteins made it difficult to assess the degree of knock down at the protein level. This, together with the short-term, heterogeneous nature of transitory transfection, meant that we could not isolate a homogenous population of affected cells on which to accurately assess the extent of knock down at the mRNA level. Thus, the transient transfection data did not allow us to distinguish between an intermediate knock down efficiency at the mRNA level in most of the cells versus a high-efficiency knock down in a fraction of the cells.
Accordingly, we sought to establish hES cell sublines stably expressing hairpin-loop siRNA as a means of obtaining homogenous populations of cells. The characteristics of a desirable protein target for such an analysis would include being selectable and being nonessential for viability, pluripotency, or differentiation. On this basis, we targeted the endogenous HPRT gene, which encodes an enzyme involved in purine metabolism [27]. Because 6-TG is metabolized to a toxic compound by HPRT, it can be used to select for and identify drug-resistant cells that are deficient in HPRT. Using this approach, we generated five hES sublines that were 6-TG resistant among 10 sublines generated by cotransfection and selection for puromycin resistance, and another five sublines that were obtained by direct 6-TG selection (Table 1; Fig. 4A). Interestingly, none of the 10 hES sublines were sensitive to medium containing hypoxanthine, aminopterin, and thymidine (HAT), which blocks DNA synthesis in cells that are HPRT deficient, suggesting that while HPRT levels are diminished sufficiently to provide 6-TG resistance, they are not reduced to a null level. Consistent with this hypothesis, the levels of HPRT mRNA (analyzed by semiquantitative RT-PCR) were diminished to the borderline of detectability in siRNA-expressing hES cells (Fig. 4B).
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). Therefore, stable expression of siRNA is also effective in differentiated cells derived from hES cells. Our analysis of HPRT gene expression in differentiated cells using semiquantitative RT-PCR revealed almost undetectable mRNA levels, whereas wild-type hES cells expressed HPRT at high levels (Fig. 5B).
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The potential for clinical application of hES cells is encouraging; however, this potential is still undergoing biological and technical evaluation. Beyond the obviously important task of controlling stem cell differentiation, realizing the clinical goal of cell-based transplantation therapies will require a means of matching donor tissues to the host immune system [32] and will also necessitate the derivation of new hES cell lines without using animal cells as feeders [33]. Each of these major tasks will benefit from robust approaches for regulating the expression of specific exogenous and endogenous genes. For example, use of siRNA could reveal the function of genes involved in the maintenance of pluripotency and thus lead to improvements in the efficiency of hES cell derivation, which would be necessary for generation of an hES cell bank. The results described here provide evidence that hES cells can undergo alteration in the expression of specific genes without effects on their pluripotency, a fundamental developmental characteristic of hES cells. Finally, because hES cells are capable of undergoing some of the differentiative events characteristic of early human development, these approaches for monitoring and modifying hES cell gene expression provide a useful model for human functional genomics. This in turn can reveal the molecular pathways of differentiation into the diversity of cells and tissues that ES cells are capable of forming.
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ACKNOWLEDGMENT |
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TopAbstractIntroductionMaterials and MethodsResults and DiscussionReferences |
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REFERENCES |
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