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a Cellular Neurobiology Branch, National Institute on Drug Abuse,
b Laboratory of Neuroscience, National Institute of Aging,
c Behavioral Neuroscience Branch, National Institute on Drug Abuse,
d Molecular Neuropsychiatry Branch, National Institute on Drug Abuse, Department of Health and Human Services, Baltimore, Maryland, USA
Key Words. Embryonic stem cells • Neuronal differentiation • Dopaminergic PA6 stromal cells • Hepatocyte growth factor
Correspondence: Xianmin Zeng, Ph.D., Development and Plasticity Section, Cellular Neurobiology Research Branch, National Institute on Drug Abuse, 333 Cassell Drive, Baltimore, Maryland 21224, USA. Telephone: 410-550-6565; Fax: 410-550-1621; e-mail: xzeng{at}intra.nida.nih.gov
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ABSTRACT |
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TH-positive cells derived from hESCs were postmitotic, as determined by bromodeoxyurindine colabeling. Differentiated cells expressed other markers of dopaminergic neurons, including the dopamine transporter, aromatic amino acid decarboxylase, and the transcription factors associated with neuronal and dopaminergic differentiation, Sox1, Nurr1, Ptx3, and Lmx1b. Neurons that had been differentiated on PA6 cells were negative for dopamine-ß-hydroxylase, a marker of noradrenergic neurons. PA6-induced neurons were able to release dopamine and 3,4-dihydroxphe-hylacetic acid (DOPAC) but not noradrenalin when depolarized by high K+.
When transplanted into 6-hydroxydopamine–treated animals, hES-derived dopaminergic cells integrated into the rat striatum. Five weeks after transplantation, surviving TH-positive cells were present but in very small numbers compared with the high frequency of TH-positive cells seen in PA6 coculture. Larger numbers of cells positive for smooth muscle actin, but no undifferentiated ES cells, were present after transplantation. Therefore, hESCs can be used to generate human dopaminergic cells that exhibit biochemical and functional properties consistent with the expected properties of mature dopaminergic neurons.
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INTRODUCTION |
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Human embryonic stem cells (hESCs), derived from the inner cell mass of preimplantation embryos, can proliferate indefinitely in culture and are able to differentiate into cell types of all three germ layers in vivo and in vitro [7]. These unique properties of hESCs make them an excellent candidate as a source of functional differentiated cells for cell replacement therapies, provided that reliable means of inducing differentiation to specific cell types can be achieved. The first step to develop such cell-based therapeutics from hESCs is to define the appropriate conditions for the in vitro differentiation of hESCs into useful somatic cell types. Indeed, many clinically relevant cell types have been generated in vitro from mouse ES cells, and functional improvements have been achieved in rodent models after transplantation of dopaminergic neurons derived from mouse ES cells [8,9]. Recently, differentiation of hESCs into multiple phenotypes, including neuronal [10–12], cardiomyogenic [13,14], hepatic [15], pancreatic [16], and hematopoietic [17] lineages, has also been described.
Although several research groups have reported the generation of neural precursors and neurons from hESCs [10–12], none of these methods produces postmitotic mesencephalic dopamine neurons at a high frequency. On the other hand, dopaminergic neurons have been efficiently generated from mouse ES cells by two different methods. One is a multiple-step method involving in embryoid body (EB) formation followed by sonic hedgehog (Shh) and fibroblast growth factor 8 (FGF8) treatment and selection for nestin-positive cells [9]. The second is a single-step method, which involves coculturing ES cells with a stromal cell line, PA6 [18]. Of the two techniques, the system of coculturing with the PA6 cell line has the advantage of simplicity and speed and can be used with cells from subhuman primates [19]. A simple and straightforward means of obtaining mature dopaminergic neurons from hESCs would be extremely valuable for both clinical application and for in vitro studies.
In this study we tested whether coculture with PA6 cells can induce differentiation of hESCs and found that a high frequency of dopaminergic neurons can be generated by this method. We show that the hES-derived cells generated appear to be authentic dopaminergic neurons of the mesencephalic type. These cells synthesize dopamine and release it under physiological stimuli, and these cells can be transplanted in a rat model of Parkinson’s disease.
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MATERIALS AND METHODS |
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Neural Differentiation
Neural differentiation of ES cells was induced by the mouse stromal cell line PA6 as described by Kawasaki et al [18], with some modifications. hESCs were cultured to form colonies on PA6 feeder cells in Glasgow minimum essential media (Invitrogen) supplemented with 10% KSR (Invitrogen), 1 mM pyruvate (Sigma), 0.1 mM nonessential amino acids, and 0.1 mM ß-mercaptoethanol. ES cell colonies were grown at a density of 1,000 colonies per 3-cm dish. The medium was changed on days 4 and 6 and every day thereafter.
Immunocytochemistry
Expression of stem cell and neuronal markers was examined by immunocytochemistry, and staining procedures were as described previously [21]. Briefly, the ES cells were fixed with 4% paraformaldehyde and permeabilized with 0.1% Triton X-100. After blocking, the cells were incubated with primary antibody. The primary antibodies and the dilution used were as follows: Nestin and bromodeoxyurindine (BrdU [BD Pharmigen, San Diego, CA, http://www.bdscience.com], 1:500 and 1:200); neural cell adhesion molecule (NCAM), synapsin, synaptophysin, and dopamine beta hydroxylase (DBH [Chemicon, Temecula, Ca, http://www.chemicon. com], 1:200, 1:20, 1:100, and 1:200); and neuron-specific class III beta tubulin (TuJ1) and tyrosine hydroxylase (TH [Sigma], 1:2000 and 1:2000, respectively). Localization of antigens was visualized by using respective secondary antibodies (Alexa fluor 594 or 488; Molecular Probes, Eugene, OR, http://www.probes.com).
For section staining, rat brains were fixed in ice-cold 4% of paraformaldehyde in phosphate-buffered saline (PBS).Cryostat sections (25 µm) were mounted onto gelatin-subbed slides. Sections were first exposed to 3% H2O2 (10 minutes) at room temperature, followed by rinsing with PBS. After blocking with 5% normal serum for 30 minutes, sections were incubated with primary antibody at 4°C overnight. Sections were then rinsed with PBS and incubated with the appropriate secondary antibody for 1 hour before visualizing.
ReverseTranscription–Polymerase Chain Reaction
Total RNA was extracted from undifferentiated or differentiated cells using RNA STAT-60 (Tel-Test Inc., Friendswood, TX). cDNA was synthesized using a reverse transcription kit (RETROscript,Ambion,Austin, TX) with 100 ng total RNA in a 20-µl reaction according to the manufacturer’s recommendations. RNase H 1 µl (Invitrogen) was added to each tube and incubated for 20 minutes at 37°C before proceeding to the reverse transcription–polymerase chain reaction (RT-PCR) analysis. The PCR primers are listed in Table 1
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Real-time PCR was used to quantify the levels of mRNA expression of Nurr1. PCR reactions were carried out using an Opticon instrument (MJ Research, Waltham, MA) and SYBR Green reagents (Roche Molecular Biochemicals, Indianapolis) according to the manufacturer’s instructions. The content of Nurr1was normalized to the content of the housekeeping gene cyclophilin. Standard curves were generated by cloning amplified products, using human cDNA as a template, into the PCR4 vector (TOPO TA cloning kit [Invitrogen]). The purified fragment solution was measured in a spectrophotometer, and the molecular number was calculated. Plasmid solutions were then used to generate serial dilutions. PCR analyses were conducted in triplicate for each sample. The primer pairs used for real-time PCR analyses were sequence verified. The acquisition temperature for each primer pair was 3°C below the determined melting point for the PCR product being analyzed.
Detection of Dopamine
BG01 cells were cultured on a PA6 cell layer for 3 weeks and rinsed twice with Hanks’ balanced salt solution (HBSS). To induce depolarization, 56 mM KCl was added into the cells for 15 minutes. The medium was then collected and stabilized with 0.1 mM EDTA and analyzed for dopamine and DOPAC. Dopamine and DOPAC levels were measured using an HPLC coupled to an ESA Coulochem II Detector (Model 5200, ESA, Inc., Chelmsford, MA) with a dual-electrode microdialysis cell. Data were analyzed using an ESA data station (Model 501). Samples (20 µl) were injected by an autosampler (CMA 280) into a C-18 reverse-phase column (3 µm; particle size, 3 x150 mm; Analytical MD-150 [ESA, Inc.]). The mobile phase for dopamine separation consisted of 75 mM NaH2PO4, 1.5 mM 1-octanesulfonic acid-sodium salt, 10 µM EDTA, and 7% acetonitrile (pH 3.0, adjusted with H3PO4). Dopamine and DOPAC were quantified using the reducing (–250 mV) and oxidizing electrodes (350 mV), respectively, and then calculated as nanomolar concentration. The limit of detection was approximately 0.3 pg per injection.
Focused Microarray Analysis
The nonradioactive GEArrayTM Q series cDNA expression array filters for human stem cell genes pathway genes and mouse cytokine genes (Hs601 and MM-003N, SuperArray Inc, http://superarray.com) [22] were used according to the manufacturer’s protocol. The biotin 2'-deoxyuridine-5'-triphosphate (dUTP)–labeled cDNA probes were specifically generated in the presence of a designed set of gene-specific primers using total RNA (4 µg per filter) and 200 U MMLV reverse transcriptase (Promega, San Luis Obispo, CA, http://www.promega.com). The array filters were hybridized with biotin-labeled probes at 60°C for 17 hours. After that, the filters were washed twice with x2 standard saline citrate (SSC)/1% SDS and then twice with x0.1 SSC/1% SDS at 60°C for 15 minutes each. Chemiluminescent detection steps were performed by incubation of the filters with alkaline phosphatase-conjugated streptavidin and CDP-Star substrate. Array membranes were exposed to X-ray film. Quantification of gene expression on the array was performed with ScionImage software. cDNA microarray experiments were done twice with new filters and RNA isolated at different times. Results from the focused array were independently confirmed, and the array itself was validated using procedures previously described [23].
Transplantation
Adult Fisher 344 rats were anesthetized with chloral hydrate (0.4 g/kg, i.p.) and injected with 6-hydroxydopamine (6-OHDA; 9 µg/4 µl in normal saline containing 0.2 mg/ml ascorbic acid) over 4 minutes into the left medial forebrain bundle (–4.4 mm anterior/posterior [AP], 1.2 mm medial/lateral [ML] relative to bregma and 7.8 mm below the dura). Five weeks after lesioning, the unilaterally 6-OHDA–lesioned rats were tested for rotational behavior in response to subcutaneous methamphetamine injections (2.5 mg/kg) in an automated rotometer. Animals that showed significant unilateral bias (>300 turns ipsilaterally) were used for transplantation studies.
ES cells were cultured on PA6 cells, and colonies were isolated after 8 (n = 8) or 22 (n = 7) days of differentiation using papain dissociation kit (Worthington Inc, Lakewood, NJ) according to the manufacturer’s instructions. In addition, two animals received transplants or undifferentiated hESCs. Isolated cells were resuspended in 50% pellet volume and 50% media. Using an UMP2 syringe pump (World Precision Instruments, Inc.), an intrastriatal (0-mm AP and 2.5-mm ML relative to bregma and 6 mm below the dura) injection of 5 µl of the cell suspension was injected over 5 minutes through a 25-µl Hamilton syringe with a pulled-glass micropipette. After injection, the needle was left for an additional 2 minutes and then slowly withdrawn. Animals were euthanized for immunostaining after 5 weeks of daily s.c. injections with 10 mg/kg of cyclosporin-A and 2 mg/kg vibramycin [24].
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RESULTS |
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Neural differentiation of BG01 cells was initiated by culturing on a feeder layer of PA6 cells. By 6 days of culture on PA6 cells, most hES colonies had generated an outgrowth of elongated cells (Fig. 1A). By 10 days, extensive process formation was observed on the edges of most of the hESC colonies (Fig. 1B). After 2 weeks of differentiation, cells that migrated out of the colonies formed a monolayer and displayed a bipolar or multipolar morphology characteristic of neurons, with extensive development of fine processes (Fig. 1C). Prominent fiber bundles formed by processes emanating from the colonies were frequently observed.
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). Nestin was expressed during neural differentiation as well as in undifferentiated hESCs (data not shown). NCAM was first detected after 5 days of differentiation and increased for the next several days. NCAM peaked at day 14, at which time 92% of the colonies were NCAM positive (Fig. 1G). Likewise, colonies positive for TuJ1 first appeared at day 7, and the percentage of positive colonies increased during the following week of differentiation, so that by day 16, approximately 92% of the colonies were positive for TuJ1 (Fig. 1G). No Oct4- or SSEA-positive colonies were found after 3 weeks of differentiation on PA6 cells (data not shown).
hESCs Differentiate into Dopaminergic Neurons
We next tested whether TH-positive cells can be generated from hESCs by coculturing with PA6 cells. After 3 weeks of differentiation, approximately 87% of colonies contained TH-positive cells, and a high percentage of the cells in most of the colonies were TH positive (Figs. 2A–2C). TH-positive cells first appeared between 8 and 10 days after induction and peaked at approximately 18 days (Fig. 1G). Many cells were also positive for the synaptic proteins synapsin and synaptophysin (Figs. 2D–2E). Less than 10% of colonies contained GAD65-positive cells (not shown).
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). BrdU incorporation for 24 hours showed that after 20 days of differentiation, most cells (72%) were not dividing. Double immunostaining of TH and BrdU showed no colocalization of TH and BrdU, indicating that the TH-positive cells were postmitotic (Fig. 2G). BrdU-positive cells were usually found outside colonies or around the edges of colonies, and very few BrdU-positive cells were present within colonies.
The expression of neuronal markers in PA6-induced cells was additionally analyzed by RT-PCR. Several dopaminergic markers, including TH, dopamine transporter (DAT), aromatic amino acid decarboxylase (AADC), and quinoid dihydropteridine reductase, were detected by RT-PCR (Fig. 3A). Receptors such as TrkB and TrkC, GFRA1, and the Shh-mediated protein smoothed (Smo) were also expressed in the PA6-induced cultures but not in undifferentiated hESCs. Several markers of non-dopaminergic neuronal sub-types were also present. The cholinergic markers choline acetyltransferase (ChAT) and vesicular acetylcholine transporter (VAChT), and the glutamatergic marker glutaminase (two isoforms, GAC and KGA) were detected in differentiated cells. DBH was not detected.
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). Most of the stem cell markers that were expressed in undifferentiated cells, including Oct4, hTERT, UTF-1, and Dppa5, were not detected or were downregulated in the differentiated cultures (Fig. 3A).
Because of the importance of Nurr1 in the generation of dopaminergic neurons, we performed quantitative RT-PCR to compare Nurr1 expression in PA6-induced neurons to undifferentiated hESCs. Nurr1 transcript was detected in both undifferentiated hESCs and PA6-induced differentiated cells, but expression of the Nurr1 transcript was threefold higher in the cultures differentiated for 3 weeks (Fig. 3B). Absolute numbers of copies of the Nurr1 transcript were 8 and 24 per 1,000 copies of the cyclophilin transcript for the undifferentiated cells and PA6-induced neurons, respectively.
Reverse-phase HPLC was used to examine the ability of hESC-derived neurons to release dopamine. After 3 weeks of differentiation, dopamine (2.8 ± 0.4 nM, from 107 to 108 cells) was released into the medium in response to a K+ depolarizing stimulus (Fig. 4), indicating that functional dopaminergic neurons were present in the culture. Noradrenalin (NA) was not detected in three of the four samples tested, although a small possible peak was seen in one sample, which is shown in Figure 4.
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, Table 2). These included 14 markers for stem and differentiated cells, 22 growth factors and receptors, adhesion molecules, and cytokines, six extracellular matrix molecules, and eight others (Fig. 5, Table 2). In particular, Sox1, Map2, TrkC, and NT3 were expressed at higher levels in the differentiated cultures, which is consistent with the results obtained by RT-PCR.
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). Greater expression of vascular endothelial growth factor (VEGF) and FGF7 mRNAs was also found in PA6 cells (Fig. 6A). RT-PCR confirmed that transcripts of HGF, VEGF, and FGF7 were increased in PA6 cells compared with MEFs (Figs. 6B, Table 3).
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Because bone morphogenic protein (BMP) and serum have been demonstrated to inhibit neural differentiation in mouse and primate ES cells, we also tested whether BMP and serum have a similar effect on neural differentiation of hESCs induced by PA6 cells. When BMP4 or FCS was added to the medium, less than 10% of the colonies were TH-positive after 3 weeks of differentiation, indicating that BMP4 and serum suppressed neural differentiation of hESCs.
Transplantation of PA6-Induced Cells into the Rat Brain
To test whether PA6-induced dopaminergic cells derived from hESCs could be integrated in vivo, we transplanted these cells into the striatum of rats that had received unilateral lesions with 6-OHDA 4 weeks before transplantation and had been tested for amphetamine-induced rotation to verify lesion completeness. Two differentiation stages were used, early neural precursors after 8 days of coculture on PA6 cells and postmitotic neurons after 22 days of coculture on PA6 cells. Two animals received transplants of undifferentiated hESCs.
Immunohistochemical analysis was performed 5 weeks after grafting, and human cells were found in all brains transplanted with either 8- or 22-day cells. Some TH-positive human cells were also identified in both groups of animals (Figs. 7A–7E). Numbers of TH-positive cells were small, but a few clearly stained cells were found throughout the graft sites. In addition, larger numbers of weakly positive cells and cells with unclear morphology were also seen (Fig. 7F). An approximation of relative numbers of TH-positive cells was made by counting the total number of TH-positive cell profiles (clear cell morphology with unstained nucleus) for 10 sections from each animal (Table 3). Greater numbers of TH-positive cells were found in the brains of animals transplanted with 22-day differentiated cells compared with the animals transplanted with 8-day differentiated cells.
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; Table 5). Larger numbers of SMA-positive cells were found in the animals that received transplants of undifferentiated cells (Fig. 7I; Table 5). Thus, coculture with PA6 cells did not preclude the differentiation of a significant number of cells with mesodermal properties. |
DISCUSSION |
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Transplantable neural precursors have recently been isolated from hESCs by two research groups [11,12]. Reubinoff et al. [11] used a method in which hESC colonies with cells containing short processes were manually extracted, followed by serum-free culture, whereas Zhang et al. [12] induced neural differentiation via EB formation followed by culturing in the presence of FGF2. Although these neural precursors differentiated into neuronal phenotypes in vivo and in vitro, only a few neurons expressed TH. Park and colleagues [24,25] also recently reported that some TH-positive cells could be generated from hESCs by a combination of factors, including brain-derived neurotrophic factor (BDNF), transforming growth factor-alpha, bFGF, and retinoic acid. Each of these methods requires multiple steps and growth factors. In contrast, the present method resulted in the differentiation of a high percentage of dopaminergic neuronal cells from hESCs, which is necessary for potential therapeutic application. Nonetheless, differentiation was not uniform, and cell types other than dopaminergic neurons were present.
The number of TH-positive cells present in the brain of the transplanted animals was small, and far greater numbers of surviving transplanted cells would presumably be required to obtain functional changes in, for example, animal models of Parkinson’s disease [26,27]. This was not simply an issue of the total number of TH-positive cells that survived after transplantation, but in addition the frequency of TH-positive cells was much lower than that seen in vitro with PA6 cell coculture. Moreover, surviving non–central nervous system cells were present in the brains of transplanted animals, so that homogeneous differentiation into TH-positive cells was not achieved by this method. The number of surviving dopaminergic neurons following transplantation to the brain was small despite the fact that many thousands of dopaminergic cells were implanted. This suggests that the transplantation procedure or the host brain environment was not ideal for the survival of transplanted human dopaminergic neurons generated by the present procedure. Fundamental improvements are likely to be required to result in a functionally effective transplantation procedure. In addition, the possibility of fusion between host and donor cells [28] was not addressed in the present study.
Dopaminergic neurons with slightly different properties are found in the mesencephalon, hypothalamus, olfactory bulb, and retina [29]. Dopaminergic neurons in general express particular and unique patterns of proteins and mRNA molecules. Among these markers are several unique transcription and differentiation factors, some of which promote the development of region-specific subtypes of dopaminergic neurons [30]. Consistent with this notion, the tyrosine hydroxylase promoter contains elements that are specifically activated in different brain regions [31].
Several factors can stimulate dopaminergic neuron development. Nurr1, Lmx1b, and Ptx3 transcription factors are mesencephalon specific, whereas the messenger molecules Shh and FGF8 seem to promote the dopaminergic phenotype irrespective of brain region [30, 32, 33]. Other transcription factors, including Sox1, influence neuronal specification per se [34]. Nurr1 is essential for development of the dopaminergic neurotransmitter function in mesencephalic neurons [35]. A second pathway involving Lmx1b and Ptx3 seems to be important for expression of other aspects of the dopaminergic neuronal phenotype [36]. Shh and FGF8 induce the dopaminergic phenotype when present in appropriate concentrations in specific brain regions [33, 37, 38]. The receptor for Shh is Smo, which is inhibited by a second protein patched (ptch) when Shh is absent [39–41]. There are several alternatively spliced forms of FGF8, which differentially interact with certain forms of FGF receptors FGFR2, FGFR3, and FGFR4 [42].
In addition to developmental pathways, dopamine neurons can be defined by neurotransmitter function and response to growth factors. Markers for mature dopamine neurons thus include proteins directly involved in dopamine neurotransmitter biosynthesis and function, especially TH, AADC, DHPR, GTPCH, and DAT. Dopamine neurons also respond to the growth factors BDNF and GDNF [43–46]. The receptor for BDNF is Trkb [47,48]. GDNF and the related molecules neurturin, artemin, and persephin interact with GFRA1, GFRA2, GFRA3, GFRA4, and cRET [49,50].
The expression of markers for dopaminergic neurons, as well as other neuronal markers, in hESC-derived differentiated cells was examined by immunocytochemistry, RT-PCR, and microarrays. The markers associated with the mature dopaminergic neuronal phenotype TH, DAT, AADC, GTPCH, PCD, DHPR, and VMAT2 were expressed. The growth factor receptors TrkA, TrkB, TrkC, GFRA1, GFRA2, GFRA3, p75R, and c-ret and the Shh receptors Ptch and Smo were also present. Transcription factors Nurr1, Ptx3, Lmx1b, and Sox-1 associated with dopaminergic and neuronal differentiation were expressed by the PA6 cell–induced cells. Nurr1 was detectable in both undifferentiated hESCs and PA6-differentiated cells, but quantitative RT-PCR verified that a threefold increase in expression was associated with differentiation. The reason that Nurr1 and other markers, which are generally associated with differentiation, were expressed in undifferentiated BG01 cells is unclear; however, it has been reported that hESC lines express some markers that are not expressed in mouse ES cells [51], and we cannot exclude the possibility that a small number of differentiated cells were present within the hESC colonies. DBH was not expressed in the TH-positive cells by immunostaining or RT-PCR, and little or no NA was released by KCl stimulation, supporting the conclusion that PA6-induced hESC-differentiated cells are dopaminergic rather than noradrenergic. In addition to dopaminergic markers, cholinergic (ChAT and VAChT) and glutamatergic (GAC and KGA) markers were detected in the induced neurons, indicating the potential for generation of multiple neuronal types by this method.
On the other hand, undifferentiated ES cell markers (hTERT, Oct3/4, Dppa5, and UTF-1) were not expressed in the differentiated cultures, indicating that undifferentiated hESCs do not persist in hESC cultures differentiated on PA6 cells. Although SMA-positive cells were present in the brain after transplantation, indicating that differentiation on PA6 cells did not preclude mesodermal differentiation, Oct4-positive cells did not persist or appear after transplantation. This is important for potential clinical use, because even a few undifferentiated ES cells could form teratomas when transplanted.
Kawasaki et al. [18] originally used the term stromal cell-derived inducing activity (SDIA) to describe the dopaminergic neuronal-inducing effect of PA6 cells. To identify the factors responsible for SDIA, a focused microarray was used to compare the expression of growth factors and cytokines between MEFs and PA6 cells. Very few growth factor messages differed between the two cell types, but the HGF transcript was sixfold higher in PA6 cells, and higher expression of FGF7 and VEGF was also seen. These growth factors alone or in combination were not, however, able to induce dopaminergic differentiation in hESCs. Therefore, PA6 cells may stimulate dopaminergic differentiation by a complex combination of growth factors, including other unidentified components. It is also possible that the supporting environment provided by PA6 cells, or an interaction between PA6 cells and hESCs, plays a role in their neuron-inducing activity. Interestingly, there has been one report that HGF stimulates the survival and differentiation of dopaminergic neurons [52]. Therefore, it remains a possibility that HGF contributes to the dopaminergic neuronal-inducing effect of PA6 cells.
In summary, coculture with PA6 cells is a simple and efficient method of generating postmitotic dopaminergic neuron-like cells from hESCs. Dopaminergic cells derived from hESCs are functionally and biochemically similar to normal dopaminergic neurons and survive transplantation for at least 5 weeks.
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REFERENCES |
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