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EMBRYONIC STEM CELLS |
aDepartment of Molecular Physiology and Biological Physics,
bThe Robert M. Berne Cardiovascular Research Center,
cBiomedical Engineering, The University of Virginia, Charlottesville, Virginia, USA;
dVeterinary Biomedical Sciences, University of Missouri, Columbia, Missouri, USA
Key Words. Embryonic stem cell • Smooth muscle • Embryoid body • Teratoma • Contraction
Correspondence: Gary K. Owens, Ph.D.,Department of Molecular Physiology and Biological Physics, University of Virginia, PO Box 800736, Charlottesville, Virginia 22908-0736, USA Telephone: 434-924-2652; Fax: 434-982-0055; email: gko{at}virginia.edu
Received January 2, 2006;
accepted for publication March 28, 2006.
First published online in STEM CELLS EXPRESS April 6, 2006.
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ABSTRACT |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionSummaryDisclosuresAcknowledgmentsReferences |
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-actin (SMA) or smooth muscle-myosin heavy chain (SM-MHC) promoter. Negative selection, either overnight or for 3 days, was then used to purify SMCs from embryoid bodies. Purified SMCs expressed multiple SMC markers by immunofluorescence, immunoblotting, quantitative reverse transcription-polymerase chain reaction, and flow cytometry and were designated APSCs (SMA-puromycin-selected cells) or MPSCs (SM-MHC-puromycin-selected cells), respectively. Both SMC lines displayed agonist-induced Ca2+ transients, expressed functional Ca2+ channels, and generated contractile force when aggregated within collagen gels and stimulated with vasoactive agonists, such as endothelin-1, or in response to depolarization with KCl. Importantly, subcutaneous injection of APSCs or MPSCs subjected to 18 hours of puromycin selection led to the formation of teratomas, presumably due to residual contamination by pluripotent stem cells. In contrast, APSCs or MPSCs subjected to prolonged puromycin selection for 3 days did not form teratomas in vivo. These studies describe for the first time a method for generating relatively pure populations of SMCs from ESCs which display appropriate excitation and contractile responses to vasoactive agonists. However, studies also indicate the potential for teratoma development in ESC-derived cell lines, even after prolonged differentiation, highlighting the critical requirement for efficient methods of separating differentiated cells from residual pluripotent precursors in future studies that use ESC derivatives, whether SMC or other cell types, in tissue engineering applications.
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INTRODUCTION |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionSummaryDisclosuresAcknowledgmentsReferences |
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Unfortunately, the study of SMC development has been hindered by the lack of suitable in vitro models wherein embryonic cells can be efficiently and reproducibly induced to differentiate into SMCs that exhibit full contractile properties. Several systems have been described in which multipotential cells give rise to SMC-like cells that express one or more SMC markers. For example, 10T1/2 cells express smooth muscle -actin (SMA) and SM22 when treated by transforming growth factor (TGF)-ß1 [3], whereas the neural crest-derived MONC-1 cells express a variety of SMC-specific genes on serum stimulation [4] or TGF-ß1 treatment [5]. In addition, we have described a system in which P19 embryonal carcinoma cells [6] or derivatives thereof [7] can be induced to form SMC lineages by treatment with all-trans retinoic acid. Although these studies certainly provide some information on the mechanisms of SMC gene expression, it is unclear whether such systems accurately model true SMC development and maturation. Indeed, major weaknesses include uncertainties regarding the developmental origins of these cells and the fact that cells fail to exhibit convincing evidence of the defining property of mature SMCs (i.e., contractility).
Embryonic stem cells (ESCs) are pluripotent because they develop into all cell types in vivo when injected into a developing blastocyst [8]. In vitro, when placed in aggregate, ESCs form an embryoid body (EB) that can recapitulate many developmental processes, and although overall morphogenesis is disrupted, they form all known cell types [9]. As part of their developmental program, EBs have been shown to form regions of visibly spontaneously contractile SMCs [10–12]. Little or no external inducers are required, and cell programming and appropriate environmental cues seem to be provided by the developing EB itself. This system is extremely powerful for investigating underlying developmental mechanisms important for SMC development. For example, we have recently used this system to show that endogenous TGF-ß1 signaling through Smads 2 and 3 was required for SMC development from ESCs [11]. Moreover, in separate studies, we used this EB system along with studies in chimeric knockout mice to show that the potent SRF (serum response factor) coactivator myocardin, which is exclusively expressed in SMCs and cardiomyocytes, is not required for SMC development [13]. These results are in marked contrast to those obtained in conventional knockout mice and suggest that the failure to develop SMCs in myocardin null mice [14] was secondary to some other defect. Indeed, a major advantage of the ESC-EB system is that because of their small size, cell lineage programming is not dependent on development of a fully functioning cardiovascular system as is the case with mouse embryos beyond embryonic day 10.5 (E10.5), which confounds the ability to distinguish primary (i.e., cell autonomous) versus secondary consequences of gene knockouts.
The cellular heterogeneity that is an intrinsic feature of the EB-ESC system is an advantage in terms of replicating the heterogeneous cell-cell and cell-matrix interactions that occur in vivo. However, the multiplicity of different cell types is a major disadvantage when investigating cell type-specific mechanisms. For instance, it is difficult to distinguish gene expression and responses to stimuli specifically in SMCs from the background response in many other cell types. It is also difficult to quantitatively assess the differentiated function of cells or tissues in this system to determine the full developmental potential for any given cell lineage. Finally, to use this system to generate SMCs for therapeutic use, it will be imperative to be able to derive a pure cell population.
The aims of this study were to develop a method of deriving a pure population of functional contractile SMCs from ESCs and to quantitatively assess their function. We generated transgenic ESC lines that stably expressed a puromycin-resistance gene under the control of SMC-specific promoters and used negative selection to isolate a pure population of SMCs from the ESC-EB system. Of major interest, the purified SMCs were shown to express multiple SMC-specific genes and contained all the cellular components required for the generation of contractile force in response to various contractile stimuli. Use of the ESC-EB system described herein should have tremendous utility in elucidating the role of a specific gene in the cellular and molecular regulation of SMC differentiation and contractile function using specific gene-null ESC lines.
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MATERIALS AND METHODS |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionSummaryDisclosuresAcknowledgmentsReferences |
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A promoter expressing puromycin-N-acetyltransferase (SMA-PAC) or a –4,200–11,600 SM-MHC promoter/PAC (SM-MHC-PAC) construct, as previously described [7, 11], were linearized, gel-purified using a Qiaex II gel purification kit (Qiagen, Valencia, CA, http://www1.qiagen.com), and cotransfected with pIRESneo (Clontech, Mountain View, CA, http://www.clontech.com), a cytomegalovirus (CMV) promoter/neomycin-resistance cassette, into D3 ESCs via electroporation as outlined in Hogan et al. [15]. Colonies derived from single cells under G418 selection were amplified and screened for the presence of the PAC transgene by polymerase chain reaction (PCR). Multiple ESC lines containing both the CMV promoter/neomycin and PAC transgenes were selected for both SMA-PAC and SM-MHC-PAC constructs.
EB Culture and SMC Isolation
ESCs were maintained in ESC medium (Dulbecco’s modified eagle’s medium supplemented with 15% fetal bovine serum [Gibco-BRL, Gaithersburg, MD, http://www.gibcobrl.com], L-glutamine, 110 µM ß-mercaptoethanol, pyruvate, nonessential amino acids, penicillin/streptomycin, and 1,000 U/ml leukemia inhibitory factor [LIF; Chemicon, Temecula, CA, http://www.chemicon.com]), and EBs were generated as previously described in Sinha et al. [11]. In brief, 800 ESCs were aggregated in a 10-µl hanging drop for 72 hours then cultured in suspension for a further 3 days in ESC-EB differentiation medium (similar to ESC media but with 20% serum, minus LIF and ß-mercaptoethanol). On day 6, ESC-EBs were plated onto a surface coated with 0.1% porcine gelatin (Sigma, St. Louis, http://www.sigmaaldrich.com) and treated with 10 nM all-trans retinoic acid from days 7 to 10. At the indicated times, SMCs were isolated by enzyme dispersion followed by selection by neomycin. Initially, 30 to 40 adherent EBs were washed twice with phosphate-buffered saline (PBS; Gibco-BRL). Then, the colonies were treated with an enzyme mixture containing 1 mg/ml collagenase (Invitrogen Collagenase Type IV) and 0.5x trypsin and incubated at 37°C for 20–30 minutes. The cells were triturated several times during the incubation to ensure that the EB aggregates were dispersed into single cells, and the enzymes were inactivated by the addition of 2–3 volumes of EB media. The cell suspension was passed through a 70-µm filter; this single-cell suspension was then plated with 0.5 mg/ml puromycin, and SMCs were selected overnight. Dead cells were removed by a PBS wash on the day after plating, and the cells were either used for experiments immediately or expanded under continuous low-dose puromycin selection (0.05 mg/ml). These SMC-like cell lines were designated as either SMA puromycin-selected cells (APSCs) or SM-MHC puromycin-selected cells (MPSCs).
Immunofluorescence and Immunoblotting
For immunofluorescence, ESC-derived, puromycin-selected cells were fixed with 4% paraformaldehyde and permeabilized using 100% ice-cold methanol. Primary antibodies were monoclonal anti-SMA-fluorescein isothiocyanate (FITC) (clone 1A4, F3777; Sigma) at 1:500 and rabbit polyclonal anti-SM-MHC (BT-562; Biomedical Technologies, Inc., Stoughton, MA, http://www.btiinc.com) at 1:100. A Cy3 (indocarbocyanine)-labeled donkey anti-rabbit secondary antibody (Jackson ImmunoResearch Laboratories, Inc., West Grove, PA, http://www.jacksonimmuno.com) used at 1:100 was used to detect anti-SM-MHC antibody localization, and images were captured using an inverted Nikon Eclipse 80i microscope (Melville, NY, http://www.nikon.com) equipped with a Melles Griot IM series argon ion confocal laser (Carlsbad, CA, http://www.mellesgriot.com). Cell lysates for immunoblotting were harvested as previously described either after overnight selection with puromycin or alternatively after four passages. Reduced and denatured samples were run on a 10% SDS gel and transferred to a PVDF (polyvinylidene difluoride) membrane. Blots were probed with an anti-chicken SM-MHC primary antibody (1:2,000) [16] previously shown to be specific for SM-MHC [17] and visualized using an enhanced chemiluminescence kit (GE Healthcare, Little Chalfont, Buckinghamshire, U.K., http://www.gehealthcare.com).
Flow Cytometry
APSCs and MPSCs were grown to confluence in a 75-cm2 flask using EB medium with 0.05 mg/ml puromycin. Rat-cultured aortic SMCs were grown to confluence and serum-starved as described [18]. Cells were trypsinized, fixed for 15 minutes in 2% paraformaldehyde, pelleted by centrifugation at 300g, and permeabilized by resuspension in ice-cold methanol. Cells were blocked in a solution of PBS, 2% bovine serum albumin, 5% donkey serum, and 0.01 mg/ml rabbit immunoglobulin G (IgG) (11–000-003; Jackson ImmunoResearch Laboratories, Inc.) for 2 hours. Cells were then stained for 1 hour with FITC-conjugated anti-SMA mouse IgG2a (F3777; Sigma) at 8 µg/ml, and Alexa 647-conjugated (A-20173; Invitrogen, Carlsbad, CA, http://www.invitrogen.com) anti-SM-MHC rabbit polyclonal IgG (BT-562; Biomedical Technologies, Inc.) at 0.25 µg/ml or IgG2a-FITC (F6522; Sigma) and Alexa 647-conjugated rabbit IgG at similar concentrations. Cells were analyzed using a FACSCalibur dual-laser benchtop cytometer (Becton, Dickinson and Company, Franklin Lakes, NJ, http://www.bd.com). Ten thousand events were collected from each experimental group and were gated using forward and side-scatter to eliminate debris and aggregates. Positive boundaries were then set so that IgG controls were less than 1% positive (online supplemental data).
Reverse Transcription-PCR
RNA extraction from ESC-derived puromycin-selected cells, EBs, precursor ESCs, and differentiated and undifferentiated A404 cells [7] was carried out using Trizol (Invitrogen) according to the manufacturer’s instructions, and reverse transcription (RT)-PCR was performed as previously described [11]. Highly sensitive and quantitative assessment of gene expression was achieved using Taqman chemistry probes on an iCycler (Bio-Rad, Hercules, CA, http://www.bio-rad.com). Probe and primer sequences are described in Table 1.
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Intracellular Ca2+ Imaging. APSCs or MPSCs isolated by puromycin selection were cultured on a 35-mm circular coverslip and loaded with the calcium-sensitive fluorophore, Fluo-4 AM (2.5 µM; Molecular Probes, Eugene, OR, http://probes.invitrogen.com), and mounted into a constant-flow superfusion chamber as previously described [19]. Observation of intracellular Ca2+ changes were performed using a DeltaVision deconvolution microscope system (Applied Precision, Issaquah, WA, http://www.api.com) through a x40/0.75 NA lens on an inverted microscope (Olympus, Tokyo, http://www.olympus-global.com). Fluo-4 was excited at 490 nm by a mercury arc lamp, and fluorescence emission was measured at 528 nm. Cells were constantly superfused with physiological saline solution (PSS) containing the following (in mM): 2 CaCl2, 143 NaCl, 1 MgCl2, 5KCl, 10 HEPES, and 10 glucose (pH 7.4). Images were acquired at 15-second intervals during PSS perfusion and endothelin-1, angiotensin II, high potassium or ionomycin exposure. Data analysis was performed with ImagePro Plus (Media Cybernetics, Inc., Silver Spring, MD). Data are expressed as F/F0, where F is the absolute fluorescence value in an area of interest during treatment and F0 is the baseline average (three images) prior to treatment.
Whole-Cell Voltage Clamp
Whole-cell Ca2+ currents were determined with a standard whole-cell voltage-clamp technique as previously described [20]. Briefly, cells were loaded into a superfusion chamber and initially superfused with PSS containing (in mM) 0.1 CaCl2, 138 NaCl, 1 MgCl2, 5 KCl, 10 HEPES, and 10 glucose (pH 7.4) during gigaseal formation with 2- to 5-M
heat-polished glass pipettes. Pipettes contained (in mM) 120 CsCl, 10 tetraethylammonia chloride (TEACl), 1 MgCl2, 20 HEPES, 5 Na2ATP, 0.5 Tris GTP, and 10 EGTA (pH 7.1). After whole-cell configuration, the superfusate was switched to PSS with TEACl substituted for NaCl and 10 mM Ba2+ as the charge carrier.
Reconstituted Smooth Muscle Fiber Preparations
Contractile function was assessed by generating reconstituted muscle fibers as described by Oishi et al. [21]. Briefly, cultured D3 ESCs, APSCs, or MPSCs were trypsinized, pelleted, and resuspended at a density of 8–10 x 106 cells per ml in an ice-cold collagen solution containing 0.8 mg/ml rat-tail collagen I (BD Biosciences, Bedford, MA, http://www.bdbiosciences.com), 0.02 M NaHCO3, and 10x Medium 199 (Sigma) at a final concentration of 1x. A 1.25-ml aliquot of the collagen-cell suspension was dispensed into a rectangular Sylgard 184 (Dow Corning, Midland, MI, http://www.dowcorning.com) mold with a well (0.8 x 5.0 x 0.5 cm deep) with two poles (2 mm in diameter) 4 cm apart and was placed into an incubator (5% CO2, 37°C) for 30 minutes or until the collagen-cell suspension was gelled. All three gelled collagen-cell suspensions (APSC, MPSC, and D3 ESC) were covered with and maintained in EB media. Incubation for 7 days led to the formation of a "dumbbell"-shaped (
0.75–1.0 mm in diameter) muscle fiber between the two poles, with the central region now having a round or oval cross-section
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1.5 mm long) were cut longitudinally with a razor knife from reconstituted muscle fibers on day 7 and transferred to HEPES-buffered Krebs’ solution. The ends of the strips were tied with silk monofilaments to wire hooks connected to a force transducer (AE 801; SensoNor A.S., Horten, Norway, http://sni.nextframe.net/index.html) and a length-adjusting device and mounted on a "bubble plate" [22]. Once mounted to the force transducer, the strip was allowed to equilibrate in HEPES-buffered Krebs’ solution at 37°C for 30 minutes prior to agonist stimulation. The bathing solution of the strip was maintained at 37°C throughout the duration of the experiment.
Phenotypic changes of the D3 ESC-, APSC- and MPSC-derived reconstituted muscle fibers were evaluated by immunofluorescence. Strips (750–800 µm wide, 1.5 mm long) were cut adjacent to the strips used for agonist-induced force measurements and fixed in 4% paraformaldehyde in PBS for 4 hours. Sucrose embedding for cryostat sectioning consisted of immersion in 5% sucrose for 8 hours, followed by 15% sucrose overnight at 4°C prior to embedding in Tissue-Tek O.C.T. (Sakura Finetek U.S.A., Inc., Torrance, CA, http://www.sakuraus.com) and rapid freezing in liquid nitrogen-cooled Freon. Longitudinal sections (10 µm thick) were cut at –24°C with a Jung Frigocut 2,800E (Leica, Deerfield, IL, http://www.leica.com), mounted on slides, and then labeled for smooth muscle markers. Briefly, longitudinal slices were rehydrated in PBS for 15 minutes, blocked with 3% bovine serum albumin in PBS for 1 hour, and labeled with rhodamine-phalloidin at 1:1,000 dilution (Molecular Probes) for 1 hour in blocking buffer.
In Vivo Injection
Prior to in vivo injection, APSCs, MPSCs, and D3 ESCs were labeled with di-I (1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate; Molecular Probes) according to manufacturer’s protocols. One x 106 cells in a 0.1 ml 50% matrigel suspension was subcutaneously injected into each hindflank of a syngeneic host. After 8 weeks of incubation, the animals were euthanized, and the site of injection was dissected out en bloc and frozen in liquid nitrogen. For immunofluorescent analysis of implanted cells, frozen tissue samples were placed in OCT compound and sections with a thickness of 10 microns were made and mounted on glass slides. For SMA immunofluorescence, tissue sections were incubated for 1 hour at room temperature with mouse anti-SMA antibody conjugated to FITC (Sigma) and mounted in Hardset mounting media containing 4,6-diamidino-2-phenylindole (DAPI) (Vector Laboratories, Burlingame, CA, http://www.vectorlabs.com). For SM-MHC immunofluorescence, sections were incubated with rabbit anti-SM-MHC overnight at 4°C in a humidified chamber and labeled with donkey anti-rabbit IgG conjugated to FITC. Fluorescent images were obtained on a Zeiss microscope (Carl Zeiss, Jena, Germany, http://www.zeiss.com) using RSImage analysis software version 1.7.3 (Roper Scientific, Inc., Duluth, GA, http://www.roperscientific.com). Images were acquired using a x40 objective field of view under the TRITC (tetramethylrhodamine B isothiocyanate)/rhodamine (300-msecond exposure time) filters, FITC filters (1.0-second exposure time), and DAPI filters (100-millisecond exposure time). Four random slides representative of two different cell implants in two different mice were evaluated for each cell type.
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RESULTS |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionSummaryDisclosuresAcknowledgmentsReferences |
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The SMA-PAC or SM-MHC-PAC constructs were co-transfected with a neomycin-resistance cassette into D3 ESCs. Multiple lines of neomycin-resistant ESC clones that stably integrated the puromycin-resistance gene under the control of promoter-enhancers of the SMC selective genes SMA or SM-MHC were amplified. EBs were generated from the transgenic ESC lines, and at day 15 or day 28 the EBs were disaggregated using enzyme digestion into a single-cell suspension and plated in the presence of puromycin, either overnight or continually for 3 days. We have previously shown that the SMA and SM-MHC promoter-enhancers are sufficient to completely recapitulate expression patterns of endogenous genes in transgenic mice [24, 25], and thus they were used to confer cell-specific puromycin resistance to developing SMCs in the EB. The surviving putative SMC lines were designated APSCs or MPSCs according to which promoter-enhancer was used for selection. The proportion of cells surviving overnight puromycin selection was estimated by counting the number of cells plated and the number of surviving adherent cells after 16 hours. For both APSCs and MPSCs, between 5% and 15% of plated cells survived overnight selection.
Immunofluorescence studies of the selected cells showed that purified APSCs stained for the SMC selective markers, SMA and SM-MHC (Fig. 1A–1F). Purified APSCs and MPCSs displayed SMC-like morphology and appearance with a well developed actin stress fiber network (Fig. 1A, 1D). Immunoblotting analyses revealed that significant amounts of the highly selective SMC protein, SM-MHC, were expressed in the purified cell population after overnight puromycin selection (Fig. 1G). Comparable levels of SM-MHC proteins were detected in both APSCs and MPSCs, and only minor reductions in protein levels were detected after four passages. These results demonstrated that the cells obtained by the described method were SMC-like and maintained this identity after initial passaging.
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A (APSC) or SM-MHC (MPSC) Promoters Showed Similar Levels of Expression of SMA and SM-MHC as Established Primary SMC LinesA and the SM-MHC promoters were able to select cell populations that expressed the highly specific SMC marker, SM-MHC. Paradoxically, SM-MHC promoter selected cells or MPSCs exhibited a bimodal SMA expression pattern with approximately 60% of cells with high levels of SMA and approximately 40% with low expression (Fig. 2B). The distinct bimodal distribution demonstrated here suggests that two distinct cell populations are isolated by the SM-MHC promoter, one similar to vascular SMCs and another with lower SMA levels.
Purified ESC-Derived SMCs Produced with the SMA (APSC) or SM-MHC (MPSC) Promoters Expressed All Known SMC Marker Genes Tested
An important benchmark for assessing SMC development is that cells must express a wide range of SMC markers. Real-time RT-PCR studies demonstrated that APSCs and MPSCs expressed multiple SMC markers at significantly increased levels compared with the day-28 mixed cell EBs they were purified from or with their precursor ESCs (Fig. 3). SMC mRNA marker expression was quantitatively comparable with differentiated A404 cells, another line of undifferentiated cells which we have previously shown to express a variety of SMC-specific genes when treated with all-trans retinoic acid [7]. In addition, these studies confirmed that there was little or no expression of cardiac, skeletal, or neuronal markers, demonstrating that the selection systems used in the present studies was highly efficacious in deriving highly enriched cultures of ESC-derived SMCs. Interestingly, myocardin expression was enriched in ESC-derived SMCs purified using the SMA but not the SM-MHC promoter. This was surprising given that expression of the SM-MHC promoter is restricted to SMCs. We speculate that this may be due to several factors. First, the lack of a "step up" in myocardin expression, but not other SMC-specific genes, may be because SMC isolation eliminates cardiomyocytes that would be an additional source of myocardin expression in the EB, but not of other SMC-specific genes. Second, it is possible that differences in the developmental timing of activation of the SM-MHC promoter versus SMA within the EB resulted in derivation of SMC populations that exhibit differential dependence on myocardin as compared with the myocardin-related transcription factors MRTFA/B (MKL1/2). Indeed, we recently showed that homozygous myocardin knockout ESCs can develop into SMCs both in vitro within EBs as well as in vivo within chimeric knockout mice [13]. In addition, several groups have shown that knockout of MRTF B is associated with selective defects in differentiation of neural crest-derived SMCs as compared with those derived from other embryological origins [26, 27].
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A (Fig. 6F) and SM-MHC (data not shown) in both APSC- and MPSC-extended puromycin selection cell implants. Thus, although it was not possible to determine in these studies whether APSCs or MPSCs that were purified by extended puromycin selection invested SMC-containing tissues, it appears that at least some of these cells retained their SMC phenotype in vivo in as much as they maintained expression of the SMC selective markers SMA and SM-MHC. In addition, results suggest that injection of purified ESC-derived SMCs alone does not result in formation of a stable SMC tissue, presumably because of a lack of appropriate local environmental or morphogenic cues at the subcutaneous injection site. |
DISCUSSION |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionSummaryDisclosuresAcknowledgmentsReferences |
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We used two different SMC-specific promoters, SM-MHC and SMA, to enrich SMC populations from mixed cell lineages induced within EBs. In vivo, the SMA promoter is expressed in all three muscle types during development but is restricted to smooth muscle by the time of birth [24]. The SM-MHC promoter is more specific to SMCs and is active only in SMCs throughout embryogenesis and in a small subset of cardiomyocytes during very early development [17, 25]. There is also evidence that expression of the SMA promoter can be highly promiscuous in many cultured cell systems, including in fibroblasts treated with TGF-ß1 [34]. Thus, there was a risk that cardiomyocytes, skeletal myocytes, and/or other cell types might have been isolated using these promoters. However, this did not appear to be the case; puromycin-resistant cells isolated in the present studies simultaneously expressed the definitive SMC marker gene SM-MHC and SMA by dual-flow cytometric assays and failed to express significant levels of markers for other cell lineages, as determined by RT-PCR in puromycin-resistant ESC-derived SMCs. These results suggest that SMA and SM-MHC expression within the developing EB exhibited a high degree of SMC specificity/selectivity and/or the unique conditions employed in the present studies selected against other cell types.
Previous studies, including our own [11, 20], on the EB-ESC system have provided information on aggregate gene expression within the entire mass of differentiating cells. However, it is difficult to determine whether changes in expression in response to an intervention are due to a change in gene expression within individual cells, change in cell numbers, or both. The ability to isolate pure populations of cells should allow an accurate estimation of how different interventions affect both gene expression and cell numbers. In addition, pure SMC populations lend themselves to SMC-specific biochemical studies that may be impossible or extremely difficult to interpret in the presence of mixed cell populations. We caution, however, that the purified cells may comprise various subpopulations of SMCs (such as vascular, gut, or other organ-specific SMC lineages) and thus the system presented in this manuscript is best suited to studies investigating mechanisms that are common to all SMCs.
The ESC-EB system described herein has major potential for directly testing the functional roles of candidate genes implicated in SMC development or function. Because ESCs are relatively amenable to genetic modification, one can readily test whether heterozygous or homozygous knockout of genes of interest either block development of SMC from ESC or, alternatively, alter functional properties of differentiated SMCs derived using this system. Indeed, this system has several major advantages to complement conventional knockout experiments in mice, including (a) relatively rapid throughput because the system can exploit any existing knockout ESC line (or ESCs derived from knockout mice) already generated either in individual labs or systematically by large collaborations such as the International Gene Trap Consortium (http://www.igtc.ca/), (b) SMC lineage determination within the EB system appears to recapitulate developmental controls in vivo yet is cheaper to use as an initial investigative modality, and (c) a more rigorous assessment of cell autonomous versus non-cell autonomous gene functions as compared with conventional knockout mice, because cell lineage programming in the ESC-EB is independent of the requirement for a fully functioning cardiovascular system.
Because stem cells are easily expanded and may be induced to form a variety of differentiated cell types, they have considerable therapeutic potential in the tissue engineering field. However, undifferentiated ESCs have the potential to form teratocarcinomas in vivo [35] and this is a major concern in considering their therapeutic application. The risk appears to be reduced if the ESCs are first differentiated prior to transplantation; as Barberi et al. [36] reported, induction of ESCs into neural precursors before central nervous system transplantation did not result in teratoma formation. However, EB differentiated up to day 15 still contained pluripotent cells resulting in teratomas when transplanted into the liver [37]. Our data extend these findings to show that pluripotential cells were present in EBs even after 28 days of differentiation and were eliminated only by prolonged negative selection for at least 3 days. Thus, these studies highlight the critical requirement for highly efficient methods of separating differentiated cells from residual pluripotent precursors in future studies that use ESC derivatives, whether SMC or other cell types, in tissue engineering applications.
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SUMMARY |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionSummaryDisclosuresAcknowledgmentsReferences |
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DISCLOSURES |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionSummaryDisclosuresAcknowledgmentsReferences |
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ACKNOWLEDGMENTS |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionSummaryDisclosuresAcknowledgmentsReferences |
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REFERENCES |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionSummaryDisclosuresAcknowledgmentsReferences |
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