Assessment of Contractility of Purified Smooth Muscle Cells Derived from Embryon - 《stemcells》干细胞学杂志干细胞之家



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发表于 2009-3-5 00:04 |只看该作者 |倒序浏览 |打印

作者：Sanjay Sinhaa,b, Brian R. Wamhoffa,b, Mark H. Hoofnaglea,b, James Thomasa,b, Ronald L. Neppla,b, Thomas Deeringa, Brian P. Helmkeb,c, Douglas K. Bowlesc,d, Avril V. Somlyoa,b, Gary K. Owensa,b作者单位：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 : s( Q( O' W9 i# @$ i8 f








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      【摘要】
   The aims of this study were to develop a method for deriving purified populations of contractile smooth muscle cells (SMCs) from embryonic stem cells (ESCs) and to characterize their function. Transgenic ESC lines were generated that stably expressed a puromycin-resistance gene under the control of either a smooth muscle -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. & m2 j4 i4 f1 d' g8 v  J  ]
      【关键词】 Embryonic stem cell Smooth muscle Embryoid body Teratoma Contraction
   INTRODUCTION

Smooth muscle cells (SMCs) play a key role in physiology and pathology because they constitute the principal layer of all SMC tissues, and they are known to play a critical role in a large number of major human diseases, including atherosclerosis, hypertension, asthma, and cancer (reviewed in . To fully understand the phenotypic and molecular changes associated with SMC-related diseases, it is essential to first have a comprehensive understanding of the mechanisms underlying normal development and maturation. These developmental mechanisms include the process by which multipotential precursor cells commit to the SMC lineage, coordinately express an array of genes that ultimately define the mature SMC phenotype, and integrate with surrounding cells and tissues to form a functional smooth muscle tissue. An appreciation of normal SMC developmental mechanisms may also contribute to novel cell-based therapies for SMC-related diseases as well as for tissue engineering and reconstruction. However, successful implementation of these strategies will require the development of methods for production of SMCs that exhibit normal contractile and other developmental/growth properties.& Z9 _% F6 [' z  Y! l
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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  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  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.0 i6 Y' r! w' `; _3 H
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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.
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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; K9 _/ g6 b' K: h& Y

Generation of Transgenic ESCs
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ESC lines that stably expressed a puromycin-resistance gene under the control of SMC-specific promoters were generated by electroporation. Briefly, DNA plasmids incorporating the ¨C2,560¨C2,784 SMA promoter expressing puromycin-N-acetyltransferase (SMA-PAC) or a ¨C4,200¨C11,600 SM-MHC promoter/PAC (SM-MHC-PAC) construct, as previously described . 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.
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EB Culture and SMC Isolation# X4 I8 j# ], ]3 g# ~9 N

ESCs were maintained in ESC medium (Dulbecco¡¯s modified eagle¡¯s medium supplemented with 15% fetal bovine serum . 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¡ãC for 20¨C30 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¨C3 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).+ m- |8 o1 X! O  x+ n

Immunofluorescence and Immunoblotting
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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)  and visualized using an enhanced chemiluminescence kit (GE Healthcare, Little Chalfont, Buckinghamshire, U.K., http://www.gehealthcare.com).7 o  G# x& w' L" u$ }

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 . 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¨C000-003; 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)., A6 h$ J9 ~$ y8 {$ M1 S# C) ?
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Reverse Transcription-PCR) G7 G+ T3 u, R% q  e

RNA extraction from ESC-derived puromycin-selected cells, EBs, precursor ESCs, and differentiated and undifferentiated A404 cells . 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.

Table 1. Probes and primer sequences used for real time RT-PCR

Measurement of Physiological Responses3 v, W& O( h. N2 Y# s

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 . 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.
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Whole-Cell Voltage Clamp; q7 y6 g# Y  ~; K5 e

Whole-cell Ca2  currents were determined with a standard whole-cell voltage-clamp technique as previously described . 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' _8 b9 M7 F6 S3 M; S; P

Contractile function was assessed by generating reconstituted muscle fibers as described by Oishi et al. . Briefly, cultured D3 ESCs, APSCs, or MPSCs were trypsinized, pelleted, and resuspended at a density of 8¨C10 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¡ãC) 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¨C1.0 mm in diameter) muscle fiber between the two poles, with the central region now having a round or oval cross-section/ w+ G2 y, _& e) B2 V$ q, e& |
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Figure 1. SMC-specific protein expression by ESC-derived puromycin-resistant cells. APSCs (A¨CC) and MPSCs (D¨CF) derived by enzyme dispersion of day-28 EB and subsequent puromycin selection were fixed and immunostained for SMA and SM-MHC. For the immunoblots (G), cell lysates were harvested from APSCs and MPSCs after either overnight selection with puromycin or after four passages in puromycin-containing medium. Tissue lysate was also obtained from mouse aorta as a positive control. Immunoblotting for SM-MHC demonstrated that both APSCs and MPSCs had significant levels of SM-MHC protein which was reduced only slightly after four passages. A second, slower migrating band in the APSC and MPSC lanes likely represents antibody cross-reactivity to non-smooth muscle myosin isoforms in the setting of high substrate concentrations. Scale bar = 50 µm. Abbreviations: APSC, smooth muscle -actin puromycin-selected cell; EB, embryoid body; ESC, embryonic stem cell; MPSC, smooth muscle-myosin heavy chain puromycin-selected cell; SMA, smooth muscle -actin; SMC, smooth muscle cell; SM-MHC, smooth muscle-myosin heavy chain.
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Small strips (0.75¨C0.80 mm wide, 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" . Once mounted to the force transducer, the strip was allowed to equilibrate in HEPES-buffered Krebs¡¯ solution at 37¡ãC for 30 minutes prior to agonist stimulation. The bathing solution of the strip was maintained at 37¡ãC throughout the duration of the experiment.$ }) T! S) c( o' t; r- Q

Phenotypic changes of the D3 ESC-, APSC- and MPSC-derived reconstituted muscle fibers were evaluated by immunofluorescence. Strips (750¨C800 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¡ãC 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 ¨C24¡ãC 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.! K# x  O! T& q; Q, ^! ?! p

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¡ãC 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.# t! W0 w8 a$ O- U$ F
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RESULTS( q% y* R5 n: L5 n

Isolation of Purified ESC-Derived SMCs

ESCs within the EB recapitulate many aspects of embryonic development and differentiate into a wide variety of cell types , which complicates further analysis of SMC-specific developmental mechanisms. Our initial goal was to develop a system that combined the development of contractile SMCs within the normal EB environment, with the ability to subsequently purify SMC populations.
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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 , 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.+ L) M( Y1 f( b$ N% S( W

Immunofluorescence studies of the selected cells showed that purified APSCs stained for the SMC selective markers, SMA and SM-MHC (Fig. 1A¨C1F). 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.5 m6 P7 y) O) D) w5 w5 S0 j8 d1 T

Figure 2. Cell sorting of ESC-derived SMCs and a primary SMC line for levels of SMC markers. APSCs (A), MPSCs (B), or rat aortic SMCs (C) were stained with FITC-conjugated SMA and Alexa 647-conjugated SM-MHC antibody and analyzed by flow cytometry. The three cell types had similar homogenous staining for the highly specific SMC marker, SM-MHC. Conversely, whereas the rat aortic SMCs and APSCs also demonstrated similar staining for SMA, the MPSCs had a bimodal distribution of SMA with approximately 40% of cells staining weakly for SMA, whereas the remainder exhibited strong staining. Thus, APSCs and MPSCs both expressed SMC-specific proteins although the MPSCs comprised at least two distinct populations. Abbreviations: APSC, smooth muscle -actin puromycin-selected cell; FACS, fluorescence-activated cell sorting; FITC, fluorescein isothiocyanate; FL,; MPSC, smooth muscle-myosin heavy chain puromycin-selected cell; SM, smooth muscle; SMA, smooth muscle -actin; SMC, smooth muscle cell; SM-MHC, smooth muscle-myosin heavy chain.

Purified ESC-Derived SMCs Produced with the SMA (APSC) or SM-MHC (MPSC) Promoters Showed Similar Levels of Expression of SMA and SM-MHC as Established Primary SMC Lines' U1 [$ G  I( V# s; Y- A2 b

Results of immunofluorescence studies suggested that virtually all puromycin-selected cells expressed SMC markers. To better quantify SMC differentiation in this system, the proportion of puromycin-purified cells expressing SMC markers and level of SMC marker gene expression was determined using dual parameter flow cytometric analyses. Expression patterns for SM-MHC were similar in APSCs, MPSCs, and a primary line of aortic SMCs (Fig. 2). Thus, both the SMA 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.! y3 c2 B6 x, m1 j

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 .
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Figure 3. mRNA expression profile of ESC-isolated cells compared with precursor ESC, EB, and a P19-derived A404 cell line. Quantitative real time reverse transcription-polymerase chain reaction was used to measure the expression of a wide variety of SMC-specific markers in APSCs, MPSCs, their precursor EBs, and ESCs. A P19-derived A404 cell line that expresses SMC-specific markers on stimulation with retinoic acid was used as a positive control. Control genes specific to cardiomyocytes (cardiac -actin and cardiac -MHC), skeletal myocytes (Myf5), and neurons (neuro D) were also measured. The data suggest an SMC identity for APSCs and MPSCs and confirm a high degree of specificity within these populations. Abbreviations: APSC, smooth muscle -actin puromycin-selected cell; EB, embryoid body; ESC, embryonic stem cell; MHC, myosin heavy chain; MPSC, smooth muscle-myosin heavy chain puromycin-selected cell; SMA, smooth muscle -actin; SMC, smooth muscle cell; SM-MHC, smooth muscle-myosin heavy chain.

ESC-Derived SMCs Showed Agonist-Induced Calcium Transients6 n* k1 _- Y& E1 ]" {

A sine qua non of mature SMCs is their ability to contract and generate force in response to vasoactive agonists. Agonists induce contraction, in part, by increasing intracellular Ca2  levels, which trigger the SMC contractile apparatus . To test whether purified ESC-derived SMCs exhibited functional Ca2  responses, APSCs and MPSCs were loaded with Fluo-4 AM, a Ca2 -sensitive dye, and their response to depolarization and vasoactive agonists was monitored using fluorescence microscopy. As depicted in Figure 4A, depolarization with 80 mM KCl to stimulate Ca2  influx through L-type voltage-gated Ca2  channels resulted in an increase in intracellular Ca2  levels as did treatment with the potent vasoconstrictor angiotensin II (1 µM) and endothelin-1 (10 nM; Fig. 4C). Figure 4D and 4E represent typical L-type voltage-gated Ca2  channel current traces and current-voltage (I-V) relationships using 10 mM Ba2  as the charge carrier. Similar results were obtained in MPSCs. However, Fluo-4 Ca2  responses were not consistent among different cells within APSC and MPSC populations, and some cells displayed no response to the agonists, suggesting that the purified cells, although expressing SMC markers, were nevertheless heterogeneous in nature.
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Figure 4. ESC-derived SMCs show changes in intracellular Ca2  in response to depolarization and vasoactive peptides and express functional L-type voltage-gated Ca2  channels. APSCs were loaded with the Ca2  indicator Fluo-4 AM, and changes in intracellular Ca2  were determined by fluorescence microscopy (A¨CC). (A): Relative changes in intracellular Ca2  (Fluo-4 intensity) of APSC exposed to 80 mM KCl, endothelin-1 (10 nM), and angiotensin II (1 µM). Relative changes in Fluo-4 intensity 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. (B, C): Raw digital fluorescent images of baseline (F0) and endothelin-1 treatment, respectively. Representative L-type voltage-gated calcium channel current traces from APSCs (, 10 mM Ba2 , external) and typical whole-cell current-voltage (I-V) relationships (E). Similar responses were obtained for MPSC (data not shown). Abbreviations: APSC, smooth muscle -actin puromycin-selected cell; ESC, embryonic stem cell; MPSC, smooth muscle-myosin heavy chain puromycin-selected cell; SMC, smooth muscle cell.* o/ X( k3 T0 u* C, ?( u7 t
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ESC-Derived SMCs Seeded Within Collagen Gels Showed Morphogenesis into an Smooth Muscle Tissue and Agonist-Induced Force Development1 B* e+ U! U3 {% ?, [1 ^

Next, we investigated whether APSCs and MPSCs were capable of contracting and generating force. Artificial muscle fibers were generated by seeding puromycin-purified cells in a collagen gel and cultured for 7 days. Strips cut from the muscle fibers were mounted on a force transducer, and the contractile response to vasoactive agonists was studied (Fig. 5A, see Materials and Methods). We found that reconstituted muscle fibers generated from either APSCs or MPSCs exhibited a K -induced contraction (depolarization, 80 mM KCl) similar to intact smooth muscle tissue . Further analysis of these artificial muscle strips by fluorescence microscopy revealed that cells near the periphery of the fiber had elongated slightly in the direction of force, whereas those in the central region appear randomly orientated in an interconnected meshwork biased toward the direction of force (Fig. 5E, 5F). Reconstituted control muscle fibers generated from undifferentiated progenitor D3 ESCs formed a dumbbell-shaped fiber by day 7 and, with the instrumentation at the most sensitive setting, did not exhibit a detectible contractile response to either K  or endothelin-1 (Fig. 5D). Furthermore, immunolabeling with rhodamine-phalloidin (Fig. 5G) indicated a random alignment within the collagen-gel matrix in comparison with the more longitudinally aligned APSC and MPSC fibers (Fig. 5E, 5F).

Figure 5. Contraction of ESC-derived SMCs in response to depolarization and endothelin-1 in reconstituted fiber preps. Artificial muscle fibers were prepared by seeding collagen gels with APSC (B, E), MPSC (C, F), or D3 progenitor cells (D, G) as depicted in (A) and described in Materials and Methods. After 7 days, small strips (1.5 mm x 0.75¨C0.80 mm) were cut from the "dumbbell"-shaped fiber and the ends tied to wire hooks of a force transducer and suspended in Krebs¡¯ solution (A). (B, C, D): Isometric tension recordings for APSC, MPSC, and D3 progenitor cell fibers, respectively. (E, F, G): Longitudinal slices of the fiber preparation stained with rhodamine-phalloidin and imaged by fluorescence microscopy in APSC, MPSC, and D3 progenitor cell fibers, respectively. Contraction was measured in response to 80 mM KCl and endothelin-1. APSC and MPSC, but not D3 progenitor cells, displayed typical SMC contractile responses in which endothelin-1-induced contraction was inhibited by the Rho kinase inhibitor Y-27632. Abbreviations: APSC, smooth muscle -actin puromycin-selected cell; ET-1, endothelin-1; MPSC, smooth muscle-myosin heavy chain puromycin-selected cell; SMC, smooth muscle cell.
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Continuous Negative Selection Is Required to Eliminate Tumorogenic Potential of Purified ESC-Derived SMCs In Vivo  j+ e; B. L9 U9 N

The preceding studies defined the APSCs and MPSCs as relatively pure and mature SMC populations. We next investigated the behavior of these cells in vivo. Purified cells obtained by an 18-hour selection from day-28 ESC-EBs were labeled with di-I, resuspended in matrigel, and injected subcutaneously into mice genetically matched to the D3 ESC line. Large subcutaneous tumors developed in these mice at the injection sites within 2¨C3 weeks (Fig. 6A, inset). Histology confirmed multiple differentiated cell types consistent with formation of teratomas (Fig. 6B). Teratoma growth at 3 weeks was eliminated by maintaining puromycin selection of APSCs or MPSCs for 72 hours before injecting the cells in vivo, as depicted in the intact small matrigel mass near the left hind limb of the mouse in Figure 6D (black outline). The image in Figure 6D represents the same region in the mouse depicted in the inset of Figure 6A (white arrow).
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Figure 6. Potential for teratoma formation in ESC-derived SMC transplantation can be eliminated by continued negative selection. APSCs were isolated from day-28 EBs by puromycin treatment for 16¨C18 hours (A¨CC) or 72 hours (D¨CF), stained with DiI, and injected subcutaneously near the hind limb of a mouse for 3 weeks. (A): Sixteen- to 18-hour selection resulted in tumor formation, as depicted by the mouse in the inset (white arrow) and gross dissection of this tumor. However, 72-hour selection did not promote tumor progression, as depicted by the higher magnification light image of the matrigel plug attached to the surface of the hind-limb musculature (, black line). The image in (D) represents the identical region of the mouse in the inset of (A), denoted by the white arrow. Sixteen- to 18-hour selection (B) and 72-hour selection (E) representative histological cross-sections of the tumor in (A) and matrigel plug in (D). (C) and (F) represent fluorescent images of DiI (red) and antibody detection of SMA (green), nuclei (blue, DAPI). The white arrows in (C) denote vessel-like structures that colocalized DiI and SMA staining. Scale bars = 1 cm (A), 0.35 cm (D), 1 mm (B, E), 200 µm (C), and 25 µm (F). Abbreviations: APSC, smooth muscle -actin puromycin-selected cell; DAPI, 4,6-diamidino-2-phenylindole; ESC, embryonic stem cell; MPSC, smooth muscle-myosin heavy chain puromycin-selected cell; SMA, smooth muscle -actin; SMC, smooth muscle cell.

We then examined whether SMC-containing tissues were present at either the teratoma-containing or teratoma-free injection sites. SMC-coated blood vessels, which frequently contained red blood cells, were identified in the teratomas by histology and immunostaining for SMC markers (Fig. 6C). Many of these vessels had mural cells derived from the di-I-labeled injected cells (Fig. 6C, white arrows). In contrast, when cells purified by extended puromycin selection were used for injection, discrete blood vessel formation was not detected in the absence of teratoma development. However, groups of cohesive spindle-shaped di-I-labeled cells were seen, some of which were positive for SMA (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.1 Q! h6 O9 ]; k% T9 f, D* G' F

DISCUSSION

Results of the present studies define a novel system for purifying ESC-derived SMC populations from the EBs that exhibit contractile properties equivalent to SMC tissues in vivo. Although a number of putative in vitro SMC differentiation systems have previously been described wherein multipotential cells are induced to express a variety of SMC marker genes upon stimulation with particular cytokines, growth factors, or extracellular matrix components  did show contractile properties in SMC-like cells derived from rat embryonic neural crest stem cells derived by primary culture. However, the expression of the neuronal progenitor marker, nestin, by the cells in these studies suggested that they were either a mixed population or that the cells were not typical SMCs and retained some stem cell properties. Thus, major advantages of the ESC-EB system as described here are that it appears to recapitulate all necessary environmental cues required for programming totipotential ESCs into fully functional SMCs and permits purification of the resulting SMCs using a stably incorporated negative selection system. Consequently, this ESC-EB system offers unique advantages for purposes of studying developmental regulation of the SMC lineage, as well as for potential SMC tissue engineering.6 u7 ?: u8 L$ {4 Y& e+ d, @8 o; x
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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 . 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.
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Previous studies, including our own , 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 . 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.$ x% H8 Z1 H$ n. R# J
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SUMMARY! S* s0 n* e/ Z0 t; E
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These studies describe for the first time a method for generating relatively pure populations of SMCs, which display appropriate excitation and contractile responses to vasoactive agonists, from ESCs and are also free from pluripotential cell contamination. This system has tremendous potential for a wide range of functional genomic studies as well as vascular tissue engineering.4 \; o9 {& F  g( U! v& O

DISCLOSURES7 W3 [9 U5 u; u/ [6 `
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The authors indicate no potential conflicts of interest.) W0 \) [; {1 U9 V9 ^) o
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ACKNOWLEDGMENTS
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S.S. and B.R.W. contributed equally to this work. This work was supported by National Institutes of Health Grants HL R01 HL57353 and R21 HL071976 to G.K.O. and P01 HL19242 to A.V.S. and G.K.O., American Heart Association (AHA) post-doctoral fellowship Grant 0120501U to S.S., and AHA scientist development grant to B.R.W. We thank Mary McCanna, Rupande Tripathi, and Diane Raines for their excellent cell culture assistance; Joanne Lannigan for cell sorting assistance; and Sherri VanHoose for histological specimen sectioning.
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