P19 Progenitor Cells Progress to Organized Contracting Myocytes After
Shared by: afawe45t3qa
J ENDOVASC THER 377 2006;13:377–388 FELLOWS’ COMPETITION, FIRST PLACE, LABORATORY SCIENCE P19 Progenitor Cells Progress to Organized Contracting Myocytes After Chemical and Electrical Stimulation: Implications for Vascular Tissue Engineering Oscar Abilez, MD1,2; Peyman Benharash, MD1,2; Emiko Miyamoto, BS1,3; Adrian Gale, BS1,4; Chengpei Xu, MD, PhD1,2; and Christopher K. Zarins, MD1,2 1Bio-X Program and Departments of 2Surgery (Division of Vascular Surgery), 3Biomedical Engineering, and 4Mechanical Engineering, Stanford University, Stanford, California, USA. Purpose: To test the hypothesis that a level of chemical and electrical stimulation exists that allows differentiation of progenitor cells into organized contracting myocytes. Methods: A custom-made bioreactor with the capability of delivering electrical pulses of varying ﬁeld strengths, widths, and frequencies was constructed. Individual chambers of the bioreactor allowed continuous electrical stimulation of cultured cells under microscopic observation. On day 0, 1% dimethylsulfoxide (DMSO), known to differentiate cells into myocytes, was added to P19 progenitor cells. Additionally, for the next 22 days, electrical pulses of varying ﬁeld strengths (0–3 V/cm), widths (2–40 ms), and frequencies (10–25 Hz) were continuously applied. On day 5, the medium containing DMSO was exchanged with regular medium, and the electrical stimulation was continued. From days 6–22, the cells were visually assessed for signs of viability, contractility, and organization. Results: P19 cells remained viable with pulsed electrical ﬁelds 3 V/cm, pulse widths 40 ms, and pulse frequencies from 10 to 25 Hz. On day 12, the ﬁrst spontaneous contractions were observed. For individual colonies, local synchronization and organization occurred; multiple colonies were synchronized with externally applied electrical ﬁelds. Conclusion: P19 progenitor cells progress to organized contracting myocytes after chem- ical and electrical stimulation. Incorporation of such cells into existing methods of pro- ducing endothelial cells, ﬁbroblasts, and scaffolds may allow production of improved tis- sue-engineered vascular grafts. J Endovasc Ther 2006;13:377–388 Key words: bioengineering, bioreactor, cell culture, chemical stimulation, electrical stim- ulation, stem cell, tissue engineering, vascular graft In 2003, cardiovascular disease afﬂicted 71 million patients annually undergo procedures million people in the United States, and the requiring arterial vascular grafts,2,3 which rep- number of inpatient cardiovascular proce- resents $2.1 billion per year for these proce- dures was about 6.8 million.1 Of these, 1.4 dures (based on the most recent data for av- Dr. Abilez was supported in part by a Stanford University Dean’s Postdoctoral Fellowship. The authors have no commercial, proprietary, or ﬁnancial interest in the products or companies described in this article. The annual ISES Endovascular Fellows’ Research Awards Competition held on February 13, 2006, at International Con- gress XIX on Endovascular Interventions (Scottsdale, Arizona, USA) evaluated participants on both their oral and written presentations. ISES congratulates the 2006 winners in the categories of Laboratory Science and Clinical Research. Address for correspondence and reprints: Oscar Abilez, MD, Stanford University Clark Center E350, MC 5431, 318 W. Campus Dr., Stanford, CA 94305-5431 USA. Fax: 1-650-725-9082; E-mail: firstname.lastname@example.org 2006 by the INTERNATIONAL SOCIETY OF ENDOVASCULAR SPECIALISTS Available at www.jevt.org 378 P19 PROGENITOR-DERIVED MYOCYTES J ENDOVASC THER Abilez et al. 2006;13:377–388 erage cost per procedure). Vascular grafts are that is known to have the potential to differ- currently used as bypass grafts, endovascular entiate into myocytes.34–38 grafts, and interposition grafts.1,3,4 However, the currently available grafts have been lim- ited by variable patency rates, material avail- METHODS ability, and immunological rejection.5–7 Complete Medium In attempts to address these limitations over the last 20 years, experimental human A complete medium was prepared from Min- and animal tissue-engineered vascular grafts imal Essential Medium Alpha ( -MEM) with ri- (TEVG) have been assembled from endothe- bonucleosides and deoxynucleosides (Invitro- lial cells (EC), smooth muscle cells (SMC), and gen, Carlsbad, CA, USA) supplemented with ﬁbroblast cells (FC)8–12; these experimental 7.5% calf bovine serum (American Type Cul- TEVGs have demonstrated favorable ture Collection [ATCC], Manassas, VA, USA) and 2.5% fetal bovine serum (GIBCO, Carls- strengths and patency rates. However, their bad, CA, USA). Next, penicillin-streptomycin main drawback has been immunological re- (GIBCO) diluted from a 100 concentration of jection during in vivo testing.8,10,13 The crea- stock solution was added to the above mix- tion of a TEVG from autologous stem cells ture to obtain a ﬁnal concentration of 1 in would potentially address these shortcom- the complete medium. Finally, -mercapto- ings and, furthermore, could potentially serve ethanol was added to a ﬁnal concentration of as the vascular source for other tissue-engi- 0.1 mM. neered materials, such as lung, heart, liver, or bone tissue.14–22 Of the several stem cell types that exist, the Cell Culture mouse embryonic stem cell (mESC) is well characterized, readily available, and has no A 1-mL vial of frozen P19 mouse embryonal carcinoma stem cells (ATCC #CRL-1825) was restrictions on its use.23 Furthermore, groups thawed in a 37 C water bath. The cells were have reported differentiating mESC into EC then re-suspended in 9 mL of new complete and SMC; in addition, FC derived from mouse medium in a 15-mL tube. The tube was spun embryos are commercially available. 24–29 in a Clinical 200 centrifuge (VWR, West Ches- However, the subsequent in vitro assembly of ter, PA, USA) at 300g (corresponding to 1750 these cell types into 3-layered blood vessels rpm) for 3 minutes. The medium was then as- has not yet been reported. In addition, it is not pirated, leaving the pellet of cells in the tube. entirely known how various stimuli affect Next, 5 mL of new fresh complete medium stem cell differentiation into these cell types. was added to the tube, and the clumped cells Furthermore, the differentiation of stem/pro- were then dissociated by pipetting up and genitor cells into myocytes for use in vascular down. The dissociated cells and new medium tissue engineering has been ill-deﬁned to were then transferred into a T-25 tissue-cul- date. Myocytes must exhibit both functional ture ﬂask (Becton Dickinson Biosciences, Bed- organization and contractility in order to serve ford, MA, USA), which was placed in a 37 C as components for tissue-engineered vascu- incubator (Fisher Scientiﬁc Isotemp, Hamp- lar grafts. Recently, groups have demonstrat- ton, NH, USA) with 5% CO2. No feeder layer ed the salutary effects of electrical stimulation was used. on primary myocyte organization and stem To ensure that they were healthy and con- cell differentiation.30–33 tinuing to grow, the cells were observed on Our purpose was to test the hypothesis that the second day of culture with a DM-IL (Leica a level of chemical and electrical stimulation Microsystems USA, Bannockburn, IL, USA) or exists that allows differentiation of progenitor TS-100F (Nikon USA, Melville, NY, USA) mi- cells into organized contracting myocytes. To croscope (magniﬁcation from 40 to 400 test our hypothesis, we applied these stimu- with Hoffman modulation contrast and phase lation signals to P19 cells, a stem cell line de- contrast optics). On the third day, the cells rived from a mouse embryonal carcinoma were fed. The original medium (usually dark J ENDOVASC THER P19 PROGENITOR-DERIVED MYOCYTES 379 2006;13:377–388 Abilez et al. yellow, indicating active cellular metabolism) was removed with a glass pipette connected to a vacuum. Care was taken not to aspirate the attached cells. Next, 5 mL of new fresh complete medium were added to the cells, and then the ﬂask was placed back in the in- cubator. On the fourth day, the cells were generally split into 10 parts, with 9 parts frozen for fu- ture use and 1 part propagated in culture. To split the cells, the medium from the ﬂask was removed. Then, 1000 L of trypsin (GIBCO) was added to the T-25 ﬂask to detach the cells from the bottom, and the ﬂask was incubated at 37 C for 5 minutes in 5% CO2. Next, the 900 L of trypsin and cells were transferred into a 15-mL tube, to which was added 9.1 mL of freezing medium [95% complete medium, 5% dimethylsulfoxide (DMSO; Sigma-Aldrich, St. Louis, MO, USA)] to inactivate the trypsin and bring the total volume to 10 mL. Pipetting the cells up and down in each tube broke apart any cell clumps. The 10 mL of freezing me- dium/cells were distributed in 1-mL aliquots to 10 cryotubes, which were placed in a 80 C freezer overnight and then transferred to a 180 C liquid nitrogen tank the following day. To the 100 L of trypsin and cells re- maining in the T-25 ﬂask, 4.9 mL of fresh com- plete medium was added, inactivating the trypsin and bringing the total volume back to 5 mL. The ﬂask was then re-incubated at 37 C Figure 1 (A) Electrical stimulation was accom- in 5% CO2. plished with a custom-made electric cell pulser. (B) The pulser delivered square waves of various volt- Electric Cell Pulser age amplitude, pulse width, and pulse frequency. (C) The electronic circuit design. Op Amp: opera- A custom-made cell pulser (Fig. 1A) was de- tional ampliﬁer, FET: ﬁeld effect transistor, VDC: signed with 4 channels to simultaneously voltage direct current, V : positive voltage, V : stimulate the P-19 cells in 4 separate biore- negative voltage, Sync-OUT: output synchroniza- actors. Each channel could deliver a square tion from timing chip. wave pulse (Fig. 1B) of varying voltage am- plitude (1–10 V), width (0.5–125 ms), and fre- quency (0.6–300 Hz). Due to technical limita- Electronics). The voltage amplitude adjust- tions, the minimum frequency obtained for ment was achieved with an LM 317 voltage these experiments was 10 Hz. regulator (Jameco Electronics). A ﬁeld effect The electronic circuit design of the cell puls- transistor (Jameco Electronics) was used in er (Fig. 1C) included an LM 556 timing chip an open collector conﬁguration. A triple-out- (Jameco Electronics, Belmont, CA, USA) to put power supply (model CPS 250; Tektronix, coordinate the manual pulse width and fre- Beaverton, OR, USA) was used to provide 15- quency adjustment. This chip also allowed volt direct current to both the timing chip and computer control of the pulse width and fre- the voltage regulator. Finally, to observe the quency via 2 operational ampliﬁers (Jameco output from the timing chip on a digital stor- 380 P19 PROGENITOR-DERIVED MYOCYTES J ENDOVASC THER Abilez et al. 2006;13:377–388 Figure 2 (A) Electrical stimulation was delivered via a custom-made 4-well bioreactor. (B, C) The experimental setup consisted of 4 bioreactors placed in an incubator. (D) The biore- actors, which were powered with an adjustable power supply, were connected to the electric cell pulser (placed on top of the incubator). age oscilloscope (model VC-6025; Hitachi, To- connectors (Jameco Electronics) and attached kyo, Japan), a synchronization channel was to the chamber with Loctite Five-Minute ep- added. oxy (Loctite-Henkel, Rocky Hill, CT, USA). Ap- plied voltages from the electric cell pulser were divided by the 1-cm distance separating Bioreactor the electrodes to obtain ﬁeld strengths in V/ Off-the-shelf items were used to assemble cm. the individual bioreactors (Fig. 2A), including Four bioreactors were used for all chemical a 4-well Lab-Tek Chamber-Slide system (Nalge and electrical stimulation experiments. The Nunc, Rochester, NY, USA) in which the bioreactors were incubated at 37 C in 5% CO2 chamber was made of polypropylene and the while they were connected to the electric cell slide of Permanox. Using a standard drill- pulser and power supply (Fig. 2B–D). A data press ﬁtted with a 1/64-inch drill bit, one hole acquisition system consisting of National In- was drilled at each end of every well (8 holes struments cFP-2000 control module hardware total). Into each hole was placed 1 cm of and LabView 7.1 software (National Instru- 99% pure gold wire (Sigma-Aldrich) to serve ments, Austin, TX, USA) was used to control as the electrodes for electrical stimulation. the pulse width and frequency of the electric The outside ends of the gold electrodes were cell pulser. The hardware was directly con- connected 1 cm apart to ﬂat ribbon computer nected to the cell pulser via BNC (Bayonet Nut wire (Jameco Electronics) via gold-plated Coupling) connectors. J ENDOVASC THER P19 PROGENITOR-DERIVED MYOCYTES 381 2006;13:377–388 Abilez et al. Figure 3 Schematic of the experimental design. To observe the daily activity in the biore- widths (2–40 ms), and frequencies (10–25 Hz) actors, a DM-IL (Leica Microsystems USA) in- were continuously applied (Table 1). On day verted microscope ﬁtted with 10 oculars 5, the medium containing DMSO was ex- and 4 , 10 , 20 , and 40 objectives was changed with complete medium (containing used to provide magniﬁcations of 40 , 100 , no DMSO), and the electrical stimulation was 200 , and 400 . Attached to the microscope continued. From days 6 to 22, the cells were was a Retiga 2000R high-speed digital CCD visually assessed for signs of viability, con- camera (QImaging, Burnaby, BC, Canada) ca- tractility, and organization. Spontaneously pable of taking single frames and/or video- contracting P19-derived myocyte colonies quality movies (30 frames/s). were counted daily by 1 observer and were documented with the image acquisition sys- tem. Finally, either the differentiation medium Chemical and Electrical Stimulation or complete medium was renewed every 3 The experimental design for chemical and days. electrical stimulation is shown in Figure 3. On day 7, P19 cells were thawed, grown, and Electrical Synchronization split as outlined above. On day 0, the P19 cells were washed 3 times with phosphate-buff- Electrical synchronization (pacing) was per- ered saline (PBS, pH 7.4) and then transferred formed on day 22 of culture on P19-derived from the complete medium to differentiation myocytes and myocyte colonies in Bioreactor medium containing 1% DMSO. This medium, 1 only because it demonstrated the most known to differentiate cells into myocytes, spontaneously contracting myocytes, which was used to chemically stimulate the P19 cells were also noted to be asynchronously con- for 5 days. tracting. The 4-channel pulser was discon- Additionally, for the next 22 days, electrical nected, and an identical single-channel pulser pulses of varying ﬁeld strengths (0–3 V/cm), was connected to the ﬂat ribbon computer TABLE 1 Electrical Stimulation Parameters for the 4 Bioreactors Bioreactor 1 2 3 4 Pulse width, ms 2 30 35 40 Field strength, V/cm 0, 1, 2, 3 0, 1, 2, 3 0, 1, 2, 3 0, 1, 2, 3 Pulse frequency, Hz 20 20 25 10 382 P19 PROGENITOR-DERIVED MYOCYTES J ENDOVASC THER Abilez et al. 2006;13:377–388 edge detection algorithm by drawing 1 line on each colony such that each line overlapped with 2 edges of each colony (Fig. 4B). The dis- placement of the colony edges with respect to the overlapping lines could then be deter- mined for each frame. The displacements cor- responded to contractions in the directions of the arrows shown in Figure 4B. The edge de- tection algorithm was applied to all the frames in an automated fashion, and the re- sulting displacements were recorded in a Mi- crosoft Excel ﬁle (Microsoft Corp, Redmond, WA, USA) for further analysis. Figure 4 (A) Two P19-derived myocyte colonies. Statistical Analysis (B) The 2 colonies (shaded areas) were electrically synchronized and their contractions were mea- Correlation coefﬁcients were calculated for sured along the lines. the electrical synchronization experiment us- ing Microsoft Excel. Correlation of contrac- tions between 2 separate P19-derived myo- wire bearing each pair of gold electrodes cyte colonies was determined before, during, from a given well of the bioreactor. The sin- and after the application of a synchronizing gle-channel pulser delivered the synchroni- electrical stimulus. Signiﬁcance of correlation zation signals, which consisted of square was determined by using the following rela- wave pulses having widths of either 2 ms or tion 10 to 100 ms (in 10-ms increments). Pulse ﬁeld strengths from 0 to 10 V/cm were applied n 2 t r in increments of 2.5 V. Pulse frequency was 1 r2 set at a constant 2 Hz (corresponding to 120 where t represents the statistical signiﬁcance contractions per minute). at n 2 degrees of freedom, n is the sample As the different pulse parameters were ap- size, and r is the calculated correlation coef- plied, the myocytes were visually monitored ﬁcient. P 0.05 was taken to be statistically via microscopy and were assessed for syn- signiﬁcant. chronization capture, which was deﬁned as coordinated contractions of all myocytes at the applied frequency of 2 Hz. At baseline, the RESULTS myocyte contraction rate ranged from zero Chemical and Electrical Stimulation (corresponding to no visually detectable con- tractions) to a maximum of 1.3 Hz (corre- Figure 5 shows a representative set of P19 sponding to 80 contractions per minute). progenitor cells exposed both to chemical Synchronization was documented with 200- and electrical stimulation. Over the course of frame movies obtained at 20 frames/s using the 22-day experiment, cell viability, as as- QCapture Pro 5.1 software (QImaging) oper- sessed by cell morphology, was inversely ating on a custom-made computer equipped proportional to pulse width and ﬁeld strength with a 3.4-GHz Pentium 4 processor, 2 GB and had no apparent dependence on pulse RAM, and a 300-GB hard drive for storage. frequency. The movie was taken before, during, and after Bioreactor 1 was exposed to 1% DMSO for synchronized contractions, then deconvolut- 5 days and to electrical stimulation of pulse ed into individual frames using Vision Assis- width 2 ms; ﬁeld strengths of 0, 1, 2, and 3 V/ tant 7.1 software (National Instruments). cm; and a pulse frequency of 20 Hz. Through- Next, using the same software, the ﬁrst frame out the experiment, the cells in all the wells (Fig. 4A) of the movie was used to create an of this bioreactor were uniform in size, at- J ENDOVASC THER P19 PROGENITOR-DERIVED MYOCYTES 383 2006;13:377–388 Abilez et al. Figure 5 These images show the qualitative analysis of the P19 progenitor cells exposed to chemical stimulation with 1% DMSO and electrical pulses of increasing pulse widths and ﬁeld strengths. Over the course of the 22-day experiment, cell viability, as assessed by cell morphology, was inversely proportional to pulse width and ﬁeld strength. tached to the bottom of the wells, and did not onstrated nuclear condensation, cytoplasmic show any nuclear or cytoplasmic changes. fragmentation, and an inability to attach. By Bioreactor 2 was exposed to 1% DMSO for day 22, the cells exposed to 2 and 3 V/cm ap- 5 days and to electrical stimulation of pulse peared non-viable; the cell suspension was width 30 ms; ﬁeld strengths of 0, 1, 2, and 3 dark. The cells exposed to 0 and 1 V/cm V/cm; and a pulse frequency also of 20 Hz. As showed some healthy cells. the experiment progressed, the cells exposed Bioreactor 4 was exposed to 1% DMSO for to ﬁeld strengths of 2 and 3 V/cm demonstrat- 5 days and to electrical stimulation of pulse ed nuclear condensation and cytoplasmic width 40 ms; ﬁeld strengths of 0, 1, 2, and 3 fragmentation; by day 22, they appeared non- V/cm; and a pulse frequency of 10 Hz. Only 2 viable. In addition, these same cells gradually days into the experiment, the cells exposed lost their ability to adhere to the bottom of the to ﬁeld strengths of 1, 2, and 3 V/cm demon- wells. The cells exposed to 0 and 1 V/cm ap- strated nuclear condensation, cytoplasmic peared healthy but did not exhibit any spon- fragmentation, and the inability to attach. By taneous contractions. day 22, all the cells except those exposed to Bioreactor 3 was exposed to 1% DMSO for 0 V/cm appeared non-viable and had turned 5 days and to electrical stimulation of 35-ms a dark brown color and were not identiﬁable. pulse width; ﬁeld strengths of 0, 1, 2, and 3 V/ Spontaneously contracting P19-derived cm; and a pulse frequency of 25 Hz. As the myocyte colonies (Movie 1, Fig. 6) appeared experiment progressed, the cells exposed to in Bioreactor 1 in all wells on day 12. The ﬁeld strengths of 1, 2, and 3 V/cm also dem- number of colonies were greatest in the cells 384 P19 PROGENITOR-DERIVED MYOCYTES J ENDOVASC THER Abilez et al. 2006;13:377–388 Figure 6 Graph showing the number of spontaneously contracting P19-derived myocyte colonies after chemical and electrical stimulation of P19 cells in Bioreactor 1. All cells were exposed to 1% DMSO for 5 days and to the electrical parameters shown. exposed to ﬁeld strengths of 1 and 2 V/cm; cient of contractions between the colonies these cells reached their maximum number during synchronization was statistically sig- on days 15 and 18, respectively. Since the col- niﬁcant (0.6, p 0.001), verifying synchroniza- onies were counted by only 1 observer, no tion. Even after synchronization, the correla- statistical results could be reported. tion coefﬁcient of contractions between the colonies was statistically signiﬁcant (0.5, p 0.001), which may be a positive by-product Electrical Synchronization of prior synchronization. For pulse widths 40 ms, capture could not be achieved at any ﬁeld strength (Table 2). Additionally, at ﬁeld strengths 5 V/cm, cap- TABLE 2 ture also could not be achieved with any Electrical Synchronization Results pulse width. The threshold for capture oc- Pulse Pulse Field Strength, Capture? curred for signals having ﬁeld strengths of 7.5 Width, ms Frequency, Hz V/cm (Y/N) and 10 V/cm, pulse widths 50 to 100 ms, and 2, 10–40 2 0 N a frequency of 2 Hz. Cells uniformly exposed 2.5 N to these parameters could be synchronized 5 N (Movie 2, Fig. 7), but this was performed for 7.5 N only a few minutes; long-term synchroniza- 10 N tion was reserved for future experiments. The 50–100 2 0 N 2.5 N correlation coefﬁcient of contractions be- 5 N tween the colonies before electrical synchro- 7.5 Y nization was 0.6, which was not statistically 10 Y signiﬁcant. In contrast, the correlation coefﬁ- J ENDOVASC THER P19 PROGENITOR-DERIVED MYOCYTES 385 2006;13:377–388 Abilez et al. Figure 7 P19-derived myocyte colony contractions before, during, and after electrical syn- chronization. #: correlation coefﬁcient 0.6 (p NS); *: correlation coefﬁcient 0.6, p 0.001; : correlation coefﬁcient 0.5, p 0.001. DISCUSSION applying the individual stimuli at various stages of differentiation are yet to be deter- In this study we have shown the effects of mined. Creating a layer of myocytes with ar- chemical and electrical stimulation on pro- chitectural and electrical organization is a crit- genitor cell differentiation and organization. ical step toward production of functional The results presented here will provide a gen- eral direction for future experiments using engineered vascular grafts. The application of chemical and electrical stimulation as differ- chemical and electrical signals to a multidi- entiation signals. mensional scaffold and assembly of different cell types may serve to generate more phys- iological vascular organization. Chemical and Electrical Stimulation For years, chemical and electrical stimuli have been noted in the early embryo.39 The Electrical Synchronization effects of electrical stimulation on myocyte organization30–32 and stem cell differentia- To our knowledge, synchronization of stem tion33 have recently been described. The work cell–derived myocytes using external pacing of Radisic et al.30 demonstrated that myocytes has not been previously reported. The ability exhibit structural, ultra-structural, and func- to synchronize multiple colonies with an ex- tional changes upon prolonged electrical ternal ﬁeld yields insights into the electro- stimulation. However, the goal of their work physiological response of these myocytes. Al- was to demonstrate these changes in primary though we did not study the effects of myocytes and not in progenitor-derived myo- long-term synchronization, one could envi- cytes. Also, in light of Deisseroth’s description sion its beneﬁcial effects with regards to cell- of neuronal stem cell differentiation with elec- cell communication and structural and ultra- trical stimulation,33 our results expand on the structural organization as suggested by the use of electrical stimulation on stem cells to work of Radisic et al.30,31 Altering the rate of derive myocytes. the synchronization signal may allow gener- Although we have demonstrated the effects ation of myocytes with more of a smooth of simultaneous application of chemical and muscle phenotype through differential ex- electrical stimulation, the consequences of pression of various types of ion channels. 386 P19 PROGENITOR-DERIVED MYOCYTES J ENDOVASC THER Abilez et al. 2006;13:377–388 This will also need to be investigated in future cells prior to exposing them to the chemical studies. and electrical stimulation. The presence of al- ready differentiated cells probably led to over- all lower yields of differentiated myocytes; Other Stimulation however, this must be conﬁrmed in future Mechanical forces have been shown to af- studies. fect organization of cell cultures and directly inﬂuence blood vessel physiology.40–44 Com- Conclusion bining these effects with chemical and elec- trical stimulation will ultimately provide a P19 progenitor cells progress to organized more realistic niche for stem cell differentia- contracting myocytes after chemical and elec- tion and organization. A by-product of electri- trical stimulation. We will use the methods cal stimulation appears to be generation of and results from this study to design addi- free radicals through hydrolysis, an issue not tional electrical stimulation experiments with addressed in the current study. Application of the goal of differentiating other progenitor ﬂow to cell cultures under electrical stimula- cells into organized myocytes. Incorporation tion may not only aid in cellular organization, of such cells into existing methods of produc- but would also mitigate the deleterious ef- ing endothelial cells, ﬁbroblasts, and scaf- fects of free radicals by continuously remov- folds may allow production of improved tis- ing them from the local environment. sue-engineered vascular grafts. Clearly, manipulation of other stimuli, such as oxygen tension, pH, and the concentration Acknowledgments: The authors would like to thank Rita of growth factors (e.g., vascular-endothelial Wedell, Maria Martinez, Shyla Barker, Deepa Basava, and various members of the Bio-X Program for their input and growth factor and transforming growth fac- assistance in this work. tor-beta), will inﬂuence differentiation and subsequent proliferation of stem cells. These stimuli, which have been studied individually REFERENCES in great detail,45–47 need to be investigated in 1. American Heart Association. Heart Disease and combination with mechanical, electrical, and Stroke Statistics–2006 Update. Circulation. other chemical stimuli. 2006;113:e85. 2. Langer R, Vacanti JP. Tissue engineering. Sci- ence. 1993;260:920–926. Limitations 3. Vacanti JP, Langer R. Tissue engineering: the One of the shortcomings of this study was design and fabrication of living replacement devices for surgical reconstruction and trans- the use of a single measurement to quantify plantation. Lancet. 1999;354:SI32–SI34. the number of spontaneously contracting 4. Weitz JI, Byrne J, Clagett GP, et al. Diagnosis P19-derived myocyte colonies, thus limiting and treatment of chronic arterial insufﬁciency the statistical analysis of this particular part of of the lower extremities: a critical review. Cir- the experiment. In addition, cell viability was culation. 1996;94:3026–3049. determined by morphological changes, such 5. Quinones-Baldrich WJ, Busuttil RW, Baker JD, as nuclear condensation, cytoplasmic frag- et al. Is the preferential use of polytetraﬂuoro- mentation, and lack of adherence. While the ethylene grafts for femoropopliteal bypass jus- changes were apparent to us, our descrip- tiﬁed? J Vasc Surg. 1988;8:219–228. tions are qualitative in nature and do not re- 6. Pevec WC, Darling RC, L’Italien GJ, et al. Fem- ﬂect the quantitative differences between cell oropopliteal reconstruction with knitted, non- velour Dacron versus expanded polytetraﬂu- populations. Use of Annexin-V immunocyto- oroethylene. J Vasc Surg. 1992;16:60–65. chemistry and propidium iodide staining to 7. Hamada Y, Kawachi K, Yamamoto T, et al. Ef- quantify degrees of apoptosis and necrosis, fect of coronary artery bypass grafting on na- respectively, would obviate this point and will tive coronary artery stenosis. Comparison of be employed in the future. internal thoracic artery and saphenous vein Finally, our study used a mixed population grafts. J Cardiovasc Surg (Torino). 2001;42: of undifferentiated and differentiated P19 159–164. J ENDOVASC THER P19 PROGENITOR-DERIVED MYOCYTES 387 2006;13:377–388 Abilez et al. 8. Weinberg CB, Bell E. A blood-vessel model logenesis and angiogenesis in embryonic- constructed from collagen and cultured vas- stem-cell-derived embryoid bodies. Develop- cular cells. Science. 1986;231:397–400. ment. 1988;102:471–478. 9. L’Heureux N, Germain L, Labbe R, et al. In vitro 25. Hirashima M, Kataoka H, Nishikawa S, et al. construction of a human blood vessel from cul- Maturation of embryonic stem cells into en- tured vascular cells: a morphologic study. J dothelial cells in an in vitro model of vasculo- Vasc Surg. 1993;17:499–509. genesis. Blood. 1999;93:1253–1263. 10. Niklason LE, Gao J, Abbott WM, et al. Func- 26. Yamashita J, Itoh H, Hirashima M, et al. Flk1- tional arteries grown in vitro. Science. 1999; positive cells derived from embryonic stem 284:489–493. cells serve as vascular progenitors. Nature. 11. Huynh T, Abraham G, Murray J, et al. Remod- 2000;408:92–96. eling of an acellular collagen graft into a phys- 27. Dinsmore J, Ratliff J, Deacon T, et al. Embry- iologically responsive neovessel. Nat Biotech- onic stem cells differentiated in vitro as a novel nol. 1999;17:1083–1086. source of cells for transplantation. Cell Trans- 12. Hoerstrup SP, Zund G, Sodian R, et al. Tissue plant. 1996;5:131–143. engineering of small caliber vascular grafts. 28. Drab M, Haller H, Bychkov R, et al. From toti- Eur J Cardiothoracic Surg. 2001;20:164–169. potent embryonic stem cells to spontaneously 13. L’Heureux N, Paquet S, Labbe R, et al. A com- contracting smooth muscle cells: a retinoic pletely biological tissue-engineered human acid and db-cAMP in vitro differentiation mod- blood vessel. FASEB J. 1998;12:47–56. el. FASEB J. 1997;11:905–915. 14. Nerem RM, Seliktar D. Vascular tissue engi- 29. Qiu H, Fujimori Y, Kai S, et al. Establishment of neering. Annu Rev Biomed Eng. 2001;3:225– mouse embryonic ﬁbroblast cell lines that pro- 243. mote ex vivo expansion of human cord blood 15. Zandstra PW, Nagy A. Stem cell bioengineer- CD34 hematopoietic progenitors. J Hema- ing. Annu Rev Biomed Eng. 2001;3:275–305. tother Stem Cell Res. 2003;12:39–46. 16. MacNeill BD, Pomerantseva I, Lowe HC, et al. 30. Radisic M, Park H, Shing H, et al. Functional Toward a new blood vessel. Vasc Med. 2002;7: assembly of engineered myocardium by elec- 241–246. trical stimulation of cardiac myocytes cultured 17. Raﬁi S, Lyden D. Therapeutic stem and progen- on scaffolds. Proc Natl Acad Sci U S A. 2004; itor cell transplantation for organ vasculariza- 101:18129–18134. tion and regeneration. Nat Med. 2003;9:702– 31. Radisic M, Yang L, Boublik J, et al. Medium 712. perfusion enables engineering of compact and 18. Matsumoto K, Yoshitomi H, Rossant J, et al. Liver organogenesis promoted by endothelial contractile cardiac tissue. Am J Physiol Heart cells prior to vascular function. Science. 2001; Circ Physiol. 2004;286:H507–H516. 294:559–563. 32. Pedrotty DM, Koh J, Davis BH, et al. Engineer- 19. Lammert E, Cleaver O, Melton D. Induction of ing skeletal myoblasts: roles of three-dimen- pancreatic differentiation by signals from sional culture and electrical stimulation. Am J blood vessels. Science. 2001;294:564–567. Physiol Heart Circ Physiol. 2005;288:H1620– 20. Rezai N, Podor TJ, McManus BM. Bone mar- H1626. row cells in the repair and modulation of heart 33. Deisseroth K, Singla S, Toda H, et al. Excita- and blood vessels: emerging opportunities in tion-neurogenesis coupling in adult neural native and engineered tissue and biomechani- stem/progenitor cells. Neuron. 2004;42:535– cal materials. Artif Organs. 2004;28:142–151. 552. 21. Merchant AM, Flake AW. Surgeons and stem 34. McBurney MW, Jones-Villeneuve EM, Edwards cells: a pragmatic perspective on shifting par- MK, et al. Control of muscle and neuronal dif- adigms. Surgery. 2004;136:975–980. ferentiation in a cultured embryonal carcinoma 22. Kannan RY, Salacinski HJ, Sales K, et al. The cell line. Nature. 1982;299:165. roles of tissue engineering and vascularisation 35. Edwards MK, Harris JF, McBurney MW. In- in the development of micro-vascular net- duced muscle differentiation in an embryonal works: a review. Biomaterials. 2005;26:1857– carcinoma cell line. Mol Cell Biol. 1983;3:2280– 1875. 2286. 23. Evans MJ, Kaufman MH. Establishment in cul- 36. McBurney MW. P19 embryonal carcinoma ture of pluripotential cells from mouse embry- cells. Int J Dev Biol. 1993;37:135–140. os. Nature. 1981;292:154–156. 37. Moore JC, Spijker R, Martens AC, et al. A 24. Risau W, Sariola H, Zerwes HG, et al. Vascu- P19Cl6 GFP reporter line to quantify cardio- 388 P19 PROGENITOR-DERIVED MYOCYTES J ENDOVASC THER Abilez et al. 2006;13:377–388 myocyte differentiation of stem cells. Int J Dev inhibits adhesion molecule expression in vas- Biol. 2004;48:47–55. cular endothelial cells induced by coculture 38. Choi SC, Yoon J, Shim WJ, et al. 5-azacytidine with smooth muscle cells. Blood. 2003;101: induces cardiac differentiation of P19 embry- 2667–2674. onic stem cells. Exp Mol Med. 2004;36:515– 44. Braddon LG, Karoyli D, Harrison DG, et al. 523. Maintenance of a functional endothelial cell 39. Nuccitelli R. Endogenous ionic currents and DC monolayer on a ﬁbroblast/polymer substrate electric ﬁelds in multicellular animal tissues. under physiologically relevant shear stress Bioelectromagnetics. 1992; Suppl 1:147–157. conditions. Tissue Eng. 2002;8:695–708. 40. Barbee KA. Role of subcellular shear-stress dis- 45. Gassmann M, Fandrey J, Bichet S, et al. Oxy- tributions in endothelial cell mechanotransduc- gen supply and oxygen-dependent gene ex- tion. Ann Biomed Eng. 2002;30:472–482. pression in differentiating embryonic stem cells. Proc Natl Acad Sci U S A. 1996;93:2867– 41. Yamamoto K, Takahashi T, Asahara T, et al. Pro- 2872. liferation, differentiation, and tube formation 46. Carmeliet P, Dor Y, Herbert JM, et al. Role of by endothelial progenitor cells in response to HIF-1 alpha in hypoxia-mediated apoptosis, shear stress. J Appl Physiol. 2003;95:2081– cell proliferation and tumour angiogenesis. Na- 2088. ture. 1998;394:485. 42. Yamamoto K, Sokabe T, Watabe T, et al. Fluid 47. Hirashima M, Ogawa M, Nishikawa S, et al. A shear stress induces differentiation of Flk-1- chemically deﬁned culture of VEGFR2 cells positive embryonic stem cells into vascular en- derived from embryonic stem cells reveals the dothelial cells in vitro. Am J Physiol Heart Circ role of VEGFR1 in tuning the threshold for Physiol. 2005;288:H1915–24. VEGF in developing endothelial cells. Blood. 43. Chiu JJ, Chen LJ, Lee PL, et al. Shear stress 2003;101:2261–2267.