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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: ojabilez@stanford.edu
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
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