BACKGROUND
embryonic stem cells  are derived from the inner cell mass  of pre-implantation embryos  <cit> .
these escs have the ability to remain undifferentiated and proliferate indefinitely in vitro, while maintaining the potential to differentiate into all three embryonic germ layers  <cit> .
an important aspect of esc research focuses on elucidating the mechanisms of differentiation from the pluripotent esc to various terminally differentiated cell types.
this differentiation capacity makes escs an attractive cell source for cell/tissue replacement therapies for the treatment of human degenerative diseases.
moreover, escs can also be used as a model system for understanding human genetic disease by elucidating the pathophysiology of specific genetic disorders, including but not limited to cardiac abnormalities.
the in vitro differentiation of escs into cardiomyocytes provides an opportunity to study the developmental aspects of cardiomyogenesis.
cardiomyocytes are terminally differentiated muscle cells in the adult mammalian heart, which do not divide.
although a small percentage of the cells may be capable of proliferation  <cit> , this is not sufficient for regeneration after myocardial injury.
the ultimate goal in cardiac regenerative medicine is to produce in large-scale, highly purified cardiomyocytes which are suitable for cell transplantation.
such cell transplantation therapies would require the successful seeding of as many as  <dig> ×  <dig> donor cardiomyocytes per patient  <cit> .
from a commercial perspective, the ability to generate such clinically relevant cell numbers through an economically viable bioprocess is a priority.
the robust generation of such large cardiomyocyte numbers could only be feasible in controlled stirred suspension bioreactors capable of maintaining high-density esc numbers.
currently, most protocols use static culture to differentiate escs into cardiomyocytes  <cit> .
although routinely used for esc culture and differentiation, static culture flasks can only support a pre-clinical research project.
alternatively, stirred suspension bioreactors offer several advantages over the conventional culture methods.
we and others have previously demonstrated that suspension bioreactors can support large-scale expansion of the escs over extended passages, while retaining their pluripotency  <cit> .
the scalable production of esc- derived cardiomyocytes in a suspension bioreactor system has previously been demonstrated using a retinoic acid based protocol  <cit> .
however, since we have previously observed that the suspension bioreactor environment enhances esc pluripotency, while suppressing differentiation efficiency  <cit> , we sought to investigate whether this phenomenon would also occur in during cardiomyocyte differentiation.
this study demonstrates that suspension bioreactor culture systems do indeed have the ability to inhibit differentiation, and even induce 'transient' pluripotency within a defined differentiation protocol, presumably due to influence of shear stress on the cells.
our data shows that in contrast to static culture, esc cultures induced to differentiate toward cardiomyocytes in suspension bioreactors retain some ability to express pluripotency markers, to form esc-like colonies, and to generate teratomas upon transplantation.
RESULTS
tumorigenicity of bioreactor-derived cardiomyocytes
the elegant study by zandstra et al., demonstrated that esc-derived cardiomyocytes could indeed be generated in a scalable stirred suspension bioreactor provided cells were lineage selected  <cit> .
however, based on our previous observation demonstrating increased tumorigenesis of esc-derived chondrocytes and osteoblasts in stirred suspension bioreactors, we were interested to determine if this effect was eliminated within the g <dig>  selected population.
using d3-mhc-neor escs from the zandstra study, we induced cardiomyocyte differentiation in static and suspension culture for teratoma analysis.
the resultant cardiomyocytes from both bioreactor and static culture  were injected into severe combined immunodeficiency  mice and after  <dig> days, were harvested and processed for histopathology studies.
interestingly, lineage selected cardiomyocytes from both bioreactor and static differentiation cultures produced no tumor growth in vivo suggesting that drug selection for terminal markers may indeed be a reliable tool for the scalable production of esc derivatives.
as expected, static and bioreactor differentiated cells without drug selection during differentiation produced teratomas; however, tumors developed from bioreactor cultured escs were significantly larger  than static derived tumors .
furthermore, teratomas formed from bioreactor escs contained cells representative of all three germ layers; whereas, cells from static culture did not.
only some gut-like epithelial cells were recognizable within the teratoma derived from static culture .
based on this result, we undertook the characterization of cardiac bodies generated in the bioreactor, in addition to an analysis of gene expression during differentiation.
expression of pluripotency markers and cardiac marker before/after drug selection in suspension and static culture condition
rt-pcr results confirmed the expression of cardiac genes in addition to the expression of pluripotency markers during differentiation in the suspension culture bioreactor.
cardiac markers were examined in non-drug selected bioreactors.
α-mhc  gene expression was up-regulated on day  <dig> through day  <dig> in ebs and decreased gradually to the end of the differentiation experiment.
the early mesodermal marker pcam- <dig> began to be expressed on day  <dig> and persisted until day  <dig>  expression of other cardiogenesis lineage genes such as alcam  and anf  showed that the escs successfully differentiated to cardiomyocytes in the suspension bioreactor.
among the examined pluripotency markers, oct <dig> was consistently expressed on all days during differentiation, surprisingly, even with drug selection present from day  <dig> of the differentiation protocol.
sox <dig> expression was absent after g <dig> selection, but was still expressed in the bioreactor without lineage selection.
nanog expression in the non-drug selected bioreactor fluctuated during the first  <dig> days of differentiation, with expression being up-regulated at day  <dig> and decreasing gradually from that time point onward .
in static culture, nanog expression was down-regulated on day  <dig> and the pluripotency markers oct <dig> and sox <dig> were completely absent on day <dig> after lif removal .
the cardiac marker α-mhc started to be expressed on day  <dig> after lif removal and persisted until the end of experiment.
quantitative rt-pcr in the non-drug selected bioreactors showed that oct <dig>  sox <dig>  and nanog expression persisted during differentiation in the bioreactor culture compared to static culture.
the maximum fold increase in expression was on day  <dig> for sox <dig> and nanog and day  <dig> for oct <dig> .
since rt-pcr is a global gene expression assay, fluorescence-activated cell sorting  analysis was used to confirm these marker results at the cellular and protein level in non-drug selected cells.
facs showed that the pluripotency marker oct <dig> was expressed during all time points of differentiation examined .
in the absence of lif and g <dig> selection, the expression of oct <dig> positive cells increased from ~20% on day  <dig> to ~55% on day  <dig> in the bioreactor.
correspondingly, expression of the cardiac marker, α-mhc , increased from ~ <dig> % on day  <dig> to ~20% by day  <dig> with a decrease to ~14% by day  <dig>  surprisingly, a small population of cells  expressed both oct <dig> and α-mhc  within bioreactors without drug selection.
these double positive cells reached a plateau on day  <dig> during differentiation .
confocal microscopy also was used to further confirm and validate the existence of oct <dig> and nanog positive cells .
interestingly, bioreactor-differentiated cells  retained their ability to form esc-like colonies on gelatin coated plates after  <dig> days differentiation.
however differentiated cells in static culture lost this ability .
confocal microscopy also confirmed the simultaneous expression of α-mhc and oct <dig> in cells within the bioreactor-differentiated ebs .
characterization of bioreactor-derived cardiomyocytes
with the observation that pluripotent cells persisted through induced differentiation and the possibility of 'bipotent' oct4+/α-mhc+ cells, it was next imperative to characterize and test the functionality of the bioreactor derived cardiomyocytes.
within the bioreactor, escs aggregated one day after the initial inoculation of single cells without lif , with aggregates increasing in size during differentiation.
the first beating ebs were seen on day  <dig>  after removal of the lif and remained for the rest of differentiation period .
moreover, the cardiac outgrowths of dissociated aggregates kept beating for almost  <dig> days with beating rates ranging from 29- <dig> beats/min.
the maximum percentage of beating ebs  was on day  <dig> after differentiation .
conversely, the appearance of the first beating ebs in the hanging drop method was on day  <dig> of ebs, even before moving to suspension culture.
after selection with g <dig>  the resistant derived- cardiomyocytes in the bioreactor had the ultrastructure phenotype of differentiated cardiomyocytes as revealed by light and electron microscope.
dissociated cells were used for light microscope visualization, as well as immunofluorescent staining.
after dissociation; the cells produced clusters of spontaneously contractile areas, which were positive for α-mhc as revealed by immunostaining with the mf- <dig> antibody .
light microscope showed rounded mononuclear cells with definite subcellular compartments.
transmission electron microscopy of whole cardiac bodies revealed the presence of cardiomyocyte-specific ultrastructure including myofibrils with distinct z-, i-, h-and a-bands, as well as cell-cell adhesion structures such as gap junctions and desmosomes .
pharmacological responses of esc-derived cardiomyocytes
spontaneously contracting cardiomyocytes were examined to check if they retain the ability to respond to pharmacological agents.
upon administration of isoprenaline , phenylephrine  and bayk <dig> , the beating frequency of cardiac bodies were enhanced.
however, the chronotropical effect of diltiazem  treatment was negative on cardiomyocyte beating rate .
the highest increased frequency was observed for bayk <dig> which was reversed to the previous beating rate about four hours after removal of the drug through changing the media .
this highlights the existence of developed functional ca2+ ion channels in cardiomyocytes.
these characterization experiments demonstrated that the cells produced were not only positive for cardiac specific markers, but also displayed the properties of functional cardiomyocytes.
discussion
the large-scale production of differentiated cells suitable for clinical transplantation is a fundamental objective of regenerative medicine research.
the replacement of damaged cells using in vitro differentiated functional cardiomyocytes has received more attention in the last decade with clinical trials on the horizon.
despite many developments in the stem cell field, we still lack the ability to produce adequate number of suitable differentiated cells that can meet clinical requirements.
we conducted this study to evaluate the tumorgenicity of bioreactor derived cardiomyocytes, since we have observed that escs differentiated in stirred suspension culture maintain their pluripotency compare to those differentiated in static culture  <cit> .
in this study we observed that by day  <dig> following the removal of lif in suspension, escs underwent efficient differentiation into cardiomyocytes in the presence of ascorbic acid and dmso.
expression of the early mesodermal marker  and cardiomyocyte specific markers   <cit> , anf  <cit> , α-mhc and β-mhc  <cit> ) clearly confirmed the differentiation of escs into cardiomyocyte in stirred suspension culture.
similarly, in static culture, cells efficiently made ebs three days after the removal of lif and the derived-cardiac bodies showed characteristics of cardiomyocytes including expression of the molecular marker α-mhc.
it has previously been shown that both aa and dmso enhance differentiation of mouse escs into cardiac myocytes.
it has been suggested that aa induces permissive changes enabling cardiomyocyte differentiation, while dmso has been shown to activate essential cardiogenic transcription factors, such as gata- <dig> and nkx- <dig> .
however, the exact related mechanism for triggering these genes is still not very well known  <cit> .
ultra-structural studies by electron microscopy revealed the specific subcellular sarcomeric organization of cardiomyocytes, such as z-banding.
h- and a-bands, and intercalated discs including desmosome and gap junction were also obvious in derived cells.
the chronotropical responses of cardiomyocytes confirmed the existence of specialized ca+ <dig> channels as well as α <dig> and β1-adrenergic receptors.
importantly, no tumors were generated after cardiomyocyte lineage selection used to eliminate cells not expressing the α-mhc-neor gene.
based on these experiments, we assume that functional cardiomyocytes had been produced that didn't pose a risk of tumor formation in vivo.
however, it should be emphasized that even in the presence of drug selection, escs differentiated in stirred suspension culture, still maintained the expression of pluripotency markers.
this trend was even more apparent in cultures not undergoing drug selection pressure, where a sub-population expressed both oct <dig> and α-mhc simultaneously in the same cell.
it is important to remember, however, that drug selection did negate tumor formation signifying that the expression of pluripotent genes is either:  not maintained once the cells are taken out of expansion, or  not sufficient to reprogram a cell that has undergone terminal differentiation.
furthermore, the results presented here show that even after differentiation toward cardiomyocytes, a sub-population of cells  still express oct <dig> demonstrating some link between oct <dig> gene expression and the environment.
this observation is further confirmed by the difference observed in teratoma formation between static and suspension.
the suspension bioreactor differentiations retained cell pluripotency to a degree that escs formed teratomas representative of all three germ layers.
in contrast, cells from static cultures only formed an unstructured cell population: although not teratomas, tumor masses were still generated.
while lineage selection using transgenic constructs is a useful research tool, this approach cannot be applied clinically.
hence, our observation that bioreactor-derived cardiomyocytes maintain hallmarks of pluripotency has significant implications.
before the large scale production of cardiomyocytes in suspension bioreactors will be possible, it will be necessary to eliminate bioreactor induced pluripotency.
we have recently evaluated the application of suspension bioreactor culture for the generation of cartilage and bone tissue  <cit> .
unlike the static culture environment, bioreactor-differentiated aggregates caused teratoma formation when implanted subcutaneously into scid mice, which implicated the existence of pluripotent cells in the bioreactor even after  <dig> days of suspension culture.
upon closer analysis of cells within aggregates, we discovered that cells on the ridges of the aggregate expressed the greatest amount of oct <dig> in locations, which would be exposed to the greatest amount of laminar fluid flow.
this result suggested that the maintenance of pluripotency within bioreactor differentiation cultures was the result of fluid shear stress.
in the current study, much like all our other bioreactor studies, we apply an agitation rate of  <dig> rpm  in order to optimize mass transfer and avoid necrosis, which otherwise occurs at lower velocities  <cit> .
we believe that liquid shear stress in the stirred suspension bioreactor plays an important mechanistic role in bioreactor induced pluripotency.
shear stress can modulate gene expression through mechanotransduction, where physical signals are sensed at the cell periphery, transduced into biochemical signals within the cell, ultimately resulting in cell responses, including changes in gene expression  <cit> .
previous studies in other cells have demonstrated that shear stress can induce the nuclear translocation of ß-catenin into the nucleus  <cit> .
as ß-catenin is an important regulator of pluripotency  <cit> , we are interested in the role of the non-canonical wnt signaling pathway in this induced pluripotency process.
recently, using a lef/tcf-gfp  reporter system, we have confirmed that ß-catenin nuclear occupancy is considerably increased over controls when cells are exposed to  <dig>  dynes/cm <dig> shear stress compared to lower levels of shear generated by lower velocities of stirring .
following nuclear translocation, β-catenin forms a complex with lef/tcf transcription factors.
this complex interacts with the specific sequence in the promoter region to activate transcription of certain genes.
recently, saha et al.
has shown that biaxial cyclic strain above a certain threshold inhibits human esc differentiation and enhances their self-renewal without selecting against growth or survival of differentiated or undifferentiated cells  <cit> .
this group later suggested that strain may induce autocrine or paracrine signaling through tgf-β superfamily ligands in human escs since the tgf-β superfamily activation of smad2/ <dig> was necessary for suppression of spontaneous differentiation under strain  <cit> .
despite the fact that stirred suspension cultures are very useful for the generation of a large number of undifferentiated cells, we have found that the addition of medium enhancers is not adequate to force complete differentiation of the population in suspension bioreactors.
by elucidating the exact mechanism by which liquid shear stress may contribute to promoting pluripotency and preventing differentiation, we will be able to create an efficient environment for both the production of large quantities of pluripotent stem cells, and their differentiated progeny.
this is an important objective for human regenerative medicine, as lineage selection using transgenes will not be possible.
CONCLUSIONS
although stirred suspension bioreactors are very advantageous for the scalable generation of undifferentiated cells, addition of medium enhancers is not sufficient to induce complete differentiation.
our observation that bioreactor-derived cardiomyocyte cultures maintain characteristics of pluripotency has significant implications for human regenerative medicine.
we believe that liquid shear stress in the stirred suspension bioreactor plays an important mechanistic role in bioreactor induced pluripotency, which needs further investigation.

 embryonic stem cells are derived from the inner cell mass of pre-implantation embryos. these escs have the ability to remain undifferentiated and proliferate indefinitely in vitro, while maintaining the potential to differentiate into all three embryonic germ layers. an important aspect of esc research focuses on elucidating the mechanisms of differentiation from the pluripotent esc to various terminally differentiated cell types. this differentiation capacity makes escs an attractive cell source for cell/tissue replacement therapies for the treatment of human degenerative diseases. moreover, escs can also be used as a model system for understanding human genetic disease by elucidating the pathophysiology of specific genetic disorders, including but not limited to cardiac abnormalities. the in vitro differentiation of escs into cardiomyocytes provides an opportunity to study the developmental aspects of cardiomyogenesis. cardiomyocytes are terminally differentiated muscle cells in the adult mammalian heart, which do not divide. although a small percentage of the cells may be capable of proliferation, this is not sufficient for regeneration after myocardial injury. the ultimate goal in cardiac regenerative medicine is to produce in large-scale, highly purified cardiomyocytes which are suitable for cell transplantation. such cell transplantation therapies would require the successful seeding of as many as × donor cardiomyocytes per patient. from a commercial perspective, the ability to generate such clinically relevant cell numbers through an economically viable bioprocess is a priority. the robust generation of such large cardiomyocyte numbers could only be feasible in controlled stirred suspension bioreactors capable of maintaining high-density esc numbers. currently, most protocols use static culture to differentiate escs into cardiomyocytes. although routinely used for esc culture and differentiation, static culture flasks can only support a pre-clinical research project. alternatively, stirred suspension bioreactors offer several advantages over the conventional culture methods. we and others have previously demonstrated that suspension bioreactors can support large-scale expansion of the escs over extended passages, while retaining their pluripotency. the scalable production of esc- derived cardiomyocytes in a suspension bioreactor system has previously been demonstrated using a retinoic acid based protocol. however, since we have previously observed that the suspension bioreactor environment enhances esc pluripotency, while suppressing differentiation efficiency, we sought to investigate whether this phenomenon would also occur in during cardiomyocyte differentiation. this study demonstrates that suspension bioreactor culture systems do indeed have the ability to inhibit differentiation, and even induce'transient' pluripotency within a defined differentiation protocol, presumably due to influence of shear stress on the cells. our data shows that in contrast to static culture, esc cultures induced to differentiate toward cardiomyocytes in suspension bioreactors retain some ability to express pluripotency markers, to form esc-like colonies, and to generate teratomas upon transplantation.  tumorigenicity of bioreactor-derived cardiomyocytes the elegant study by zandstra et al., demonstrated that esc-derived cardiomyocytes could indeed be generated in a scalable stirred suspension bioreactor provided cells were lineage selected. however, based on our previous observation demonstrating increased tumorigenesis of esc-derived chondrocytes and osteoblasts in stirred suspension bioreactors, we were interested to determine if this effect was eliminated within the g selected population. using d3-mhc-neor escs from the zandstra study, we induced cardiomyocyte differentiation in static and suspension culture for teratoma analysis. the resultant cardiomyocytes from both bioreactor and static culture were injected into severe combined immunodeficiency mice and after days, were harvested and processed for histopathology studies. interestingly, lineage selected cardiomyocytes from both bioreactor and static differentiation cultures produced no tumor growth in vivo suggesting that drug selection for terminal markers may indeed be a reliable tool for the scalable production of esc derivatives. as expected, static and bioreactor differentiated cells without drug selection during differentiation produced teratomas; however, tumors developed from bioreactor cultured escs were significantly larger than static derived tumors. furthermore, teratomas formed from bioreactor escs contained cells representative of all three germ layers; whereas, cells from static culture did not. only some gut-like epithelial cells were recognizable within the teratoma derived from static culture. based on this result, we undertook the characterization of cardiac bodies generated in the bioreactor, in addition to an analysis of gene expression during differentiation. expression of pluripotency markers and cardiac marker before/after drug selection in suspension and static culture condition rt-pcr results confirmed the expression of cardiac genes in addition to the expression of pluripotency markers during differentiation in the suspension culture bioreactor. cardiac markers were examined in non-drug selected bioreactors. α-mhc gene expression was up-regulated on day through day in ebs and decreased gradually to the end of the differentiation experiment. the early mesodermal marker pcam- began to be expressed on day and persisted until day expression of other cardiogenesis lineage genes such as alcam and anf showed that the escs successfully differentiated to cardiomyocytes in the suspension bioreactor. among the examined pluripotency markers, oct was consistently expressed on all days during differentiation, surprisingly, even with drug selection present from day of the differentiation protocol. sox expression was absent after g selection, but was still expressed in the bioreactor without lineage selection. nanog expression in the non-drug selected bioreactor fluctuated during the first days of differentiation, with expression being up-regulated at day and decreasing gradually from that time point onward. in static culture, nanog expression was down-regulated on day and the pluripotency markers oct and sox were completely absent on day after lif removal. the cardiac marker α-mhc started to be expressed on day after lif removal and persisted until the end of experiment. quantitative rt-pcr in the non-drug selected bioreactors showed that oct sox and nanog expression persisted during differentiation in the bioreactor culture compared to static culture. the maximum fold increase in expression was on day for sox and nanog and day for oct. since rt-pcr is a global gene expression assay, fluorescence-activated cell sorting analysis was used to confirm these marker results at the cellular and protein level in non-drug selected cells. facs showed that the pluripotency marker oct was expressed during all time points of differentiation examined. in the absence of lif and g selection, the expression of oct positive cells increased from ~20% on day to ~55% on day in the bioreactor. correspondingly, expression of the cardiac marker, α-mhc, increased from ~ % on day to ~20% by day with a decrease to ~14% by day surprisingly, a small population of cells expressed both oct and α-mhc within bioreactors without drug selection. these double positive cells reached a plateau on day during differentiation. confocal microscopy also was used to further confirm and validate the existence of oct and nanog positive cells. interestingly, bioreactor-differentiated cells retained their ability to form esc-like colonies on gelatin coated plates after days differentiation. however differentiated cells in static culture lost this ability. confocal microscopy also confirmed the simultaneous expression of α-mhc and oct in cells within the bioreactor-differentiated ebs. characterization of bioreactor-derived cardiomyocytes with the observation that pluripotent cells persisted through induced differentiation and the possibility of'bipotent' oct4+/α-mhc+ cells, it was next imperative to characterize and test the functionality of the bioreactor derived cardiomyocytes. within the bioreactor, escs aggregated one day after the initial inoculation of single cells without lif, with aggregates increasing in size during differentiation. the first beating ebs were seen on day after removal of the lif and remained for the rest of differentiation period. moreover, the cardiac outgrowths of dissociated aggregates kept beating for almost days with beating rates ranging from 29- beats/min. the maximum percentage of beating ebs was on day after differentiation. conversely, the appearance of the first beating ebs in the hanging drop method was on day of ebs, even before moving to suspension culture. after selection with g the resistant derived- cardiomyocytes in the bioreactor had the ultrastructure phenotype of differentiated cardiomyocytes as revealed by light and electron microscope. dissociated cells were used for light microscope visualization, as well as immunofluorescent staining. after dissociation; the cells produced clusters of spontaneously contractile areas, which were positive for α-mhc as revealed by immunostaining with the mf- antibody. light microscope showed rounded mononuclear cells with definite subcellular compartments. transmission electron microscopy of whole cardiac bodies revealed the presence of cardiomyocyte-specific ultrastructure including myofibrils with distinct z-, i-, h-and a-bands, as well as cell-cell adhesion structures such as gap junctions and desmosomes. pharmacological responses of esc-derived cardiomyocytes spontaneously contracting cardiomyocytes were examined to check if they retain the ability to respond to pharmacological agents. upon administration of isoprenaline, phenylephrine and bayk, the beating frequency of cardiac bodies were enhanced. however, the chronotropical effect of diltiazem treatment was negative on cardiomyocyte beating rate. the highest increased frequency was observed for bayk which was reversed to the previous beating rate about four hours after removal of the drug through changing the media. this highlights the existence of developed functional ca2+ ion channels in cardiomyocytes. these characterization experiments demonstrated that the cells produced were not only positive for cardiac specific markers, but also displayed the properties of functional cardiomyocytes. discussion the large-scale production of differentiated cells suitable for clinical transplantation is a fundamental objective of regenerative medicine research. the replacement of damaged cells using in vitro differentiated functional cardiomyocytes has received more attention in the last decade with clinical trials on the horizon. despite many developments in the stem cell field, we still lack the ability to produce adequate number of suitable differentiated cells that can meet clinical requirements. we conducted this study to evaluate the tumorgenicity of bioreactor derived cardiomyocytes, since we have observed that escs differentiated in stirred suspension culture maintain their pluripotency compare to those differentiated in static culture. in this study we observed that by day following the removal of lif in suspension, escs underwent efficient differentiation into cardiomyocytes in the presence of ascorbic acid and dmso. expression of the early mesodermal marker and cardiomyocyte specific markers, anf, α-mhc and β-mhc ) clearly confirmed the differentiation of escs into cardiomyocyte in stirred suspension culture. similarly, in static culture, cells efficiently made ebs three days after the removal of lif and the derived-cardiac bodies showed characteristics of cardiomyocytes including expression of the molecular marker α-mhc. it has previously been shown that both aa and dmso enhance differentiation of mouse escs into cardiac myocytes. it has been suggested that aa induces permissive changes enabling cardiomyocyte differentiation, while dmso has been shown to activate essential cardiogenic transcription factors, such as gata- and nkx-. however, the exact related mechanism for triggering these genes is still not very well known. ultra-structural studies by electron microscopy revealed the specific subcellular sarcomeric organization of cardiomyocytes, such as z-banding. h- and a-bands, and intercalated discs including desmosome and gap junction were also obvious in derived cells. the chronotropical responses of cardiomyocytes confirmed the existence of specialized ca+ channels as well as α and β1-adrenergic receptors. importantly, no tumors were generated after cardiomyocyte lineage selection used to eliminate cells not expressing the α-mhc-neor gene. based on these experiments, we assume that functional cardiomyocytes had been produced that didn't pose a risk of tumor formation in vivo. however, it should be emphasized that even in the presence of drug selection, escs differentiated in stirred suspension culture, still maintained the expression of pluripotency markers. this trend was even more apparent in cultures not undergoing drug selection pressure, where a sub-population expressed both oct and α-mhc simultaneously in the same cell. it is important to remember, however, that drug selection did negate tumor formation signifying that the expression of pluripotent genes is either: not maintained once the cells are taken out of expansion, or not sufficient to reprogram a cell that has undergone terminal differentiation. furthermore, the results presented here show that even after differentiation toward cardiomyocytes, a sub-population of cells still express oct demonstrating some link between oct gene expression and the environment. this observation is further confirmed by the difference observed in teratoma formation between static and suspension. the suspension bioreactor differentiations retained cell pluripotency to a degree that escs formed teratomas representative of all three germ layers. in contrast, cells from static cultures only formed an unstructured cell population: although not teratomas, tumor masses were still generated. while lineage selection using transgenic constructs is a useful research tool, this approach cannot be applied clinically. hence, our observation that bioreactor-derived cardiomyocytes maintain hallmarks of pluripotency has significant implications. before the large scale production of cardiomyocytes in suspension bioreactors will be possible, it will be necessary to eliminate bioreactor induced pluripotency. we have recently evaluated the application of suspension bioreactor culture for the generation of cartilage and bone tissue. unlike the static culture environment, bioreactor-differentiated aggregates caused teratoma formation when implanted subcutaneously into scid mice, which implicated the existence of pluripotent cells in the bioreactor even after days of suspension culture. upon closer analysis of cells within aggregates, we discovered that cells on the ridges of the aggregate expressed the greatest amount of oct in locations, which would be exposed to the greatest amount of laminar fluid flow. this result suggested that the maintenance of pluripotency within bioreactor differentiation cultures was the result of fluid shear stress. in the current study, much like all our other bioreactor studies, we apply an agitation rate of rpm in order to optimize mass transfer and avoid necrosis, which otherwise occurs at lower velocities. we believe that liquid shear stress in the stirred suspension bioreactor plays an important mechanistic role in bioreactor induced pluripotency. shear stress can modulate gene expression through mechanotransduction, where physical signals are sensed at the cell periphery, transduced into biochemical signals within the cell, ultimately resulting in cell responses, including changes in gene expression. previous studies in other cells have demonstrated that shear stress can induce the nuclear translocation of ß-catenin into the nucleus. as ß-catenin is an important regulator of pluripotency, we are interested in the role of the non-canonical wnt signaling pathway in this induced pluripotency process. recently, using a lef/tcf-gfp reporter system, we have confirmed that ß-catenin nuclear occupancy is considerably increased over controls when cells are exposed to dynes/cm shear stress compared to lower levels of shear generated by lower velocities of stirring. following nuclear translocation, β-catenin forms a complex with lef/tcf transcription factors. this complex interacts with the specific sequence in the promoter region to activate transcription of certain genes. recently, saha et al. has shown that biaxial cyclic strain above a certain threshold inhibits human esc differentiation and enhances their self-renewal without selecting against growth or survival of differentiated or undifferentiated cells. this group later suggested that strain may induce autocrine or paracrine signaling through tgf-β superfamily ligands in human escs since the tgf-β superfamily activation of smad2/ was necessary for suppression of spontaneous differentiation under strain. despite the fact that stirred suspension cultures are very useful for the generation of a large number of undifferentiated cells, we have found that the addition of medium enhancers is not adequate to force complete differentiation of the population in suspension bioreactors. by elucidating the exact mechanism by which liquid shear stress may contribute to promoting pluripotency and preventing differentiation, we will be able to create an efficient environment for both the production of large quantities of pluripotent stem cells, and their differentiated progeny. this is an important objective for human regenerative medicine, as lineage selection using transgenes will not be possible.  although stirred suspension bioreactors are very advantageous for the scalable generation of undifferentiated cells, addition of medium enhancers is not sufficient to induce complete differentiation. our observation that bioreactor-derived cardiomyocyte cultures maintain characteristics of pluripotency has significant implications for human regenerative medicine. we believe that liquid shear stress in the stirred suspension bioreactor plays an important mechanistic role in bioreactor induced pluripotency, which needs further investigation. 
the development of successful esc differentiation protocols within suspension bioreactors demands a more complete understanding of the impacts of shear forces on the regulation of pluripotency and differentiation in pluripotent stem cells. this study demonstrates that although cardiomyocyte differentiation can be achieved in stirred suspension bioreactors, the addition of medium enhancers is not adequate to force complete differentiation as fluid shear forces appear to maintain a subpopulation of cells in a transient pluripotent state. the effect of mechanical forces and shear stress on esc pluripotency and differentiation has yet to be clarified. remarkably, however, our results demonstrated that the bioreactor differentiated escs retained their ability to express pluripotency markers, to form esc-like colonies, and to generate teratomas upon transplantation, whereas the cells differentiated in adherent culture lost these characteristics. embryonic stem cells can proliferate endlessly and are able to differentiate into all cell lineages that make up the adult organism. our results showed that the bioreactor differentiated cells displayed the characteristics of fully functional cardiomyocytes. under particular in vitro culture conditions, escs can be expanded and induced to differentiate into cardiomyocytes in stirred suspension bioreactors. murine d3-mhc-neor escs formed embyroid bodies and differentiated into cardiomyocytes over days in static culture and suspension bioreactors. the purpose of this study was to investigate the impact of the suspension culture environment on esc pluripotency during cardiomyocyte differentiation.
 embryonic stem cells can proliferate endlessly and are able to differentiate into all cell lineages that make up the adult organism. under particular in vitro culture conditions, escs can be expanded and induced to differentiate into cardiomyocytes in stirred suspension bioreactors. however, in using these systems we must be cognizant of the mechanical forces acting upon the cells. the effect of mechanical forces and shear stress on esc pluripotency and differentiation has yet to be clarified. the purpose of this study was to investigate the impact of the suspension culture environment on esc pluripotency during cardiomyocyte differentiation.  murine d3-mhc-neor escs formed embyroid bodies and differentiated into cardiomyocytes over days in static culture and suspension bioreactors. g was used in both systems from day to enrich for cardiomyocytes by eliminating non-resistant, undifferentiated cells. treatment of ebs with mm ascorbic acid and % dimethyl sulfoxide from day markedly increased the number of beating ebs, which displayed spontaneous and cadenced contractile beating on day in the bioreactor. our results showed that the bioreactor differentiated cells displayed the characteristics of fully functional cardiomyocytes. remarkably, however, our results demonstrated that the bioreactor differentiated escs retained their ability to express pluripotency markers, to form esc-like colonies, and to generate teratomas upon transplantation, whereas the cells differentiated in adherent culture lost these characteristics.  this study demonstrates that although cardiomyocyte differentiation can be achieved in stirred suspension bioreactors, the addition of medium enhancers is not adequate to force complete differentiation as fluid shear forces appear to maintain a subpopulation of cells in a transient pluripotent state. the development of successful esc differentiation protocols within suspension bioreactors demands a more complete understanding of the impacts of shear forces on the regulation of pluripotency and differentiation in pluripotent stem cells. 