BACKGROUND
human umbilical cord blood contains a subset of stem cells that can differentiate into cells representative of all three germline layers  <cit> .
we follow kögler, the first to describe the multilineage capacity of these cells in vivo, in calling them "unrestricted somatic stem cells"   <cit> .
a number of studies demonstrate the therapeutic potential of usscs in bone healing, as accessory cells to reduce graft-versus-host disease, in the repair of myocardial infarcts and as vehicles for gene therapy  <cit> .
although usscs are rare compared to haematopoietic stem cells in cord blood, they can be expanded rapidly to yield large numbers of cells for study or transplantation.
culturing of usscs depends on growth in medium containing a high concentration of specific batches of fetal calf serum.
when cultured in fetal calf serum, usscs form an adherent monolayer, do not require a feeder layer, and can be cultured without differentiating or losing their proliferative capacity.
before usscs can be used therapeutically, a method for large scale amplification of cells under good manufacturing practice  conditions is necessary, preferably using defined medium free of xenobiotic components.
fetal calf serum contains undefined factors, which may vary from batch to batch, and that may activate or inhibit stem cell differentiation.
the growth of cells in defined medium not only reduces variability, but also eliminates the need for costly and time consuming fetal calf serum batch testing.
additionally, medium that is free of non-human products reduces concerns about transmission of infectious cross-species pathogens , and possible immunogenic responses to non-human proteins  <cit> .
here we investigate the capacity of three different stem cell culture media hescgro™, psm and ussc growth mediumacf to enable maintenance of usscs in serum-free conditions.
psm is of interest as it contains sphingosine-1-phosphate  and platelet-derived growth factor , factors known to enable the survival and proliferation of human embryonic stem  cells in an undifferentiated state for a prolonged period of time  <cit> .
s1p is a bioactive sphingolipid present in serum that binds to its own g protein-coupled receptors, sphingosine-1-phosphate receptor  <dig> to  <dig> ), and activates various intracellular signaling pathways depending on the cell type  <cit> .
s1p regulates key biological processes such as cell proliferation, motility, migration and survival in both adult and embryonic stem cell types  <cit> .
pdgf is a growth factor, also present in serum, that is widely described as a potent mitogen  <cit> .
pdgf is comprised of homo- and hetero-dimeric isoforms of polypeptide chains a, b, c and d  <cit> .
the response of a cell to a given isoform of pdgf is dependent on the signaling pathways activated by the two platelet-derived growth factor receptors , and the capacity of the cell to respond to these signals  <cit> .
hescgro™ is a commercial serum- and animal component-free, defined medium known to support the growth and pluripotency maintenance of hes cells.
the media contains fgf <dig>  that, like pdgf, has been reported as having proliferative and differentiation effects on various cell types  <cit> , and figures quite prominently in numerous other adult  <cit> , or embryonic-derived stem cell media formulations  <cit> .
RESULTS
usscs form spheres in ussc growth mediumacf
we have analyzed the growth of usscs in three serum-free media formulations; hescgro, psm and ussc growth mediumacf.
figures 1a clearly demonstrate that cells cultured in hescgro and psm do not grow, whereas cells grown in ussc growth mediumacf proliferate at the same rate as cells grown in stem cell proliferation medium containing 30% fetal calf serum.
however, after one to three passages in ussc growth mediumacf cells start to form small clusters, which become tight spheres after a further  <dig> hours .
these spheres are initially heterogeneous in shape, but with continued culturing, rounded spheres with defined borders are observed.
seven days after their first appearance, the size of the spheres varies.
figure 1c shows that the majority of spheres  are small in diameter , 31% are medium  and the remaining spheres  are large .
the smallest sphere observed was  <dig> μm, and the largest was  <dig> μm.
as the culturing of stem cells as spheres is important for a number of applications, we set out to confirm whether usscs could form spheres using one of the standard sphere formation methods  <cit> .
using the serum-free hanging drop method, we cultured cells either in ussc growth mediumacf, or stem cell proliferation media without fetal calf serum.
figure 2a shows that cells grown in stem cell proliferation media without fetal calf serum in the presence or absence of bfgf do not form spheres, but cells grown in ussc growth mediumacf always form spheres.
there also appears to be a differential effect on the morphology of the sphere depending on whether bfgf is used, in that spheres derived using bfgf are smaller, but tighter, and have less of a halo.
to determine their structure, spheres were serially sectioned and stained with haematoxylin and eosin.
figure 2b shows representative images of a serial section of a sphere.
the cells in the spheres appear homogenous in size and morphology and 48% of the spheres also contained cavities.
altogether, these data suggest that ussc growth mediumacf enables ussc proliferation, that this growth occurs simultaneously with the loss of the cells' ability to adhere to plastic, and that these non-adherent cells can continue to grow in suspension as spheres.
ussc-derived spheres can revert to monolayer growth
the ability of mechanically dissociated spheres to revert to monolayer growth and continue to proliferate was studied after culture in ussc growth mediumacf on fibronectin or gelatin coated surfaces, or in the presence of stem cell proliferation media containing fetal calf serum.
figure 1d shows that the cells from the spheres re-adhere and morphologically resemble the parental usscs in the presence of fetal calf serum, or when the surface is coated with extracellular matrix proteins.
these cells were cultured for up to two weeks, during which they were passaged three times and expanded up to six-fold.
this confirms that cells within the spheres, which have been cultured in ussc growth mediumacf, can revert to monolayer growth and continue to proliferate.
usscs maintain their multipotentiality when cultured as spheres
to determine if day seven spheres express pluripotency-associated markers, the expression of oct <dig> and sox <dig> was determined by rt-pcr.
figure  <dig> shows that usscs grown in adherent culture do not express oct <dig>  but do express sox <dig>  and that usscs grown as spheres maintain this expression profile.
in view of recent reports questioning accurate detection of oct <dig> mrna expression  <cit> , we confirmed that the primers used in this study do not bind to oct4b  and are specific to the cdna of the nuclear-localized oct4a .
we also confirmed this result using antibody staining.
the antibody staining clearly detects nuclear-localized oct <dig> in hes cells, but expression could not be detected in usscs nor seven day spheres .
to determine if the cells in the spheres initiate a differentiation program, we analysed markers associated with commitment of stem cells.
figures  <dig> and  <dig> show there is no change in the expression profile of markers associated with ectodermal , mesodermal  and endodermal  differentiation in day seven spheres when compared to usscs.
this expression profile is also maintained after five passages as determined by rt-pcr for sox <dig>  pax <dig>  gata <dig>  and brachyury .
spheres can be differentiated to show expression of markers representative of the three germline layers
to confirm that the differentiation capacity of the cells in the spheres had been maintained; seven day spheres were dissociated and incubated in lineage-specific differentiation medium.
figure  <dig> shows that the cells within the spheres maintain the ability to differentiate and express markers representative of the three germline layers.
cells expressing neuronal-specific β-tubulin iii , that stain positive for mineralization as detected by alizarin red s staining  and express epithelial-specific surfactant protein-c  were all observed.
the differentiation capacity of the cells is also maintained after five passages in ussc growth mediumacf .
discussion
with the goal of deriving clinically safe usscs, we aimed to culture established usscs in the serum- and animal component-free medium, ussc growth mediumacf.
we observe that usscs continue to proliferate in ussc growth mediumacf, but after one to three passages, the cells aggregate and grow in suspension as spheres.
we show that spheres can be dissociated and can continue to grow for five passages, as long as they are dissociated before the sphere becomes cystic.
we also show the spheres can revert to monolayer growth when provided with extracellular matrix support or when plated in medium containing fetal calf serum.
cells passaged in uscc growth mediumacf maintained their gene expression profile as judged by sox <dig>  brachyury, pax <dig> and gata <dig> expression.
cells also maintained their capacity to form bone-like alizarin red positive cells, spc positive epithelial cells and β-tubulin iii positive neuronal cells after directed differentiation.
this is the first report of a serum-free medium that enables proliferation of usscs and the first to report that usscs can be grown in suspension as spheres, without losing their differentiation capacity.
growing stem cells as spheres has been the initial step in a wide range of differentiation studies, as well as studies in pharmacogenetics, teratogenesis and tumourigenesis  <cit> .
sphere formation can be facilitated by growing cells in specific medium , on non-adherent plates, in bioreactors, in agar, as hanging drops, or by centrifugation  <cit> .
despite these many studies, the significance of sphere formation is yet to be fully understood.
while it could reflect a non-specific event, it could alternatively be an active process essential for continued development and differentiation.
the differentiation potential of stem cells in spheres appears to be dependent upon the starting cell population and the medium in which the spheres are cultured.
for example, human bone marrow stromal cells can be conditioned to give rise to neurospheres, which lose their stem cell characteristics and become committed to neuronal development  <cit> .
to characterize the spheres derived in this study further, we investigated a number of markers expressed in pluripotent cells and in cells representative of the three germline layers.
we show that oct4-negative usscs give rise to spheres, and that cells within these spheres continue to express sox <dig>  analysis of differentiation-associated markers  by rt-pcr confirms that cells cultured in ussc growth mediumacf do not initiate a program of differentiation.
the ability of ussc spheres to maintain an undifferentiated state makes them distinct from other spheres derived from cells capable of multilineage differentiation.
the most studied of these spheres are embryoid bodies, defined as spherical clusters of both pluripotent and committed stem cells that can organize in a developmental-specific manner  <cit> .
unlike embryoid bodies, ussc spheres do not commit to any differentiation programs when cultured in ussc growth mediumacf.
the cavities detected in ussc spheres are also distinct from cavities observed in embryoid bodies.
the formation of cavities in embryoid bodies is an active process, characterized by a thickening of the wall around the cavity, and associated with the expression of endodermal-specific markers such as cytokeratin  <dig>  <cit> .
while ussc spheres can have cavities, we show that the cells surrounding the cavities and the outer layer of the sphere do not express the endodermal marker cytokeratin  <dig>
as marker analysis is insufficient to infer function, we also analyzed the ability of the cells to respond to differentiation cues.
when ussc spheres are reverted to monolayer growth and differentiated in lineage-specific medium, surfactant protein c-positive epithelial-like cells, β-tubulin iii-positive neuronal-like cells and alizarin red s-positive bone-like cells are observed after one to five passages, confirming that the cells in the spheres are still multipotent.
however, derivation of spheres from a single cell and validation that each cell in the sphere retains these stem cell properties is still essential.
CONCLUSIONS
we present a new method to grow usscs in suspension without losing their multi-lineage differentiation capacity in short term assays.
the ability to culture these cells in a three-dimensional spherical structure in a defined animal component-free environment is a first step towards improving protocols for tissue engineering, and expansion of these cells in vitro.
although there are a number of serum-free media marketed for culturing other types of stem cells, ussc growth mediumacf is unique because it enables the short term expansion of usscs in suspension, the formation of spheres, and the maintenance of the multilineage differentiation capacity of usscs.
suspension culturing of cells facilitates large scale expansion, but we note that it will still be necessary to validate the genetic stability and safety of cells after long term cell growth before they can be used therapeutically.
the latter is also heightened by two recent publications using human cord to derive ipsc  <cit>  which would further expand ussc multipotentiality for therapeutic utility, including drug discovery.

 human umbilical cord blood contains a subset of stem cells that can differentiate into cells representative of all three germline layers. we follow kögler, the first to describe the multilineage capacity of these cells in vivo, in calling them "unrestricted somatic stem cells". a number of studies demonstrate the therapeutic potential of usscs in bone healing, as accessory cells to reduce graft-versus-host disease, in the repair of myocardial infarcts and as vehicles for gene therapy. although usscs are rare compared to haematopoietic stem cells in cord blood, they can be expanded rapidly to yield large numbers of cells for study or transplantation. culturing of usscs depends on growth in medium containing a high concentration of specific batches of fetal calf serum. when cultured in fetal calf serum, usscs form an adherent monolayer, do not require a feeder layer, and can be cultured without differentiating or losing their proliferative capacity. before usscs can be used therapeutically, a method for large scale amplification of cells under good manufacturing practice conditions is necessary, preferably using defined medium free of xenobiotic components. fetal calf serum contains undefined factors, which may vary from batch to batch, and that may activate or inhibit stem cell differentiation. the growth of cells in defined medium not only reduces variability, but also eliminates the need for costly and time consuming fetal calf serum batch testing. additionally, medium that is free of non-human products reduces concerns about transmission of infectious cross-species pathogens, and possible immunogenic responses to non-human proteins. here we investigate the capacity of three different stem cell culture media hescgro™, psm and ussc growth mediumacf to enable maintenance of usscs in serum-free conditions. psm is of interest as it contains sphingosine-1-phosphate and platelet-derived growth factor, factors known to enable the survival and proliferation of human embryonic stem cells in an undifferentiated state for a prolonged period of time. s1p is a bioactive sphingolipid present in serum that binds to its own g protein-coupled receptors, sphingosine-1-phosphate receptor to ), and activates various intracellular signaling pathways depending on the cell type. s1p regulates key biological processes such as cell proliferation, motility, migration and survival in both adult and embryonic stem cell types. pdgf is a growth factor, also present in serum, that is widely described as a potent mitogen. pdgf is comprised of homo- and hetero-dimeric isoforms of polypeptide chains a, b, c and d. the response of a cell to a given isoform of pdgf is dependent on the signaling pathways activated by the two platelet-derived growth factor receptors, and the capacity of the cell to respond to these signals. hescgro™ is a commercial serum- and animal component-free, defined medium known to support the growth and pluripotency maintenance of hes cells. the media contains fgf that, like pdgf, has been reported as having proliferative and differentiation effects on various cell types, and figures quite prominently in numerous other adult, or embryonic-derived stem cell media formulations.  usscs form spheres in ussc growth mediumacf we have analyzed the growth of usscs in three serum-free media formulations; hescgro, psm and ussc growth mediumacf. figures 1a clearly demonstrate that cells cultured in hescgro and psm do not grow, whereas cells grown in ussc growth mediumacf proliferate at the same rate as cells grown in stem cell proliferation medium containing 30% fetal calf serum. however, after one to three passages in ussc growth mediumacf cells start to form small clusters, which become tight spheres after a further hours. these spheres are initially heterogeneous in shape, but with continued culturing, rounded spheres with defined borders are observed. seven days after their first appearance, the size of the spheres varies. figure 1c shows that the majority of spheres are small in diameter, 31% are medium and the remaining spheres are large. the smallest sphere observed was μm, and the largest was μm. as the culturing of stem cells as spheres is important for a number of applications, we set out to confirm whether usscs could form spheres using one of the standard sphere formation methods. using the serum-free hanging drop method, we cultured cells either in ussc growth mediumacf, or stem cell proliferation media without fetal calf serum. figure 2a shows that cells grown in stem cell proliferation media without fetal calf serum in the presence or absence of bfgf do not form spheres, but cells grown in ussc growth mediumacf always form spheres. there also appears to be a differential effect on the morphology of the sphere depending on whether bfgf is used, in that spheres derived using bfgf are smaller, but tighter, and have less of a halo. to determine their structure, spheres were serially sectioned and stained with haematoxylin and eosin. figure 2b shows representative images of a serial section of a sphere. the cells in the spheres appear homogenous in size and morphology and 48% of the spheres also contained cavities. altogether, these data suggest that ussc growth mediumacf enables ussc proliferation, that this growth occurs simultaneously with the loss of the cells' ability to adhere to plastic, and that these non-adherent cells can continue to grow in suspension as spheres. ussc-derived spheres can revert to monolayer growth the ability of mechanically dissociated spheres to revert to monolayer growth and continue to proliferate was studied after culture in ussc growth mediumacf on fibronectin or gelatin coated surfaces, or in the presence of stem cell proliferation media containing fetal calf serum. figure 1d shows that the cells from the spheres re-adhere and morphologically resemble the parental usscs in the presence of fetal calf serum, or when the surface is coated with extracellular matrix proteins. these cells were cultured for up to two weeks, during which they were passaged three times and expanded up to six-fold. this confirms that cells within the spheres, which have been cultured in ussc growth mediumacf, can revert to monolayer growth and continue to proliferate. usscs maintain their multipotentiality when cultured as spheres to determine if day seven spheres express pluripotency-associated markers, the expression of oct and sox was determined by rt-pcr. figure shows that usscs grown in adherent culture do not express oct but do express sox and that usscs grown as spheres maintain this expression profile. in view of recent reports questioning accurate detection of oct mrna expression, we confirmed that the primers used in this study do not bind to oct4b and are specific to the cdna of the nuclear-localized oct4a. we also confirmed this result using antibody staining. the antibody staining clearly detects nuclear-localized oct in hes cells, but expression could not be detected in usscs nor seven day spheres. to determine if the cells in the spheres initiate a differentiation program, we analysed markers associated with commitment of stem cells. figures and show there is no change in the expression profile of markers associated with ectodermal, mesodermal and endodermal differentiation in day seven spheres when compared to usscs. this expression profile is also maintained after five passages as determined by rt-pcr for sox pax gata and brachyury. spheres can be differentiated to show expression of markers representative of the three germline layers to confirm that the differentiation capacity of the cells in the spheres had been maintained; seven day spheres were dissociated and incubated in lineage-specific differentiation medium. figure shows that the cells within the spheres maintain the ability to differentiate and express markers representative of the three germline layers. cells expressing neuronal-specific β-tubulin iii, that stain positive for mineralization as detected by alizarin red s staining and express epithelial-specific surfactant protein-c were all observed. the differentiation capacity of the cells is also maintained after five passages in ussc growth mediumacf. discussion with the goal of deriving clinically safe usscs, we aimed to culture established usscs in the serum- and animal component-free medium, ussc growth mediumacf. we observe that usscs continue to proliferate in ussc growth mediumacf, but after one to three passages, the cells aggregate and grow in suspension as spheres. we show that spheres can be dissociated and can continue to grow for five passages, as long as they are dissociated before the sphere becomes cystic. we also show the spheres can revert to monolayer growth when provided with extracellular matrix support or when plated in medium containing fetal calf serum. cells passaged in uscc growth mediumacf maintained their gene expression profile as judged by sox brachyury, pax and gata expression. cells also maintained their capacity to form bone-like alizarin red positive cells, spc positive epithelial cells and β-tubulin iii positive neuronal cells after directed differentiation. this is the first report of a serum-free medium that enables proliferation of usscs and the first to report that usscs can be grown in suspension as spheres, without losing their differentiation capacity. growing stem cells as spheres has been the initial step in a wide range of differentiation studies, as well as studies in pharmacogenetics, teratogenesis and tumourigenesis. sphere formation can be facilitated by growing cells in specific medium, on non-adherent plates, in bioreactors, in agar, as hanging drops, or by centrifugation. despite these many studies, the significance of sphere formation is yet to be fully understood. while it could reflect a non-specific event, it could alternatively be an active process essential for continued development and differentiation. the differentiation potential of stem cells in spheres appears to be dependent upon the starting cell population and the medium in which the spheres are cultured. for example, human bone marrow stromal cells can be conditioned to give rise to neurospheres, which lose their stem cell characteristics and become committed to neuronal development. to characterize the spheres derived in this study further, we investigated a number of markers expressed in pluripotent cells and in cells representative of the three germline layers. we show that oct4-negative usscs give rise to spheres, and that cells within these spheres continue to express sox analysis of differentiation-associated markers by rt-pcr confirms that cells cultured in ussc growth mediumacf do not initiate a program of differentiation. the ability of ussc spheres to maintain an undifferentiated state makes them distinct from other spheres derived from cells capable of multilineage differentiation. the most studied of these spheres are embryoid bodies, defined as spherical clusters of both pluripotent and committed stem cells that can organize in a developmental-specific manner. unlike embryoid bodies, ussc spheres do not commit to any differentiation programs when cultured in ussc growth mediumacf. the cavities detected in ussc spheres are also distinct from cavities observed in embryoid bodies. the formation of cavities in embryoid bodies is an active process, characterized by a thickening of the wall around the cavity, and associated with the expression of endodermal-specific markers such as cytokeratin. while ussc spheres can have cavities, we show that the cells surrounding the cavities and the outer layer of the sphere do not express the endodermal marker cytokeratin as marker analysis is insufficient to infer function, we also analyzed the ability of the cells to respond to differentiation cues. when ussc spheres are reverted to monolayer growth and differentiated in lineage-specific medium, surfactant protein c-positive epithelial-like cells, β-tubulin iii-positive neuronal-like cells and alizarin red s-positive bone-like cells are observed after one to five passages, confirming that the cells in the spheres are still multipotent. however, derivation of spheres from a single cell and validation that each cell in the sphere retains these stem cell properties is still essential.  we present a new method to grow usscs in suspension without losing their multi-lineage differentiation capacity in short term assays. the ability to culture these cells in a three-dimensional spherical structure in a defined animal component-free environment is a first step towards improving protocols for tissue engineering, and expansion of these cells in vitro. although there are a number of serum-free media marketed for culturing other types of stem cells, ussc growth mediumacf is unique because it enables the short term expansion of usscs in suspension, the formation of spheres, and the maintenance of the multilineage differentiation capacity of usscs. suspension culturing of cells facilitates large scale expansion, but we note that it will still be necessary to validate the genetic stability and safety of cells after long term cell growth before they can be used therapeutically. the latter is also heightened by two recent publications using human cord to derive ipsc which would further expand ussc multipotentiality for therapeutic utility, including drug discovery. 
this is the first demonstration that usscs can grow in a serum- and animal component-free medium and that usscs can form spheres. analysis of markers associated with pluripotency and differentiation confirms that cells in the spheres maintain their gene expression profile. for the first one to three passages, cells grown in ussc growth mediumacf proliferate and maintain their morphology, but with continued passaging the cells form spherical cell aggregates. a major obstacle to their clinical use is the fact that in vitro expansion is still dependent upon fetal calf serum, which could be a source of pathogens. dissociated cells can also revert to monolayer growth when cultured on extracellular matrix support, or in medium containing fetal calf serum. the cells in the spheres also retain the ability to differentiate in vitro to form cells representative of the three germline layers after five passages. in this study, we investigate the capacity of three different stem cell culture media to support usscs in serum-free conditions; hescgro™, psm and ussc growth mediumacf. our findings demonstrate that usscs do not grow in hescgro™ or psm, but we were able to isolate, proliferate and maintain multipotency of three ussc lines in ussc growth mediumacf. these data suggest that ussc growth mediumacf maintains usscs in an undifferentiated state and supports growth in suspension. human umbilical cord blood-derived unrestricted somatic stem cells, which are capable of multilineage differentiation, are currently under investigation for a number of therapeutic applications.
 human umbilical cord blood-derived unrestricted somatic stem cells, which are capable of multilineage differentiation, are currently under investigation for a number of therapeutic applications. a major obstacle to their clinical use is the fact that in vitro expansion is still dependent upon fetal calf serum, which could be a source of pathogens. in this study, we investigate the capacity of three different stem cell culture media to support usscs in serum-free conditions; hescgro™, psm and ussc growth mediumacf. our findings demonstrate that usscs do not grow in hescgro™ or psm, but we were able to isolate, proliferate and maintain multipotency of three ussc lines in ussc growth mediumacf.  for the first one to three passages, cells grown in ussc growth mediumacf proliferate and maintain their morphology, but with continued passaging the cells form spherical cell aggregates. upon dissociation of spheres, cells continue to grow in suspension and form new spheres. dissociated cells can also revert to monolayer growth when cultured on extracellular matrix support, or in medium containing fetal calf serum. analysis of markers associated with pluripotency and differentiation confirms that cells in the spheres maintain their gene expression profile. the cells in the spheres also retain the ability to differentiate in vitro to form cells representative of the three germline layers after five passages.  these data suggest that ussc growth mediumacf maintains usscs in an undifferentiated state and supports growth in suspension. this is the first demonstration that usscs can grow in a serum- and animal component-free medium and that usscs can form spheres. 