BACKGROUND
mesenchymal stem cells  are extensively used as the cell source for regenerative medicine because of their capacities to differentiate into different lineages and expand while maintaining their undifferentiated state.
mscs are commonly cultured as two-dimensional  monolayer by traditional tissue culture techniques.
however, the 2d culture method has difficulty in overcoming the replicative senescence and maintaining the self-renewal properties, which results in cells of low differentiation capacity  <cit> .
a three-dimensional  culture environment is considered more favorable than 2d monolayer culture for cell-cell contacts.
previous studies have developed several methods to generate 3d msc spheroids.
many of these methods involve the use of cell suspension system or non-adherent surface to induce spheroid formation .
in general, these 3d msc spheroids were reported to have greater differentiation capacities.
chitosan  is the deacetylated derivative of chitin which is abundant in shell of shrimp or crap, and in fungus, and the content is only next to cellulose in nature.
cs has a main structure composed of β linked d-glucosamine residues with different amounts of n-acetyl-glucosamine group.
owing to its biocompatibility and biodegradability, cs has been widely studied as a scaffolding material for tissue engineering.
recent findings have revealed that mscs attached on the membranes made of cs can form self-assembled 3d cellular spheroids .
during the process, mscs attach and spread on cs membranes before they retract their pseudopodia to form multicellular spheroids.
this self-assembly process is quite different from that occurs in suspension or hanging drop systems, or on non-adherent polymer surfaces.
several genes/proteins have been referred to participate in the process of spheroid formation on cs, including cadherin molecules  <cit> , rho/rho-associated kinase   <cit> , and the wnt molecule  <cit> .
activations of these proteins were not as evident for spheroids on non-adherent surfaces.
in addition, it was observed that the surface-bound calcium on cs substrates may be transported into mscs and play a role in spheroid formation as well as gene regulation  <cit> .
although a few changes in gene/protein expression were observed, the exact mechanism for spheroid formation on cs is still far from being elucidated.
therefore, a more comprehensive understanding of the genomic profile for cs-derived msc spheroids is essential for further revealing the substrate-dependent nature of these unique msc spheroids.
the technique of microarray has been developed to detect the changes within cells and is a powerful tool by which many genes can be probed simultaneously.
dalby et al.
have reported the genomic expression profile of human mscs responding to the shape of their environment by the messenger rna  microarray  <cit> .
the antiinflammatory properties of human msc spheroids generated by hanging drop have also been compared to those of the adherent msc monolayer by surveying with mrna microarray  <cit> .
furthermore, recent advances in microrna , a class of non-coding small rna, have identified a few important modulators in stem cell proliferation and differentiation.
they can bind to the cognate mrna to repress the expression of target genes.
simultaneous analyses of the mrna and mirna expression profiles may help narrowing down the signaling events involved in the behavior change of the cells  <cit> .
in this study, we examined both mrna and mirna expression profiles of the cs substrate-induced 3d spheroids of human mscs isolated from the umbilical cord, using 2d mscs on tissue culture polystyrene  as a control.
cross-correlation analysis of the results from these two microarrays was further confirmed with the quantitative reverse transcriptase-polymerase chain reaction  analysis to identify the critical signaling events for substrate-derived msc spheroids during the spheroid forming process.
RESULTS
characteristics of human umbilical cord mscs
the expression profile of cell surface markers analyzed by flow cytometry is shown in additional file 1: figure s <dig>  human umbilical cord mscs were positive for specific antigen markers of mscs such as cd <dig>  cd <dig>  cd <dig>  cd <dig>  cd <dig>  cd <dig>  cd <dig>  cd <dig>  cd <dig>  cd <dig>  and hla-abc, and negative for specific markers of endothelial cells and haematopoietic cells including cd <dig>  cd <dig>  cd <dig>  cd <dig>  and hla-dr. besides, cells showed positive expression of cd <dig> and low expression of cd <dig>  which was consistent with that described for human umbilical cord mscs in literatures  <cit> .
surface properties of cs membranes
the different surface properties of cs membranes and tcps are summarized in figure  <dig>  the static water contact angle of cs membranes was  <dig> ± <dig> °  and that of tcps was  <dig> ± <dig> ° .
the greater contact angle of cs vs. tcps revealed the slightly higher hydrophobicity of cs, which may be attributed to the rearrangement of hydrophobic n-acetyl groups of cs to move to the surface and reduce the surface free energy while exposed to atmosphere.
the surface zeta potential of cs was relatively neutral , distinct from the negatively charged tcps .
previous literature suggested an isoelectric point at ph  <dig>  for cs arising from the deprotonation of positive amino group  <cit> .
another study indicated that the amino groups of cs could chelate calcium ion to form cs/calcium ion complexes   <cit> .
the amount of surface-bound calcium on cs membranes  was significantly greater than that on tcps after either  <dig> h or  <dig> h. the consistent values observed at  <dig> h and  <dig> h suggested a saturation of surface-bound calcium.figure  <dig>
surface properties of cell culture substrates.
the static contact angle of cs and tcps.
the surface zeta potential of cs and tcps.
the amount of surface-bound calcium on cs or tcps after being soaked in the culture medium for  <dig> h without cells.
* p <  <dig>  among the indicated groups .
phenotype of mscs grown on cs or tcps.
the morphologies of mscs on cs or tcps after  <dig> h,  <dig> h and  <dig> h.  the cell viability of msc spheroids on cs after  <dig> h analyzed by flow cytometry.
the percentage of cells without being stained by pi was defined as the cell viability.
spheroid formation for mscs growth on cs membranes
the morphology of mscs grown on cs membranes and tcps is shown in figure  <dig>  as was expected, mscs attached to tcps with fibroblast-like morphology.
on the other hand, mscs on cs remained attached to the surface before  <dig> h and formed spheroids afterwards.
the average diameter of the msc spheroids on cs membranes was  <dig>  ±  <dig>  μm at  <dig> h .
the spheroid size was relatively stable within  <dig> h .
the cell viability for the msc spheroids was  <dig> % at  <dig> h.
cross-correlation analysis between mrna and mirna microarrays
based on the screening of the mrna microarray,  <dig> upregulated genes and  <dig> downregulated genes showed significant difference  for mscs on cs vs. tcps.
on the other hand, screening based on the mirna microarray showed that there were  <dig> targeted genes corresponding to the downregulated mirna, and  <dig> targeted genes corresponding to the upregulated mirna.
cross-correlation analysis of these two microarray data have helped narrowing down the critical genes involved in the behavioral changes of these cells.
screening based on the cross-correlation analysis with the software, agilent.twocolor.
<dig>  revealed that there were  <dig> upregulated genes and  <dig> downregulated genes.
the results of microarray analyses are shown in figure  <dig>  in addition, the screened genes as well as their expression ratio are listed in additional file  <dig> figure  <dig>
the results of microarrays for mscs grown on cs or tcps.
the screening of the mirna microarray for mscs grown on cs or tcps.
the numbers of genes that were subjected to cross-correlation analysis of mrna and mirna microarrays and the numbers of screened genes based on the cross-correlation analysis.
a complete list of screened genes as well as their expression ratios is shown in additional file  <dig>
the biological significance behind the list of screened genes was further analyzed by the david  program to evaluate the gene enrichment in gene ontology  and kyoto encyclopedia of genes and genomes  pathway maps .
the results of gene enrichment in kegg pathway maps suggested that several pathways were regulated for mscs on cs vs. tcps including calcium signaling pathway , focal adhesion , regulation of actin cytoskeleton , adherens junction , gap junction , ecm-receptor interaction , p <dig> signaling pathway , cytokine-cytokine receptor interaction , tryptophan metabolism , and tgf-β signaling pathway .
among the  <dig> genes, we picked up the ones with high expression ratio, the critical ones in certain signaling pathways, or the ones which had been reported in literature to be associated with cell adhesion, migration, or fate decision.
they were then further confirmed by qrt-pcr.
regulation of calcium-associated genes for mscs on cs
a number of calcium-associated genes were noted and listed in table  <dig> including those of calcium channels, receptors, and intracellular signaling proteins.
atp2b <dig> and atp2b <dig>  are highly regulated ca2+ extrusion pumps and provide fine-tuning of intracellular calcium level  <cit> .
slc8a <dig>  is a na+-ca2+ exchanger which also modulates ca2+ extrusion system from the cells  <cit> .
trpa <dig> and trpc <dig> belong to the transient receptor potential  channel superfamily that regulates the mechanism for ca2+ entry  <cit> .
the upregulation of atp2b <dig> and trpa <dig> and downregulation of atp2b <dig>  slc8a <dig>  and trpc <dig> for mscs on cs vs. tcps suggested that the process for intracellular calcium homeostasis was turned on.
modulation of these genes may cause an elevation in the intracellular calcium level.table  <dig>
the group of calcium signaling-associated genes screened from microarrays
ma: based on microarray data.
qrt-pcr: based on quantitative rt-pcr analysis.
*p <  <dig>  between cs and tcps groups .
a few genes of calcium-associated receptors were upregulated for mscs growth on cs vs. tcps.
htr2a and htr <dig>  are the receptors for serotonin which is a well-characterized neurotransmitter with regulative function in multiple physiological aspects  <cit> .
gpr <dig>  is a proton-sensing receptor that can modulate the level of intracellular calcium  <cit> .
pdgfra  and f2r  are also intracellular calcium modulators  <cit> .
the activation of these calcium-associated receptors may directly enhance the level of intracellular calcium and lead further to cytoskeleton rearrangement.
several intracellular signaling genes were also upregulated.
these genes included those of map3k <dig> , itpr <dig>  rasgrp <dig> , pla2g4a .
map3k <dig> is required for the transduction of signals initiated by par <dig> and other g-coupled receptors, which promote actin reorganization and cell migration.
map3k <dig> can also mediate signal-induced increases in cytoplasmic ca2+ through the activation of phospholipase c  <cit> .
on the other hand, itpr <dig> is a ca2+-release channel located on intracellular membranes, especially the endoplasmic reticulum .
the ip <dig> receptor has an affinity for ip <dig> in the low nanomolar range.
moreover, cytosolic ca2+ is considered as a co-agonist of the ip <dig> receptor, as it strongly increases the ip <dig> receptor activity at concentrations up to about  <dig> nm  <cit> .
rasgrp <dig> is a calcium and dag-regulated ras guanyl releasing protein which can activate small gtpases such as ras and rap <dig>  <cit> .
pla2g4a is a member of phospholipases a <dig>  superfamily, which regulates the release of arachidonic acid   <cit> .
although calmodulin is also a family of ca2+ binding proteins and mediates many important cellular processes  <cit> , calm <dig>  gene was downregulated.
the regulation of various calcium-associated genes suggested the critical role of calcium signaling in the cs-derived msc spheroids, which has not yet been reported in any other spheroid systems.
regulation of cell adhesion and migration/cytoskeleton-associated genes for mscs on cs
genes that were screened out for mscs on cs and associated with cell adhesion, migration, or cytoskeleton reorganization are displayed in table  <dig>  mmp <dig>  is a kind of interstitial collagenase, and its activity was enhanced in highly migrating mscs compared with poorly migrating mscs  <cit> .
mmp <dig> and mmp <dig> were also upregulated which have similar substrate specificity.
mmp <dig> is correlated with neuronal migration and neurite outgrowth and is able to activate mmp <dig>  <cit> .
the migration ability of cs-derived msc spheroids has been mentioned  <cit> .
the upregulation of these cell adhesion/migration-associated genes agreed with the high cell mobility of mscs on cs.table  <dig>
the group of adhesion and migration/cytoskeleton-associated genes screened from microarrays
*p <  <dig>  between cs and tcps groups .
on the other hand, a variety of genes that controls cell-to-cell adhesion were upregulated for mscs on cs, including cadherins, cell adhesion molecules , notch, and ephrin receptor.
cdh <dig>  is a ca2+-dependent cell-cell adhesion molecule, and expresses in the central nervous system  <cit> .
pcdh <dig>  is also a member of cadherin family, and has a role in embryo development  <cit> .
pecam <dig>  is an endothelial cell marker  <cit> .
the expression of notch <dig>  and its ligand, dll <dig> , were both enhanced for msc growth on cs.
notch signaling pathway is critical for cell fate decisions including proliferation, lineage commitment, and terminal differentiation in many adult stem cell types  <cit> .
epha <dig>  can bind to cell surface-associated ephrin ligands on neighboring cells to generate bidirectional signals that affect both the receptor-expressing and ephrin-expressing cells  <cit> .
based on the literature, epha <dig> is an axon guidance receptor important for the development of cortical circuits  <cit> .
the enhancement in these cell-cell adhesion genes may provide better cell-cell communication and coordination during spheroid formation.
the upregulation of chemokines and their receptors for mscs grown on cs vs. tcps is of particular interest.
among them, the gene encoding cmklr <dig>  was upregulated superbly .
cmklr <dig> was reported as a multifunctional receptor which can bind with the proinflammatory chemokine, chemerin, or with the anti-inflammatory lipid mediator, resolving e <dig>   <cit> .
chemerin/cmklr <dig> interaction was also reported to promote adipogenesis and angiogenesis  <cit> .
other upregulated chemokine receptors or ligands included the cxcr <dig> and cxcr <dig> , which are the receptors of stromal derived factor- <dig> .
cxcr <dig> is one of the most studied chemokine receptors that play an important role in cell migration, proliferation, and differentiation  <cit> .
the cxcl <dig>  is a ligand for another cxc motif chemokine receptor, cxcr <dig>  which was reported to crosstalk with cxcr <dig> and cxcr <dig>  <cit> .
the ccl <dig> and ccl <dig>  are important homing factors for mscs  <cit> .
cell fate decision in msc spheroids
the gene expression for a group of growth factors and receptors was modulated in mscs on cs vs. tcps, as listed in table  <dig>  these included tgf-β <dig> , bmp <dig> , hgf , igf1r , kdr  and kit .
the regulation of these genes may influence the function and fate of stem cells.table  <dig>
the group of development-associated genes screened from microarrays
* p <  <dig>  between cs and tcps groups .
several wnt signaling related genes were also enhanced for mscs on cs including the wls , wnt <dig> , lef <dig> , tcf <dig> , daam <dig> , and cxxc <dig> .
the wnt proteins and wnt signaling pathway are known to control cell specification and fate during embryonic development and adult tissue homeostasis.
wnt <dig>  a member of wnt family, can promote the earliest aspects of lung airway smooth muscle development  <cit> , and accelerate cardiac myocyte differentiation from es-cell derived mesodermal cells through the non-canonical wnt pathway  <cit> .
wls is a multipass transmembrane protein, and was found to control the secretion of wnt proteins  <cit> .
the tcf/lef family is the downstream proteins in the canonical wnt/ β-catenin pathway.
in response to wnt signals, tcf/lef members present as a switch to modulate the transcription of numerous target genes from repression to activation as binding with β-catenin  <cit> .
daam <dig> was identified as a downstream protein interacting with dishevelled , which mediates the non-canonical wnt/planar cell polarity  signaling pathway.
a study indicated that daam <dig> may play a crucial role in regulating the actin cytoskeleton and tissue morphogenesis  <cit> .
on the other hand, one of the negative regulators of wnt/ β-catenin signaling pathway, cxxc <dig>  was also enhanced for mscs on cs  <cit> .
two other development-related genes worth mentioning are rarb  and egr <dig> .
rarb is a nuclear receptor for retinoic acid  which is a vitamin a-derived, non-peptidic, small lipophilic molecule.
the ra signaling during embryo development has been extensively investigated  <cit> .
egr <dig> is a zinc-finger transcription factor of the early growth response gene  family that has critical functions in hindbrain development and myelination of the peripheral nervous system  <cit> .
the gene expression of egr <dig> may be regulated by tgf-β <dig>  <cit> .
antiinflammatory and antitumor properties of mscs on cs
genes upregulated for mscs growth on cs and encoding cytokines or their receptors are listed in table  <dig>  among them, a small number of proinflammatory cytokines were upregulated, which included il1a , il1b , il <dig>   <cit> , and tnfsf13b   <cit> .
many antiinflammatory genes were enhanced to even higher expression levels as compared with those of proinflammatory ones.
these genes include the il1rn   <cit> , il4i <dig>   <cit> , lif   <cit>  and its common receptor subunit, il6st   <cit> , and tnfaip <dig>   <cit> .
the tnfsf <dig>  was also upregulated which can mediate both immune suppression and immune stimulation through the cd <dig> receptor/ligand system  <cit> .
another upregulated antiinflammatory-associated gene was ptgs <dig> , which is the rate-limiting enzyme for arachidonic acid metabolic transformation into prostanoids during eicosanoid synthesis in response to inflammatory stimuli  <cit> .
besides, the above-mentioned lif, tgf-β and hgf, are considered as factors associated with the immunomodulatory property of mscs  <cit> .
this aspect of mscs is important in clinical application for graft-versus-host and autoimmune diseases.table  <dig>
the group of antiinflammatory and antitumor genes screened from microarrays
*p <  <dig>  between cs and tcps groups .
a few antitumor genes were upregulated for mscs on cs.
of interest to note is the high gene expression level of il <dig>  observed for mscs grown on cs vs. tcps.
il <dig> is considered as a tumor suppressor and can selectively induce apoptosis in cancer cells without affecting normal cells  <cit> .
tp <dig>  is another upregulated tumor suppressor gene that involves in various critical cellular functions such as proliferation, cell cycle arrest, apoptosis, and dna repair mechanisms  <cit> .
the members of tnfα-induced protein  <dig>  family were upregulated.
tnfaip <dig> was reported as an apoptosis regulator, while the gene encoding tnfaip8l <dig>  is still unknown for its biological function  <cit> .
regulation of aryl hydrocarbon receptor  pathway
the upregulation of aryl hydrocarbon receptor  system was also observed and displayed in table  <dig>  ahr is a ligand-activated transcription factor activated by endogenous physical ligands.
ahr is involved in a variety of toxicity mechanisms as well as in endogenous biological functions.
arnt  was also upregulated, which can transduce the ahr signaling and promote the expression of target genes   <cit> .
cytochrome p450s  have been identified as the functional enzymes that catalyze the metabolic activation and detoxification of a variety of xenobiotics  <cit> .
the expressions of cyp1b <dig> , cyp3a <dig> , and cyp19a <dig>  were upregulated for mscs on cs.
cyps play a critical role in drug metabolism, and therefore cells with appreciated cyp activities can be used for risk assessment of drug-induced hepatotoxicity  <cit> .
a recent study showed that the expression and activities of cyps were enhanced by culturing the transfected human dermal fibroblasts as spheroids  <cit> , which was in line with our results.table  <dig>
other significant genes screened from microarrays
* p <  <dig>  between cs and tcps groups .
substrate-dependent nature of gene upregulation for 3d spheroids
the expressions of special interested genes for msc spheroids grown on cs and those derived on a non-adherent  substrate were compared by qrt-pcr.
the mechanism driving spheroid formation on the non-adherent pva is similar to that in suspension culture .
the self-made pva substrate, however, is more chemically defined.
the results  showed significantly higher expression levels of genes including lif, il <dig>  tp <dig>  tgf-β <dig>  pdgfra, and ptgs <dig> for mscs grown on cs vs. pva.
the enhanced gene expressions may be attributed to the greater cell-substrate interaction for mscs on cs through the upregulation of the calcium-associated genes.
furthermore, the high expression levels for the above-mentioned genes in cs-derived spheroids were maintained or even further enhanced at  <dig> h as compared with those at  <dig> h. the sustained regulation of these genes by cs vs. the transient upregulation by pva suggested the importance of cell-substrate interaction in gene regulation.
the differential gene expression in different substrate-derived msc spheroids also suggested substrate-dependent gene regulation and the critical role of culture substrates in influencing the cell functions and fates even in 3d spheroids.
discussion and
CONCLUSIONS
the gene expression profiles of cs substrate-derived 3d msc spheroids vs. 2d cultured mscs were cross-analyzed by mrna as well as mirna microarrays and confirmed by qrt-pcr measurement.
the critical role of calcium signaling in cs substrate-derived msc spheroids was justified by the upregulation of various calcium-associated genes, which has not yet been reported in any other spheroid systems.
the unique role of calcium in self-assembled spheroids may be related to the observation that the surface-bound calcium on cs may be translocated into mscs  <cit> .
several kinds of integrin subunit which participate in the processing of cell adhesion or migration were modulated.
besides, members of matrix metalloproteinases  were upregulated for mscs grown on cs.
mmps are proteolytic enzymes that degrade various components of the extracellular matrix .
the proteolytic effects of mmps play an important role in vascular remodeling, cellular migration, and the processing of ecm proteins and adhesion molecules  <cit> .
the modified chemotactic function of the migrating msc spheroids was also verified by the upregulation of many chemokines and their receptors.
since the chemotactic function is critical for the therapeutic performance of mscs, the migration and chemotaxis of msc spheroids and their link to homing phenomena deserve further investigations.
the multilineage differentiation capacities as well as antiinflammatory and antitumor properties of mscs may be enhanced after forming spheroids on cs.
the antiinflammatory and antitumor properties have been reported in msc spheroids generated by hanging drop  <cit> , including the upregulated gene expression of tnfaip <dig> and il <dig>  which were also observed in cs-derived msc spheroids.
the favorable chondrogenic and osteogenic differentiation capacities previously demonstrated for cs-derived msc spheroids  <cit>  may be associated with the induced expression of tgf-β <dig> and bmp <dig> genes.
on the other hand, hgf, egr <dig>  mmp <dig>  and epha <dig> are involved in the development of nervous system  <cit> .
the upregulation of these genes may suggest the enhanced transdifferentiation ability of cs-derived msc spheroids.
most of all, the upregulation of wnt related genes suggested a profound influence of cs on the fate decision of mscs.
the regulatory changes in the expression of these genes were significantly greater for msc spheroids derived on cs substrates than those derived on the non-adherent pva substrates.
the critical importance of substrates in stem cell culture, even in the circumstance of 3d spheroid culture, was substantiated in this study.
the distinct gene expression profiles on different substrates were in line with the significant substrate-dependent alterations in cell-cell interaction  and cell-substrate interaction  based on the gene analysis.
finally, it has been mentioned that cells in the core of a 3d spheroid may be exposed to mild hypoxia  <cit> .
the hypoxic environment may resemble the natural niche of mscs  more than the normal culture condition   <cit> .
a recent literature has demonstrated that mscs can benefit from hypoxia to inhibit the senescence, increase the proliferation, and enhance the differentiation potential along the mesenchymal lineages  <cit> .
the hypoxia-inducible factor 1α signaling pathway was proposed to be involved in the modulation mechanism of hypoxia effect  <cit> .
spheroids generated by suspension culture could precondition the human adipose-derived stromal cells  <cit>  and umbilical vein endothelial cells  <cit>  to hypoxia environment, leading to upregulations of hif-1α and angiogenesis.
another recent literature showed that forming 3d spheroids of human gingiva-derived mscs by suspension culture upregulated many hypoxia-responsive genes, such as hif-1α, vegf, sdf-1α, and cxcr <dig>  <cit> .
the apoptosis signal-regulating kinase  <dig>  and its downstream proteins, the p38α mitogen-activated protein kinase  family, act as sensors of oxidative stress  <cit> .
in our study, the upregulation of cxcr <dig> was observed for msc spheroids grown on cs.
on the other hand, neither the oxidative stress-sensitive genes  nor the oxidative stress-associated microrna  were screened out by the mrna and mirna microarrays.
these results suggested that the oxidative stress and the associated genes may not be activated during the formation of msc spheroids on cs, and the modulating mechanism of the development-associated genes such as cxcr <dig> in cs-derived spheroids may be distinct from that in spheroids derived on a non-adherent substrate.
this finding reinforces the uniqueness of culture substrates as a microenvironment to predefine the properties of 3d stem cell spheroids.
although mscs also formed spheroids on non-adherent substrate , the forming process and the gene regulation profile were not the same as those on cs.
on the other hand, mscs showed similar features of spheroid formation and calcium-related cell behavior on cs substrates even when they were isolated from different tissue sources  or species   <cit> .
the capacity of mscs to form spheroids on cs was more influenced by their stemness  <cit> , and the surface-bound calcium on the substrate  <cit> .
in this study, we observed that a few genes were regulated during spheroid formation which may participate in calcium signaling pathway.
however, the critical genes that turn on the mechanism as well as the link between calcium regulation and the genes involved in different cell functions  remains unknown.
these issues are interesting and worthy of further investigations.
the gene regulation profile screened by the cross-correlation analysis described here may provide helpful information for studying these unique substrate-induced msc spheroids.

 mesenchymal stem cells are extensively used as the cell source for regenerative medicine because of their capacities to differentiate into different lineages and expand while maintaining their undifferentiated state. mscs are commonly cultured as two-dimensional monolayer by traditional tissue culture techniques. however, the 2d culture method has difficulty in overcoming the replicative senescence and maintaining the self-renewal properties, which results in cells of low differentiation capacity. a three-dimensional culture environment is considered more favorable than 2d monolayer culture for cell-cell contacts. previous studies have developed several methods to generate 3d msc spheroids. many of these methods involve the use of cell suspension system or non-adherent surface to induce spheroid formation. in general, these 3d msc spheroids were reported to have greater differentiation capacities. chitosan is the deacetylated derivative of chitin which is abundant in shell of shrimp or crap, and in fungus, and the content is only next to cellulose in nature. cs has a main structure composed of β linked d-glucosamine residues with different amounts of n-acetyl-glucosamine group. owing to its biocompatibility and biodegradability, cs has been widely studied as a scaffolding material for tissue engineering. recent findings have revealed that mscs attached on the membranes made of cs can form self-assembled 3d cellular spheroids. during the process, mscs attach and spread on cs membranes before they retract their pseudopodia to form multicellular spheroids. this self-assembly process is quite different from that occurs in suspension or hanging drop systems, or on non-adherent polymer surfaces. several genes/proteins have been referred to participate in the process of spheroid formation on cs, including cadherin molecules, rho/rho-associated kinase, and the wnt molecule. activations of these proteins were not as evident for spheroids on non-adherent surfaces. in addition, it was observed that the surface-bound calcium on cs substrates may be transported into mscs and play a role in spheroid formation as well as gene regulation. although a few changes in gene/protein expression were observed, the exact mechanism for spheroid formation on cs is still far from being elucidated. therefore, a more comprehensive understanding of the genomic profile for cs-derived msc spheroids is essential for further revealing the substrate-dependent nature of these unique msc spheroids. the technique of microarray has been developed to detect the changes within cells and is a powerful tool by which many genes can be probed simultaneously. dalby et al. have reported the genomic expression profile of human mscs responding to the shape of their environment by the messenger rna microarray. the antiinflammatory properties of human msc spheroids generated by hanging drop have also been compared to those of the adherent msc monolayer by surveying with mrna microarray. furthermore, recent advances in microrna, a class of non-coding small rna, have identified a few important modulators in stem cell proliferation and differentiation. they can bind to the cognate mrna to repress the expression of target genes. simultaneous analyses of the mrna and mirna expression profiles may help narrowing down the signaling events involved in the behavior change of the cells. in this study, we examined both mrna and mirna expression profiles of the cs substrate-induced 3d spheroids of human mscs isolated from the umbilical cord, using 2d mscs on tissue culture polystyrene as a control. cross-correlation analysis of the results from these two microarrays was further confirmed with the quantitative reverse transcriptase-polymerase chain reaction analysis to identify the critical signaling events for substrate-derived msc spheroids during the spheroid forming process.  characteristics of human umbilical cord mscs the expression profile of cell surface markers analyzed by flow cytometry is shown in additional file 1: figure s human umbilical cord mscs were positive for specific antigen markers of mscs such as cd cd cd cd cd cd cd cd cd cd and hla-abc, and negative for specific markers of endothelial cells and haematopoietic cells including cd cd cd cd and hla-dr. besides, cells showed positive expression of cd and low expression of cd which was consistent with that described for human umbilical cord mscs in literatures. surface properties of cs membranes the different surface properties of cs membranes and tcps are summarized in figure  the static water contact angle of cs membranes was ± ° and that of tcps was ± °. the greater contact angle of cs vs. tcps revealed the slightly higher hydrophobicity of cs, which may be attributed to the rearrangement of hydrophobic n-acetyl groups of cs to move to the surface and reduce the surface free energy while exposed to atmosphere. the surface zeta potential of cs was relatively neutral, distinct from the negatively charged tcps. previous literature suggested an isoelectric point at ph for cs arising from the deprotonation of positive amino group. another study indicated that the amino groups of cs could chelate calcium ion to form cs/calcium ion complexes. the amount of surface-bound calcium on cs membranes was significantly greater than that on tcps after either h or h. the consistent values observed at h and h suggested a saturation of surface-bound calcium.figure surface properties of cell culture substrates. the static contact angle of cs and tcps. the surface zeta potential of cs and tcps. the amount of surface-bound calcium on cs or tcps after being soaked in the culture medium for h without cells. * p <  among the indicated groups. phenotype of mscs grown on cs or tcps. the morphologies of mscs on cs or tcps after h, h and h. the cell viability of msc spheroids on cs after h analyzed by flow cytometry. the percentage of cells without being stained by pi was defined as the cell viability. spheroid formation for mscs growth on cs membranes the morphology of mscs grown on cs membranes and tcps is shown in figure  as was expected, mscs attached to tcps with fibroblast-like morphology. on the other hand, mscs on cs remained attached to the surface before h and formed spheroids afterwards. the average diameter of the msc spheroids on cs membranes was  ±  μm at h. the spheroid size was relatively stable within h. the cell viability for the msc spheroids was % at h. cross-correlation analysis between mrna and mirna microarrays based on the screening of the mrna microarray, upregulated genes and downregulated genes showed significant difference for mscs on cs vs. tcps. on the other hand, screening based on the mirna microarray showed that there were targeted genes corresponding to the downregulated mirna, and targeted genes corresponding to the upregulated mirna. cross-correlation analysis of these two microarray data have helped narrowing down the critical genes involved in the behavioral changes of these cells. screening based on the cross-correlation analysis with the software, agilent.twocolor. revealed that there were upregulated genes and downregulated genes. the results of microarray analyses are shown in figure  in addition, the screened genes as well as their expression ratio are listed in additional file figure the results of microarrays for mscs grown on cs or tcps. the screening of the mirna microarray for mscs grown on cs or tcps. the numbers of genes that were subjected to cross-correlation analysis of mrna and mirna microarrays and the numbers of screened genes based on the cross-correlation analysis. a complete list of screened genes as well as their expression ratios is shown in additional file the biological significance behind the list of screened genes was further analyzed by the david program to evaluate the gene enrichment in gene ontology and kyoto encyclopedia of genes and genomes pathway maps. the results of gene enrichment in kegg pathway maps suggested that several pathways were regulated for mscs on cs vs. tcps including calcium signaling pathway, focal adhesion, regulation of actin cytoskeleton, adherens junction, gap junction, ecm-receptor interaction, p signaling pathway, cytokine-cytokine receptor interaction, tryptophan metabolism, and tgf-β signaling pathway. among the genes, we picked up the ones with high expression ratio, the critical ones in certain signaling pathways, or the ones which had been reported in literature to be associated with cell adhesion, migration, or fate decision. they were then further confirmed by qrt-pcr. regulation of calcium-associated genes for mscs on cs a number of calcium-associated genes were noted and listed in table  including those of calcium channels, receptors, and intracellular signaling proteins. atp2b and atp2b are highly regulated ca2+ extrusion pumps and provide fine-tuning of intracellular calcium level. slc8a is a na+-ca2+ exchanger which also modulates ca2+ extrusion system from the cells. trpa and trpc belong to the transient receptor potential channel superfamily that regulates the mechanism for ca2+ entry. the upregulation of atp2b and trpa and downregulation of atp2b slc8a and trpc for mscs on cs vs. tcps suggested that the process for intracellular calcium homeostasis was turned on. modulation of these genes may cause an elevation in the intracellular calcium level.table the group of calcium signaling-associated genes screened from microarrays ma: based on microarray data. qrt-pcr: based on quantitative rt-pcr analysis. *p <  between cs and tcps groups. a few genes of calcium-associated receptors were upregulated for mscs growth on cs vs. tcps. htr2a and htr are the receptors for serotonin which is a well-characterized neurotransmitter with regulative function in multiple physiological aspects. gpr is a proton-sensing receptor that can modulate the level of intracellular calcium. pdgfra and f2r are also intracellular calcium modulators. the activation of these calcium-associated receptors may directly enhance the level of intracellular calcium and lead further to cytoskeleton rearrangement. several intracellular signaling genes were also upregulated. these genes included those of map3k, itpr rasgrp, pla2g4a. map3k is required for the transduction of signals initiated by par and other g-coupled receptors, which promote actin reorganization and cell migration. map3k can also mediate signal-induced increases in cytoplasmic ca2+ through the activation of phospholipase c. on the other hand, itpr is a ca2+-release channel located on intracellular membranes, especially the endoplasmic reticulum. the ip receptor has an affinity for ip in the low nanomolar range. moreover, cytosolic ca2+ is considered as a co-agonist of the ip receptor, as it strongly increases the ip receptor activity at concentrations up to about nm. rasgrp is a calcium and dag-regulated ras guanyl releasing protein which can activate small gtpases such as ras and rap. pla2g4a is a member of phospholipases a superfamily, which regulates the release of arachidonic acid. although calmodulin is also a family of ca2+ binding proteins and mediates many important cellular processes, calm gene was downregulated. the regulation of various calcium-associated genes suggested the critical role of calcium signaling in the cs-derived msc spheroids, which has not yet been reported in any other spheroid systems. regulation of cell adhesion and migration/cytoskeleton-associated genes for mscs on cs genes that were screened out for mscs on cs and associated with cell adhesion, migration, or cytoskeleton reorganization are displayed in table  mmp is a kind of interstitial collagenase, and its activity was enhanced in highly migrating mscs compared with poorly migrating mscs. mmp and mmp were also upregulated which have similar substrate specificity. mmp is correlated with neuronal migration and neurite outgrowth and is able to activate mmp. the migration ability of cs-derived msc spheroids has been mentioned. the upregulation of these cell adhesion/migration-associated genes agreed with the high cell mobility of mscs on cs.table the group of adhesion and migration/cytoskeleton-associated genes screened from microarrays *p <  between cs and tcps groups. on the other hand, a variety of genes that controls cell-to-cell adhesion were upregulated for mscs on cs, including cadherins, cell adhesion molecules, notch, and ephrin receptor. cdh is a ca2+-dependent cell-cell adhesion molecule, and expresses in the central nervous system. pcdh is also a member of cadherin family, and has a role in embryo development. pecam is an endothelial cell marker. the expression of notch and its ligand, dll, were both enhanced for msc growth on cs. notch signaling pathway is critical for cell fate decisions including proliferation, lineage commitment, and terminal differentiation in many adult stem cell types. epha can bind to cell surface-associated ephrin ligands on neighboring cells to generate bidirectional signals that affect both the receptor-expressing and ephrin-expressing cells. based on the literature, epha is an axon guidance receptor important for the development of cortical circuits. the enhancement in these cell-cell adhesion genes may provide better cell-cell communication and coordination during spheroid formation. the upregulation of chemokines and their receptors for mscs grown on cs vs. tcps is of particular interest. among them, the gene encoding cmklr was upregulated superbly. cmklr was reported as a multifunctional receptor which can bind with the proinflammatory chemokine, chemerin, or with the anti-inflammatory lipid mediator, resolving e. chemerin/cmklr interaction was also reported to promote adipogenesis and angiogenesis. other upregulated chemokine receptors or ligands included the cxcr and cxcr, which are the receptors of stromal derived factor-. cxcr is one of the most studied chemokine receptors that play an important role in cell migration, proliferation, and differentiation. the cxcl is a ligand for another cxc motif chemokine receptor, cxcr which was reported to crosstalk with cxcr and cxcr. the ccl and ccl are important homing factors for mscs. cell fate decision in msc spheroids the gene expression for a group of growth factors and receptors was modulated in mscs on cs vs. tcps, as listed in table  these included tgf-β, bmp, hgf, igf1r, kdr and kit. the regulation of these genes may influence the function and fate of stem cells.table the group of development-associated genes screened from microarrays * p <  between cs and tcps groups. several wnt signaling related genes were also enhanced for mscs on cs including the wls, wnt, lef, tcf, daam, and cxxc. the wnt proteins and wnt signaling pathway are known to control cell specification and fate during embryonic development and adult tissue homeostasis. wnt a member of wnt family, can promote the earliest aspects of lung airway smooth muscle development, and accelerate cardiac myocyte differentiation from es-cell derived mesodermal cells through the non-canonical wnt pathway. wls is a multipass transmembrane protein, and was found to control the secretion of wnt proteins. the tcf/lef family is the downstream proteins in the canonical wnt/ β-catenin pathway. in response to wnt signals, tcf/lef members present as a switch to modulate the transcription of numerous target genes from repression to activation as binding with β-catenin. daam was identified as a downstream protein interacting with dishevelled, which mediates the non-canonical wnt/planar cell polarity signaling pathway. a study indicated that daam may play a crucial role in regulating the actin cytoskeleton and tissue morphogenesis. on the other hand, one of the negative regulators of wnt/ β-catenin signaling pathway, cxxc was also enhanced for mscs on cs. two other development-related genes worth mentioning are rarb and egr. rarb is a nuclear receptor for retinoic acid which is a vitamin a-derived, non-peptidic, small lipophilic molecule. the ra signaling during embryo development has been extensively investigated. egr is a zinc-finger transcription factor of the early growth response gene family that has critical functions in hindbrain development and myelination of the peripheral nervous system. the gene expression of egr may be regulated by tgf-β. antiinflammatory and antitumor properties of mscs on cs genes upregulated for mscs growth on cs and encoding cytokines or their receptors are listed in table  among them, a small number of proinflammatory cytokines were upregulated, which included il1a, il1b, il, and tnfsf13b. many antiinflammatory genes were enhanced to even higher expression levels as compared with those of proinflammatory ones. these genes include the il1rn, il4i, lif and its common receptor subunit, il6st, and tnfaip. the tnfsf was also upregulated which can mediate both immune suppression and immune stimulation through the cd receptor/ligand system. another upregulated antiinflammatory-associated gene was ptgs, which is the rate-limiting enzyme for arachidonic acid metabolic transformation into prostanoids during eicosanoid synthesis in response to inflammatory stimuli. besides, the above-mentioned lif, tgf-β and hgf, are considered as factors associated with the immunomodulatory property of mscs. this aspect of mscs is important in clinical application for graft-versus-host and autoimmune diseases.table the group of antiinflammatory and antitumor genes screened from microarrays *p <  between cs and tcps groups. a few antitumor genes were upregulated for mscs on cs. of interest to note is the high gene expression level of il observed for mscs grown on cs vs. tcps. il is considered as a tumor suppressor and can selectively induce apoptosis in cancer cells without affecting normal cells. tp is another upregulated tumor suppressor gene that involves in various critical cellular functions such as proliferation, cell cycle arrest, apoptosis, and dna repair mechanisms. the members of tnfα-induced protein family were upregulated. tnfaip was reported as an apoptosis regulator, while the gene encoding tnfaip8l is still unknown for its biological function. regulation of aryl hydrocarbon receptor pathway the upregulation of aryl hydrocarbon receptor system was also observed and displayed in table  ahr is a ligand-activated transcription factor activated by endogenous physical ligands. ahr is involved in a variety of toxicity mechanisms as well as in endogenous biological functions. arnt was also upregulated, which can transduce the ahr signaling and promote the expression of target genes. cytochrome p450s have been identified as the functional enzymes that catalyze the metabolic activation and detoxification of a variety of xenobiotics. the expressions of cyp1b, cyp3a, and cyp19a were upregulated for mscs on cs. cyps play a critical role in drug metabolism, and therefore cells with appreciated cyp activities can be used for risk assessment of drug-induced hepatotoxicity. a recent study showed that the expression and activities of cyps were enhanced by culturing the transfected human dermal fibroblasts as spheroids, which was in line with our results.table other significant genes screened from microarrays * p <  between cs and tcps groups. substrate-dependent nature of gene upregulation for 3d spheroids the expressions of special interested genes for msc spheroids grown on cs and those derived on a non-adherent substrate were compared by qrt-pcr. the mechanism driving spheroid formation on the non-adherent pva is similar to that in suspension culture. the self-made pva substrate, however, is more chemically defined. the results showed significantly higher expression levels of genes including lif, il tp tgf-β pdgfra, and ptgs for mscs grown on cs vs. pva. the enhanced gene expressions may be attributed to the greater cell-substrate interaction for mscs on cs through the upregulation of the calcium-associated genes. furthermore, the high expression levels for the above-mentioned genes in cs-derived spheroids were maintained or even further enhanced at h as compared with those at h. the sustained regulation of these genes by cs vs. the transient upregulation by pva suggested the importance of cell-substrate interaction in gene regulation. the differential gene expression in different substrate-derived msc spheroids also suggested substrate-dependent gene regulation and the critical role of culture substrates in influencing the cell functions and fates even in 3d spheroids. discussion and  the gene expression profiles of cs substrate-derived 3d msc spheroids vs. 2d cultured mscs were cross-analyzed by mrna as well as mirna microarrays and confirmed by qrt-pcr measurement. the critical role of calcium signaling in cs substrate-derived msc spheroids was justified by the upregulation of various calcium-associated genes, which has not yet been reported in any other spheroid systems. the unique role of calcium in self-assembled spheroids may be related to the observation that the surface-bound calcium on cs may be translocated into mscs. several kinds of integrin subunit which participate in the processing of cell adhesion or migration were modulated. besides, members of matrix metalloproteinases were upregulated for mscs grown on cs. mmps are proteolytic enzymes that degrade various components of the extracellular matrix. the proteolytic effects of mmps play an important role in vascular remodeling, cellular migration, and the processing of ecm proteins and adhesion molecules. the modified chemotactic function of the migrating msc spheroids was also verified by the upregulation of many chemokines and their receptors. since the chemotactic function is critical for the therapeutic performance of mscs, the migration and chemotaxis of msc spheroids and their link to homing phenomena deserve further investigations. the multilineage differentiation capacities as well as antiinflammatory and antitumor properties of mscs may be enhanced after forming spheroids on cs. the antiinflammatory and antitumor properties have been reported in msc spheroids generated by hanging drop, including the upregulated gene expression of tnfaip and il which were also observed in cs-derived msc spheroids. the favorable chondrogenic and osteogenic differentiation capacities previously demonstrated for cs-derived msc spheroids may be associated with the induced expression of tgf-β and bmp genes. on the other hand, hgf, egr mmp and epha are involved in the development of nervous system. the upregulation of these genes may suggest the enhanced transdifferentiation ability of cs-derived msc spheroids. most of all, the upregulation of wnt related genes suggested a profound influence of cs on the fate decision of mscs. the regulatory changes in the expression of these genes were significantly greater for msc spheroids derived on cs substrates than those derived on the non-adherent pva substrates. the critical importance of substrates in stem cell culture, even in the circumstance of 3d spheroid culture, was substantiated in this study. the distinct gene expression profiles on different substrates were in line with the significant substrate-dependent alterations in cell-cell interaction and cell-substrate interaction based on the gene analysis. finally, it has been mentioned that cells in the core of a 3d spheroid may be exposed to mild hypoxia. the hypoxic environment may resemble the natural niche of mscs more than the normal culture condition. a recent literature has demonstrated that mscs can benefit from hypoxia to inhibit the senescence, increase the proliferation, and enhance the differentiation potential along the mesenchymal lineages. the hypoxia-inducible factor 1α signaling pathway was proposed to be involved in the modulation mechanism of hypoxia effect. spheroids generated by suspension culture could precondition the human adipose-derived stromal cells and umbilical vein endothelial cells to hypoxia environment, leading to upregulations of hif-1α and angiogenesis. another recent literature showed that forming 3d spheroids of human gingiva-derived mscs by suspension culture upregulated many hypoxia-responsive genes, such as hif-1α, vegf, sdf-1α, and cxcr. the apoptosis signal-regulating kinase and its downstream proteins, the p38α mitogen-activated protein kinase family, act as sensors of oxidative stress. in our study, the upregulation of cxcr was observed for msc spheroids grown on cs. on the other hand, neither the oxidative stress-sensitive genes nor the oxidative stress-associated microrna were screened out by the mrna and mirna microarrays. these results suggested that the oxidative stress and the associated genes may not be activated during the formation of msc spheroids on cs, and the modulating mechanism of the development-associated genes such as cxcr in cs-derived spheroids may be distinct from that in spheroids derived on a non-adherent substrate. this finding reinforces the uniqueness of culture substrates as a microenvironment to predefine the properties of 3d stem cell spheroids. although mscs also formed spheroids on non-adherent substrate, the forming process and the gene regulation profile were not the same as those on cs. on the other hand, mscs showed similar features of spheroid formation and calcium-related cell behavior on cs substrates even when they were isolated from different tissue sources or species. the capacity of mscs to form spheroids on cs was more influenced by their stemness, and the surface-bound calcium on the substrate. in this study, we observed that a few genes were regulated during spheroid formation which may participate in calcium signaling pathway. however, the critical genes that turn on the mechanism as well as the link between calcium regulation and the genes involved in different cell functions remains unknown. these issues are interesting and worthy of further investigations. the gene regulation profile screened by the cross-correlation analysis described here may provide helpful information for studying these unique substrate-induced msc spheroids. 
based on the study, the culture substrates used to prepare 3d msc spheroids may predefine their properties through cell-substrate interaction. results revealed the regulation of a significant number of calcium-associated genes, which suggested the crucial role of calcium signaling in cs-derived msc spheroids. recent literatures have documented that mscs can self-assemble into 3d spheroids with a greater capacity for differentiation into various cell types when grown on chitosan, a biopolymer. in this study, 3d spheroids self-assembled from human umbilical cord mscs grown on cs membranes were analyzed by mrna as well as microrna microarrays, which helped identify the critical signaling events that may alter the cellular functions during the spheroid forming process. the genetic modulation was significantly more remarkable and endured longer for msc spheroids derived on cs substrates compared to those derived on a non-adherent substrate. in addition, many genes associated with the multilineage differentiation capacities and those associated with the antiinflammatory and antitumor properties of mscs were upregulated. three-dimensional multicellular spheroids of mesenchymal stem cells are generally regarded to have beneficial properties over mscs in monolayer. mesenchymal stem cells cellular spheroidschitosancalcium signalinggene profilemicroarrayissue-copyright-statement© biomed central ltd 2014 the genomic modulation occurring in these msc spheroids is thus of essential importance for understanding their uniqueness and therapeutic potentials.
 three-dimensional multicellular spheroids of mesenchymal stem cells are generally regarded to have beneficial properties over mscs in monolayer. recent literatures have documented that mscs can self-assemble into 3d spheroids with a greater capacity for differentiation into various cell types when grown on chitosan, a biopolymer. the genomic modulation occurring in these msc spheroids is thus of essential importance for understanding their uniqueness and therapeutic potentials. in this study, 3d spheroids self-assembled from human umbilical cord mscs grown on cs membranes were analyzed by mrna as well as microrna microarrays, which helped identify the critical signaling events that may alter the cellular functions during the spheroid forming process.  genes screened from mrna and microrna cross-correlation analyses were further confirmed with the quantitative reverse transcriptase-polymerase chain reaction analysis. results revealed the regulation of a significant number of calcium-associated genes, which suggested the crucial role of calcium signaling in cs-derived msc spheroids. in addition, many genes associated with the multilineage differentiation capacities and those associated with the antiinflammatory and antitumor properties of mscs were upregulated. the genetic modulation was significantly more remarkable and endured longer for msc spheroids derived on cs substrates compared to those derived on a non-adherent substrate.  based on the study, the culture substrates used to prepare 3d msc spheroids may predefine their properties through cell-substrate interaction. electronic supplementary material the online version of this article contains supplementary material, which is available to authorized users. keywords mesenchymal stem cells cellular spheroidschitosancalcium signalinggene profilemicroarrayissue-copyright-statement© biomed central ltd 2014 