BACKGROUND
escs are derived from the inner cell mass  of blastocyst-stage embryos  <cit> .
self-renewal and pluripotency enable escs to be a renewable and versatile model to study developmental biology.
moreover, ecss have potential applications in regenerative medicine.
rhesus macaques  are a well-studied primates, and with genetic and physiological similarities to humans, rhesus macaques have become an ideal model for esc-based therapies  <cit> .
however, the study and application of rescs are lacking compared with those of mouse and human escs due to limited resc line availability and a need for systematic analyses of fundamental resc characteristics.
mirnas are small endogenous non-coding transcripts  with diverse roles in development, differentiation and oncogenesis.
mirnas bind to complementary sites within cognate mrna 3' utrs, resulting in degradation, deadenylation or translational repression, which provide a crucial level of post-transcriptional regulation  <cit> .
moreover, tissue- and cell type-specific mirna expression patterns have been described  <cit> , which elucidate various mirna functions in specific conditions.
mirnas also play important roles in escs as demonstrated by deletion of dicer or dgcr <dig> in mouse escs resulting in proliferation and differentiation defects  <cit> .
previous studies of mirna expression patterns in mouse and human escs have revealed a unique mirna set that is distinct from other cell types and tissues  <cit> .
several mirnas preferentially expressed in human and mouse escs, and down-regulated in differentiated cells are key regulators of 'stemness'  <cit> .
however, the mirna expression profile of rescs is unknown.
esc lines derived from the same species may contain distinct mirna profiles and share only a small number of mirnas  <cit> .
this observation is likely caused by various esc culture conditions rather than inherent genetic variation within embryos used for esc derivation  <cit> .
another factor is the use of numerous analyses for detecting mirna patterns due to the limited resolution of techniques such as microarray analysis  <cit> .
however, recent advancements in next-generation sequencing technology provide an ideal tool for analyzing the mirna transcriptome with high resolution to identify novel mirnas  <cit> .
in this study, we isolated and characterized three resc lines, and performed mirna profiling using solexa sequencing.
our mirna study of rescs and cross-species comparison may assist future studies for understanding and modulating esc regulatory networks.
RESULTS
isolation and characterization of resc lines
thirteen expanded rhesus macaque blastocysts with a prominent icm were selected by immunosurgery and  <dig> icms were isolated and plated onto feeder cells.
icms attached to feeder cells within  <dig> h and three esc-like icm outgrowths appeared after 7- <dig> days.
icm outgrowths were manually dissociated into 4- <dig> smaller clumps using a microscalpel, excised from feeder cells and replated onto fresh mouse embryonic fibroblasts .
clones with distinct boundaries and high nuclear to cytoplasm ratios were selected for further propagation.
three resc lines were established and designated as ivf <dig> , ivf <dig>  and ivf <dig> .
ivf <dig>  and ivf <dig>  were derived using the same sperm and oocyte donors.
rescs shared common morphologies with other primate escs such as being flat with a distinct boundary against feeder cells.
cells showed high nuclear to cytoplasm ratios and prominent nucleoli .
ivf <dig>  and ivf <dig>  were cultured for > <dig> passages and ivf <dig>  for > <dig>  pluripotency markers were highly expressed in all resc lines including oct- <dig>  nanog, ssea- <dig>  tra-1- <dig> and tra-1- <dig> .
other crucial transcription factors, such as sox- <dig> and rex- <dig>  were also detected by rt-pcr .
all resc lines showed strong alkaline phosphatase activity  and could spontaneously differentiate into cell lineages from the three embryonic germ layers.
after  <dig> days in suspension culture, resc clones formed embryoid bodies .
after  <dig> days, cavities were observed in ebs, which are termed cystic ebs.
ebs attached to gelatin-coated dishes and were cultured for 5- <dig> days until various differentiated cells appeared including neuron-like cells, contractive cardiomyocytes and endoderm-like cells .
teratomas were formed 6- <dig> weeks after resc injection into the hind leg muscles of scid mice.
histological characterization revealed that ectoderm , mesoderm  and endoderm  like structures were present in teratomas .
these results demonstrated that resc lines could differentiate into all three embryonic germ layers in vitro and in vivo.
detailed g-banding analysis revealed that all resc lines were karyotypically normal with a diploid set of  <dig> chromosomes , even after long-term culture in vitro and repeated freeze-thaw procedures.
ivf <dig>  and ivf <dig>  were derived from male blastocysts  and ivf <dig>  was derived from a female blastocyst .
small rna sequencing
newly established rescs were tested for purity using oct- <dig> staining, which resulted in >95% oct-4-positive cells .
a small rna library from each cell line was then constructed for sequencing.
small rna library sequencing yielded  <dig>  ×  <dig>   <dig>  × 106and  <dig>  ×  <dig> raw reads from ivf <dig> , ivf <dig>  and ivf <dig> , respectively.
after filtering,  <dig>  ×  <dig> ,  <dig>  ×  <dig>  and  <dig>  ×  <dig>  clean reads  were obtained.
the length distribution of clean reads centered at 22- <dig> nt, coinciding with the mirna length range.
sequence alignment with the rhesus macaque genome  using the short oligonucleotide alignment program   <cit>  demonstrated that  <dig>  ×  <dig> ,  <dig>  ×  <dig>  and  <dig>  ×  <dig>  sequences were mapped to the genome .
mirnas were the major component of small rna libraries from rescs  being 60% of sequences, which were matched to reference mirnas in each sample.
the proportions of other annotated rnas was <15%.
notably, ~25% of sequences could not be annotated because of low coverage  and relatively poor annotations of the available rhesus macaque genome.
unknown sequences were further analyzed to identify novel mirnas.
mirna expression profiles of resc lines
after matching to  <dig> reference rhesus macaque mirnas,  <dig>   <dig> and  <dig> known mirnas were detected in ivf <dig> , ivf <dig>  and ivf <dig> , respectively, with absolute counts ranging from 1- <dig> , <dig> .
among them,  <dig> mirnas were shared by the three pools and remaining mirnas were expressed at low levels with < <dig> counts.
from a genome-wide perspective, very low mirnas were likely sequencing artifacts or mirna machinery byproducts  <cit> .
therefore, an absolute count of  <dig> was set for each pool as the minimum threshold for further analyses.
to compare mirna expression patterns between resc lines, we evaluated the expression variability  and coefficient of variation  of mirna clusters .
the results revealed similar expression patterns of mirna clusters in resc lines.
we also compared the expression of total mirnas using a differential index  and kappa statistical analyses.
for the d.i., most mirnas were consistently expressed , whereas a slight deviation existed in ivf <dig>  due to low-level expressed mirnas.
for kappa statistical analysis, there was high repeatability between any two cell lines .
taken together, mirna expression patterns in resc lines were highly consistent.
identification of novel mirnas
we predicted novel mirna genes in unannotated sequences using mireap .
briefly, according to the characteristic hairpin structure of mirna precursors, unannotated small rna sequences mapped to genome were downloaded with flanking sequences and analyzed for secondary structure, dicer cleavage sites and minimum free energy .
after analysis,  <dig>  reads that contained  <dig> novel mirna precursors with > <dig> reads were identified.
there were  <dig> novel mirna precursors  shared between the three resc lines  and  <dig>  were homologous to reference mirnas  in other species .
of the  <dig> mirna precursors with homologs,  <dig> precursors were only conserved in humans and other primates, but not rodents.
mirna target gene prediction and functional classification
three programs were used to predict mirna target genes.
using the targetscan program, a custom set of perl codes were downloaded from the targetscan database .
conserved and non-conserved predicted patterns of mirna targets were considered separately.
for pita and miranda, we used  <dig> expressed mirnas  and rhesus macaque 3'utrs orthologous to annotated human 3'utrs as inputs to predict mirna targets.
over-representation of predicted mirna targets for both conserved and non-conserved patterns based on the targetscan program in the kyoto encyclopedia of genes and genomes  pathway is shown in table s <dig> in additional file  <dig> .
gene enrichment patterns obtained by various methods were similar to each other, indicating that mirnas correlated to functionally critical roles in rescs.
for example, predicted targets involved in the mapk , wnt  and tgf-beta signaling pathways , and pathways involved in cancer  were significantly enriched .
mirna-target interaction network analysis
a systematic survey of mirnas that interacted with targets may provide a better understanding of mirna regulation in stem cells.
thus, we analyzed potential interactions between mirnas and their targets, which is termed as the mirna-target network .
all mirnas and their targets were used to generate a bipartite graph of mirna-target interactions.. nodes represent mirnas or target genes and edges correspond to interactions between mirnas with target genes.
node degree in the mt network is the number of connections or edges with other nodes.
for each target gene node, degrees represent the number of mirnas targeting the gene.
globally, the mt network was comprised of  <dig>  nodes with  <dig>  edges.
using mt network degree analysis , the degree of genes regulated by mirnas was negatively correlated  with their evolutionary rate  .
similarly, a previous study reported a negative correlation between the degree and evolutionary rate of proteins in a protein-protein interaction network  <cit> .
to exclude factors with potential effects on the observed negative correlation, we tested the effect of 3'utr length.
after length normalization, the negative correlation remained .
another factor was a potential ascertainment bias caused by mirna target prediction and identification of orthologous macaque 3'utrs.
to evaluate this possibly, we randomized the scale-free mt network based on barabasi-albert model.
the degree of mirna and target genes in the random network was significantly lower  compared with that observed in the mt network .
a significant correlation was not observed in the random network, which further supported the negative correlation in the mt network.
comparison of mirna profiles from mouse, human and rhesus macaque
to compare esc mirna profiles between species, we retrieved previously reported mouse and human esc mirna profiles.
to avoid a cross-talking bias due to various sequencing techniques and data sizes, we selected solexa sequencing datasets from mouse and human escs  <cit> .
mirna expression frequencies were normalized to each other by determining the expected frequency in mapped reads per million.
mirna expression levels were calculated as the mean of three samples.
the three species expressed  <dig> common mirnas.
clustering analysis based on the  <dig> shared mirnas revealed that mirna expression profiles were more similar between human and rhesus macaque , which was consistent with their evolutionary relationship.
a number of mirnas were stably and highly expressed between species, such as the mir- <dig> family, mir- <dig> and mir- <dig>  which suggested conserved functions in escs during evolution.
of the  <dig> common mirnas the 'stemness' mir-290- <dig> cluster  <cit>  was the highest expressed in mouse escs.
however, primate homologs the mir-290- <dig> and mir-371- <dig> clusters were not highly expressed in human and rescs.
instead, the mir- <dig> cluster was the highest expressed in human and rhesus macaque  <cit> , indicating the functional divergence of stemness mirna clusters in primate lineages.
the mir-290- <dig> cluster contains multiple mature mirnas with seed sequences similar or identical to those in the mir- <dig> cluster  <cit> .
mir-290- <dig> and mir- <dig> clusters are transcriptionally regulated by oct-4/sox <dig>  <cit> .
taken together, these observations suggest that these two clusters are similarly expressed, regulate common target genes and are functionally conserved in escs derived from various species.
there are three mirna clusters differentially expressed in mouse, human and rescs.
the chromosome  <dig> mirna cluster , a primate-specific mirna cluster  <cit> , was enriched in human escs, but almost absent in rescs .
a conserved mirna cluster within the imprinted dlk1-dio <dig> region was highly expressed in mouse and rhesus macaque escs, but rarely expressed in human escs , which was consistent with results from four other human esc lines  <cit> .the mir- <dig> cluster in the sfmbt <dig> gene intron region was only expressed in mouse escs , embryos and newborn ovaries  <cit> , but not in human and rhesus macaque escs, consistent with previous reports  <cit> .
discussion
we established three resc lines  and profiled mirna expression using solexa sequencing.
mirna expression profiles were generally conserved between the three resc lines, sharing  <dig> % of expressed mirnas.
ivf <dig>  and ivf <dig>  were more similar because of derivation from identical sperm and ovum donors.
similarly, navara et al.
<cit>  reported that pedigreed rescs express homogeneous gene profiles.
compared with known mirnas in our data set, most novel mirnas were moderately expressed.
novel mirna biological functions and association with stemness are yet to be elucidated.
we predicted mirna targets in rescs using three independent algorithms.
targets involved in tgf-β, mapk and wnt signaling pathways were significantly enriched.
these pathways play important roles in mouse and human esc differentiation  <cit> .
this observation suggests that in addition to transcriptional level regulation by stemness-associated factors such as oct <dig> and nanog, mirnas may also play an important role in maintaining esc self-renewal and pluripotency at the post-transcriptional level.
moreover, over-represented mirna targets involved in cancer pathways support the observation that cancer and escs share pathways for self-renewal and proliferation  <cit> .
our systematic investigation of the mt network suggested that the degree of genes regulated by mirnas was negatively correlated with their evolutionary rate in rescs.
genes with more mirna regulators evolve more slowly.
this was anticipated in accordance to a rule of "the more conserved, the deeper regulated" has been proposed in protein-protein interaction networks, in which hubs or central proteins are functionally essential and usually lethal after gene knockout  <cit> .
therefore in the mt network, hub targets should be more regulated due to selective pressure, which indicates the fine regulation of mirnas in escs.
the cross-species comparison indicated that the majority of shared mirnas were stably expressed between species and consistent with mirna sequence-level conservation.
this observation suggested that mirna expression is under stabilizing selection due to functional constraints on mirnas in escs.
despite this close relationship, mirna expression differences existed between human and rhesus macaque escs.
the chromosome  <dig> mirna cluster  is a primate-specific mirna cluster and contains > <dig> mature mirnas.
these clustered mirnas are expressed in human escs, placenta and fetal brain  <cit> , suggesting that they are involved in embryogenesis.
however, the c19mc cluster is almost absent in rescs.
this expression difference suggests that even between closely related species, there may be a significant mirna expression divergence and regulatory differences during embryogenesis between rhesus macaques and humans.
in addition, the mirna cluster in the imprinted dlk1-dio <dig> region was conserved at the sequence level between mice, rhesus macaques and humans.
liu et al reported that the expression of this cluster is positively correlated to mouse stem cell pluripotencies   <cit> .
we found that this cluster was enriched in mouse and rhesus macaque escs, but rare in human escs.
recently, it was reported that diverse temporal origins may be responsible for observed differences between mouse and human escs  <cit> .
our data suggests that mirna expression patterns may provide a new strategy for investigating interspecies differences in esc pluripotency.
CONCLUSIONS
our results indicate that mirna profiles are highly conserved and consistent between the three resc lines, and these mirnas may play critical roles in the maintenance of self-renewal and pluripotency in rescs, particularly in differentiation pathways.
further analysis of the mirna-target network indicates that the degree of genes regulated by mirnas is negatively correlated with their evolutionary rate in rescs.
this observation suggests important roles of mirnas in post-transcriptional regulation.
moreover, cross-species comparison of escs revealed an overall conservation of mirna expression patterns, indicating stabilizing selection on mirna expression in escs during evolution.
however, we identified three mirna clusters  that show distinct differences between mouse, human and rhesus macaque escs, which suggests species and/or lineage specific mirna regulatory changes during evolution.

 escs are derived from the inner cell mass of blastocyst-stage embryos. self-renewal and pluripotency enable escs to be a renewable and versatile model to study developmental biology. moreover, ecss have potential applications in regenerative medicine. rhesus macaques are a well-studied primates, and with genetic and physiological similarities to humans, rhesus macaques have become an ideal model for esc-based therapies. however, the study and application of rescs are lacking compared with those of mouse and human escs due to limited resc line availability and a need for systematic analyses of fundamental resc characteristics. mirnas are small endogenous non-coding transcripts with diverse roles in development, differentiation and oncogenesis. mirnas bind to complementary sites within cognate mrna 3' utrs, resulting in degradation, deadenylation or translational repression, which provide a crucial level of post-transcriptional regulation. moreover, tissue- and cell type-specific mirna expression patterns have been described, which elucidate various mirna functions in specific conditions. mirnas also play important roles in escs as demonstrated by deletion of dicer or dgcr in mouse escs resulting in proliferation and differentiation defects. previous studies of mirna expression patterns in mouse and human escs have revealed a unique mirna set that is distinct from other cell types and tissues. several mirnas preferentially expressed in human and mouse escs, and down-regulated in differentiated cells are key regulators of'stemness'. however, the mirna expression profile of rescs is unknown. esc lines derived from the same species may contain distinct mirna profiles and share only a small number of mirnas. this observation is likely caused by various esc culture conditions rather than inherent genetic variation within embryos used for esc derivation. another factor is the use of numerous analyses for detecting mirna patterns due to the limited resolution of techniques such as microarray analysis. however, recent advancements in next-generation sequencing technology provide an ideal tool for analyzing the mirna transcriptome with high resolution to identify novel mirnas. in this study, we isolated and characterized three resc lines, and performed mirna profiling using solexa sequencing. our mirna study of rescs and cross-species comparison may assist future studies for understanding and modulating esc regulatory networks.  isolation and characterization of resc lines thirteen expanded rhesus macaque blastocysts with a prominent icm were selected by immunosurgery and icms were isolated and plated onto feeder cells. icms attached to feeder cells within h and three esc-like icm outgrowths appeared after 7- days. icm outgrowths were manually dissociated into 4- smaller clumps using a microscalpel, excised from feeder cells and replated onto fresh mouse embryonic fibroblasts. clones with distinct boundaries and high nuclear to cytoplasm ratios were selected for further propagation. three resc lines were established and designated as ivf, ivf and ivf. ivf and ivf were derived using the same sperm and oocyte donors. rescs shared common morphologies with other primate escs such as being flat with a distinct boundary against feeder cells. cells showed high nuclear to cytoplasm ratios and prominent nucleoli. ivf and ivf were cultured for > passages and ivf for > pluripotency markers were highly expressed in all resc lines including oct- nanog, ssea- tra-1- and tra-1-. other crucial transcription factors, such as sox- and rex- were also detected by rt-pcr. all resc lines showed strong alkaline phosphatase activity and could spontaneously differentiate into cell lineages from the three embryonic germ layers. after days in suspension culture, resc clones formed embryoid bodies. after days, cavities were observed in ebs, which are termed cystic ebs. ebs attached to gelatin-coated dishes and were cultured for 5- days until various differentiated cells appeared including neuron-like cells, contractive cardiomyocytes and endoderm-like cells. teratomas were formed 6- weeks after resc injection into the hind leg muscles of scid mice. histological characterization revealed that ectoderm, mesoderm and endoderm like structures were present in teratomas. these results demonstrated that resc lines could differentiate into all three embryonic germ layers in vitro and in vivo. detailed g-banding analysis revealed that all resc lines were karyotypically normal with a diploid set of chromosomes, even after long-term culture in vitro and repeated freeze-thaw procedures. ivf and ivf were derived from male blastocysts and ivf was derived from a female blastocyst. small rna sequencing newly established rescs were tested for purity using oct- staining, which resulted in >95% oct-4-positive cells. a small rna library from each cell line was then constructed for sequencing. small rna library sequencing yielded × × 106and × raw reads from ivf, ivf and ivf, respectively. after filtering, ×, × and × clean reads were obtained. the length distribution of clean reads centered at 22- nt, coinciding with the mirna length range. sequence alignment with the rhesus macaque genome using the short oligonucleotide alignment program demonstrated that ×, × and × sequences were mapped to the genome. mirnas were the major component of small rna libraries from rescs being 60% of sequences, which were matched to reference mirnas in each sample. the proportions of other annotated rnas was <15%. notably, ~25% of sequences could not be annotated because of low coverage and relatively poor annotations of the available rhesus macaque genome. unknown sequences were further analyzed to identify novel mirnas. mirna expression profiles of resc lines after matching to reference rhesus macaque mirnas, and known mirnas were detected in ivf, ivf and ivf, respectively, with absolute counts ranging from 1-,. among them, mirnas were shared by the three pools and remaining mirnas were expressed at low levels with < counts. from a genome-wide perspective, very low mirnas were likely sequencing artifacts or mirna machinery byproducts. therefore, an absolute count of was set for each pool as the minimum threshold for further analyses. to compare mirna expression patterns between resc lines, we evaluated the expression variability and coefficient of variation of mirna clusters. the results revealed similar expression patterns of mirna clusters in resc lines. we also compared the expression of total mirnas using a differential index and kappa statistical analyses. for the d.i., most mirnas were consistently expressed, whereas a slight deviation existed in ivf due to low-level expressed mirnas. for kappa statistical analysis, there was high repeatability between any two cell lines. taken together, mirna expression patterns in resc lines were highly consistent. identification of novel mirnas we predicted novel mirna genes in unannotated sequences using mireap. briefly, according to the characteristic hairpin structure of mirna precursors, unannotated small rna sequences mapped to genome were downloaded with flanking sequences and analyzed for secondary structure, dicer cleavage sites and minimum free energy. after analysis, reads that contained novel mirna precursors with > reads were identified. there were novel mirna precursors shared between the three resc lines and were homologous to reference mirnas in other species. of the mirna precursors with homologs, precursors were only conserved in humans and other primates, but not rodents. mirna target gene prediction and functional classification three programs were used to predict mirna target genes. using the targetscan program, a custom set of perl codes were downloaded from the targetscan database. conserved and non-conserved predicted patterns of mirna targets were considered separately. for pita and miranda, we used expressed mirnas and rhesus macaque 3'utrs orthologous to annotated human 3'utrs as inputs to predict mirna targets. over-representation of predicted mirna targets for both conserved and non-conserved patterns based on the targetscan program in the kyoto encyclopedia of genes and genomes pathway is shown in table s in additional file. gene enrichment patterns obtained by various methods were similar to each other, indicating that mirnas correlated to functionally critical roles in rescs. for example, predicted targets involved in the mapk, wnt and tgf-beta signaling pathways, and pathways involved in cancer were significantly enriched. mirna-target interaction network analysis a systematic survey of mirnas that interacted with targets may provide a better understanding of mirna regulation in stem cells. thus, we analyzed potential interactions between mirnas and their targets, which is termed as the mirna-target network. all mirnas and their targets were used to generate a bipartite graph of mirna-target interactions.. nodes represent mirnas or target genes and edges correspond to interactions between mirnas with target genes. node degree in the mt network is the number of connections or edges with other nodes. for each target gene node, degrees represent the number of mirnas targeting the gene. globally, the mt network was comprised of nodes with edges. using mt network degree analysis, the degree of genes regulated by mirnas was negatively correlated with their evolutionary rate. similarly, a previous study reported a negative correlation between the degree and evolutionary rate of proteins in a protein-protein interaction network. to exclude factors with potential effects on the observed negative correlation, we tested the effect of 3'utr length. after length normalization, the negative correlation remained. another factor was a potential ascertainment bias caused by mirna target prediction and identification of orthologous macaque 3'utrs. to evaluate this possibly, we randomized the scale-free mt network based on barabasi-albert model. the degree of mirna and target genes in the random network was significantly lower compared with that observed in the mt network. a significant correlation was not observed in the random network, which further supported the negative correlation in the mt network. comparison of mirna profiles from mouse, human and rhesus macaque to compare esc mirna profiles between species, we retrieved previously reported mouse and human esc mirna profiles. to avoid a cross-talking bias due to various sequencing techniques and data sizes, we selected solexa sequencing datasets from mouse and human escs. mirna expression frequencies were normalized to each other by determining the expected frequency in mapped reads per million. mirna expression levels were calculated as the mean of three samples. the three species expressed common mirnas. clustering analysis based on the shared mirnas revealed that mirna expression profiles were more similar between human and rhesus macaque, which was consistent with their evolutionary relationship. a number of mirnas were stably and highly expressed between species, such as the mir- family, mir- and mir- which suggested conserved functions in escs during evolution. of the common mirnas the'stemness' mir-290- cluster was the highest expressed in mouse escs. however, primate homologs the mir-290- and mir-371- clusters were not highly expressed in human and rescs. instead, the mir- cluster was the highest expressed in human and rhesus macaque, indicating the functional divergence of stemness mirna clusters in primate lineages. the mir-290- cluster contains multiple mature mirnas with seed sequences similar or identical to those in the mir- cluster. mir-290- and mir- clusters are transcriptionally regulated by oct-4/sox. taken together, these observations suggest that these two clusters are similarly expressed, regulate common target genes and are functionally conserved in escs derived from various species. there are three mirna clusters differentially expressed in mouse, human and rescs. the chromosome mirna cluster, a primate-specific mirna cluster, was enriched in human escs, but almost absent in rescs. a conserved mirna cluster within the imprinted dlk1-dio region was highly expressed in mouse and rhesus macaque escs, but rarely expressed in human escs, which was consistent with results from four other human esc lines.the mir- cluster in the sfmbt gene intron region was only expressed in mouse escs, embryos and newborn ovaries, but not in human and rhesus macaque escs, consistent with previous reports. discussion we established three resc lines and profiled mirna expression using solexa sequencing. mirna expression profiles were generally conserved between the three resc lines, sharing % of expressed mirnas. ivf and ivf were more similar because of derivation from identical sperm and ovum donors. similarly, navara et al. reported that pedigreed rescs express homogeneous gene profiles. compared with known mirnas in our data set, most novel mirnas were moderately expressed. novel mirna biological functions and association with stemness are yet to be elucidated. we predicted mirna targets in rescs using three independent algorithms. targets involved in tgf-β, mapk and wnt signaling pathways were significantly enriched. these pathways play important roles in mouse and human esc differentiation. this observation suggests that in addition to transcriptional level regulation by stemness-associated factors such as oct and nanog, mirnas may also play an important role in maintaining esc self-renewal and pluripotency at the post-transcriptional level. moreover, over-represented mirna targets involved in cancer pathways support the observation that cancer and escs share pathways for self-renewal and proliferation. our systematic investigation of the mt network suggested that the degree of genes regulated by mirnas was negatively correlated with their evolutionary rate in rescs. genes with more mirna regulators evolve more slowly. this was anticipated in accordance to a rule of "the more conserved, the deeper regulated" has been proposed in protein-protein interaction networks, in which hubs or central proteins are functionally essential and usually lethal after gene knockout. therefore in the mt network, hub targets should be more regulated due to selective pressure, which indicates the fine regulation of mirnas in escs. the cross-species comparison indicated that the majority of shared mirnas were stably expressed between species and consistent with mirna sequence-level conservation. this observation suggested that mirna expression is under stabilizing selection due to functional constraints on mirnas in escs. despite this close relationship, mirna expression differences existed between human and rhesus macaque escs. the chromosome mirna cluster is a primate-specific mirna cluster and contains > mature mirnas. these clustered mirnas are expressed in human escs, placenta and fetal brain, suggesting that they are involved in embryogenesis. however, the c19mc cluster is almost absent in rescs. this expression difference suggests that even between closely related species, there may be a significant mirna expression divergence and regulatory differences during embryogenesis between rhesus macaques and humans. in addition, the mirna cluster in the imprinted dlk1-dio region was conserved at the sequence level between mice, rhesus macaques and humans. liu et al reported that the expression of this cluster is positively correlated to mouse stem cell pluripotencies. we found that this cluster was enriched in mouse and rhesus macaque escs, but rare in human escs. recently, it was reported that diverse temporal origins may be responsible for observed differences between mouse and human escs. our data suggests that mirna expression patterns may provide a new strategy for investigating interspecies differences in esc pluripotency.  our results indicate that mirna profiles are highly conserved and consistent between the three resc lines, and these mirnas may play critical roles in the maintenance of self-renewal and pluripotency in rescs, particularly in differentiation pathways. further analysis of the mirna-target network indicates that the degree of genes regulated by mirnas is negatively correlated with their evolutionary rate in rescs. this observation suggests important roles of mirnas in post-transcriptional regulation. moreover, cross-species comparison of escs revealed an overall conservation of mirna expression patterns, indicating stabilizing selection on mirna expression in escs during evolution. however, we identified three mirna clusters that show distinct differences between mouse, human and rhesus macaque escs, which suggests species and/or lineage specific mirna regulatory changes during evolution. 
numerous studies have revealed human and mouse esc mirna profiles. mirna profiles were highly conserved between resc lines and predicted target genes were significantly enriched in differentiation pathways. however, studies on rhesus macaque escs are lacking due to limited resc availability and a need for systematic analyses of fundamental resc characteristics. moreover, a cross-species comparison revealed an overall conservation of mirna expression patterns between human, mouse and rhesus macaque escs. as a model for human-related study, the rhesus macaque is ideal for delineating the regulatory mechanisms of mirnas in escs. we established three resc lines and profiled microrna using solexa sequencing resulting in known and novel mirnas. further analysis of the mirna-target network indicated that gene expression regulated by mirnas was negatively correlated to their evolutionary rate in rescs. rescs share a unique mirna set that may play critical roles in self-renewal and pluripotency. however, we identified three mirna clusters that showed clear interspecies differences. micrornas play important roles in embryonic stem cell self-renewal and pluripotency. mirna expression patterns are generally conserved between species.
 micrornas play important roles in embryonic stem cell self-renewal and pluripotency. numerous studies have revealed human and mouse esc mirna profiles. as a model for human-related study, the rhesus macaque is ideal for delineating the regulatory mechanisms of mirnas in escs. however, studies on rhesus macaque escs are lacking due to limited resc availability and a need for systematic analyses of fundamental resc characteristics.  we established three resc lines and profiled microrna using solexa sequencing resulting in known and novel mirnas. mirna profiles were highly conserved between resc lines and predicted target genes were significantly enriched in differentiation pathways. further analysis of the mirna-target network indicated that gene expression regulated by mirnas was negatively correlated to their evolutionary rate in rescs. moreover, a cross-species comparison revealed an overall conservation of mirna expression patterns between human, mouse and rhesus macaque escs. however, we identified three mirna clusters that showed clear interspecies differences.  rescs share a unique mirna set that may play critical roles in self-renewal and pluripotency. mirna expression patterns are generally conserved between species. however, species and/or lineage specific mirna regulation changed during evolution. 