in recent years , studies in embryonic and somatic stem cells have provided basic insight into the molecular and cellular properties of stem cells . it is recognized that pluripotent stem cells such as embryonic stem cells ( esc ) can replenish differentiated cell types and achieve long - term tissue reconstitution . similar to esc , adult stem cells also have the capacity for self - renewal and multilineage differentiation . adult stem cells constitute ~12% of the total cell population within a specific tissue and are essential for maintaining homeostasis . these cells are normally quiescent and are held in an undifferentiated state within their niche until they receive a signal to self - renew or differentiate . they reside in various tissue types including the bone marrow , brain , digestive system , skin , retina , muscles , pancreas , and liver . hematopoietic stem cells ( hsc ) are the most widely studied and characterized adult stem cells . they play an essential role in sustaining the formation of the blood and the immune system . these adult stem cells reside in the bone marrow along with mesenchymal stem cells ( msc ) . it was previously thought that adult stem cells were lineage restricted ; however , recent studies have shown that hsc and mesenchymal stem cells have enormous plasticity . moreover , they offer several advantages including ease of manipulation , lack of serious ethical issues and , in the autologous setting , the absence of immunogenicity . micrornas ( mirnas ) are short single - stranded rna molecules of about 1725 nucleotides that control gene expression post - transcriptionally in many different cellular processes either by blocking translation or by inducing mrna degradation through sequence complementation . although numerous signaling pathways , transcription factors and epigenetic changes are essential cellular regulators in determining stem cell fate , recent studies have shown that mirnas are also implicated in coordinating the necessary changes in gene expression . typically , mirnas are capable of controlling the fate of cells in a time and tissue specific manner through regulation of cellular differentiation , developmental patterning , and morphogenesis . therefore , manipulation of mirnas could potentially be a useful approach to developing strategies for stem cell therapy . in this study , we investigated the role of mirnas in an adherent subpopulation of cd34 cells isolated from granulocyte colony - stimulating factor mobilized peripheral blood cells . we identified several clusters of mirnas that were differentially expressed in these stem cells by mirna profiling . further analysis of one of the clusters revealed a mirna that targets the stem cell gene nanog . our data provide evidence that mir-181a * targets nanog in a subpopulation of cd34 cells . we performed mirna profiling on a subpopulation of adherent cd34 cells mobilized peripheral blood cells . using a 466 mirna chip , we compared the mirna signatures of adherent and nonadherent cd34 cells ( figure 1a ) . these two subpopulations of cd34 cells have previously been established as having distinct morphology , immunophenotype , and gene expression profile . moreover , the adherent cd34 cells display greater plasticity than the nonadherent population as they have the potential to express determinants specific to liver , pancreas , heart , muscle , and nerve cell differentiation . hierarchical clustering revealed eight unique mirna clusters that were differentially expressed in the adherent and nonadherent populations . this finding is not entirely surprising since the biological roles of mirnas are known to vary according to their expression in distinct cell populations in normal tissues . interestingly , there were several mirnas that have not been previously characterized for human in cluster 8 ( table 1 ) . we were particularly interested in the mirnas in cluster 8 because among the mirnas in this cluster , mir-181a * ( mir-181a-3p ) was highly upregulated and studies have also shown that the mir-181 family are associated with regulation of cd34 cells . however , the mrna targets are largely unknown . we did identify some conserved regions within the mature human mir-181 family of sequences which consist of mir-181a , mir-181b , mir-181c , and mir-181d ( figure 1b ) . we have taken the first step toward better understanding the role of mir-181a * in cd34 cells . higher alkaline phosphatase activity is generally associated with stem cells that are in a less differentiated state . we found increasing levels of alkaline phosphatase activity in cells transfected with mir-181a * inhibitor when compared to transfection with mir-181a * mimic ( figure 1c ) . the data raises the possibility that these stem cell population could be maintained in a less differentiated state by inhibiting mir-181a * expression . we next applied an in - house genomic - bioinformatics database to identify and validate potential candidate target messages for mir-181a*. being important factors in stem cell maintenance , the genomic loci of nanog , stat3 , hic1 , sox2 , hoxb4 , and pou5f1 ( oct4 ) were scanned for putative target sites that was complementary to the mir-181a * sequence ( 5-accaucgaccguugauuguacc-3 ) . specifically , the annotated 3 utr sequences of these genes were scanned for seed sites with perfect reverse - complementarity to mirna-181a seed sequence ( nucleotides 27 from the 5 end ) and sites with strong overall complementarity to mirna-181a . of the putative target genes , we discovered that mir-181a * formed the most stable base pairing at the nanog 3utr ( figure 2a ) . since nanog is an important regulator of stem cell maintenance and self - renewal , we determined whether mir-181a * targeted nanog transcripts . using endpoint reverse transcription - pcr ( rt - pcr ) ( figure 2b ) and quantitative rt - pcr ( figure 2c ) in transfected cd34 stem cells , we found that inhibiting mir-181a * significantly upregulates transcript levels of nanog . it has been reported that there exists a sensitive feedback loop that maintains the differentiation of stem cells through the control of essential stem cell genes such as nanog . this loop enables where by environmental or molecular cues to influence the expression levels of genes , leading to a binary decision to switch downstream differentiation genes on or off . the data raises the possibility that mir-181a * may be involved in directly regulating nanog expression in cd34 stem cells . to gain more insight into the functional interaction between mir-181a * and nanog mrna , we further interrogated the entire sequence alignment between the two rna molecules . we discovered a high degree of base pairing at the 3 region ( 7 nucleotides ) and a weak base pairing at the 5 seed site of mir-181a * ( figure 3a , top panel ) . in general most mirna target sites have a strong base pairing at the 5 seed site ( 78 nucleotides ) . however , the 5 seed site rule is not always the case and studies have shown that the 3 region of mirna also play an important role in targeting mrna but are rare events . in addition to the 3 sites , a recent report has identified another site called the centre site suggesting that mirna targeting is more complex and diverse . in our case , because the base pairing at the 5 seed site of mir-181a * was weak , we speculated that the strong complementarity pairing at the 3 region would compensate and may follow the 3-compensatory site rule similar to one of the earlier mirna that was discovered such as let-7 ( figure 3a , bottom panel ) . both mir-181a * and let-7 mirnas , in this setting show a very similar overall structural identity in the way they bind to their targets . to determine whether the 3 region of mir-181a * was important , we generated , using rnahybrid modeling , the most energy stable complementarity base pairing between mir-181a * and nanog 3 utr mrna ( figure 3b , top panel ) as well as determining the best mutations that would disrupt the 3 region base pairing . this was generated by replacing two bases at the 3utr of nanog ( figure 3b , bottom panel ) . to test this modeling we generated a dual - luciferase reporter construct for mir-181a * and nanog 3 utr mrna . we inserted , upstream of the firefly luciferase gene , either the wild - type 3utr of nanog or a mutant variant containing the two point mutations predicted to abolish mir-181a * targeting ( figure 3b , bottom panel ) . transfecting the wild - type nanog 3utr reporter construct by itself or with the mir-181a * mimic into cd34 stem cells significantly reduced expression of luciferase ( figure 3c ) . transfecting mir-181a * inhibitor caused a significant reversal in suppression of luciferase expression indicating that binding of mir-181a * to the 3utr of nanog was suppressed thus allowing expression of the luciferase reporter . by contrast , transfection of the mutant nanog 3utr reporter alone or in combination with mir-181a * mimic or inhibitor had no repressive effect on expression of the luciferase reporter ( figure 3c ) . this is the first attempt to characterize mir-181a * in cd34 cells and we have identified nanog as its potential target . we performed mirna profiling on a subpopulation of adherent cd34 cells mobilized peripheral blood cells . using a 466 mirna chip , we compared the mirna signatures of adherent and nonadherent cd34 cells ( figure 1a ) . these two subpopulations of cd34 cells have previously been established as having distinct morphology , immunophenotype , and gene expression profile . moreover , the adherent cd34 cells display greater plasticity than the nonadherent population as they have the potential to express determinants specific to liver , pancreas , heart , muscle , and nerve cell differentiation . hierarchical clustering revealed eight unique mirna clusters that were differentially expressed in the adherent and nonadherent populations . this finding is not entirely surprising since the biological roles of mirnas are known to vary according to their expression in distinct cell populations in normal tissues . interestingly , there were several mirnas that have not been previously characterized for human in cluster 8 ( table 1 ) . we were particularly interested in the mirnas in cluster 8 because among the mirnas in this cluster , mir-181a * ( mir-181a-3p ) was highly upregulated and studies have also shown that the mir-181 family are associated with regulation of cd34 cells . however , the mrna targets are largely unknown . we did identify some conserved regions within the mature human mir-181 family of sequences which consist of mir-181a , mir-181b , mir-181c , and mir-181d ( figure 1b ) . we have taken the first step toward better understanding the role of mir-181a * in cd34 cells . higher alkaline phosphatase activity is generally associated with stem cells that are in a less differentiated state . we found increasing levels of alkaline phosphatase activity in cells transfected with mir-181a * inhibitor when compared to transfection with mir-181a * mimic ( figure 1c ) . the data raises the possibility that these stem cell population could be maintained in a less differentiated state by inhibiting mir-181a * expression . we next applied an in - house genomic - bioinformatics database to identify and validate potential candidate target messages for mir-181a*. being important factors in stem cell maintenance , the genomic loci of nanog , stat3 , hic1 , sox2 , hoxb4 , and pou5f1 ( oct4 ) were scanned for putative target sites that was complementary to the mir-181a * sequence ( 5-accaucgaccguugauuguacc-3 ) . specifically , the annotated 3 utr sequences of these genes were scanned for seed sites with perfect reverse - complementarity to mirna-181a seed sequence ( nucleotides 27 from the 5 end ) and sites with strong overall complementarity to mirna-181a . of the putative target genes , we discovered that mir-181a * formed the most stable base pairing at the nanog 3utr ( figure 2a ) . since nanog is an important regulator of stem cell maintenance and self - renewal , we determined whether mir-181a * targeted nanog transcripts . using endpoint reverse transcription - pcr ( rt - pcr ) ( figure 2b ) and quantitative rt - pcr ( figure 2c ) in transfected cd34 stem cells , we found that inhibiting mir-181a * significantly upregulates transcript levels of nanog . it has been reported that there exists a sensitive feedback loop that maintains the differentiation of stem cells through the control of essential stem cell genes such as nanog . this loop enables where by environmental or molecular cues to influence the expression levels of genes , leading to a binary decision to switch downstream differentiation genes on or off . the data raises the possibility that mir-181a * may be involved in directly regulating nanog expression in cd34 stem cells . to gain more insight into the functional interaction between mir-181a * and nanog mrna , we further interrogated the entire sequence alignment between the two rna molecules . we discovered a high degree of base pairing at the 3 region ( 7 nucleotides ) and a weak base pairing at the 5 seed site of mir-181a * ( figure 3a , top panel ) . in general most mirna target sites have a strong base pairing at the 5 seed site ( 78 nucleotides ) . however , the 5 seed site rule is not always the case and studies have shown that the 3 region of mirna also play an important role in targeting mrna but are rare events . in addition to the 3 sites , a recent report has identified another site called the centre site suggesting that mirna targeting is more complex and diverse . in our case , because the base pairing at the 5 seed site of mir-181a * was weak , we speculated that the strong complementarity pairing at the 3 region would compensate and may follow the 3-compensatory site rule similar to one of the earlier mirna that was discovered such as let-7 ( figure 3a , bottom panel ) . both mir-181a * and let-7 mirnas , in this setting show a very similar overall structural identity in the way they bind to their targets . to determine whether the 3 region of mir-181a * was important , we generated , using rnahybrid modeling , the most energy stable complementarity base pairing between mir-181a * and nanog 3 utr mrna ( figure 3b , top panel ) as well as determining the best mutations that would disrupt the 3 region base pairing . this was generated by replacing two bases at the 3utr of nanog ( figure 3b , bottom panel ) . to test this modeling we generated a dual - luciferase reporter construct for mir-181a * and nanog 3 utr mrna . we inserted , upstream of the firefly luciferase gene , either the wild - type 3utr of nanog or a mutant variant containing the two point mutations predicted to abolish mir-181a * targeting ( figure 3b , bottom panel ) . transfecting the wild - type nanog 3utr reporter construct by itself or with the mir-181a * mimic into cd34 stem cells significantly reduced expression of luciferase ( figure 3c ) . transfecting mir-181a * inhibitor caused a significant reversal in suppression of luciferase expression indicating that binding of mir-181a * to the 3utr of nanog was suppressed thus allowing expression of the luciferase reporter . by contrast , transfection of the mutant nanog 3utr reporter alone or in combination with mir-181a * mimic or inhibitor had no repressive effect on expression of the luciferase reporter ( figure 3c ) . this is the first attempt to characterize mir-181a * in cd34 cells and we have identified nanog as its potential target . understanding the cellular and molecular mechanisms responsible for the multi - pluripotency of stem cells will be critical for their practical use in therapy . micrornas have been shown to be important and necessary for proper stem cell regulation . in this study , we generated a mirna profile from a subpopulation of adherent cd34 cells isolated from g- colony - stimulating factor mobilized peripheral blood and demonstrated a marked difference in expression patterns for a small cluster of mirna . using bioinformatics , we identified nanog as a potential target for mir-181a*. interestingly , there have been works published regarding other potential targets for the mir-181 family . for example , mir-181b has been shown to be an important regulator of nuclear factor-b signaling in the vascular endothelium by targeting importin-3 . in another cell type , mir-181b has been shown to target tcl1 oncogene in chronic lymphocytic leukemia . similar to mir-181b , mir-181a also has several targets such as prox1 in lymphatic endothelial cells , c2h2 zinc - finger proteins , and multiple phosphatases such as shp2 , ptpn22 , dusp5 , and dusp6 . although mir-181a*s binding site within the 3utr of nanog displayed a weak canonical 5 mirna seed pairing , it also showed strong complementarity at the mirna 3 end and at the central ago2 catalytic site 1011 nucleotides from the 5 mirna site . importantly , by introducing two point mutations to disrupt the 3 and central complementary sites , we abolished mir-181a * regulation of a reporter construct containing the 3utr of nanog . our studies highlight a new stem cell - related target for the mir-181 family and show that mir-181a * directly targets nanog . the hematopoietic blood samples were obtained with informed patient consent and approved by the hammersmith hospital research ethics committee . samples of granulocyte - colony stimulating factor mobilized peripheral blood progenitor cells were processed by leukapheresis at the stem cell laboratory at the hammersmith hospital . briefly , human mobilized peripheral blood samples were diluted in a ratio of 1:4 in hanks'-buffered saline solution ( gibco , paisley , uk ) , the mononuclear cells were separated by centrifugation over a lymphoprep ( dundee , scotland ) density gradient at 1,800  rpm for 30 minutes . the mononuclear cell fraction at the interface was aspirated and washed twice with hanks'-buffered saline solution , and finally with macs buffer ( phosphate - buffered saline solution ) at ph 7.2 supplemented with 0.5% bovine serum albumin and 2  mmol / l edta ) . cd34 cells were isolated using a cd34 isolation kit ( miltenyi biotec , cologne , germany ) according to the manufacturer 's protocol . briefly , isolated cd34 cells were added to 24-well or 35-mm tissue - culture treated dishes ( nunc , roskilde , denmark ) at a density of 2.55  10 cells in -minimum essential medium to isolate the adherent cd34 cell population . after 30-minute incubation , nonadherent cells were removed and adherent cells were rinsed three times with phosphate - buffered saline or -minimum essential medium . the adherent cd34 cell population was grown in a serum - free medium ( cellgro ; cellgenix , freiburg , germany ) containing three cytokines : 250  ng / ml of stem cell factor , 250  ng / ml of interleukin-6 , and 250  ng / ml of interleukin-3 ( invitrogen , paisley , uk ) in 0.5% penicillin / streptomycin antibiotics . total viable cells were counted using the trypan exclusion assay . for the alkaline phosphatase assay , ~2.5  10 progenitor cells in 24-wells were transfected twice with the oligonucleotides at 100 nmol / l and the cells were harvested 72 hours later as described in the alkaline phosphatase assay kit ( cell biolabs , san diego , ca and millipore , billerica , ma ) . equal protein load at 100 g of protein was used for the assays and the reaction was measured after 1-hour incubation at 37  c at 405 adherent and nonadherent cells were added into a pcr tube and heat - treated at 95  c for 5 minutes . then the mirnas were reverse transcribed into cdnas by 460 mirna - specific stem - looped primers . then these mirna cdnas were amplified by 18 cycles of pcr by 460-specific forward primers and a universal reverse primer . finally , the cdnas were split and individual mirna was measured by taqman probe - directed real - time pcr . the pcr was done as following by ab7900 with 384-well plates : first , 95  c for 10 minutes to activate the taq polymerase . then 40 cycles of 95  c for 15 seconds ( for denaturation ) and 60  c for 1 minute ( for annealing and extension ) were performed . two independent allogenic donor samples were used in the analysis . in order to obtain sufficient total rnas , the mirna-181a * mimic and inhibitor were purchased from dharmacon , lafayette , co. the oligonucleotides were transfected ( at day 0 and again at 24 hours ) at 100 nmol / l into 1  10 adherent cd34 stem cells using nanofectamine reagent following the manufacturer 's recommendation ( paa , pasching , austria ) . total rna was harvested using the rnaqueous - micro kit ( ambion , paisley , uk ) 72 hours post - transfection . five hundred nanogram of total rna from each sample was reverse transcribed using the one - step rt - pcr kit from qiagen ( hilden , germany ) following the manufacturer 's recommendation . expression of human nanog was measured semiquantitatively by pcr using primer pair from r&d systems ( minneapolis , mn ) after 32 cycles at 94  c for 45 seconds . gapdh primers : forward ( 5gtgaaggtcggagtcaacg3 ) and reverse ( 5ggtgaagacgccagtggactc3 ) was used as a loading control after 30 cycles at 94  c for 45 seconds . the samples were separated on agarose gel and analyzed using uvp geldoc system ( uvp , upland , ca ) . four sets of band intensity across three independent experiments were used for semiquantitative analysis . for the quantitative real - time rt - pcr , 500  ng of total extracted rna ( as described above ) was processed for elimination of genomic dna followed by reverse transcription using the quantitect reverse transcription kit from qiagen . the first strand cdna synthesis was then amplified for quantitative analysis of human nanog ( nm_024865 ) and the reference gene , human gluceraldehyde-3-phosphate - dehydrogenase ( gapdh ) ( nm_002046 ) using quantifast sybr green pcr kit from qiagen . amplification was performed using applied biosystems 7900ht fast - real - time system with 40 cycle conditions at 95 c15 seconds and 60 c45 seconds with a total volume of 25 l per sample . sequence annotations and data were downloaded from version hg18 of the ucsc table browser 's refseq genes track . the genomic loci nanog , stat3 , hic1 , sox2 , hoxb4 , and pou5f1 ( oct4 ) were scanned for putative target sites for mir-181a * ( 5-accaucgaccguugauuguacc-3 ) . more specifically , the genes ' annotated 3 utr sequences were scanned for ( i ) seed sites with perfect reverse - complementarity to the oligonucleotide 's seed sequence ( nucleotides 27 from the 5 end ) and ( ii ) sites with strong overall complimentarily to the oligonucleotide . such sites could be target sites for mirna - like translational suppression and mrna degradation , and small interfering rna - like mrna cleavage . the former scan used a custom python script , whereas the second scan used rnahybrid with default parameter settings to evaluate potential hybridization between the oligo and target sequence . none of the five genes ' 3 utrs contained mirna - like seed sites for the oligonucleotide . of the sites that formed the most stable interactions with the oligonucleotide , the nanog site was the most stable site that contained paired bases at the putative ago2 cleavage site . the ago2 cleavage site would presumably be between nucleotides 10 and 11 from the 5 end . nanog 3 utr was amplified from hela genomic dna by nested pcr using two different forward primers and a single reverse primer ( primers are listed below ) . for both pcrs , 50 l sample volumes were prepared using 0.3 mol / l each of forward and reverse primers , 1 accuprime pfx reaction mix ( invitrogen ) , and 1.0 unit of accuprime pfx dna polymerase ( invitrogen ) . one hundred nanogram of genomic dna and 0.5 l of pcr product were used as templates in the first and the second pcr , respectively . the forward 2 and the reverse primers each contained two restriction enzyme cut sites ; xhoi and sgfi for forward 2 and noti and ecori for reverse . the nanog 3 utr construct was cloned into the psicheck 2.2 vector ( promega , madison , wi ) , using the unique restriction enzyme cut sites for noti and xhoi . two point mutations were inserted in the nanog 3utr target site of hsa - mir-181a * using two mutagenic primers and psicheck 2.2 with nanog 3 utr insert as template . a 25.5 l pcr sample volume was prepared using 10  ng of template , 0.5 mol / l of each primer , 1 reaction buffer ( agilent technologies , santa clara , ca ) , dntp mix ( 0.2 mol / l each , finnzymes ) , and 1.25 units of pfuturbo dna polymerase ( stratagene ) . sequence of mutated hsa - mir-181a * target site ( mutations in bold and underlined ) : gtgaagtggcgaggtcttggc . list of primer sequences : forward 1 , agcaaccagacccagaacatccag ; forward 2,gcgatcgctcgagagatgagtgaaactgatattactcaatttcagtctgg;reverse , gaattcgcggccgcatgtttaagctgtatatttactcattgaaacactcgg ; psicheck , aggacgctccagatgaaatg ; nanog seq , tcactgcaagctccgtctcc ; rev psicheck , caaaccctaaccaccgctta ; nanog mut fr , ggctggagtgaagtggcgaggtcttggctc ; nanog mut rev , gagccaagacctcgccacttcactccagcc . for the reporter luciferase reporter assay , 100  nmol / l of mir181a * mimic or its inhibitor ( dharmacon ) were transfected into cd34 progenitor cells ( 2.5  10 cells / well ) in a 24-well plate for 24 hours . the nanog 3utr wild - type or mutant with two point mutations was cloned downstream of the renilla translational stop codon in psicheck-2 vector ( promega ) . 100  ng of the 3-utr - nanog ( 3-utr - nanog - psi - check-2 ) or its mutated variant was cotransfected into the cells pre - conditioned with mir181a * mimic or inhibitor for further 24 hours before harvesting . empty ( psicheck-2 ) vector was used for normalization and 3-utr - nanog alone was used as a control . renilla and firefly luciferase assay was carried out using the dual - luciferase reporter assay system ( promega ) . readings were measured the victor plate reader ( waltham , ma ) ( perkin - elmer victor ) . statistical analysis .