a novel lentivirus for quantitative assessment of gene knockdown in stem cell differentiation

REVIEW

A novel lentivirus for quantitative assessment ofgene knockdown in stem cell differentiationS Alimperti1, P Lei1, J Tian1,4 and ST Andreadis1,2,3

Loss of gene function is a valuable tool for screening genes in cellular processes including stem cell differentiationdifferentiation. However, the criteria for evaluating gene knockdown are usually based on end-point analysis and real-time,dynamic information is lacking. To overcome these limitations, we engineered a shRNA encoding LentiViral Dual Promotervector (shLVDP) that enabled real-time monitoring of mesenchymal stem (MSC) differentiation and simultaneous gene knockdown.In this vector, the activity of the alpha-smooth muscle actin (aSMA) promoter was measured by the expression of a destabilizedgreen fluorescent protein, and was used as an indicator of myogenic differentiation; constitutive expression of discosoma redfluorescent protein was used to measure transduction efficiency and to normalize aSMA promoter activity; and shRNA was encodedby a doxycycline (Dox)-regulatable H1 promoter. Importantly, the normalized promoter activity was independent of lentivirus titerallowing quantitative assessment of gene knockdown. Using this vector, we evaluated 11 genes in the TGF-b1 or Rho signalingpathway on SMC maturation and on MSC differentiation along the myogenic lineage. As expected, knockdown of genes such asSmad2/3 or RhoA inhibited myogenic differentiation, while knocking down the myogenic differentiation inhibitor, Klf4, increasedaSMA promoter activity significantly. Notably, some genes for example, Smad7 or KLF4 showed differential regulation of myogenicdifferentiation in MSC from different anatomic locations such as bone marrow and hair follicles. Finally, Dox-regulatable shRNAexpression enabled temporal control of gene knockdown and provided dynamic information on the effect of different geneson myogenic phenotype. Our data suggests that shLVDP may be ideal for development of lentiviral microarrays to deciphergene regulatory networks of complex biological processes such as stem cell differentiation or reprogramming.

Gene Therapy (2012) 19, 1123--1132; doi:10.1038/gt.2011.208; published online 12 January 2012

Keywords: lentivirus arrays; dynamic gene expression; regulatable gene knockdown; stem cells; screening gene function

INTRODUCTIONSince its discovery, RNAi has become a powerful tool forunderstanding gene function through loss-of-function screening.For example, gene silencing was used to identify tumor-suppressor genes,1 genes essential for maintaining stemness2 orcontrolling cell differentiation.3,4 In addition to understandingbiological processes, RNAi has been proposed as a novel strategyfor cancer therapy, antiviral therapy, treatment of geneticor metabolic disorders as well as cell reprogramming forpersonalized regenerative medicine.5

In addition to single gene knockdown, investigators developedRNAi libraries to discover gene pathways controlling variouscellular processes and facilitate global understanding of cellularbehavior.6 -- 9 This approach was used to identify genes that areinvolved in proteasome-mediated proteolysis,10 virus infectionand replication,11 -- 14 cytokinesis,8,9,15,16 endocytosis,17 stem cellself-renewal18 or cancer cell proliferation and survival.19,20

RNAi knockdown requires a good selection strategy to linkgenetic information to cellular phenotype. In many cases, suchreadout is based on cell proliferation or expression of markerproteins that are detected by antibody staining in order to score a‘hit’. These procedures are usually destructive and therefore, theyare employed as end-point analyses. In addition, quantitativecomparison among samples suffers from differences in gene

transfer efficiency owing to differences in the titer of viralpreparations. To overcome these limitations, we engineered ashRNA encoding LentiViral Dual Promoter (shLVDP) vector thatcontains three transcriptional units. The first encodes for unstableZsGreen (ZsG-DR) under a promoter of interest and is used asreadout to monitor the effect of gene knockdown on cellularphenotype. The second encodes for discosoma red fluorescentprotein (DsRed)-Express under a constitutive promoter forexample, human phosphoglycerate kinase promoter (hPGK)and is used to assess gene transfer efficiency as well as for datanormalization to obtain quantitative data independent of thegene transfer efficiency.21,22 Finally, the third unit encodes forshRNA under a tetracycline-regulatable H1 promoter in the viralLTR. This vector allows shRNA expression and at the same timeprovides quantitative real-time monitoring of the effect of geneknockdown on cellular phenotype.

Using the shLVDP vector, we measured the effects of severalgenes in the TGF-b1 and Rho pathways on maturation of humanaortic smooth muscle cells (HASMC) and in myogenic differentia-tion of mesenchymal stem cells from bone marrow (BM-MSCs) andhair follicles (HF-MSC). TGF-b1 has been shown to upregulate theexpression of alpha-smooth muscle actin (aSMA) in SMC23

and MSC,24,25 and RhoA is known to affect actin reorganizationand contractile activity.26 Myogenic differentiation was monitored

Received 21 June 2011; revised 26 September 2011; accepted 10 October 2011; published online 12 January 2012

1Bioengineering Laboratory, Department of Chemical and Biological Engineering, University at Buffalo, State University of New York, Amherst, NY, USA; 2Department ofBiomedical Engineering, University at Buffalo, State University of New York, Amherst, NY, USA and 3Center of Excellence in Bioinformatics and Life Sciences, Buffalo, NY, USA.Correspondence: Dr ST Andreadis, Bioengineering Laboratory, 908 Furnas Hall, Department of Chemical and Biological Engineering, University at Buffalo, State University ofNew York, Amherst, NY 14260-4200, USA.E-mail: [email protected] address: Life Technologies, Grand Island, NY 14072, USA.

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by the aSMA promoter activity and confirmed by western blot andimmunostaining. Normalized promoter activity rendered theresults independent of gene transfer efficiency and allowedcomparison of the relative effect of each gene on myogenicdifferentiation of different cell types. Finally, Dox-inducible shRNAexpression enabled temporal control of gene knockdown andprevented toxic effects associated with loss of genes that areimportant in cell spreading or cytoskeletal reorganization.

RESULTSGeneration of shLVDP vectorRecently, our laboratory developed a LVDP that provides a highexpression level of two genes from independent promoters inmammalian cells including MSCs.22 A constitutive promoter drivesexpression of one marker gene (for example, DsRed) and is usedto measure transduction efficiency. The promoter of interestdrives expression of another marker (for example, greenfluorescent protein (EGFP)) and is used to monitor the biologicalprocess of interest. Polyadenylation, terminator and insulatorsequences were inserted between the two transcription units toeliminate promoter interference yielding promoter activity thatwas similar to that exhibited by vectors containing a singletranscription unit. This vector was used to generate lentiviralmicroarrays to monitor the dynamics of gene expression in realtime in response to external stimuli.21

In this study, we modified this vector to also express shRNAfrom a tetracycline-regulatable H1 promoter, thereby allowingassessment of genes that may be involved in a process, whichcan be monitored in real time by the activity of a promoter orresponse element (RE) (Figure 1a). To this end, the 30LTR of ourLVDP vector was substituted by the 30LTR of pLVTHM vector,which was engineered to express shRNA under a tetracycline-regulatable H1 promoter.27,28 During reverse transcription of the

lentiviral RNA, the 30LTR replaces the 50LTR, thereby allowingexpression of two shRNA copies for each integrated lentivirusgenome. Overall, this modified vector expresses two genes andone shRNA and was termed shLVDP.

Signal normalization shows that promoter activity is independentof lentivirus titerThe shLVDP allows measurement of promoter activity throughchanges in green fluorescent intensity (GFI), which depends onthe number of infected cells and the number of transgene copiesper cell. However, quantitative measurements of promoter activitymay suffer by lack of consistency due to differences in genetransfer efficiency among different lentivirus preparations eachexpressing a different shRNA. As shLVDP also encodes for DsRedunder a constitutive promoter we hypothesized that normal-ization of GFI by the red fluorescence intensity (RFI) may beindependent of the gene transfer efficiency, thereby yieldingquantitative data.

To address this hypothesis, we employed shLVDP encoding forEGFP under the hPGK promoter, DsRed under the cytomegaloviruspromoter (CMV) promoter and scramble shRNA under the H1promoter to transduce HASMCs with two MOI that differed by10-fold, MOI¼ 1 and MOI¼ 10. As expected, RFI and GFI increasedwith increasing virus concentration as measured by fluorescencemicroscopy (Figures 1b and c) or flow cytometry (Figures 1e and f),likely due to increased fraction of transduced cells and/orincreased number of gene copies per cell. However, the normal-ized fluorescence intensity (GFI/RFI) remained the same regardlessof the analysis method (Figures 1d and g), suggesting that theintrinsic promoter activity was independent of virus titer. Thisresult suggests that internal signaling normalization can be usedto eliminate disparity owing to differences in transductionefficiency, thereby enabling acquisition of quantitative data.

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Figure 1. Normalized promoter activity of shLVDP vector is independent of virus titer. (a) Schematic of shLVDP vector. (a) Red fluorescenceintensity (RFI) and (c) green fluorescence intensity (GFI) of cells transduced with shLVDP vector as determined by microscopy. (d) Thenormalized fluorescence intensity was calculated as ratio of GFI/RFI. (e) Red fluorescence intensity (RFI) and (f) green fluorescence intensity(GFI) of cells transduced with shLVDP virus as determined by flow cytometry. (g) The normalized fluorescence intensity was calculated as ratioof GFI/RFI. *Significant difference from the MOI¼ 10 (*Po0.05). cHS4, chicken hypersensitive site 4; H1, H1 promotersMAR8, synthetic MARsequence; SPA, synthetic polyA; Tactb, transcriptional termination of human b-globin; tetO, tetracycline repressor-binding site; TKpA, herpessimplex virus thymidine kinase poly(A).

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shLVDP can be used to monitor smooth muscle cell maturationNext, we modified this vector to enable monitoring of geneexpression from any promoter or RE. As REs usually requireaddition of a minimal CMV promoter to achieve high expressionlevels, we substituted the CMV promoter-driving DsRed with thehPGK promoter to avoid the potential of homologous recombina-tion due to repeated sequences, as previously reported forlentiviral vectors.29 We also replaced EGFP with ZsG-DR, a brighterand destabilized (t1/2¼ 8 -- 12 h) green fluorescent protein to bettercapture the kinetics of gene expression. Finally, we cloned thepromoter for the well-known early myogenic marker, aSMAupstream of ZsG-DR, to monitor smooth muscle cell maturation inresponse to TGF-b1 treatment. The resulting vector (Figure 2a) wasused to produce recombinant lentivirus to transduce HASMCs.Three days after transduction, the cells were cultured either ingrowth medium (GM:SMGS) or differentiation medium (DM:SMDSþ 10 ng ml -- 1 TGF-b1) in the presence or absence of TGF-b1receptor inhibitor, SB431542 (SB4). Fluorescence intensity wasmeasured 3 days later using fluorescence microscopy and flowcytometry.

Under growth conditions, cells displayed basal aSMA promoteractivity (normalized intensity, NI¼ 0.94), which increased by3-fold (NI¼ 2.8) in the presence of TGF-b1 (Figure 2b). Additionof the TGF-b1 inhibitor, SB431542, significantly reduced the aSMApromoter activity by 16-fold (NI¼ 0.17). Similarly, flow cytometryshowed that the normalized intensity (GFI/RFI) increased by2.5-fold (NI¼ 2.45) upon treatment with TGF-b1, whereas additionof the TGF-b1 receptor inhibitor, SB431542, reduced GFI/RFI by18-fold (NI¼ 0.05) (Figure 2c). In addition to the normalizedintensity, the percentage of ZsG-DRþ cells was 3.3±0.78,26.1±1.56 or 0.17±0.06% in GM, DM or DMþ SB431542,respectively (Figure 2d). In agreement, western blot showed thataSMA protein level increased by 3.35±0.3-fold in DM (Figure 2e),which was further verified by immunostaining (Figure 2f). Inparticular, under GM aSMA expression was weak and diffuse but

upon TGF-b1 treatment, aSMA was organized in filaments--anindication of contractile myogenic phenotype (Figure 2g).Taken together, these results suggested that our shLVDPcan be used to monitor TGF-b1-mediated myogenic differ-entiation.

Normalized promoter activity is independent of the extent of genesilencingThe ability to monitor cell differentiation using our lentiviral vectorsuggested that this system could be used to examine the effectsof various genes/pathways in myogenesis using shRNA knock-down from the same vector as shown in Figure 3a. As the extentof gene silencing may vary with the transduction efficiency, weexamined whether differential shRNA expression might affect thelevel of promoter activity.

To address this issue, we expressed shRNAs-representing genesthat are known to affect myogenic differentiation (Klf4 andSmad4) from the H1 promoter of the shLVDP vector shown inFigure 3a. The same vector also encodes for ZsG-DR under theaSMA promoter and DsRed under the hPGK promoter. Asexpected, higher virus titer resulted in higher knockdown of theKlf4 or Smad4 genes as shown by the decreased mRNA levels(Figure 3b). To evaluate promoter activity upon gene silencing,HASMC were transduced with the shLVDP at the indicated virusMOI and cultured in the presence of TGF-b1 to induce myogenicdifferentiation. Three days later, aSMA promoter activity wasdetermined by fluorescence microscopy.

As expected, higher virus concentrations resulted in highertransduction efficiency as indicated by higher RFI values(Figure 3c). Similarly, the level of GFI---an indicator of aSMApromoter activity---depended strongly on the transduction effi-ciency (Figure 3d). Specifically, for scramble shRNA that did notaffect aSMA promoter activity, GFI increased with increasinglentivirus concentration. Knockdown of the myogenic inhibitor,Klf4 also enhanced GFI, whereas knockdown of pro-myogenic

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Figure 2. Optimization of smooth muscle cell differentiation system. (a) Schematic of smooth muscle-specific shLVDP vector carrying scrambleshRNA (ZsG-DR: destabilized Green protein). Transduced HASMC were cultured in GM:SMGS, DM(SMDSþ 10 ngml -- 1 TGF-b1) orDMþ SB431542. (b) Normalized intensity (GFI/RFI) of transduced cells was determined by fluorescence microscopy. (c) Normalized intensity(GFI/RFI) of transduced cells was determined by flow cytometry. (d) The percent of positive ZsG-DR cells (%ZsG-DRþ ) was measured by flowcytometry. (e) Protein level of aSMA was measured by western blot. Immunostaining of HASMC for aSMA (green) after 3 days of culture in(f) GM; or (g) DM. Cell nuclei were counterstained with Hoechst dye (blue). Scale bar¼ 50 mm. *,**Significant difference compared withGM (*Po0.05, **Po0.1). The color reproduction of this figure is available at the Gene Therapy journal online.


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genes, for example, RhoA or Smad4 decreased GFI in a lentivirusconcentration-dependent manner. In contrast to GFI, the normal-ized aSMA promoter activity (GFI/RFI) was independent of thelentivirus concentration for the range of MOI tested (Figure 3e),strongly suggesting that the normalized promoter activity wasindependent of the extent of gene silencing.

Evaluating gene knockdown on TGF-b1-induced aSMApromoter activityWe used TGF-b1 to induce SMC maturation as a model system todemonstrate the utility of the shLVDP vector in measuringpromoter activity with simultaneous gene silencing. To this end,we employed the shLVDP vector encoding ZsG-DR under theaSMA promoter, DsRed under the PGK promoter and shRNAunder the H1 promoter targeting the indicated genes. The genesthat were selected for knockdown are known effectors of TGF-b1signaling including Smad2, Smad3, Smad4 and Smad7; theRhoA pathway such as RhoA, Rock1, Mrtf-B/Mlk2, Pkn1; as wellas other transcription factors that are known to be importantin myogenic differentiation such as Srf, Klf4 and MyoCD. Theknockdown efficiency of each shRNA sequence was firstmeasured by real time-PCR in transiently transfected 293T cellsand only shRNAs showing at least 60% knockdown were chosenfor further experiments (Supplementary Figure S1; Supplemen-tary Material).

Next, HASMC were transduced with shLVDP each encoding forthe indicated shRNA (Figure 4a). Three days later, modified cellswere coaxed into a mature SMC phenotype by switching to DMand aSMA activity was measured using a Zeiss Observer.Z1microscope (Carl Zeiss Inc., Thornwood, NY, USA) equipped withenvironmental room with temperature, CO2 and humidity control.Image analysis was performed using the Cell Profiler software (seeMethods section) and GFI was normalized to RFI to reportpromoter activity independent of gene transfer efficiency.

As shown in Figure 4b, silencing myogenic transcription factors(Srf, MyoCD), TGF-b or Rho pathway effectors (for example, Smad2,Smad3, Smad4, RhoA, Rock1, Mrtf-B/Mlk2, Pkn1) decreased aSMApromoter activity significantly as compared with control (scramble

shRNA in DM). On the other hand, knocking down the myogenicdifferentiation inhibitor Klf4 increased aSMA promoter activitysignificantly by almost 2-fold. These measurements were verifiedby flow cytometry that showed a significant 3.40±0.41-foldincrease in the fraction of ZsG-DRþ cells upon Klf4 knockdown(Figure 4c).

In addition, immunostaining showed that aSMA promoteractivity correlated with aSMA protein intensity and organization(Figure 5). Specifically, HASMC transduced by scramble or Klf4-shRNA spread more and expressed higher level of aSMA, whichwas also organized in filaments, indicating mature myogenicphenotype. In contrast, RhoA knockdown induced cell roundingand abolished aSMA protein expression in agreement with thedecline in aSMA promoter activity. Finally, cells that were modifiedwith Smad4 shRNA became elongated with diffused aSMAstaining and no filamentous organization. Taken together,these results demonstrate that the effect of shRNA on aSMApromoter activity correlated with protein expression and fibrousorganization.

shLVDP for monitoring MSC differentiation to the myogeniclineageTo demonstrate the application of this approach in stem celldifferentiation, we employed BM-MSCs and HF-MSC. The cellswere transduced with the same group of lentiviruses, coaxed todifferentiate into SMC in DM and 3 days later GFI and RFI weremeasured by fluorescence microscopy. Similar to HASMC, aSMApromoter activity decreased significantly by knocking downTGF-b1 or Rho pathway effectors and increased by knocking downKlf4 (Figures 6a and b). Interestingly, although knocking down Klf4increased aSMA activity (GFI/RFI) in HASMC (1.68±0.07; Po0.05)and BM-MSC (2.03±0.42; Po0.05), it had no effect on HF-MSC(1.35±0.19; P40.05). On the other hand, Smad7 knockdown hada small effect on HASMC but decreased aSMA activity in BM-MSC(0.722±0.06; P¼ 0.065) and to a much larger extent in HF-MSC(0.11±0.03; Po0.05). These results suggest that Klf4 and Smad7may differentially regulate aSMA expression in HF-MSC comparedwith BM-MSC and HASMC.

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Figure 3. Normalized promoter activity is independent of the extent of gene silencing. (a) Schematic of lentiviral vector encoding for shRNAtargeting Smad4, Klf4 genes or no gene (scramble). (b) Quantitative RT-PCR analysis of Klf4 and Smad4 mRNA expression in 293T cellstransduced with lentivirus encoding for Klf4 or Smad4 shRNA, respectively, at the indicated MOI. (c) Red fluorescence intensity (RFI); (d) Greenfluorescence intensity (GFI) and (e) normalized promoter activity (GFI/RFI) of HASMC transduced with lentivirus at the indicated MOI.


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We also verified MSC differentiation into the SMC lineage usinga gel compaction assay, an indicator of force generation ability,the quintessential property of SMC. To this end, HF-MSCs or

BM-MCS were induced to differentiate into SMC in DM andthen embedded into fibrin hydrogels (106 cells ml -- 1). Afterpolymerization was complete (1 h), the gels were released from

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Figure 4. shLVDP cell array to assess the effects of the indicated genes on SMC maturation in response to TGF-b1. (a) Schematic of HASMCcell array. Each well was infected with lentivirus encoding for shRNA targeting the indicated gene. (b) Normalized aSMA promoter activity(GFI/RFI) when each of the indicated genes was knocked down. (c) The percentage of ZsG-DRþ cells was measured by flow cytometryand normalized to that of control (scramble/DM). *Significant difference as compared with scramble control (*Po0.05).

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Figure 5. aSMA promoter activity correlates with protein expression. HASMC were transduced with shRNA targeting: (a, e, i) scramble;(b, f, j) RhoA; (c, g, k) Smad4; and (d, g, l) Klf4 lentiviral vector and cultured in DM. (a --d) RFI; (e --h) GFI; (i -- l) Immunostaining for aSMA (green)and counterstaining with the nuclear dye Hoeschst (blue). (a --h): Bar¼ 200 mm; (i -- l): Bar¼ 50mm. The color reproduction of this figure isavailable at the Gene Therapy journal online.


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the wells and allowed to contract. At 48 h, the gels werephotographed and the gel area was measured using Image Jsoftware. As shown in Supplementary Figure S2A, HF-MSCscompacted the hydrogels by B70% in DM but only by B20%in GM and compaction was diminished in the presence ofSB431542. Similar results were observed with BM-MSCs (Supple-mentary Figure S2B). In agreement with aSMA activity, this datasuggested that treatment with DM-induced HF-MSC and BM-MSCdifferentiation into contractile SMCs.

Dox-inducible shRNA expression during MSC differentiationTo study MSC differentiation with regulated shRNA expression,HF-MSC were transduced with a tet-pLKO-puro lentivirus encod-ing for Tet repressor (TetR), selected with puromycin andexpanded. These cells were then transduced with shLVDPencoding for shRNA against Rock1, Smad4 or Klf4 and coaxed todifferentiate toward SMC lineage in the presence of DM. Threedays later, Dox was applied to trigger shRNA expression and aSMAactivity was measured as a function of time following addition ofDox. As expected, knockdown of genes that are important in SMCcontractility decreased promoter activity as compared with controlcells (scramble-shRNA) (Figure 7). Specifically, the level of aSMAactivity was diminished within 12 h after the expression of Rock1shRNA and decreased more slowly upon expression of Smad4shRNA---down to 40% and 25% after 12 and 36 h, respectively.In contrast, Klf4 shRNA had no effect on aSMA promoter activity.These data demonstrated that shLVDP can capture the kinetics ofpromoter activity following Dox-inducible gene knockdown.

DISCUSSIONIn this study, we sought to develop a strategy to monitor cellularactivity in real time and at the same time examine how this

activity is affected by certain genes or pathways. To this end, wegenerated a lentiviral vector with three transcriptional units toenable quantitative monitoring of gene expression with con-comitant gene knockdown. Using this vector, we generated alentiviral array, whereby each virus encoded for a different shRNA,and examined myogenic differentiation by monitoring aSMApromoter activity in response to gene knockdown. Our strategyyielded quantitative information of gene expression in humanprimary cells and stem cells, thereby providing a useful platformfor deciphering gene regulatory networks of complex biologicalprocesses such as stem cell differentiation.

Total fluorescent intensity of each spot on the array depends onthe number of infected cells and the number of transgene copiesper cell. These factors, however, might change significantly owingto variations in the viral titer making it difficult to obtain trulyquantitative data. This may be particularly important for high-throughput experiments that require a large number of lentiviralvectors because it is very difficult to obtain lentivirus preparationsof equal potency. As a result, the difference in signal intensity mayreflect differences in gene transfer efficiency rather than theeffect of gene knockdown. Although, in transfection methodsco-transfection with a second plasmid constitutively encoding fora second reporter gene, for example, Renilla luc has been used forsignal normalization, it is difficult to ensure similar transfectionefficiency for both plasmids. In the present case, this strategywould be further complicated by the requirement for threeplasmid co-transfection. On the other hand, expressing two genesand shRNA from the same vector provides an internal control forsignal normalization enabling acquisition of quantitative data.

Indeed, our results showed that the novel shLVDP vector addressedthis issue as the normalized fluorescence intensity (GFI/RFI) wasindependent of the gene transfer efficiency, for the range ofviral titers tested in this study. Specifically, although increasingthe viral titer increased the efficiency of gene knockdown as wellas GFI and RFI, the normalized aSMA promoter activity (GFI/RFI)remained unaffected. As expected, knockdown of genes thatpromote contractility, for example, RhoA decreased normalizedpromoter activity significantly, whereas knockdown of myogenic

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Figure 6. shLVDP cell array to assess the effects of the indicatedgenes on MSC differentiation into SMC. (a) HF-MSC or (b) BM-MSCswere transduced with shLVDP encoding for the indicated shRNA.Normalized fluorescence intensity (GFI/RFI) indicates the effect ofgene knockdown on aSMA promoter activity. *Significant differenceas compared with scramble control (*Po0.05).

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Figure 7. Kinetics of aSMA promoter activity in response toDox-induced gene knockdown. TetR expressing HF-MSCs weretransduced with shLVDP vector encoding for Klf4, Smad4, Rock1or scramble shRNA and coaxed to differentiate with TGF-b1(10 ngml -- 1). Three days later, Dox (10mgml -- 1) was added in themedium and the normalized aSMA promoter activity (GFI/RFI) wasmeasured as a function of time using fluorescence microscopy.


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inhibitors, fpr example, Klf4 increased promoter activity ascompared with scramble control. However, in all cases thenormalized activity remained independent of the efficiency ofgene knockdown owing to variations in viral titer, suggesting thatshLVDP enabled quantitative assessment of gene knockdown onthe activity of a promoter that can be used to monitor a biologicalprocess, for example, stem cell differentiation.

We chose myogenic differentiation as an example to obtainproof-of-concept that the shLVDP can be used to examine howdifferent pathways may affect MSC differentiation. To this end, weemployed aSMA promoter as an indicator of myogenic pheno-type30 and selected several genes that are known to regulatecontractile function, through the Rho or TGF-b1 pathways. Inagreement with previous studies,31,32 TGF-b1 induced MSCdifferentiation toward the myogenic lineage as shown byenhanced aSMA promoter activity and verified at the proteinlevel by western blot and immunostaining. Blocking TGF-b1receptor activity or knocking down the TGF-b1 mediators Smad2,Smad3 or Smad4 significantly decreased aSMA activity. Inaddition, non-Smad pathways that are induced by TGF-b1 suchas RhoA and its downstream effector Rock1(refs 33,34) are importantfor myogenic differentiation. Similarly, MyoCD and myocardinrelated transcription factors, Mrtf-A and Mrtf-B are necessary fordevelopment of contractile phenotype through their role asSRF co-activators.35 -- 38 As a result, knocking down any one ofthose factors severely impaired aSMA activity. On the other hand,knockdown of the MyoCD antagonist, Klf4 increased aSMAactivity.39 These results are in agreement with the literature andsuggest that the normalized aSMA promoter activity accuratelyreflects the effect of well-known pathways on myogenicdifferentiation.

Surprisingly, we found that Smad7 knockdown hadno significant effect in HASMC and BM-MSC but significantlydecreased aSMA activity in HF-MSC, suggesting that Smad7 maydifferentially regulate myogenesis in different cell types. Asinhibitor of Smad2/3, Smad7 is expected to decrease aSMAactivity. However, Smad7 has also been implicated in myogenic-promoting pathways. For example, Smad3 was shown to interactwith SRF,40 whereas Smad7 activated the small GTPase Cdc4241

and interacted with myocardin in the nucleus-promoting myo-genesis.42 Therefore, our results may suggest that in HASMCs andBM-MSCs, Smad7 may participate in both anti- and pro-myogenicinteractions, so that their overall action balances out the influenceof these pathways. On the other hand, in HF-MSC, SMAD7 mayact predominantly in a pro-myogenic fashion, for example, byinteracting with MyoCD, so that when it is knocked down, aSMAactivity is reduced significantly. Although the mechanism remainselusive, our results suggest that gene knockdown with concomi-tant monitoring of promoter activity may reveal interestingdifferences between stem cells of different tissue origin as theydifferentiate along a common pathway.

The shLVDP can be adapted to a chemically-inducible shRNAexpression system by introducing a second vector (tet-pLKO-puro)encoding for TetR, which binds to the TRE and inhibits shRNAexpression. Upon addition of Dox, transcription is resumed as Doxbinds to TetR releasing it from the promoter.43 This systemenables temporal control of shRNA expression at preferred timesduring the experiment, which is essential for studying genesthat may have adverse effect on cell viability or other biologicalprocesses, for example, cytoskeletal re-organization.44 For example,during myogenic differentiation, we observed a considerablelevel of cytotoxicity in cells actively expressing Rock1 shRNA (datanot shown), making data interpretation difficult. Use of theinducible shLVDP enabled TGF-b1-induced differentiation beforeshRNA expression, thereby allowing us to assess the effect ofRock1 on aSMA promoter activity instead of viability. This systemmay be useful in tissue engineering where temporal control ofshRNA expression may enable engineering of tissues lacking

expression of genes that may be essential during a particularstage of tissue development.

For the inducible shRNA experiments, the cells were transducedwith two lentiviruses: shLVDP and a virus encoding for TetR,raising the concern that any variability in the transductionefficiency of the TetR-encoding lentivirus might affect datanormalization due to potential variability in shRNA induction.To minimize this possibility, HF-MSCs were first transduced withTetR lentivirus and selected with puromycin before they weretransduced with the indicated shLVDP viruses. Most important,any variability in TetR concentration should be eliminated whenthe Dox concentration is high enough to block all the availablerepressor, thereby rendering shRNA induction solely dependenton the H1 promoter of the shLVDP vector. Indeed, preliminaryexperiments employing Smad4 shRNA showed that at 10 mg ml -- 1

of Dox the aSMA promoter response reached steady state,thereby suggesting that at that concentration Dox eliminated anyvariability in shRNA induction between cells (data not shown).For this reason, all experiments in this study were conducted withDox at 10 mg ml -- 1.

The shLVDP vector may be ideal for generating shRNA librariesto discover new genes that are necessary for commitment ofadult, embryonic or induced pluripotent stem cells to a particularlineage. Although several lentiviral constructs such as pLKO.1,pENTR or pLVTHM have been applied for generation of RNAilibraries,8,45,46 the shLVDP provides a means for monitoringdifferentiation in real time, thereby facilitating selection of livecells. Moreover, by enabling quantitative activity measurementsshLVDP libraries may enable development of quantitative criteriato evaluate the relative importance of different genes or pathwaysin stem cell differentiation and select cells that meet these criteria.This system would be useful in evaluating differentiation ofembryonic or induced pluripotent stem cells, which are limited bylow differentiation efficiency resulting in a heterogeneous mixtureof differentiated, partially differentiated and undifferentiated cells.

In conclusion, the shLVDP lentivirus can be used to evaluate theeffects of gene networks on complex biological processes such asstem cell differentiation, in live cells and in real time. Libraries ofshLVDP vectors containing carefully selected transcription factor-binding sites (REs) and shRNAs may provide quantitative data thatmay be amenable to mathematical modeling for reverseengineering the gene networks controlling stem cell fate decisionsor other complex biological processes.

MATERIALS AND METHODSCell culture293T/17 cells (ATCC, Manassas, VA, USA) were cultured in Dulbecco’sModified Eagle Medium (DMEM; GIBCO BRL, Grand Island, NY, USA)supplemented with 10% (v/v) fetal bovine serum (GIBCO) and 1% (v/v)antibiotic-antimycotic (GIBCO). BM-MSCs were purchased from StemCell Technologies (Vancouver, Canada) and HF-MSCs were isolated asdescribed previously.47,48 Both cells were cultured in GM constitutedof DMEM supplemented with 5% (v/v) fetal bovine serum and 1% (v/v)antibiotic-antimycotic and 1.0 ng ml -- 1 basic fibroblast growth factor (BDBiosciences, San Jose, CA, USA). The GM for HASMCs (GIBCO) constituted of231 Medium (GIBCO) that was supplemented with smooth muscle growthsupplement (SMGS; GIBCO).

Lentiviral vector construction strategy and lentiviralvector preparationThe lentiviral vectors used in this study were generated as follows. First, weused the shRNA cassette from pLVTHM (Addgene, Cambridge, MA, USA),which encodes for shRNA under the Dox-regulatable H1 promoterbetween MluI and ClaI restriction sites in the 30LTR of the vector.An EcoRV site was added immediately downstream of MluI for subsequentcloning. Afterwards, the 30LTR of pLVTHM was amplified by PCR with


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primers THM-for: 50-TCTTCCGCGTCTTCGCCTTC-30 and THM-rev: 50-AATTCTAGATGCTGCTAGAG-30 . The PCR product was used to replace the 30LTR inthe pCSCG (Addgene) between the KpnI and PmeI sites. The resultingvector was digested with PpuMI and Bsu36I to remove CMV-GFP andreplace it with the dual promoter cassettes: sMAR8_TKpA_DRE2_CMV_cHS4_Tactb_SPA_EGFP_hPGK, or sMAR8_TKpA_DRE2_hPGK_cHS4_Tactb_SPA_ZsG-DR_MCS from our previously reported LVDP vectors in whichDsRed2 was replaced by DsRed-Express2 (Clontech, Mountain View,CA, USA). These sequences were cloned in trans to the viral LTRs to avoidpremature termination of viral mRNA by the inserted polyAs during virusproduction in packaging cells as reported previously.22 The resultingshRNA encoding LentiViral Dual Promoter vectors were termed shLVDPvectors.

shRNAs were cloned into this vector between the EcoRV and ClaI sitesimmediately downstream of the H1 promoter in the 30LTR. Briefly, the twocomplementary oligos for each shRNA were mixed at 1:1 ratio to a finalconcentration of 50 mM and annealed under the following conditions: 951Cfor 30 s, 721C for 2 min, 37 1C for 2 min and 25 1C for 2 min. All clonedsequences were confirmed by sequencing with ABI PRISM 3130XL GeneticAnalyzer (Applied Biosystems, Foster City, CA, USA). All siRNA sequencesare listed in Table 1.

Finally, the aSMA promoter sequence was cloned into the multiplecloning site (MCS) to drive ZsG-DR expression. To this end, the promoterwas amplified from human genomic DNA by PCR with primers: hACTA2F:50-ACAACAGTTAACCTTCCTGTCAAGGAGGTTAG-30 and hACTA2R: 50-ACAACAACCGGTCGGGTAATTAAAAGAGCCAC-30 . Phusion High-Fidelity DNA Poly-merase (New England Biolabs, Ipswich, MA, USA) was used for PCRaccording to the manufacturer’s protocol and the PCR product was ligatedinto the MCS between the HpaI and AgeI sites.

For lentivirus production, 293T/17 cells were transfected with threeplasmids: (16.8mg each lentiviral vector, 15 mg psPAX2, and 5 mg pMD.G),using the standard calcium phosphate precipitation method. Virus washarvested 24 h post transfection, filtered through 0.45 mm filter (Millipore,Bedford, MA, USA), pelleted by ultracentrifugation (50 000 g at 4 1C for 2 h)and resuspended in fresh medium. The virus titer was determined usingHASMCs cells and was used for subsequent experiments at an MOIB5.

Infection and differentiation protocolHASMCs (12 000 cells per well), BM-MSCs (12 000 cells per well) or HF-MSC(4000 cells per well) were plated in 96-well tissue culture treated plates(NUNC, Rochester, NY, USA). The cells were transduced with differentviruses carrying different shRNAs in the presence of 8 mg ml -- 1 polybreneunder GM. Viruses were removed 2 h after infection and fresh cell culturemedium was added. After 3 days, the cells were induced to differentiate bytreatment with 231 medium supplemented with SMDS (GIBCO), 5% (v/v)fetal bovine serum and 10 ng ml -- 1 TGF-b1 (Biolegends, San Diego, CA,USA), (DM). DM for BM-MSCs or HF-MSCs constituted of DMEM containing5% (v/v) fetal bovine serum, 30mg ml -- 1 heparin (App Pharmaceuticals,Schaumburg, IL, USA) and 10 ng ml -- 1 TGF-b1. After 3 days, the cells were

imaged and 2 days later they were procesed for flow cytometry asdescribed previously.22

Cell transfection and gene knockdown293T cells (8� 105 per well) were seeded in 6-well tissue culture-treatedplates and transfection was done using Lipofectamine 2000 (Invitrogen,Carlsbad, CA, USA), following the manufacturer’s instructions. Transfectionwas performed for 24 h and cells were replenished with fresh medium.The cells were cultured for 3 days and gene-knockdown efficiency wasassessed by qRT-PCR. Briefly, total mRNA was isolated from the cells withRNeasy Mini Kit (Qiagen, Valencia, CA, USA) and cDNA was synthesizedfrom the isolated mRNA using Superscript III cDNA Synthesis Kit(Invitrogen). The cDNA was subjected to real-time PCR using SYBR GreenKit (Bio-Rad, Hercules, CA, USA). Primers for real-time PCR were listedin Table 2.

ImmunostainingTransduced HAMCS cells were cultured in DM for 3 days. Afterwards, thecells were washed three times with phosphate-buffered saline (PBS), andfixed in a mixture of methanol and acetone (75:25, v/v) for 10 min at roomtemperature (RT). After fixation, the cells were washed with PBS twice andpermeabilized with 0.1% (v/v) Triton X 100 in PBS for 10 min at RT.Afterwards, the cells were blocked with 0.01% (v/v) Triton X 100 and 1%(w/v) bovine serum albumin (EMD Chemicals, San Diego, CA, USA) in PBSfor 1 h at RT. Thereafter, the cells were incubated with primary antibody(1:50 dilution, mouse anti-human aSMA; Serotec, Raleigh, NC, USA) inblocking buffer overnight at 4 1C. The cells were washed for three timeswith PBS and Alexa 488-conjugated goat-anti-mouse IgG (20mg ml -- 1,Molecular Probes, Grand Island, NY, USA) in blocking buffer was applied for1 h at RT. The cells were washed three times with PBS and the cell nucleiwere counterstained with Hoechst 33258 (25mg ml -- 1 in TNE buffer:10 mmol l -- 1 Tris, 2 mol l -- 1 NaCl, 1 mmol l -- 1 EDTA, pH 7.4; Molecular Probes)for 10 min at RT. Finally, the cells were washed for three times andfluorescent images were obtained using an inverted fluorescencemicroscope Zeiss Observer.Z1.

Compaction of fibrin hydrogelsCompaction of three-dimensional fibrin hydrogels was described else-where.47 -- 49 Briefly, fibrin gels were prepared with HF-MSCs or BM-MCScells (106 cells ml -- 1) and allowed to polymerize each in a well of a 24-well

Table 1. Cloning of different shRNAs

Accessionnumber

Gene Sense sequence (50 -- 30)

Scramble CAACAAGATGAAGAGCACCAANM_005359 SMAD4_2 GTGGCTGGTCGGAAAGGATTTNM_005359 SMAD4_1 CCCAGGATCAGTAGGTGGAATNM_005901 SMAD2 CTCTTCTGGCTCAGTCTGTTANM_005904 SMAD7 AGTCAAGAGGCTGTGTTGCTGTGAANM_001664 RHOA TGTTTGAGAACTATGTGGCAGATATNM_005406 ROCK1 CCTTCAAGCTCGAATTACATCTTTANM_003131 SRF GCGTGAAGATCAAGATGGAGTTCATNM_153604 MYOCD CCGACTTGGTTAATATGCACATACTNM_014048 MRTFB/MLK2 GACTCAGCCTCAGATAGCAACTGCTNM_00274 PKN1 CACTGTGCTTAAGCTGGATAACACANM_005902 SMAD3 GAGAAACCAGTGACCACCAGATGAANM_004235 KLF4 GATGGAAATTCGCCCGCTCAGATGA

Table 2. Primers for qRT-PCR

Accessionnumber

Gene Sense sequence (50 -- 30)

NM_005359 SMAD4 For: CTTAGTGACCACGCGGTCTTRev: GCTGCTGCATCTGTCGATGA

NM_005901 SMAD2 For:CCGAAATGCCACGGTAGARev: GAATTTTACACACTGTTGCAGG

NM_005904 SMAD7 For: TCTCAGGCATTCCTCGGAAGRev: GCAGAGTCGGCTAAGGTGAT

NM_005902 SMAD3 For: CTTCGCAGAGTGCCTCAGTGRev: CTGGAACAGCGGATGCTTGG

NM_005406 ROCK1 For: GTGCCACAGAGATCACTTAGRev: CCAGATGGTGGATTCTTAGG

NM_001664 RHOA For: TCTTCAGCAAGGACCAGTTCRev: GCCTCAGGCGATCATAATCT

NM_003131 SRF For: AGCCTGAGCGAGATGGAGATRev: TGGAGAGTCTGGCGAGTTGA

NM_153604 MYOCD For: TCCAACGGCTTCTACCACTTRev: AGCGGCTTCTTCTCTGAACA

NM_014048 MRTFB/MLK2 For: CATCGTGGCAGTGTCATCATRev: AGAACTGTGAAGGCGACATC

NM_00274 PKN1 For: CAGCGTGCTAGGCAGATGAARev: CAGAGCCTTGATGGCGAACA

NM_004235 KLF4 For: GTCAGCGTCAGCCTCCTCTTRev: GAGACCGCCTCCTGCTTGAT


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plate to form gel with final concentration of fibrinogen and thrombin at2.5 mg ml -- 1 and 2.5 U ml -- 1, respectively. After 1 h, the gels were releasedfrom the walls and incubated in GM, DM or DM plus SB431542 and 48 hthe gels were photographed at a fixed distance using a gel documentationimaging system (UVP). The area of each gel was measured using Image Jand normalized to the initial area (t¼ 0 h).

Induced regulated shRNA expression protocolHF-MSCs (5� 104 cells per well) were plated in 6-well plate. The cells weretransduced with tet-pLKO-puro (Addgene) lentivirus encoding forthe Tetracycline Repressor (TetR) in the presence of 8 mg ml -- 1 polybrene.Thereafter, transduced cells were selected with 0.5mg ml -- 1 puromycin(Sigma, St Louis, MO, USA) for 5days. At the end of selection, HF-MSC(4� 103 cells per well) were plated in 96-well tissue culture-treated plates.Cells in each well were transduced with virus carrying the indicated shRNAin the presence of 8 mg ml -- 1 polybrene. The virus was removed at 2 h postinfection and replaced with fresh cell culture medium. After 3 days, thecells were induced to differentiate in DM. Three days later, the cells weretreated with Dox (10mg ml -- 1; BD Biosciences, Palo Alto, CA, USA) and cellimages were taken every 12 h.

Image acquisition and analysisThe image-analysis protocol was described previously.2 Briefly, fluores-cence images of the cells were acquired at 3 days after induction of celldifferentiation using a Zeiss Observer.Z1 inverted fluorescence microscope.The integral total fluorescence intensity of cells in each well wasdetermined using Cellprofiler (http://www.cellprofiler.org/).

Statistical analysisStatistical analysis of the data was performed using a two-tailed Student’st-test (a¼ 0.05) in Microsoft Excel (Microsoft, Redwood, CA, USA). Thesample size was n¼ 3 and each experiment was repeated two to threetimes unless indicated otherwise.

CONFLICT OF INTERESTThe authors declare no conflict of interest.

ACKNOWLEDGEMENTSThis work was supported by grants from the National Heart and Lung Institute (R01HL086582), the National Science Foundation (BES-0853993) and the New York StemCell Science Fund (NYSTEM, Contract# C024315) to STA.

REFERENCES1 Zhou LL, Zhao Y, Ringrose A, DeGeer D, Kennah E, Lin AE et al. AHI-1 interacts with

BCR-ABL and modulates BCR-ABL transforming activity and imatinib response ofCML stem/progenitor cells. J Exp Med 2008; 205: 2657 -- 2671.

2 Velkey JM, O’Shea KS. Oct4 RNA interference induces trophectoderm differentia-tion in mouse embryonic stem cells. Genesis 2003; 37: 18 -- 24.

3 Zou GM, Chan RJ, Shelley WC, Yoder MC. Reduction of Shp-2 expression by smallinterfering RNA reduces murine embryonic stem cell-derived in vitro hemato-poietic differentiation. Stem Cells 2006; 24: 587 -- 594.

4 Zou GM, Luo MH, Reed A, Kelley MR, Yoder MC. Ape1 regulates hematopoieticdifferentiation of embryonic stem cells through its redox functional domain.Blood 2007; 109: 1917 -- 1922.

5 Angaji SA, Hedayati SS, Poor RH, Madani S, Poor SS, Panahi S. Application of RNAinterference in treating human diseases. J Genet 2010; 89: 527 -- 537.

6 Paddison PJ, Silva JM, Conklin DS, Schlabach M, Li M, Aruleba S et al. A resourcefor large-scale RNA-interference-based screens in mammals. Nature 2004; 428:427 -- 431.

7 Foley E, O’Farrell PH. Functional dissection of an innate immune response by agenome-wide RNAi screen. PLoS Biol 2004; 2: E203.

8 Moffat J, Grueneberg DA, Yang X, Kim SY, Kloepfer AM, Hinkle G et al. A lentiviralRNAi library for human and mouse genes applied to an arrayed viral high-contentscreen. Cell 2006; 124: 1283 -- 1298.

9 Kittler R, Surendranath V, Heninger AK, Slabicki M, Theis M, Putz G et al.Genome-wide resources of endoribonuclease-prepared short interfering RNAs forspecific loss-of-function studies. Nat Methods 2007; 4: 337 -- 344.

10 Silva JM, Mizuno H, Brady A, Lucito R, Hannon GJ. RNA interference microarrays:high-throughput loss-of-function genetics in mammalian cells. Proc Natl Acad SciUSA 2004; 101: 6548 -- 6552.

11 Brass AL, Dykxhoorn DM, Benita Y, Yan N, Engelman A, Xavier RJ et al.Identification of host proteins required for HIV infection through a functionalgenomic screen. Science 2008; 319: 921 -- 926.

12 Konig R, Zhou Y, Elleder D, Diamond TL, Bonamy GM, Irelan JT et al. Globalanalysis of host-pathogen interactions that regulate early-stage HIV-1 replication.Cell 2008; 135: 49 -- 60.

13 Krishnan MN, Ng A, Sukumaran B, Gilfoy FD, Uchil PD, Sultana H et al.RNA interference screen for human genes associated with West Nile virusinfection. Nature 2008; 455: 242 -- 245.

14 Yeung ML, Houzet L, Yedavalli VS, Jeang KT. A genome-wide short hairpin RNAscreening of jurkat T-cells for human proteins contributing to productive HIV-1replication. J Biol Chem 2009; 284: 19463 -- 19473.

15 Echard A, Hickson GR, Foley E, O0Farrell PH. Terminal cytokinesis eventsuncovered after an RNAi screen. Curr Biol 2004; 14: 1685 -- 1693.

16 Mukherji M, Bell R, Supekova L, Wang Y, Orth AP, Batalov S et al. Genome-widefunctional analysis of human cell-cycle regulators. Proc Natl Acad Sci USA 2006;103: 14819 -- 14824.

17 Collinet C, Stoter M, Bradshaw CR, Samusik N, Rink JC, Kenski D et al.Systems survey of endocytosis by multiparametric image analysis. Nature 2010;464: 243 -- 249.

18 Zhang JZ, Gao W, Yang HB, Zhang B, Zhu ZY, Xue YF. Screening for genesessential for mouse embryonic stem cell self-renewal using a subtractive RNAinterference library. Stem Cells 2006; 24: 2661 -- 2668.

19 Ngo VN, Davis RE, Lamy L, Yu X, Zhao H, Lenz G et al. A loss-of-function RNAinterference screen for molecular targets in cancer. Nature 2006; 441: 106 -- 110.

20 Sheng Z, Li L, Zhu LJ, Smith TW, Demers A, Ross AH et al. A genome-wide RNAinterference screen reveals an essential CREB3L2-ATF5-MCL1 survival pathway inmalignant glioma with therapeutic implications. Nat Med 2010; 16: 671 -- 677.

21 Tian J, Alimperti S, Lei P, Andreadis ST. Lentiviral microarrays for real-timemonitoring of gene expression dynamics. Lab Chip 2010; 10: 1967 -- 1975.

22 Tian J, Andreadis ST. Independent and high-level dual-gene expression in adultstem-progenitor cells from a single lentiviral vector. Gene Therapy 2009; 16:874 -- 884.

23 Hautmann MB, Madsen CS, Owens GK. A transforming growth factor beta(TGFbeta) control element drives TGFbeta-induced stimulation of smooth musclealpha-actin gene expression in concert with two CArG elements. J Biol Chem 1997;272: 10948 -- 10956.

24 Kinner B, Zaleskas JM, Spector M. Regulation of smooth muscle actin expressionand contraction in adult human mesenchymal stem cells. Exp Cell Res 2002; 278:72 -- 83.

25 Wang D, Park JS, Chu JS, Krakowski A, Luo K, Chen DJ et al. Proteomic profiling ofbone marrow mesenchymal stem cells upon transforming growth factor beta1stimulation. J Biol Chem 2004; 279: 43725 -- 43734.

26 Mack CP, Somlyo AV, Hautmann M, Somlyo AP, Owens GK. Smooth muscledifferentiation marker gene expression is regulated by RhoA-mediated actinpolymerization. J Biol Chem 2001; 276: 341 -- 347.

27 Szulc J, Wiznerowicz M, Sauvain MO, Trono D, Aebischer P. A versatile tool forconditional gene expression and knockdown. Nat Methods 2006; 3: 109 -- 116.

28 Wiznerowicz M, Trono D. Conditional suppression of cellular genes: lentivirusvector-mediated drug-inducible RNA interference. J Virol 2003; 77: 8957 -- 8961.

29 ter Brake O, t Hooft K, Liu YP, Centlivre M, von Eije KJ, Berkhout B. Lentiviral vectordesign for multiple shRNA expression and durable HIV-1 inhibition. Mol Ther 2008;16: 557 -- 564.

30 Owens GK, Kumar MS, Wamhoff BR. Molecular regulation of vascular smoothmuscle cell differentiation in development and disease. Physiol Rev 2004; 84:767 -- 801.

31 Massague J. Receptors for the TGF-beta family. Cell 1992; 69: 1067 -- 1070.32 Ng F, Boucher S, Koh S, Sastry KS, Chase L, Lakshmipathy U et al. PDGF, TGF-beta,

and FGF signaling is important for differentiation and growth of mesenchymalstem cells (MSCs): transcriptional profiling can identify markers and signalingpathways important in differentiation of MSCs into adipogenic, chondrogenic,and osteogenic lineages. Blood 2008; 112: 295 -- 307.

33 Mack CP, Somlyo AV, Hautmann M, Somlyo AP, Owens GK. Smooth muscledifferentiation marker gene expression is regulated by RhoA-mediated actinpolymerization. J Biol Chem 2001; 276: 341 -- 347.

34 Zhang YE. Non-Smad pathways in TGF-beta signaling. Cell Res 2009; 19: 128 -- 139.35 Larsson E, Zhou X, Akyurek LM. RhoA-dependent vascular smooth muscle

cell-specific transcription: adding diaphanous formins to the puzzle. ArteriosclerThromb Vasc Biol 2007; 27: 448 -- 449.

36 Wang D, Chang PS, Wang Z, Sutherland L, Richardson JA, Small E et al. Activationof cardiac gene expression by myocardin, a transcriptional cofactor for serumresponse factor. Cell 2001; 105: 851 -- 862.


1131


http://www.cellprofiler.org/

37 Wang DZ, Li S, Hockemeyer D, Sutherland L, Wang Z, Schratt G et al. Potentiationof serum response factor activity by a family of myocardin-related transcriptionfactors. Proc Natl Acad Sci USA 2002; 99: 14855 -- 14860.

38 Wang DZ, Olson EN. Control of smooth muscle development by the myocardinfamily of transcriptional coactivators. Curr Opin Genet Dev 2004; 14: 558 -- 566.

39 Liu Y, Sinha S, McDonald OG, Shang Y, Hoofnagle MH, Owens GK. Kruppel-likefactor 4 abrogates myocardin-induced activation of smooth muscle geneexpression. J Biol Chem 2005; 280: 9719 -- 9727.

40 Qiu P, Feng XH, Li L. Interaction of Smad3 and SRF-associated complex mediatesTGF-beta1 signals to regulate SM22 transcription during myofibroblastdifferentiation. J Mol Cell Cardiol 2003; 35: 1407 -- 1420.

41 Edlund S, Landstrom M, Heldin CH, Aspenstrom P. Smad7 is required for TGF-beta-induced activation of the small GTPase Cdc42. J cell sci 2004; 117: 1835 -- 1847.

42 Miyake T, Alli NS, McDermott JC. Nuclear function of Smad7 promotesmyogenesis. Mol cell biol 2010; 30: 722 -- 735.

43 Wiederschain D, Wee S, Chen L, Loo A, Yang G, Huang A et al. Single-vector induciblelentiviral RNAi system for oncology target validation. Cell Cycle 2009; 8: 498 -- 504.

44 Dickins RA, Hemann MT, Zilfou JT, Simpson DR, Ibarra I, Hannon GJ et al. Probingtumor phenotypes using stable and regulated synthetic microRNA precursors. NatGenet 2005; 37: 1289 -- 1295.

45 Beronja S, Livshits G, Williams S, Fuchs E. Rapid functional dissection of geneticnetworks via tissue-specific transduction and RNAi in mouse embryos. Nat Med2010; 16: 821 -- 827.

46 Pongratz C, Yazdanpanah B, Kashkar H, Lehmann MJ, Krausslich HG, Kronke M.Selection of potent non-toxic inhibitory sequences from a randomizedHIV-1 specific lentiviral short hairpin RNA library. PloS one 2010; 5: e13172.

47 Liu JY, Peng HF, Andreadis ST. Contractile smooth muscle cells derived fromhair-follicle stem cells. Cardiovasc Res 2008; 79: 24 -- 33.

48 Liu JY, Peng HF, Gopinath S, Tian J, Andreadis ST. Derivation of functionalsmooth muscle cells from multipotent human hair follicle mesenchymal stemcells. Tissue Eng Part A 2010; 16: 2553 -- 2564.

49 Han J, Liu JY, Swartz DD, Andreadis ST. Molecular and functional effects oforganismal ageing on smooth muscle cells derived from bone marrowmesenchymal stem cells. Cardiovasc Res 2010; 87: 147 -- 155.

Supplementary Information accompanies the paper on Gene Therapy website (http://www.nature.com/gt)


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http://www.nature.com/gt

a novel lentivirus for quantitative assessment of gene knockdown in stem cell differentiation

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