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Proc Natl Acad Sci U S A. Nov 25, 2008; 105(47): 18372–18377.
Published online Nov 14, 2008. doi:  10.1073/pnas.0809807105
PMCID: PMC2587615
Cell Biology

Human multipotent stromal cells from bone marrow and microRNA: Regulation of differentiation and leukemia inhibitory factor expression

Abstract

We observed that microRNAs (miRNAs) that regulate differentiation in a variety of simpler systems also regulate differentiation of human multipotent stromal cells (hMSCs) from bone marrow. Differentiation of hMSCs into osteoblasts and adipocytes was inhibited by using lentiviruses expressing shRNAs to decrease expression of Dicer and Drosha, two enzymes that process early transcripts to miRNA. Expression analysis of miRNAs during hMSC differentiation identified 19 miRNAs that were up-regulated during osteogenic differentiation and 20 during adipogenic differentiation, 11 of which were commonly up-regulated in both osteogenic and adipogenic differentiation. In silico models predicted that five of the up-regulated miRNAs targeted leukemia inhibitory factor (LIF) expression. The prediction was confirmed for two of the miRNAs, hsa-mir 199a and hsa-mir346, in that over-expression of the miRNAs decreased LIF secretion by hMSCs. The results demonstrate that differentiation of hMSCs is regulated by miRNAs and that several of these miRNAs target LIF.

Keywords: hsa-mir199a, hsa-mir346, stem cells, plasticity

Human multipotent stromal cells (hMSCs) from bone marrow, also known as mesenchymal stem cells, are progenitor cells capable of differentiating into a variety of mature tissues (1). hMSCs can be isolated easily as single cell clones and differentiated into osteoblasts and adipocytes as well as other cellular phenotypes in culture. The cells hold great promise for therapy but the molecular mechanisms that govern the plasticity and differentiation remain unclear. Recently, the existence and function of a class of small, noncoding RNA molecules known as microRNAs (miRNAs) have gained attention as regulatory molecules. These genomically encoded RNAs undergo several modifications before being converted into mature 21–23 base pair transcripts capable of gene silencing. Biogenesis of functional miRNAs involves several enzymes, including Dicer and Drosha. Previous studies demonstrated that decreased expression of Dicer in Drosophila, zebrafish, and mice restricts the differentiation potential of stem cells (25). Studies have also demonstrated that specific miRNAs regulate gene expression during germ line development and cellular differentiation (26), including playing key roles in hematopoiesis as well as myogenic and neurogenic function (711). However, much of the work on miRNAs has focused on simpler organisms with very little data on human miRNAs. In this study we investigated the role of miRNAs in hMSCs, focusing first on the need for miRNA processing enzymes in hMSC differentiation. Then, we identified the key miRNAs that may regulate differentiation of hMSCs. Finally, we demonstrated that two of the miRNAs target leukemia inhibitory factor (LIF), the expression of which decreases as hMSCs differentiate.

Results

Generation of a Stable Knockdown of Dicer and Drosha in hMSCs.

As a first step toward identifying the role of miRNAs in the differentiation of hMSCs, we analyzed the effect of decreasing the expression of key proteins including the cytoplasmic enzyme Dicer and the nuclear enzyme Drosha. We used a tetracycline inducible vector that contained a short hairpin RNA (shRNA) targeting either Dicer or Drosha to prepare stable cell lines of hMSCs that could be induced to reduce the expression of either enzyme [see supporting information (SI) Fig. S1 a and b]. Transduced cells showed a specific knockdown of Dicer or Drosha at the protein level when induced with doxycycline as compared with transduced hMSCs cultured without doxycycline in the media. Donor matched control cells showed no variation in the expression of these enzymes when cultured in the presence or absence of doxycycline (Fig. S1c).

Dicer or Drosha Knockdown Inhibits Osteogenic Differentiation of hMSCs.

We then tested the effect of impaired miRNA processing and global miRNA knockdown on the ability of hMSCs to differentiate into an osteogenic lineage. The hMSCs transduced with an shRNA targeting Dicer or Drosha were grown to subconfluent levels and then placed in osteogenic media in the presence or absence of doxycycline. Passage matched hMSCs from the same donor, and from the same preparation as the transduced cells, were grown in the same differentiation conditions in the presence or absence of doxycycline as controls. After 28 days, gross and microscopic visualization of transduced hMSCs grown in the presence of doxycycline showed undetectable mineralization, as demonstrated by staining monolayers with alizarin red stain (ARS) (Fig. 1A and Fig. S2a). Both transduced hMSCs grown in the absence of doxycycline as well as untransduced cells grown in the presence and absence of doxycycline showed robust mineralization (Fig. 1A and Fig. S2a). Quantitation of mineralization was performed by measuring absorbance of ARS extracted from stained cultures. Osteogenic monolayers from transduced hMSCs grown in the presence of doxycycline showed a >95% reduction (P < 10−6) in ARS incorporation compared with transduced cells grown in the absence of doxycycline and control cells grown in the presence and absence of doxycycline (Fig. 1B and Fig. S2b). To further evaluate the effect of Dicer knockdown on osteogenic differentiation of hMSCs, we assayed alkaline phosphatase (ALP) activity in monolayers grown in osteogenic conditions for 15 days. Transduced hMSCs grown in the presence of doxycycline showed a >95% reduction (P < 10−6) of ALP activity as compared with transduced cells grown in the absence of doxycycline and control cells grown in the presence and absence of doxycycline (Fig. 1C). The level of ALP activity in transduced hMSCs grown in the presence of doxycycline was slightly lower than the level of activity of hMSCs grown in nondifferentiating culture conditions for 15 days.

Fig. 1.
Dicer knockdown inhibits osteogenic differentiation of hMSCs. hMSCs transduced (T) with a tetracycline inducible shRNA targeting Dicer, as well as control cells (C), were incubated in osteogenic differentiation media in the presence (D) or absence of ...

We then assessed the level of osteopontin and osteocalcin, two late markers of osteogenesis, in cells that had been differentiated for 21 days as compared with undifferentiated cells. hMSCs transduced with an shRNA targeting Dicer differentiated in the presence of doxycycline had a small increase in osteopontin and osteocalcin (3.95- and 2.8-fold, respectively), when compared with undifferentiated cells (Fig. 1D). In contrast, transduced hMSCs grown in the absence of doxycycline had significantly greater changes in mRNA levels (P < 0.0001) with a 34.3-fold increase in osteopontin and 14.8-fold increase in osteocalcin, as compared with undifferentiated cells.

Dicer or Drosha Knockdown Inhibits Adipogenic Differentiation of hMSCs.

A similar approach was used to assess the ability of hMSCs to differentiate into an adipogenic lineage after inhibition of Dicer or Drosha. Transduced hMSCs were grown to subconfluent levels and then placed in adipogenic media in the presence or absence of doxycycline along with passage matched controls. After 21 days, bright field microscopic inspection of oil red-o (ORO) stained cultures demonstrated that hMSCs transduced with either an shRNA targeting Dicer or Drosha grown in the presence or absence of doxycycline and control hMSCs grown in the presence and absence of doxycycline were able to form intracellular lipid droplets (Fig. 2A and Fig. S3a). The intracellular lipid accumulation in transduced hMSCs grown in the presence of doxycycline was sparse, often containing small droplets that did not coalesce, a characteristic of early lipid formation. Transduced hMSCs grown in the absence of doxycycline and control hMSCs grown in the presence and absence of doxycycline showed accumulation of larger lipid droplets, characteristic of later stage adipogenesis. Quantitation of lipid formation in the Dicer knockdown model, by measuring absorbance of ORO extracted from stained cultures, showed a 70% reduction (P < 0.001) in transduced hMSCs grown in the presence of doxycycline when compared with transduced hMSCs grown in the absence of doxycycline and control cells grown in the presence and absence of doxycycline (Fig. 2B). In the Drosha knockdown model, a >95% reduction of lipid droplet formation (P < 10−6) was observed (Fig. S3b). To further evaluate the effect of Dicer knockdown on adipogenic differentiation of hMSCs, we evaluated hormone sensitive lipase (HSL) activity in monolayers grown in adipogenic conditions for 21 days using a technique first described by Wieland (12). Both transduced hMSCs grown in the absence of doxycycline and control hMSCs grown in the presence of doxycycline showed lipolytic activity that increased when increasing concentrations of isoproterenol were added to the cellular monolayers (Fig. 2C). In contrast, the transduced hMSCs with an shRNA targeting Dicer grown in the presence of doxycycline showed significantly less baseline lipolytic activity (P = 0.03). Furthermore, lipolytic activity was unaffected by increased isoproterenol concentration, with significantly lower lipolytic activity in Dicer knockdown hMSCs after stimulation with 5 nanomoles of isoproterenol (P < 0.0001) (Fig. 2C). Thus, despite the ability of hMSCs to form some lipid droplets with impaired miRNA processing, the hormone sensitive lipolytic functional activity characteristic of adipocytes remained very low if not absent, suggesting adipogenic differentiation was either disrupted or severely retarded.

Fig. 2.
Dicer knockdown inhibits adipogenic differentiation of hMSCs. hMSCs transduced (T) with a tetracycline inducible shRNA targeting Dicer, as well as control cells, were incubated in adipogenic differentiation media in the presence (D) or absence of doxycycline. ...

We then assessed the level of adiponectin and FABP4 transcripts, two late markers of adipogenesis, in cells that had been differentiated for 21 days as compared with undifferentiated cells. hMSCs transduced with an shRNA targeting Dicer differentiated in the presence of doxycycline had a 5.6-fold increase in adiponectin with a 50% decrease in FABP4 when compared with undifferentiated cells (Fig. 2D). In contrast, transduced hMSCs differentiated in the absence of doxycycline had a significantly larger increase in both mRNA levels (P < 0.0001) with a 250-fold increase in adiponectin and 6.4-fold increase in FABP4 compared with undifferentiated cells.

miRNA Expression During Differentiation of hMSCs.

Next, we performed miRNA expression analysis of hMSCs from three preparations from each of three donors as they differentiated into both adipogenic and osteogenic lineages. We used a new bead based flowcytometric method to analyze 435 known miRNAs, demonstrated by Lu and colleagues to be specific to a 1 nucleotide mutation (13). Of the 435 miRNAs tested, 58 were expressed in all three donor cell populations in undifferentiated hMSCs (Table S1). Stringent expression analysis to exclude changes reflecting proliferation rate and cell cycle status showed 19 miRNAs were up-regulated during osteogenic differentiation (Fig. 3A Right) and 20 were up-regulated during adipogenic differentiation (Fig. 3A Left). Of the miRNAs that were up-regulated during differentiation, 11 were up-regulated under both conditions (Fig. S4). Only one miRNA decreased in expression during adipogenic differentiation.

Fig. 3.
miRNA expression during hMSC differentiation. (A) Heat map of miRNA expression during differentiation toward osteogenic and adipogenic lineages (Red, high expression; Blue, low expression). Three different donors were cultured in specific differentiation ...

We then searched for mRNA targets that were known to be important in regulating cellular differentiation and that may be regulated by the candidate miRNAs. Using three in silico prediction algorithms (1416) designed to identify miRNAs that target the 3′ UTR of mature transcripts, we discovered that five of the miRNAs that were up-regulated in hMSCs during differentiation were predicted to target LIF: hsa-mir 26a, hsa-mir 125a, hsa-mir 125b, hsa-mir 199a, and hsa-mir 346 (Fig. 3B). When hMSCs were cultured in media was supplemented with exogenous LIF, we observed a 46% reduction (P < 0.05) in lipid accumulation on day 21 of adipogenic differentiation, compared with adipogenic cultures without supplemental LIF (Fig. S5 a and b). We also observed a 43% reduction (P < 0.05) of ALP activity on Day 7 of osteogenic cultures containing supplemental LIF, compared cultures without supplemental LIF (Fig. S5c).

To confirm that the five candidate miRNAs that targeted LIF were up-regulated during differentiation of hMSCs, we performed real time RT-PCR by using a stem loop priming method (17). All five miRNAs were up-regulated at day 15 of differentiation in both osteogenic and adipogenic conditions when compared with a subconfluent undifferentiated sample (P < 0.001) (Fig. 3C). In addition a sixth miRNA, hsa-mir23a, that has been demonstrated to regulate stromal derived factor 1 (SDF1) (18) was up-regulated during both osteogenic and adipogenic conditions (Fig. 3C). Furthermore, cells deficient in Dicer also showed a significantly lower level of all 6 miRNAs when compared with the undifferentiated sample of hMSCs (P < 0.001), indicating that the cells deficient in Dicer also showed a decrease in miRNA processing (Fig. 3C).

miRNAs Target LIF During Differentiation of hMSCs.

Next, we examined functional modulation of LIF in differentiating hMSCs. First, we analyzed the level of LIF secreted in culture media as hMSCs differentiated, which correlates with increase in miRNA expression. As hMSCs differentiated, the level of secreted LIF in the culture media decreased (Fig. 4A). Furthermore, hMSCs transduced with an shRNA targeting Dicer in the presence of doxycycline showed significantly higher level of LIF expression during differentiation than transduced cells incubated in the absence of doxycycline. These data demonstrated that the LIF decrease during differentiation is coincident with the increase in miRNAs predicted to target LIF; and that when the miRNA processing mechanism is disrupted these changes in LIF secretion are attenuated.

Fig. 4.
miRNA regulation of LIF in hMSCs. (A) ELISAs of LIF in hMSC conditioned media during osteogenic and adipogenic differentiation. hMSCs were incubated for 48 h to condition media during the specified time points. The average of four culture replicates is ...

To prove a direct role of specific miRNAs in the modulation of LIF expression, we transfected hMSCs with synthetic forms of all five miRNAs predicted to target LIF, hsa-mir199a hsa-mir346, hsa-mir26a, hsa-mir125a, and hsa-mir125b. Following transfection, the secretion of LIF was significantly decreased in cells transfected with miRNA hsa-mir199a and hsa-mir346 (Fig. 4B) (p <. 0001). Cells transfected with hsa-mir26a, hsa-mir125a, and hsa-mir125b showed no significant alteration in LIF secretion. When hsa-mir199a and hsa-mir346 were cotransfected at half the concentration into the same hMSC population, a greater reduction in LIF secretion occurred than with either synthetic miRNA alone (P < 0.001) (Fig. 4B). The reduction of LIF secretion was confirmed by performing immunofluorescent analysis of cellular LIF levels in cells transfected with both hsa-mir199a and hsa-mir346 (Fig. 4C). Next, we tested the role of hsa-mir199a and hsa-mir346 in targeting the 3′UTR of LIF using a luciferase reporter gene with LIF 3′UTR sequences. Cotransfection of hsa-mir199a or hsa-mir346 and a luciferase reporter construct containing the 3′ UTR of LIF, resulted in a >80% reduction of luciferase activity (P < 0.001), compared with control cells (Fig. 4D).

Discussion

To our knowledge this is the first demonstration that the miRNA processing machinery is essential for differentiation of hMSCs into adult cellular phenotypes, specifically osteogenic and adipogenic lineages. Partial functional disruption of the miRNA processing machinery was sufficient to greatly reduce the differentiation capability of hMSCs suggesting that precise levels of mature miRNAs are critical for proper stem cell differentiation. This finding is consistent with several other studies that demonstrated poor differentiation of hematopoietic cells into mature tissue when they lack major enzymes involved in miRNA processing (19, 20). Furthermore, studies in germ line stem cells of both Drosophila and zebrafish indicated that miRNAs are not essential for poorly differentiated tissues to survive but are required for differentiation and tissue specific gene expression later in life (24, 10). Thus, it is not surprising that we identified a number of miRNAs that are up-regulated as hMSCs differentiated, whereas only one miRNA showed a decrease in expression. The miRNAs identified as increasing during differentiation are consistent with other studies that used clonal analysis to examine miRNA expression during adipogenic differentiation from preadipocytes (21), particularly, miRNA hsa-mir143 that regulates Erk5 was up-regulated specifically in adipogenic differentiation (Fig. 3A). Other studies have identified miRNAs that are important for hematopoiesis (7, 9), as well as muscle differentiation (8, 11). These specific miRNAs were not identified as being differentially expressed during hMSC differentiation. Several members of the miRNA let7 family were significantly up-regulated during both adipogenic and osteogenic differentiation. The let7 family has been shown to regulate differentiation in other models, including breast cancer and neurogenesis (22, 23), indicating a potential role of let7 family role in hMSC differentiation.

LIF has been associated with uncommitted state of both embryonic and adult stem cells. Removal of LIF from the cultures was shown to induce differentiation of embryonic stem cells (24, 25). It is also known to be essential for hematopoietic stem cell maintenance and growth but does not support their differentiation (2628). Several studies demonstrated that during differentiation and passage of hMSCs, LIF decreases as plasticity decreases (2931). A recent study from our center also demonstrates that LIF is a marker for hMSC multipotentiality (32). Here, we identified two miRNAs, hsa-mir 199 and hsa-mir346, which regulate the cytokine LIF during hMSC differentiation, and demonstrated that these miRNAs can act synergistically to regulate gene expression.

Future studies of miRNA function during hMSC differentiation will focus on specific pathways regulated by miRNAs. It is important to note that Dicer or Drosha disruption causes global changes in miRNAs and therefore effects numerous pathways within the cell. Considering the number of miRNAs expressed in hMSCs and the number of potential targets each miRNA can regulate it is unlikely that one or even a few miRNAs are the essential molecules regulating hMSC differentiation. The data presented here corroborates most of the published studies using Dicer knockout models (3, 4), which indicate that the enzyme is critical during early embryogenesis and its function may be limited to specific differentiation pathways in adult tissue (19). As such, a focus on specific pathways will more clearly elucidate the function of miRNAs in hMSC differentiation and cellular function.

Experimental Procedures

Isolation and Culture of hMSCs.

hMSCs from bone marrow aspirates were obtained from the National Institutes of Health-funded National Center for Research Resources (NCRR) Tulane Center for the Preparation and Distribution of Adult Stem Cells (http://www.som.tulane.edu/gene_therapy/distribute.shtml). The cells were obtained as frozen vials of passage one cells that were shown to be multipotent for differentiation. The cells were negative for hematopoietic markers (CD34, CD36, CD117, and CD45), and positive for CD29 (95%), CD44 (>93%), CD49c (99%), CD49f (>70%), CD59(>99%), CD90 (>99%), CD105 (>99%), and CD166 (>99%). The hMSCs (≈1 million per vial) were thawed, plated in a 15-cm diameter dish and incubated overnight to recover adherent, viable cells. The cells were then lifted with trypsin/EDTA (0.25% Trypsin/1 mM EDTA; GIBCO/BRL) and replated at 500 cells per cm2. All cultures were incubated in complete culture medium (CCM): α-MEM (GIBCO/BRL) containing 17% (vol/vol) FBS (lot-selected for rapid growth of MSCs; Atlanta Biologicals) and 2 or 4 mM l-glutamine (GIBCO/BRL) at 37°C with 5% humidified CO2, unless otherwise noted.

Mineralization Quantification.

Mineralization was assessed by incorporation of ARS (Sigma-Aldrich) into monolayers of differentiated hMSCs. ARS incorporation was quantified with cetylpyridium chloride (CPC) (Sigma-Aldrich) extraction as previously described (33). Briefly, 10% (wt/vol) CPC solution was prepared in Na2PO4 (pH 7.0). Stained monolayers were incubated in 1 ml of CPC solution for 45 min. Aliquots of the extracted dye were then transferred to 96-well plates. The absorbance at 550 nm of each aliquot was then measured by using a 96-well plate reader (Fluostar Optima, BMG Labtech).

ALP Activity.

ALP activity of monolayers was determined by measuring conversion of p-nitro-phenol phosphate (PNPP) to p-nitro-phenol.

Intracytoplasmic Lipid Quantification.

Lipid formation was assessed by incorporation of ORO (Sigma-Aldrich) into monolayers of differentiated hMSCs. Quantitation of ORO incorporation was performed as previously described (34).

Hormone Sensitive Lipolysis Quantification.

hMSC monolayers were washed three times with PBS and then incubated for 3 h in α-MEM (GIBCO/BCR) with 4% fatty acid free BSA (Sigma-Aldrich) to remove any residual fatty acids. Monolayers were then incubated in fresh α-MEM with 4% fatty acid free BSA, along with varying concentrations of DL-isoproterenol dihydrochloride, (Sigma-Aldrich) for 2 h. The amount of NADH released was measured at OD350 (SmartSpec 3000, Bio-Rad). A standard curve was prepared with pure glycerol (Sigma-Aldrich).

Generation of Stable Cell Lines with Dicer and Drosha Knockdown.

The lentivirus construct containing a tetracycline-response element, with an shRNA targeting Dicer, was prepared by using the pPrime vector (35), a gift of the Stephen J. Elledge laboratory (Cambridge, MA). The lentiviral construct targeting Drosha (36) containing the tetracycline activator and the tetracycline-response element with an shRNA targeting Drosha, was a gift of the John Rossi laboratory (Duarte, CA). With both vectors, virus was generated by the lentiviral Vector Core of the Louisiana Cancer Research Consortium (37).

miRNA Expression Profiles and Data Analysis.

miRNA expression profiling was preformed as described by Lu et al. (13). The data from differentiation samples (adipogenesis and osteogenesis) was divided to early (days 0, 1, and 3) and late (days 7 and 14) differentiation. The data were filtered separately for adipogenesis and osteogenesis samples by using ANOVA with differentiation time (early or late) as a factor with P value <0.01. This resulted in 21 miRNAs for adipogenesis and 19 for osteogenesis that changed significantly between early and late time points. These miRNAs were used for hierarchical clustering in dChip.

Real Time RT-PCR Analysis.

Quantitative RT-PCR for miRNA was performed according to the manufacturer's protocol using Taqman miRNA assays for hsa-mir 26a, hsa-mir 125a, hsa-mir 125b, hsa-mir 199a, and hsa-mir 346 (Applied Biosystems). The process is described in detail by Chen and colleagues (17). Similarly, quantitative RT-PCR for mRNA was performed according to the manufacturer's protocol using Taqman gene expression assays (Applied Biosystems). Fold change was calculated by using the ΔΔCt method of relative quantification.

Synthetic miRNA Transfection.

A mixture of 50 μl of siPORT NeoFX transfection agent (Ambion), 400 μl of Opti-MEM (GIBCO/BCR) and the appropriate synthetic miRNAs (Ambion) or scrambled siRNA (Ambion) was prepared according to Ambion's siRNA transfection protocol. Passage two hMSCs were harvested with trypsin/EDTA, washed with PBS and counted using a hemocytometer. The transfection mixture was mixed with 105 cells, then one quarter of the entire cell and transfection mixture was transferred to a 12-well plate or a 4 cm2 chamber slide (Nunc) containing 500 μl of CCM. After 24 h the CCM/transfection media was replaced with CCM and appropriate experiments were completed.

Luciferase Assay.

The pMIR REPORT vector (Ambion) containing the LIF 3′UTR was cotransfected into A549 cells with premir synthetic miRNA constructs or a scrambled siRNA, using Lipofectamine 2000 (Invitrogen) according to the manufacturer's protocol for cotransfection of DNA and RNAi. Cells were harvested 48 h after transfection, and the luciferase activity in the cellular lysate was assayed by using the Luciferase Assay System (Promega) according to the manufacturer's protocol. Light intensity for each sample was measured by using a 96-well plate reader (Fluostar Optima, BMG Labtech).

ELISA.

ELISAs were performed by using a 96-well LIF colorimetric based assay (R&D Systems) according to the manufacturer's protocol.

Western Blots and Immunocytochemistry.

The following primary antibody dilutions were used for Western blotting: mouse monoclonal anti-Dicer antibody 1:200 (clone 13D6; Abcam); goat polyclonal anti-Drosha 1:200 (Santa Cruz Biotechnology); HRP conjugated rabbit polyclonal anti-GAPDH 1:200 (Abcam). Immunocytochemistry was performed by using a rabbit anti-LIF polyclonal antibody at a dilution of 1:200 (Santa Cruz Biotechnology) followed by an Alexa Fluor 488 (green)-conjugated anti-rabbit secondary antibody (Invitrogen), at a concentration of 1:1,000. Alexa Fluor 594 (red) conjugated phalloidin (Invitrogen) was used to stain actin filaments at a concentration of 1:1000.

Statistical Analysis.

All statistical analysis was performed using Student's t test using a two-sample method assuming unequal variances with P values generated for two tails. Sample sizes are noted in figure legends and specific P values are included in the text. A table of values used for key statistical analysis is included (Table S2).

For additional information, please see SI Materials and Methods.

Supplementary Material

Supporting Information:

Acknowledgments.

Human MSCs used in this work were provided by the Tulane Center for Gene Therapy through Grant P40RR017447 from National Center for Research Resources of the National Institutes of Health. The tetracycline inducible shRNA construct in pPrime vector (Stegmeier F) was a kind gift from the Stephen J. Elledge laboratory in Cambridge, MA. The tetracycline inducible shRNA construct targeting Drosha (Aagaard L) was a kind gift from the John Rossi laboratory in Duarte, CA. This work was supported in part by National Institutes of Health Grants AR 47796 and AR 48323, the Oberkotter Foundation, HCA the Healthcare Company, and the Louisiana Gene Therapy Research Consortium (D.J.P.).

Footnotes

The authors declare no conflict of interest.

This article contains supporting information online at www.pnas.org/cgi/content/full/0809807105/DCSupplemental.

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