TY - JOUR
AU - Kassem, Moustapha
AB - Abstract Development of novel approaches to enhance bone regeneration is needed for efficient treatment of bone defects. Protein kinases play a key role in regulation of intracellular signal transduction pathways, and pharmacological targeting of protein kinases has led to development of novel treatments for several malignant and nonmalignant conditions. We screened a library of kinase inhibitors to identify small molecules that enhance bone formation by human skeletal (stromal or mesenchymal) stem cells (hMSC). We identified H-8 (known to inhibit protein kinases A, C, and G) as a potent enhancer of ex vivo osteoblast (OB) differentiation of hMSC, in a stage- and cell type-specific manner, without affecting adipogenesis or osteoclastogenesis. Furthermore, we showed that systemic administration of H-8 enhances in vivo bone formation by hMSC, using a preclinical ectopic bone formation model in mice. Using functional screening of known H-8 targets, we demonstrated that inhibition of protein kinase G1 (PRKG1) and consequent activation of RhoA-Akt signaling is the main mechanism through which H-8 enhances osteogenesis. Our studies revealed PRKG1 as a novel negative regulator of OB differentiation and suggest that pharmacological inhibition of PRKG1 in hMSC implanted at the site of bone defect can enhance bone regeneration. Stem Cells 2015;33:2219–2231 Human skeletal (mesenchymal) stem cells, Osteoblast differentiation, Bone formation, Kinase inhibitor, Akt signaling Introduction Developing novel approaches for enhancing bone tissue regeneration is required for optimal treatment of a number of common clinical conditions such as repair of critical size bone defects following trauma, infection, or tumor resection [1]. Stem cell-based therapy is a promising new approach where local implantation of skeletal (also known as marrow stromal or mesenchymal) stem cells (MSC) together with functionalized scaffolds containing agents enhancing osteoblast (OB) differentiation are carried out at sites of bone defects [2]. However, identification of agents that enhance OB differentiation of MSC and in vivo bone regeneration remains a challenge [1, 3]. Protein phosphorylation is known as the most common type of post-translational modification of proteins, and it is estimated that around 30% of cellular proteins are phosphorylated on at least one residue [4, 5]. Around 518 protein kinases with a wide range of structures, functions, and subcellular localizations have been identified [6-8], making protein kinases one of the largest gene families comprising approximately 2% of the human genome [4]. Many kinases have been identified to regulate osteoblastic cell functions. Several growth factors with known significant effects on OB differentiation and bone formation have cognate receptors with intrinsic kinase activity, for example, bone morphogenetic proteins and insulin-like growth factor 1. In addition, several kinases are known to regulate OB functions through direct activation or inactivation of key osteoblastic transcription factors, for example, Runx2 [9], Osterix [10], and ATF4 [11]. A number of kinases have also been identified as activators or inhibitors of intracellular proteins that regulate important signaling pathways in OB biology, for example, P300. Akt phosphorylates P300 at Ser-1834 and promotes its function as a coactivator of Runx2; the master regulator of osteogenesis [12-14], whereas phosphorylation of P300 at Ser-89 by protein kinase C inhibits its function [15]. Small molecule protein kinase inhibitors have been developed as novel drugs for treatment of malignant and nonmalignant diseases [16-18]. The USA food and drug administration has approved several kinase inhibitor drugs alleviating concerns about their safe use as therapeutic agents [17, 19, 20]. Using small molecule protein kinase inhibitors for targeting human MSC and enhancing bone formation has not been previously explored. The aim of this study was to identify small molecule kinase inhibitors that enhance differentiation of hMSC to osteoblastic cells and test their ability for enhancing in vivo bone formation. Thus, we screened a small molecule kinase inhibitor library containing 80 known small molecule protein kinase inhibitors that cover a wide spectrum of signaling pathways. Our data identified H-8 as a potent stimulator of in vitro OB differentiation and in vivo bone formation of hMSC. Materials and Methods Cell Culture We used a well-characterized immortalized hMSC cell line at low passage (hMSC-TERT4) that is generated by overexpressing human telomerase reverse transcriptase gene [21, 22]. For simplicity, hMSC-TERT4 cells will hereafter be referred to as hMSC. Primary hMSC cultures were established from bone marrow aspirates and adipose tissue of different healthy donors as described [23]. The cells were cultured in a standard growth medium containing minimal essential medium (MEM) (Gibco, Grand Island, NY, http://www.invitrogen.com) supplemented with 10% batch-tested fetal bovine serum (FBS) (South America origin) (Gibco, U.K.) and 1% penicillin/streptomycin (Gibco). Cells were incubated at 37°C in a humidified atmosphere containing 5% CO2. OB Differentiation of hMSC For osteogenic differentiation, cells were plated at a density of 18,000 cells per centimeter square in six-well plates in standard growth medium. At 70% confluence, the medium was replaced with osteoblastic induction medium (OIM) consisting of standard growth medium supplemented with 5 mM β-glycerophosphate (Calbiochem, Germany, http://www.merckmillipore.com), 50 µg/mL l-ascorbic acid (Sigma, Denmark, http://www.sigmaaldrich.com), 10 nM dexamethasone (Sigma), and 10 nM 1,25-dihydroxy vitamin D3 (LEO Pharma, Denmark, http://www.leo-pharma.com). Alkaline Phosphatase Activity Assay Alkaline phosphatase (ALP) activity was measured by using p-nitrophenyl phosphate (Fluka, U.K., http://www.sigmaaldrich.com) as substrate and normalization to cell viability was used to correct for differences in cell number, as described before [24]. Briefly, CellTiter-Blue reagent (Promega, Madison, WI, http://www.promega.com) was added to culture medium, incubated at 37°C for 1 hour, and fluorescent intensity (560EX/590EM) was measured using FLUOstar Omega multimode microplate reader (BMG Labtech, Germany, http://www.bmglabtech.com). Cells were then washed with Tris-buffered saline, fixed in 3.7% formaldehyde, 90% ethanol for 30 seconds at room temperature, incubated with substrate (1 mg/ml of p-nitrophenyl phosphate in 50 mM NaHCO3, pH 9.6 and 1 mM MgCl2) at 37°C for 20 minutes, and the absorbance was measured at 405 nm, using FLUOstar Omega multimode microplate reader. Alizarin Red Staining In vitro mineralization was assessed by performing alizarin red S (AR-S) staining, as described previously [25]. Briefly, cells were induced into OB differentiation as described earlier for 14 days. Cells were then washed in phosphate buffered saline (PBS), fixed in 70% ethanol at −20°C for 1 hour, rinsed in dH2O, and stained with 40 mM AR-S (Sigma-Aldrich, St. Louis, MO), pH 4.2, for 10 minutes with rotation. Stained cultures were then rinsed twice with dH2O, followed by washing three times with PBS to reduce nonspecific staining. The amount of mineralized matrix (Bound stain) was quantified by elution of the Alizarin red stain, using 20 minutes incubation of the cultures in 10% (w/v) cetylpyridinium chloride solution on a shaker (100 rpm) at room temperature. The absorbance of the eluted dye was measured at 570 nM, using FLUOstar Omega multimode microplate reader. Small Molecule Kinase Inhibitors Screening We screened a commercially available small molecule kinase inhibitor library (Screen-Well Kinase Inhibitor Library from Biomol International, USA, http://www.enzolifesciences.com). This library contains 80 known inhibitors and covers a wide variety of signaling pathways. hMSC cells were plated in 96-well plates (18,000 cells per centimeter square) in culture media. The day after, culture media was replaced with OIM. In order to determine the kinase inhibitors that have the potential to enhance the ALP activity, each kinase inhibitor was added individually to the OIM at 1 µM and 10 µM concentration. Media was renewed every third day and 6 days after induction of differentiation, ALP activity was quantified as described before. In each 96-well plate, a noninduced sample as well as the samples that had only the OIM or OIM plus the vehicle were included. Adipocyte Differentiation of hMSC For adipogenic differentiation of hMSC, cells were plated at high densities (40 × 103 cells pre centimeter square) in six-well plates in standard growth medium. To induce adipocyte differentiation, one day after seeding the cells, the medium was changed to adipogenic inducing media (AIM) consisting of standard growth medium supplemented with 10% horse serum (Sigma), 100 nM dexamethasone (Sigma-Aldrich, Denmark), 450 μM 1-methyl-3-isobutylxanthine (Sigma), 1 μM rosiglitazone (BRL49653) (Cayman Chemical, USA, http://www.caymanchem.com), and 3 μg/ml Insulin (Sigma) [26-28]. The medium was changed every other day, and on day 15, cells were visualized for adipocytes by oil red O staining. Oil Red O Staining Oil red O is a fat-soluble dye which is used for staining of neutral triglycerides and lipids. Adipogenic cultures of day 15 were fixed with 4% paraformaldehyde for 10 minutes at room temperature, rinsed with 3% isopropanol solution, and stained with oil red O (Sigma) solution (25 mg oil red O dye, 5 ml of 100% isopropanol, and 3.35 ml of H2O) for 1 hour at room temperature. The bound dye was eluted by 100% isopropanol, and its absorbance was measured at 490 nM, using FLUOstar Omega multimode microplate reader. Isolation and Osteoclast Differentiation of CD14+ Mononuclear Cells Osteoclast (OC) differentiation of human peripheral blood mononuclear cells (PBMC) was performed as described previously [29]. Briefly, human OC precursors were isolated from the blood of healthy donors provided anonymously by the blood bank of Odense University Hospital. PBMC were first separated by centrifugation on Ficoll-Paque PLUS; then, CD14+ cells (monocytes) were isolated by magnetic cell sorting according to the manufacturer's instruction. Briefly, PBMCs were resuspended in PBS containing 2% FBS, incubated (15 minutes at 4°C) with biotinylated anti-human CD14 goat antibody (R&D Systems, Minneapolis, http://www.rndsystems.com), and then incubated with Magcellect streptavidin ferrofluid (R&D Systems) (15 minutes at 4°C). A magnetic device was used to retain the tagged cells, and negative cells were discarded by extensive washes in PBS containing 2% FBS. Sorted cells were cultured in culture medium containing α-MEM supplemented with 10% FBS and 30 ng/ml Recombinant human Macrophage Colony-Stimulating Factor (rhM-CSF) for 3 days at 37°C. For OC differentiation, adherent monocytes were trypsinized and reseeded in 96-well plates (250,000 cells per well), in culture medium supplemented with 30 ng/ml rhM-CSF. The day after, media was replaced by OC differentiation medium containing both 30 ng/ml rhM-CSF and 30 ng/ml rhRANKL with replacement of medium every second day. To monitor OC differentiation, tartrate resistant acid phosphatase (TRAP) staining was performed 5 days after induction of OC differentiation [29]. Briefly, cells were fixed with 4% formaldehyde and stained for TRAP using the leukocyte acid phosphatase kit (Sigma) according to the manufacturer's protocol. TRAP-positive multinucleated cells with more than four nuclei were scored as OC. Total RNA Extraction and Reverse Transcription Quantitative Polymerase Chain Reaction Total RNA was isolated using TRIzol according to the manufacturer's instructions. First-strand complementary cDNA was synthesized using a revertAid H minus first-strand cDNA synthesis kit (Fermentas, St. Leon-Rot, Germany, https://www.lifetechnologies.com) according to the manufacturer's instructions. Quantitative polymerase chain reaction (qPCR) was performed using the StepOnePlus qPCR system and Fast SYBR Green master mix as a double strand DNA-specific binding dye. The comparative threshold cycle (CT) between target genes and the reference genes was used to measure the expression level of each target gene using the formula (1/(2ΔCT)) in which ΔCT is the difference between the CT value of the target gene and the CT value of the reference genes. Following minimum information for publication of quantitative real-time PCR experiments guidelines [30], two reference genes, β2m and TBP were used for normalization of RT-qPCR data. Supporting Information Table 1 shows the primers used for reverse transcriptase qPCR (RT-qPCR). Western Blot Analysis of Proteins Cells were lysed using RIPA buffer (Sigma) containing phosphatase inhibitor cocktail (Sigma) and protease inhibitor cocktail (Sigma). Cell lysates were centrifuged at 12,000g for 10 minutes at 4°C. Total protein concentrations were measured using Bradford assay (Thermo Fisher Scientific, USA, http://www.thermofisher.com), and equal amount of protein was loaded on a 10% polyacrylamide gel (Invitrogen, Carlsbad, CA, http://www.invitrogen.com). Blotted Polyvinylidene fluoride (PVDF) membranes were incubated overnight at 4°C with antibodies against P-Akt (Ser473, Cell Signaling, Beverly, MA, http://www.cellsignal.com), P-RhoA (Ser188, Abcam, Cambridge, U.K., http://www.abcam.com), p-mTOR (Ser2448, Cell Signaling, USA, http://www.cellsignal.com), and Actin (Sigma). Membranes were incubated with horseradish peroxidase (HRP)-conjugated secondary antibody (Santa Cruz Biotechnology, Santa Cruz, CA, http://www.scbt.com) for 45 minutes at room temperature, and protein bands were visualized using Amersham ECL chemiluminescence detection system (GE Healthcare Bio-Sciences, USA, http://www.gelifesciences.com). Western blot band intensities were measured by image J and presented as relative to the control. All antibodies were used at a 1:1,000 dilution in 5% blotting grade milk solution prepared in PBST (PBS supplemented with 0.1% Tween 20). In Vivo Bone Regeneration Assays in Immunodeficient Mice For ectopic bone formation assay, hMSC (5 × 105) were mixed with hydroxyapatite-tricalcium phosphate ceramic powder (HA-TCP, 40 mg; Zimmer Scandinavia, Denmark, http://www.zimmer.com) and transplanted subcutaneously into the dorsal surface of 2-month-old female NOD/SCID mice (NOD/LtSz-Prkdcscid), as described previously [25, 31]. The implants were removed after 8 weeks and transferred to 4% neutral buffered formalin for 24 hours; afterward, formic acid was added for 3 days. Using standard histopathologic methods, the HA-TCP implants were embedded in paraffin, and tissue sections (4 mm thick) were cut and stained with hematoxylin and eosin. The total bone volume per total volume was quantified as described previously [25, 31]. Healos is an osteoconductive carrier that is composed of crosslinked type I collagen fibers fully coated with HA and has been used in clinical trials before [32, 33]. For critical-size calvarial defect model, hMSC (15,000 per centimeter square) were seeded on Healos scaffolds (DePuy Spine, USA, https://www.depuysynthes.com) and treated with vehicle or H-8 (25 µM) for 5 days, before implantation into mouse calvarial defects. Calvarial defects (3 mm) were created in the right and left parietal of 2-month-old female NOD/SCID mice, using a biopsy punch, as described previously [34]. In each mouse, one defect was implanted with vehicle-treated and the other with H-8-treated cells. Survival and localization of the implanted hMSC were evaluated using bioluminescent imaging of the animals as described before [35]. To evaluate bone formation, microcomputed tomographical (μCT) scanning images were obtained from mice at 1, 4, and 6 weeks after the surgery, using a VivaCT40 scanner (Scanco Medical AG, Bassersdorf, Switzerland, www.scanco.ch). KINOMEscan Kinase Assay Inhibition of PRKG1 by H-8 was determined using KINOMEscan kinase assay, performed at the LeadHunter Discovery Services (DiscoveRx Corporation, USA, http://www.discoverx.com). KINOMEscan is a novel and proprietary active site-directed competition binding assay that directly and quantitatively measures the interactions between test compounds and kinases, by determining binding of the small molecule kinase inhibitors to the kinase ATP binding site [36]. KINOMEscan assay do not require ATP and thereby report true thermodynamic interaction affinities, as opposed to IC50 values, which can depend on the ATP concentration. In addition, the assay has a wide dynamic range and can measure bindings at concentrations as low as 1–10 pM. More information about KINOMEscan assay can be found at www.discoverx.com. G-LISA RhoA Activation Assay RhoA activity was determined using the active RhoA colorimetric ELISA assay (Cytoskeleton, Sweden, http://www.cytoskeleton.com) according to the manufacturer's instructions. Briefly, cells were serum starved for 24 hours at 70% confluence and then stimulated with either vehicle or H-8 (10 µM) for 15 minutes. Cells were then lysed using the provided lysis buffer (Cytoskeleton, Sweden) supplemented with 0.001% protease inhibitor cocktail (Cytoskeleton). Protein concentrations were determined using Precision Red Advanced Protein Assay Reagent (Cytoskeleton). Lysate concentrations were equilibrated to 2 mg/ml. The amount of active RhoA was determined using the absorbance based G-LISA RhoA activation assay kit (Cytoskeleton). In this assay, the plate wells are coated with a Rho-GTP binding protein that binds active (GTP-bound) RhoA. 50 µL of cell lysate and blank were pipetted, in triplicate, into wells coated with a Rho-GTP binding protein and active RhoA levels were determined using incubation with the provided anti-RhoA primary antibody and the HRP-conjugated secondary antibody, and measurement of absorbance (490 nm) was done using FLUOstar Omega multimode microplate reader. Small interfering RNA (siRNA) Transfections For siRNA transfections, we used the nontargeting control siRNA#1 and #2 (Ambion, Austin, TX, http://www.ambion.com) as negative control. All siRNAs were Silencer Select siRNA (Ambion) that are chemically modified with locked nucleic acid residues that results in higher stability, less off-target effects, and less immune-stimulatory effects. Supporting Information Table 2 shows the sequence of the targeting siRNAs. We used a reverse transfection protocol using Lipofectamine 2000 according to the manufacturer's instructions (Invitrogen). Briefly, 70% confluent hMSC cultures were trypsinized and 18,000 cells per centimeter square were reverse-transfected with siRNAs (25 nM) using MEM supplemented with 10% FBS, and after 8 hours, the transfection media was replaced with normal culture media (CM). Two days after transfection, OIM was added to hMSC cultures. For the screening study, each of the three independent siRNAs against each target were reverse-transfected separately, and changes in OB differentiation were determined using ALP activity quantitation on day 6 of OB differentiation. The screening experiment was performed twice, and the average ALP activity of the three independent siRNAs from two biological replicates was used for the analysis. To avoid selection of a false positive hit and to ensure the specificity of the effects observed by PRKG1 siRNAs, we performed follow-up studies using a new siRNA targeting a different region of PRKG1 mRNA, compared to the siRNAs used in the screening study. RT-qPCR analysis of PRKG1 expression on days 2, 6, and 12 after siRNA transfection was used to determine the knockdown efficiency. Changes in OB differentiation and mineralization were determined using ALP activity quantitation (day 6 of OB differentiation), alizarin red staining (day 12 of OB differentiation), and RT-qPCR analysis of OB marker gene expression (day 4 of OB differentiation). Statistical Analysis Data are represented as mean ± SD of at least three independent experiments with at least three replicates for each biological replicate, unless otherwise stated. Differences between variables were calculated using standard two-tailed unpaired student t tests. p < .05 was considered statistically significant. *, p ≤ .05; **, p ≤ .01; ***, p ≤ .001. Results Screening of the Kinase Inhibitor Library Identified H-8 as Inducer of ALP Activity To identify novel molecules that enhance OB differentiation of hMSC, we screened a library of small molecules kinase inhibitors (Screen-Well Kinase Inhibitor Library, Biomol international). We used a well-characterized telomerized hMSC line: hMSC-TERT4; for simplicity referred to as hMSC [21]. Each kinase inhibitor was added to OIM at concentration of 1 and 10 µM, since majority of the kinase inhibitors included in this library have been used before in different model systems at this range of concentrations [37]. ALP activity was determined at day 6 postinduction (Supporting Information Fig. S1A, S1B). H-8 which is known to inhibit protein kinases A, C, and G (compound #1 in Supporting Information Fig. S1A, S1B) induced maximal increase of ALP activity at both concentrations. The specificity of the H-8 effects was revealed by the absence of significant effects on ALP activity of closely related compounds: H-7 and H-9, which belong to the same family of kinase inhibitors (Supporting Information Fig. S1C, S1D) [38-40]. H-8 Enhanced Ex Vivo OB Differentiation of hMSC In order to confirm the results obtained from the initial screen and to determine the concentration range at which H-8 enhances OB differentiation, we incubated hMSC and human primary MSC (hpMSC) obtained from bone marrow aspirate of a healthy young donor, in OIM supplemented with H-8 (dose range 1–30 µM) or vehicle. H-8 increased ALP activity (Fig. 1A) and ex vivo matrix mineralization (Fig. 1B) at different doses. Adding H-8 to hMSC cultures showed maximal enhancing effects on ALP activity and matrix mineralization at 20 µM and 30 µM, respectively, H-8 was used at 25 µM in the follow-up experiments, unless another concentration is stated. Enhanced ALP activity was also observed, when osteogenic cultures of primary MSC isolated from bone marrow of three healthy donors were treated with H-8 (25 µM) (Supporting Information Fig. S2A). To determine whether H-8 enhances OB differentiation of MSC-like cells derived from tissues other than bone marrow, we added H-8 to the primary hMSC cultures obtained from adipose tissue of a healthy donor (hAT-pMSC) and observed enhanced ALP activity at 10, 20, and 30 µM (Supporting Information Fig. S2B) and increased formation of mineralized matrix (tested at 25 µM) (Supporting Information Fig. S2C). H-8 (25 µM) also enhanced ALP activity in hMSC cultures in the absence of osteogenic inducers, or when individual components were omitted from OIM (Supporting Information Fig. S3A). Moreover, adding H-8 (25 µM) to primary MSC isolated from bone marrow of three healthy donors, in the absence of OIM, showed enhanced ALP activity (Supporting Information Fig. S3B). H-8 (25 µM) treatment of hMSC cultures also enhanced early and late osteoblastic genes expression during the course of ex vivo OB differentiation (Fig. 1C). Open in new tabDownload slide H-8 enhanced ex vivo osteoblast differentiation. (A): Quantitation of ALP activity on day 6 and (B) Alizarin red staining of mineralized matrix on day 12 of osteoblast differentiation of telomerized hMSC and hpMSC treated with vehicle or different doses: 1, 10, 20, or 30 μM of H-8. (C): Reverse transcriptase quantitative polymerase chain reaction analysis of osteoblastic gene expression during 7-days in vitro differentiation of hMSC, in the presence of H-8 (25 µM). Data were corrected for variation using average expression of β2M and TBP as housekeeping genes. Results presented from three independent experiments, Error bars represent SD. **, p ≤ .01; ***, p ≤ .001. Abbreviations: ALP, alkaline phosphatase; hMSC, human skeletal stem cell; hpMSC, human primary MSC. Open in new tabDownload slide H-8 enhanced ex vivo osteoblast differentiation. (A): Quantitation of ALP activity on day 6 and (B) Alizarin red staining of mineralized matrix on day 12 of osteoblast differentiation of telomerized hMSC and hpMSC treated with vehicle or different doses: 1, 10, 20, or 30 μM of H-8. (C): Reverse transcriptase quantitative polymerase chain reaction analysis of osteoblastic gene expression during 7-days in vitro differentiation of hMSC, in the presence of H-8 (25 µM). Data were corrected for variation using average expression of β2M and TBP as housekeeping genes. Results presented from three independent experiments, Error bars represent SD. **, p ≤ .01; ***, p ≤ .001. Abbreviations: ALP, alkaline phosphatase; hMSC, human skeletal stem cell; hpMSC, human primary MSC. H-8-Induced OB Differentiation is Stage- and Cell Type-Specific To determine differentiation-stage specific effects, H-8 was added to the OIM at different time points during OB differentiation. Quantification of ALP activity and mineralized matrix formation revealed that H-8 exerted the most pronounced effects when added at day 1 to day 3 (D1–D3), that is, early post OB induction (Fig. 2A, 2B). To determine the cell type specificity, human skin fibroblasts were treated with H-8 (25 µM) in the presence of basal CM or OIM. H-8 did not induce ALP activity (Fig. 2C) or affect cell viability of the human skin fibroblasts (Fig. 2D). Open in new tabDownload slide Stage- and cell type-specific effect of H-8 on osteoblast (OB) differentiation. H-8 (25 µM) was added at different time points during OB differentiation of human skeletal stem cell (hMSC). (A): Quantification of ALP activity on day 6 and (B) quantification of mineralized matrix formation on day 12 of OB differentiation (results presented from two to three independent experiments). H-8 (25 µM) was added to normal human skin fibroblasts and the effects on (C) ALP activity and (D) cell viability were determined (n = 6 technical replicates). Error bars represent SD. *, p ≤ .05; **, p ≤ .01; N.S., not statistically significant difference detected, D = day. Abbreviation: ALP, alkaline phosphatase. Open in new tabDownload slide Stage- and cell type-specific effect of H-8 on osteoblast (OB) differentiation. H-8 (25 µM) was added at different time points during OB differentiation of human skeletal stem cell (hMSC). (A): Quantification of ALP activity on day 6 and (B) quantification of mineralized matrix formation on day 12 of OB differentiation (results presented from two to three independent experiments). H-8 (25 µM) was added to normal human skin fibroblasts and the effects on (C) ALP activity and (D) cell viability were determined (n = 6 technical replicates). Error bars represent SD. *, p ≤ .05; **, p ≤ .01; N.S., not statistically significant difference detected, D = day. Abbreviation: ALP, alkaline phosphatase. H-8 Did Not Affect Adipogenesis or Osteoclastogenesis H-8 (25 µM) added to the AIM did not lead to significant changes in adipocyte differentiation of hMSC as shown by quantification of oil red O staining (Fig. 3A, 3B) and adipogenic gene expression (Fig. 3C). Moreover, H-8 treatment (dose range 1–30 µM) did not change the TRAP-positive multinucleated osteoclastic cell formation in human peripheral blood-CD14+ mononuclear cell cultures [29] (Fig. 3D, 3E). Open in new tabDownload slide H-8 treatment did not affect adipogenesis or osteoclastogenesis. Human skeletal stem cells were cultured in adipocyte induction medium in the presence of H-8 (25 µM). (A): Oil-red-O staining of adipocytes, (B) quantitation of oil-red-O staining on day 15, (C) reverse transcriptase quantitative polymerase chain reaction analysis of the adipogenic genes: ADN, LPL, and AP2 on day 10 of differentiation. Data were corrected for variation in average expression of β2M and TBP as housekeeping genes. (D): H-8 (Dose range 1–30 µM) was added to osteoclastogenic cultures of human CD14+ mononuclear cells obtained from peripheral blood of young healthy donors. Osteoclasts were identified by being multinucleated and positive for tartrate-resistant acid phosphatase staining. Results presented from two to three independent experiments. Error bars represent SD, N.S., not statistically significant differences detected. Scale bar = 200 µm. Abbreviations: ADN, adiponectin; AP2, adipocyte lipid-binding protein; LPL, lipoprotein lipase. Open in new tabDownload slide H-8 treatment did not affect adipogenesis or osteoclastogenesis. Human skeletal stem cells were cultured in adipocyte induction medium in the presence of H-8 (25 µM). (A): Oil-red-O staining of adipocytes, (B) quantitation of oil-red-O staining on day 15, (C) reverse transcriptase quantitative polymerase chain reaction analysis of the adipogenic genes: ADN, LPL, and AP2 on day 10 of differentiation. Data were corrected for variation in average expression of β2M and TBP as housekeeping genes. (D): H-8 (Dose range 1–30 µM) was added to osteoclastogenic cultures of human CD14+ mononuclear cells obtained from peripheral blood of young healthy donors. Osteoclasts were identified by being multinucleated and positive for tartrate-resistant acid phosphatase staining. Results presented from two to three independent experiments. Error bars represent SD, N.S., not statistically significant differences detected. Scale bar = 200 µm. Abbreviations: ADN, adiponectin; AP2, adipocyte lipid-binding protein; LPL, lipoprotein lipase. H-8 Enhanced the In Vivo Bone Regeneration Capacity of hMSC To determine the effect of in vivo administration of H-8 on the ability of hMSC to form heterotopic bone [41], hMSC were mixed with HA/TCP as osteoconductive carrier and implanted subcutaneously in NOD/SCID mice. The mice received H-8 as intraperitoneal injections every other day for 4 weeks at a fixed dose of either 5, 15, 30, or 90 mg/kg per day. The administered in vivo doses were chosen based on the observation that the maximal in vitro effects of H-8 were exerted at approximately 20 µM. Considering the mice body weight, 5 mg/kg was calculated to be a rough estimate of the dose providing an approximate concentration of 20 µM in body fluids following systemic injection. However, due to lack of information about the H-8 biodistribution and clearance rate, higher concentrations were also used. Histological analysis of the implants after 8 weeks revealed around two fold increase in the amount of bone formed at 90 mg/kg per day dose compared to vehicle-treated controls (p < .001) (Fig. 4A, 4B). Positive staining for human specific vimentin confirmed that the newly formed bone was of human origin (Fig. 4C). Necropsy of the injected mice did not reveal any adverse organ effects following H-8 administration (data not shown). In addition, histological analysis of the kidneys from injected mice did not show any toxic effects of H-8 administration (Supporting Information Fig. S4). Open in new tabDownload slide H-8 enhances in vivo bone formation. Human skeletal stem cells (hMSC) were mixed with hydroxyapatite/ticalcium phosphate and implanted subcutaneously in immune deficient mice that received intraperitoneal injections of H-8 (dose range: 5–90 mg/kg per day) for 4 weeks. (A): Histological analysis of the implants showed normal lamellar bone formation by hMSC. Scale bar = 500 µm. (B): Volume of the newly formed heterotopic bone expressed as bone area/total area (%), Scale bar = 500 µm (N = 8 implants per treatment). (C): Human specific Vimentin staining confirmed that the formed heterotopic bone is of human origin. Arrows show hydroxyapatite, Arrow heads show bone. (D): Quantitation of ALP activity in hMSC seeded on Healos scaffold and treated with H-8 (25 µM) or vehicle for 5 days (N = 3 independent experiments). (E): Bioluminescent imaging of the Luciferase expressing hMSC, 1 and 4 weeks after implantation into mouse critical-size calvarial defect. Left defects contain vehicle-treated hMSC and right defects contain H-8 treated hMSC. (F): Microcomputed tomography analysis of bone formation in the calvarial defects, 1, 4, and 6 weeks after the surgery. (G): Human specific Vimentin staining confirmed that the newly formed bone is of human origin. Scale bar = 1 mm (N = 4 per treatment group). Error bars represent SD, ***, p ≤ .001. Abbreviation: ALP, alkaline phosphatase. Open in new tabDownload slide H-8 enhances in vivo bone formation. Human skeletal stem cells (hMSC) were mixed with hydroxyapatite/ticalcium phosphate and implanted subcutaneously in immune deficient mice that received intraperitoneal injections of H-8 (dose range: 5–90 mg/kg per day) for 4 weeks. (A): Histological analysis of the implants showed normal lamellar bone formation by hMSC. Scale bar = 500 µm. (B): Volume of the newly formed heterotopic bone expressed as bone area/total area (%), Scale bar = 500 µm (N = 8 implants per treatment). (C): Human specific Vimentin staining confirmed that the formed heterotopic bone is of human origin. Arrows show hydroxyapatite, Arrow heads show bone. (D): Quantitation of ALP activity in hMSC seeded on Healos scaffold and treated with H-8 (25 µM) or vehicle for 5 days (N = 3 independent experiments). (E): Bioluminescent imaging of the Luciferase expressing hMSC, 1 and 4 weeks after implantation into mouse critical-size calvarial defect. Left defects contain vehicle-treated hMSC and right defects contain H-8 treated hMSC. (F): Microcomputed tomography analysis of bone formation in the calvarial defects, 1, 4, and 6 weeks after the surgery. (G): Human specific Vimentin staining confirmed that the newly formed bone is of human origin. Scale bar = 1 mm (N = 4 per treatment group). Error bars represent SD, ***, p ≤ .001. Abbreviation: ALP, alkaline phosphatase. Moreover, hMSC-loaded Healos scaffolds were treated with H-8 (25 µM) for 5 days. Quantification of ALP activity showed that H-8 treatment enhances the OB differentiation of hMSC loaded on scaffolds (Fig. 4D). To study the effect of H-8 on bone tissue regeneration by hMSC, luciferase-labeled hMSC were cultured on Healos scaffolds for 5 days, in the presence or absence of H-8 (25 µM), followed by implantation into mouse critical-size calvarial defect (3 mm). Luminescent imaging 1 and 4 weeks after implantation showed the survival of H-8-treated hMSC in vivo (Fig. 4E), and µCT analysis of the calvarial defects after 4 weeks showed enhanced onset of bone formation by H-8 treated hMSC. However, bone regeneration was also observed by vehicle-treated hMSC after 6 weeks, and the difference in regeneration of the defects was not as clear as week 4 (Fig. 4F). Positive staining for human specific vimentin confirmed that the newly formed bone in the defects is of human origin (Fig. 4G). H-8 Stimulates Akt Phosphorylation Through Inhibition of PRKG1 and Activation of RhoA Signaling To identify the molecular mechanisms of H-8 effects, we conducted Western blot analysis that revealed enhanced Akt phosphorylation (Ser473) following H-8 (25 µM) treatment of hMSC (Fig. 5A, 5B). Enhanced Akt phosphorylation was also observed when hpMSC isolated from bone marrow of three healthy donors were treated with H-8 (25 µM, 15 minutes) (Fig. 5C, 5D). Pharmacological inhibition of Akt signaling using Triciribine hydrate (TCN) (0.2 µM) abolished the enhancing effect of H-8 on ALP activity (Fig. 5E) and matrix mineralization (Fig. 5F). In addition, TCN (0.2 µM) treatment of hpMSC osteogenic cultures inhibited H-8-induced ALP activity (Supporting Information Fig. S5A). To identify the protein kinase targeted by H-8 upstream of Akt, we used siRNA-mediated loss of function studies in the presence of OIM (N = 3 siRNAs for each target) for different isoforms of PKA, PKC, and PKG, which are known to be inhibited by H-8. To assess the effect of each siRNA on OB differentiation, activity of ALP was quantified on day 6 of OB differentiation, and hMSC transfected with siRNA against ALP were used as positive control for siRNA transfection. Among known targets of H-8, PRKG1 inhibition led to highest stimulation of ALP activity (Fig. 6A). An additional siRNA against PRKG1 (siPRKG1 #4) was used for the follow-up studies to confirm the observed effect with the screening siRNAs. RT-qPCR analysis showed that siPRKG1 reduced the expression of PRKG1 by 80%, 81%, and 50%, on days 2, 6, and 12 after siRNA transfection, respectively (Fig. 6B). siPRKG1 enhanced the activity of ALP (Fig. 6C) and formation of mineralized matrix (Fig. 6D) at day 6 and day 12 post-OB differentiation of hMSC, respectively. RT-qPCR analysis showed enhanced expression of OB marker genes (ALP, Col1, RUNX2, OSX, BSP) in hMSC transfected with siPRKG1, at day 4 of OB differentiation (Fig. 6E). Moreover, in the presence of H-8 at submaximal concentration (10 µM), siPRKG1 enhanced H-8-induced ALP activity (Fig. 6F). PRKG1 is known to phosphorylate and thereby inhibit the activity of the RhoA small GTPase [42-44]. Activation of Akt signaling by RhoA has been reported [45]. We hypothesized that activation of Akt signaling by H-8 is mediated through inhibition of PRKG1 and activation of RhoA signaling. Quantitative measurement of H-8 binding to PRKG1 active site using KINOMEscan kinase assay revealed direct binding of H-8 to PRKG1 and consequent inhibition of its activity to 5% of vehicle-treated controls (Fig. 6G). Furthermore, G-LISA RhoA activity assay showed increased RhoA activity 15 minutes following H-8 (10 µM) treatment (Fig. 6H). siRNA-mediated knockdown of PRKG1 mimicked the effect of H-8 by decreasing P-RhoA and increasing P-mTOR and P-Akt (Fig. 6I–6K). Open in new tabDownload slide H-8 treatment of hMSC stimulates Akt phosphorylation. (A, B): Western blot analysis of Akt phosphorylation (S473) and quantification of band intensities after H-8 treatment of hMSC (25 µM) for 10, 15, and 20 minutes. (C, D): Western blot analysis of Akt phosphorylation (S473) and quantification of band intensities after H-8 treatment (25 µM, for 15 minutes) of hpMSC isolated from bone marrow of three healthy donors. (E): Effect of selective inhibitor of Akt signaling; Triciribine hydrate (0.2 µM) on H-8-induced ALP activity and (F) matrix mineralization of hMSC during osteoblast differentiation. Western blot band intensities are measured by image J and presented as relative to control (vehicle treated) group. Results presented from three independent experiments, Error bars represent SD. **, p ≤ .01; ***, p ≤ .001. Abbreviations: ALP, alkaline phosphatase; hpMSC, human primary MSC; hMSC, human skeletal stem cell. Open in new tabDownload slide H-8 treatment of hMSC stimulates Akt phosphorylation. (A, B): Western blot analysis of Akt phosphorylation (S473) and quantification of band intensities after H-8 treatment of hMSC (25 µM) for 10, 15, and 20 minutes. (C, D): Western blot analysis of Akt phosphorylation (S473) and quantification of band intensities after H-8 treatment (25 µM, for 15 minutes) of hpMSC isolated from bone marrow of three healthy donors. (E): Effect of selective inhibitor of Akt signaling; Triciribine hydrate (0.2 µM) on H-8-induced ALP activity and (F) matrix mineralization of hMSC during osteoblast differentiation. Western blot band intensities are measured by image J and presented as relative to control (vehicle treated) group. Results presented from three independent experiments, Error bars represent SD. **, p ≤ .01; ***, p ≤ .001. Abbreviations: ALP, alkaline phosphatase; hpMSC, human primary MSC; hMSC, human skeletal stem cell. Open in new tabDownload slide H-8 enhances OB differentiation through inhibition of PRKG1 and activation of RhoA-Akt signaling. (A): Quantification of ALP activity on day 6 of osteoblast (OB) differentiation, following siRNA-mediated knockdown of known H-8 targets (N = 3 siRNAs for each target). siALP was used as control for siRNA transfection. (B): Reverse transcriptase quantitative polymerase chain reaction (RT-qPCR) analysis of PRKG1 expression, 2, 6, and 12 days after transfection of human skeletal stem cell (hMSC) with a new siRNA against PRKG1 (siPRKG1). Effect of siPRKG1 on OB differentiation of hMSC, assessed by (C) quantitation of ALP activity on day 6, (D) quantitation of matrix mineralization on day 12 of OB differentiation, and (E) RT-qPCR analysis of OB marker genes' expression (ALP, Col1, RUNX2, OSX, DLX5, BSP) in hMSC transfected with siPRKG1 on day 4 of OB differentiation. (F): Effect of siPRKG1 on ALP activity, in the presence of H-8 at submaximal concentration (10 µM). (G): Inhibition of PRKG1 function by H-8 shown by KINOMEscan kinase assay. (H): Effect of H-8 on RhoA activity, measured by G-LISA RhoA activation assay. (I–K): Western blot analysis, showing the effect of H-8 and siPRKG1 on phospho (P)-RhoA, P-mTOR, and P-Akt, and quantification of band intensities. Western blot band intensities are measured by image J and presented as relative to control (vehicle or siCtrl treated) group. Results presented from three independent experiments. Error bars represent SD. *, p ≤ .05; ***, p ≤ .001. Abbreviations: ALP, alkaline phosphatase; CM, culture media; LF2000, Lipofectamine 2000; NT, nontransfected; OIM, osteogenic induction media; PRKG1, protein kinase G1. Open in new tabDownload slide H-8 enhances OB differentiation through inhibition of PRKG1 and activation of RhoA-Akt signaling. (A): Quantification of ALP activity on day 6 of osteoblast (OB) differentiation, following siRNA-mediated knockdown of known H-8 targets (N = 3 siRNAs for each target). siALP was used as control for siRNA transfection. (B): Reverse transcriptase quantitative polymerase chain reaction (RT-qPCR) analysis of PRKG1 expression, 2, 6, and 12 days after transfection of human skeletal stem cell (hMSC) with a new siRNA against PRKG1 (siPRKG1). Effect of siPRKG1 on OB differentiation of hMSC, assessed by (C) quantitation of ALP activity on day 6, (D) quantitation of matrix mineralization on day 12 of OB differentiation, and (E) RT-qPCR analysis of OB marker genes' expression (ALP, Col1, RUNX2, OSX, DLX5, BSP) in hMSC transfected with siPRKG1 on day 4 of OB differentiation. (F): Effect of siPRKG1 on ALP activity, in the presence of H-8 at submaximal concentration (10 µM). (G): Inhibition of PRKG1 function by H-8 shown by KINOMEscan kinase assay. (H): Effect of H-8 on RhoA activity, measured by G-LISA RhoA activation assay. (I–K): Western blot analysis, showing the effect of H-8 and siPRKG1 on phospho (P)-RhoA, P-mTOR, and P-Akt, and quantification of band intensities. Western blot band intensities are measured by image J and presented as relative to control (vehicle or siCtrl treated) group. Results presented from three independent experiments. Error bars represent SD. *, p ≤ .05; ***, p ≤ .001. Abbreviations: ALP, alkaline phosphatase; CM, culture media; LF2000, Lipofectamine 2000; NT, nontransfected; OIM, osteogenic induction media; PRKG1, protein kinase G1. Treating hMSC with PRKG selective activator, 8-pCPT-cGMP (10 µM) [46], in the presence of H-8 at submaximal concentration (10 µM), abolished the effects of H-8 on P-RhoA, P-Akt (Fig. 7A–7C), and ALP activity (Fig. 7D). 8-pCPT-cGMP (10 µM) treatment also inhibited H-8-induced ALP activity in osteogenic cultures of hpMSC (Supporting Information Fig. S5B). Rho kinase (ROCK) is one of the effector proteins that functions downstream of RhoA. Y-27632 is known to be a selective inhibitor of ROCK activity [47]. We found that inhibition of ROCK activity blunted the enhancing effect of H-8 on P-Akt (Fig. 7E, 7F) as well as ALP activity (Fig. 7G). Moreover, treatment of hpMSC osteogenic cultures with Y-27632 (10 µM) showed inhibitory effect on H-8-induced ALP activity (Supporting Information Fig. S5C). These data demonstrate that the enhancing effect of H-8 on OB differentiation of hMSC is mediated through inhibition of PRKG1 activity and consequent activation of RhoA-Akt signaling (Supporting Information Fig. S6). Open in new tabDownload slide Activation of protein kinase G (PRKG) and inhibition of Rho kinase (ROCK) blunts the effect of H-8 on human skeletal stem cell (hMSC). Selective activator of PRKG (8-pCPT-cGMP) (10 µM), in the presence of H-8 at submaximal concentration (10 µM), abolished the effect of H-8 on (A–C) P-RhoA, P-Akt (shown by Western blot analysis and quantification of band intensities), and (D) ALP activity on day 6 of osteoblast (OB) differentiation. Selective inhibitor of ROCK (Y-27632) (10 µM), in the presence of H-8 at submaximal concentration (10 µM) rescued the (E, F) H-8-induced P-Akt (shown by Western blot analysis and quantification of band intensities), and (G) ALP activity on day 6 of OB differentiation. Western blot band intensities are measured by image J and presented as relative to control (vehicle treated) group. Results presented from two to three independent experiments. Error bars represent SD, *, p ≤ .05; **, p ≤ .01; ***, p ≤ .001. Abbreviation: ALP, alkaline phosphatase. Open in new tabDownload slide Activation of protein kinase G (PRKG) and inhibition of Rho kinase (ROCK) blunts the effect of H-8 on human skeletal stem cell (hMSC). Selective activator of PRKG (8-pCPT-cGMP) (10 µM), in the presence of H-8 at submaximal concentration (10 µM), abolished the effect of H-8 on (A–C) P-RhoA, P-Akt (shown by Western blot analysis and quantification of band intensities), and (D) ALP activity on day 6 of osteoblast (OB) differentiation. Selective inhibitor of ROCK (Y-27632) (10 µM), in the presence of H-8 at submaximal concentration (10 µM) rescued the (E, F) H-8-induced P-Akt (shown by Western blot analysis and quantification of band intensities), and (G) ALP activity on day 6 of OB differentiation. Western blot band intensities are measured by image J and presented as relative to control (vehicle treated) group. Results presented from two to three independent experiments. Error bars represent SD, *, p ≤ .05; **, p ≤ .01; ***, p ≤ .001. Abbreviation: ALP, alkaline phosphatase. Discussion Use of small molecule protein kinase inhibitors for development of novel therapies for diseases other than cancer is gaining momentum due to the ease of modifying their chemical structure depending on the clinical needs, their adaptability to large scale production, and possible oral delivery [17, 48]. In this study, we identified H-8 as a small molecule kinase inhibitor that targets hMSC, enhances their ex vivo OB differentiation and in vivo bone regeneration. Although the trend of enhanced mineralized matrix formation, after H-8 treatment of primary hMSC osteogenic cultures was similar to immortalized hMSC, differences were observed in dose-response kinetics of enhanced mineralization that may be attributed to the presence of higher capacity of mineralized matrix formation by early passage primary osteoprogenitor cultures [49]. We found that H-8 effects on hMSC are specific. First, two closely related molecules, H-7 and H-9, which belong to the same family of isoquinoline sulfonamide kinase inhibitors exerted no effects on hMSC. Second, no biological effects of H-8 were observed in human skin fibroblasts, which is a closely related cell type to hMSC. Third, H-8 lacked biological effects on adipocyte differentiation, and OC formation. Therefore, H-8 has the potential to be developed as a drug for enhancing bone regeneration through targeting hMSC and without affecting other cellular components of the skeleton. The most pronounced effects of H-8 on hMSC were observed when exposed to H-8 during the early phases of hMSC differentiation. The effects were not related to increased cell proliferation but due to enhanced commitment of hMSC to osteoblastic lineage and increased extracellular matrix production. This observation has a clinical relevance, since defective differentiation of OB progenitor cells is the rate limiting step for optimal bone regeneration [50, 51]. Ectopic bone formation by hMSC in immune deficient mice is a well-established preclinical model to study the in vivo bone formation [31]. We found that systemic H-8 administration at 90 mg/kg per day for 4 weeks enhanced the in vivo bone formation capacity of hMSC, without toxic effects on other organs. However, the µM range of biologically effective dose of H-8 may not be suitable for systematic administration in humans and synthesizing H-8 analogs with lower effective molar concentration is desirable. Developing efficient treatment of nonhealed bone defects and complicated fractures is a major challenge in orthopedic surgery [52]. One of the promising approaches is bone tissue engineering in which osteoblastic cells are loaded on osteoconductive scaffolds and implanted at the site of injury [52]. As a clinically relevant model, mouse critical-size calvarial defect has been used extensively to identify novel strategies for efficient bone regeneration [27, 34]. Using this model, we showed that 4 weeks after implantation, hMSC treated in vitro with H-8 show enhanced in vivo survival and earlier onset of bone regeneration as compared to vehicle treated hMSC. However, 6 weeks after implantation, the difference in regeneration of the defects was not as clear as week 4. It is plausible that H-8 may accelerate specifically early stages of OB differentiation and in vivo osteogensis. Alternatively, it is possible that the observed short term effect of H-8 in vivo was caused by lack of prolonged hMSC stimulation. Developing functionalized scaffolds for continuous local release of H-8 over a longer period of time would probably result in significant enhancement of bone formation [2]. We used RNAi-mediated knockdown of gene expression to identify the kinase(s) that mediate the effect of H-8 on hMSC. We showed that siRNAs against PRKG1 and protein kinase C-γ (PRKCG) enhanced ALP activity, whereas protein kinase C-β (PRKCB) and protein kinase A catalytic subunit-γ (PRKACG) siRNAs reduced ALP activity. The role of PRKCG and PRKCB in regulation of OB differentiation of hMSC is not well-understood. However, activation of PRKA pathway has been shown to enhance in vitro OB differentiation and in vivo bone formation of hMSC [53], corroborating our observation that siPRKACG reduced ALP activity. However, our data suggest that PRKG1 is the main kinase mediating the effects of H-8 on OB differentiation of hMSC. KINOMEscan kinase assay showed that H-8 binds to PRKG1 and inhibits binding of PRKG1 active site to its substrate and siRNA-mediated down regulation of PRKG1 in hMSC mimicked the effects of H-8 treatment and the opposite effects were observed using 8-pCPT-cGMP which is a selective activator of PRKG [54]. PRKG1 is a serine/threonine protein kinase that activates nitric oxide/cGMP signaling pathway known to translate mechanical strain to biological signals [42, 55]. PRKG1 phosphorylates and inactivates the RhoA small GTPase [42-44], which has been shown to regulate OB differentiation and bone formation of hMSC [56-59]. Our observation that H-8 reduced RhoA phosphorylation in hMSC was in line with increased RhoA activity, shown by RhoA activity assay. Involvement of RhoA in regulation of responses to mechanical stimuli [56] suggests that H-8 may be useful in preventing bone loss in clinical conditions that are caused by immobilization or microgravity. Conclusions We identified H-8 as a small molecule kinase inhibitor that enhanced ex vivo OB differentiation of hMSC, without affecting adipogenesis or osteoclastogenesis. In a preclinical model of ectopic bone formation, systemic H-8 administration led to increased bone formation by hMSC. Local implantation of hMSC cultured on a functionalized scaffold containing H-8 may represent a novel approach to enhance local bone regeneration needed for treatment of localized bone defects and nonhealed fractures. The enhancing effect of H-8 on OB differentiation of hMSC is mediated through inhibition of PRKG1 and consequent activation of RhoA-Akt signaling. PRKG1 is a novel negative regulator of OB differentiation and bone formation by hMSC and pharmacological targeting of PRKG1 represents a novel possible approach to enhance bone formation. Acknowledgments We thank Dr. Henrik Daa Shrøder (Department of Pathology, University of Southern Denmark) for help with histopathological analysis of mouse kidneys, Dr. Charles Frary for establishing hpMSC cultures, Dr. Stephen Cohen, and Dr. Hung Thanh Nguyen (University of Copenhagen) for helpful discussions, and Lone Christiansen for excellent technical assistance. The work was supported by a grant from University of Southern Denmark and local government of Southern Denmark (project SDU647-68), NovoNordisk foundation's exploratory pre-seed grant (project 1015746), Lundbeck foundation, Danish Arthritis association (Gigtforeningen, project A1562), and Simon Fougner Hartmanns family foundation. Author Contributions A.J.: conception and design, collection of data, data analysis and interpretation, and manuscript writing; M.S.: collection of data, data analysis and interpretation, and manuscript writing; L.C., D.Q., and W.Z.: collection of data; B.A.: collection of data and data analysis and interpretation; M.K.: conception and design, data analysis and interpretation, manuscript writing, and final approval of manuscript. Disclosure of Potential Conflicts of Interest Use of H-8 for enhancing bone regeneration is patent pending and the intellectual property rights belong to University of Southern Denmark and Region of Southern Denmark. References 1 Dawson JI , Kanczler J, Tare R et al. Concise review: Bridging the gap: Bone regeneration using skeletal stem cell-based strategies—Where are we now? Stem Cells 2014 ; 32 : 35 – 44 . 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TI - Pharmacological Inhibition of Protein Kinase G1 Enhances Bone Formation by Human Skeletal Stem Cells Through Activation of RhoA-Akt Signaling
JO - Stem Cells
DO - 10.1002/stem.2013
DA - 2015-07-01
UR - https://www.deepdyve.com/lp/oxford-university-press/pharmacological-inhibition-of-protein-kinase-g1-enhances-bone-cPOYB5cQW4
SP - 2219
EP - 2231
VL - 33
IS - 7
DP - DeepDyve
ER -