Corrigendum to "Establishment and functional characterization of the reversibly immortalized mouse glomerular podocytes (imPODs)" [Genes & Diseases 5 (2018) 137-149].

[This corrects the article DOI: 10.1016/j.gendis.2018.04.003.].

Corrigendum to "The development of a sensitive fluorescent protein-based transcript reporter for high throughput screening of negative modulators of lncRNAs" [Genes & Diseases 5 (2018) 62-74].

[This corrects the article DOI: 10.1016/j.gendis.2018.02.001.].

OUHP: an optimized universal hairpin primer system for cost-effective and high-throughput RT-qPCR-based quantification of microRNA (miRNA) expression.

MicroRNAs (miRNAs or miRs) are single-stranded, ∼22-nucleotide noncoding RNAs that regulate many cellular processes. While numerous miRNA quantification technologies are available, a recent analysis of 12 commercial platforms revealed high variations in reproducibility, sensitivity, accuracy, specificity and concordance within and/or between platforms. Here, we developed a universal hairpin primer (UHP) system that negates the use of miRNA-specific hairpin primers (MsHPs) for quantitative reverse transcription PCR (RT-qPCR)-based miRNA quantification. Specifically, we analyzed four UHPs that share the same hairpin structure but are anchored with two, three, four and six degenerate nucleotides at 3'-ends (namely UHP2, UHP3, UHP4 and UHP6), and found that the four UHPs yielded robust RT products and quantified miRNAs with high efficiency. UHP-based RT-qPCR miRNA quantification was not affected by long transcripts. By analyzing 14 miRNAs, we demonstrated that UHP4 closely mimicked MsHPs in miRNA quantification. Fine-tuning experiments identified an optimized UHP (OUHP) mix with a molar composition of UHP2:UHP4:UHP6 = 8:1:1, which closely recapitulated MsHPs in miRNA quantification. Using synthetic LET7 isomiRs, we demonstrated that the OUHP-based qPCR system exhibited high specificity and sensitivity. Collectively, our results demonstrate that the OUHP system can serve as a reliable and cost-effective surrogate of MsHPs for RT-qPCR-based miRNA quantification for basic research and precision medicine.

Long transcripts minus touchdown qPCR (LTMT-qPCR): a simplified and convenient method for the screening and quantification of microRNA profiles.

Due to the short length and differences in abundance of microRNAs, microRNA profile screening and quantification is challenging. In this study, we found that size selection magnetic beads could be employed to easily and efficiently remove long RNA transcripts. After removing the long transcripts, the remaining small RNAs could be concentrated and then reverse-transcribed using universal stem-loop primers (USLP), with six randomized nucleotides at the 3' end region. The efficiency of reverse transcription decreased when the number of randomized nucleotides was reduced. In addition, we found that touchdown qPCR improved microRNA profile detection, with lower CT values and better detection efficiency than the regular qPCR protocol, especially for those low-abundance microRNAs. Finally, we incorporated these observations to create a new protocol we named long transcripts minus touchdown qPCR (LTMT-qPCR). We performed a side-by-side comparison of LTMT with USLP and traditional stem-loop primer (TSLP) protocols. We found that LTMT has higher detection efficiency than USLP, especially for the detection of low-abundance microRNAs. Although LTMT was equivalent to TSLP in terms of microRNA profile detection, LTMT is more convenient, user-friendly, and cost-effective. Taken together, the present data indicate that LTMT is a simple, rapid, and user-friendly approach that has higher precision, accuracy, and sensitivity than the previously described methods, making it more suitable for microRNA profile screening and quantification.

LncRNA HOXA-AS2 Promotes Tumor Progression by Suppressing miR-567 Expression in Oral Squamous Cell Carcinoma.

INTRODUCTION: Growing evidence suggests that long non-coding RNAs (lncRNAs), such as lncRNA HOXA-AS2, are critical regulators involved in human cancer. However, the biological functions and detailed mechanisms underlying how lncRNA HOXA-AS2 affects oral squamous cell carcinoma (OSCC) remain unexplored. METHODS: The expression of lncRNA HOXA-AS2 and miR-567 was determined in OSCC cell lines and clinical tissues by quantitative real-time PCR (qRT-PCR). Target site prediction and luciferase report assays were used to explore their potential interaction and binding sites between lncRNA HOXA-AS2 and miR-567. Overexpression or silencing expression of lncRNA HOXA-AS2 was performed to confirm that miR-567 was suppressed by lncRNA HOXA-AS2. WST-1 assay, crystal staining assay, and cell cycle analysis were used to assess the cell viability and proliferation ability. The target gene of miR-567 was predicted by Targetscan and validated by luciferase report assay as well as qRT-PCR and Western Blot. Xenograft nude mice model was done to demonstrate that lncRNA HOXA-AS2 promoted cell proliferation via targeting miR-567/CDK8 in vivo. RESULTS: LncRNA HOXA-AS2 was up-regulated in OSCC cells and tissues with the expression of miR-567 decreased. The tissue lncRNA HOXA-AS2 expression was found to positively correlate with the TNM stage and lymph node metastasis of OSCC patients. In terms of the mechanism, we found that lncRNA HOXA-AS2 negatively regulates miR-567 expression via a direct interaction. Functionally, overexpression of lncRNA HOXA-AS2 significantly promoted OSCC cell proliferation, while knockdown of lncRNA HOXA-AS2 significantly inhibited it. We also observed that miR-567 directly targets the 3' UTR of CDK8. Moreover, silencing lncRNA HOXA-AS2 inhibited tumor growth with the expression of miR-567 increased and CDK8 decreased in vivo. CONCLUSION: LncRNA HOXA-AS2 was up-regulated in OSCC, and its up-regulation correlated with poor clinical outcomes. The lncRNA also promoted OSCC cell proliferation by directly binding to miR-567, leading to an increase in CDK8 expression. The potential prognostic value of lncRNA HOXA-AS2 should be explored in future studies.

Development of a simplified and inexpensive RNA depletion method for plasmid DNA purification using size selection magnetic beads (SSMBs).

Plasmid DNA (pDNA) isolation from bacterial cells is one of the most common and critical steps in molecular cloning and biomedical research. Almost all pDNA purification involves disruption of bacteria, removal of membrane lipids, proteins and genomic DNA, purification of pDNA from bulk lysate, and concentration of pDNA for downstream applications. While many liquid-phase and solid-phase pDNA purification methods are used, the final pDNA preparations are usually contaminated with varied degrees of host RNA, which cannot be completely digested by RNase A. To develop a simple, cost-effective, and yet effective method for RNA depletion, we investigated whether commercially available size selection magnetic beads (SSMBs), such as Mag-Bind® TotalPure NGS Kit (or Mag-Bind), can completely deplete bacterial RNA in pDNA preparations. In this proof-of-principle study, we demonstrated that, compared with RNase A digestion and two commercial plasmid affinity purification kits, the SSMB method was highly efficient in depleting contaminating RNA from pDNA minipreps. Gene transfection and bacterial colony formation assays revealed that pDNA purified from SSMB method had superior quality and integrity to pDNA samples cleaned up by RNase A digestion and/or commercial plasmid purification kits. We further demonstrated that the SSMB method completely depleted contaminating RNA in large-scale pDNA samples. Furthermore, the Mag-bind-based SSMB method costs only 5-10% of most commercial plasmid purification kits on a per sample basis. Thus, the reported SSMB method can be a valuable and inexpensive tool for the removal of bacterial RNA for routine pDNA preparations.

Notch signaling: Its essential roles in bone and craniofacial development.

Notch is a cell-cell signaling pathway that is involved in a host of activities including development, oncogenesis, skeletal homeostasis, and much more. More specifically, recent research has demonstrated the importance of Notch signaling in osteogenic differentiation, bone healing, and in the development of the skeleton. The craniofacial skeleton is complex and understanding its development has remained an important focus in biology. In this review we briefly summarize what recent research has revealed about Notch signaling and the current understanding of how the skeleton, skull, and face develop. We then discuss the crucial role that Notch plays in both craniofacial development and the skeletal system, and what importance it may play in the future.

Long non-coding RNA (LncRNA) HOTAIR regulates BMP9-induced osteogenic differentiation by targeting the proliferation of mesenchymal stem cells (MSCs).

Long non-coding RNAs are important regulators of biological processes, but their roles in the osteogenic differentiation of mesenchymal stem cells (MSCs) remain unclear. Here we investigated the role of murine HOX transcript antisense RNA (mHotair) in BMP9-induced osteogenic differentiation of MSCs using immortalized mouse adipose-derived cells (iMADs). Touchdown quantitative polymerase chain reaction analysis found increased mHotair expression in bones in comparison with most other tissues. Moreover, the level of mHotair in femurs peaked at the age of week-4, a period of fast skeleton development. BMP9 could induce earlier peak expression of mHotair during in vitro iMAD osteogenesis. Silencing mHotair diminished BMP9-induced ALP activity, matrix mineralization, and expression of osteogenic, chondrogenic and adipogenic markers. Cell implantation experiments further confirmed that knockdown of mHotair attenuated BMP9-induced ectopic bone formation and mineralization of iMADs, leading to more undifferentiated cells. Crystal violet staining and cell cycle analysis revealed that silencing of mHotair promoted the proliferation of iMAD cells regardless of BMP9 induction. Moreover, ectopic bone masses developed from mHotair-knockdown iMAD cells exhibited higher expression of PCNA than the control group. Taken together, our results demonstrated that murine mHotair is an important regulator of BMP9-induced MSC osteogenesis by targeting cell cycle and proliferation.

FAMSi: A Synthetic Biology Approach to the Fast Assembly of Multiplex siRNAs for Silencing Gene Expression in Mammalian Cells.

RNA interference (RNAi) is mediated by an ∼21-nt double-stranded small interfering RNA (siRNA) and shows great promise in delineating gene functions and in developing therapeutics for human diseases. However, effective gene silencing usually requires the delivery of multiple siRNAs for a given gene, which is often technically challenging and time-consuming. In this study, by exploiting the type IIS restriction endonuclease-based synthetic biology methodology, we developed the fast assembly of multiplex siRNAs (FAMSi) system. In our proof-of-concept experiments, we demonstrated that multiple fragments containing three, four, or five siRNA sites targeting common Smad4 and/or BMPR-specific Smad1, Smad5, and Smad8 required for BMP9 signaling could be assembled efficiently. The constructed multiplex siRNAs effectively knocked down the expression of Smad4 and/or Smad1, Smad5, and Smad8 in mesenchymal stem cells (MSCs), and they inhibited all aspects of BMP9-induced osteogenic differentiation in bone marrow MSCs (BMSCs), including decreased expression of osteogenic regulators/markers, reduced osteogenic marker alkaline phosphatase (ALP) activity, and diminished in vitro matrix mineralization and in vivo ectopic bone formation. Collectively, we demonstrate that the engineered FAMSi system provides a fast-track platform for assembling multiplexed siRNAs in a single vector, and thus it may be a valuable tool to study gene functions or to develop novel siRNA-based therapeutics.

A reverse transcriptase-mediated ribosomal RNA depletion (RTR2D) strategy for the cost-effective construction of RNA sequencing libraries.

RNA sequencing (RNA-seq)-based whole transcriptome analysis (WTA) using ever-evolving next-generation sequencing technologies has become a primary tool for coding and/or noncoding transcriptome profiling. As WTA requires RNA-seq data for both coding and noncoding RNAs, one key step for obtaining high-quality RNA-seq data is to remove ribosomal RNAs, which can be accomplished by using various commercial kits. Nonetheless, an ideal rRNA removal method should be efficient, user-friendly and cost-effective so it can be adapted for homemade RNA-seq library construction. Here, we developed a novel reverse transcriptase-mediated ribosomal RNA depletion (RTR2D) method. We demonstrated that RTR2D was simple and efficient, and depleted human or mouse rRNAs with high specificity without affecting coding and noncoding transcripts. RNA-seq data analysis indicated that RTR2D yielded highly correlative transcriptome landscape with that of NEBNext rRNA Depletion Kit at both mRNA and lncRNA levels. In a proof-of-principle study, we found that RNA-seq dataset from RTR2D-depleted rRNA samples identified more differentially expressed mRNAs and lncRNAs regulated by Nutlin3A in human osteosarcoma cells than that from NEBNext rRNA Depletion samples, suggesting that RTR2D may have lower off-target depletion of non-rRNA transcripts. Collectively, our results have demonstrated that the RTR2D methodology should be a valuable tool for rRNA depletion.

Microvesicles (MIVs) secreted from adipose-derived stem cells (ADSCs) contain multiple microRNAs and promote the migration and invasion of endothelial cells.

Extracellular vesicles (EVs) such as microvesicles (MIVs) play an important role in intercellular communications. MIVs are small membrane vesicles sized 100-1000 nm in diameter that are released by many types of cells, such as mesenchymal stem cells (MSCs), tumor cells and adipose-derived stem cells (ADSC). As EVs can carry out autocrine and paracrine functions by controlling multiple cell processes, it is conceivable that EVs can be used as delivery vehicles for treating several clinical conditions, such as to improve cardiac angiogenesis after myocardial infarction (MI). Here, we seek to investigate whether ADSC-derived MIVs contain microRNAs that regulate angiogenesis and affect cell migration of endothelial cells. We first characterized the ADSC-derived MIVs and found that the MIVs had a size range of 100-300 nm, and expressed the MIV marker protein Alix. We then analyzed the microRNAs in ADSCs and ADSC-derived MIVs and demonstrated that ADSC-derived MIVs selectively released a panel of microRNAs, several of which were related to angiogenesis, including two members of the let-7 family. Furthermore, we demonstrated that ADSC-derived MIVs promoted the cell migration and invasion of the HUVEC endothelial cells. The PKH26-labeled ADSC-derived MIVs were effectively uptaken into the cytoplasm of HUVEC cells. Collectively, our results demonstrate that the ADSC-derived MIVs can promote migration and invasion abilities of endothelial cells, suggesting pro-angiogenetic potential. Future studies should focus on investigating the roles and mechanisms through which ADSC-derived MIVs regulate angiogenesis.

Highly expressed BMP9/GDF2 in postnatal mouse liver and lungs may account for its pleiotropic effects on stem cell differentiation, angiogenesis, tumor growth and metabolism.

Bone morphogenetic protein 9 (BMP9) (or GDF2) was originally identified from fetal mouse liver cDNA libraries. Emerging evidence indicates BMP9 exerts diverse and pleiotropic functions during postnatal development and in maintaining tissue homeostasis. However, the expression landscape of BMP9 signaling during development and/or in adult tissues remains to be analyzed. Here, we conducted a comprehensive analysis of the expression landscape of BMP9 and its signaling mediators in postnatal mice. By analyzing mouse ENCODE transcriptome datasets we found Bmp9 was highly expressed in the liver and detectable in embryonic brain, adult lungs and adult placenta. We next conducted a comprehensive qPCR analysis of RNAs isolated from major mouse tissues/organs at various ages. We found that Bmp9 was highly expressed in the liver and lung tissues of young adult mice, but decreased in older mice. Interestingly, Bmp9 was only expressed at low to modest levels in developing bones. BMP9-associated TGFß/BMPR type I receptor Alk1 was highly expressed in the adult lungs. Furthermore, the feedback inhibitor Smads Smad6 and Smad7 were widely expressed in mouse postnatal tissues. However, the BMP signaling antagonist noggin was highly expressed in fat and heart in the older age groups, as well as in kidney, liver and lungs in a biphasic fashion. Thus, our findings indicate that the circulating BMP9 produced in liver and lungs may account for its pleiotropic effects on postnatal tissues/organs although possible roles of BMP9 signaling in liver and lungs remain to be fully understood.

Leptin Potentiates BMP9-Induced Osteogenic Differentiation of Mesenchymal Stem Cells Through the Activation of JAK/STAT Signaling.

Mesenchymal stem cells (MSCs) are multipotent progenitors that have the ability to differentiate into multiple lineages, including bone, cartilage, and fat. We previously demonstrated that the least known bone morphogenetic protein (BMP)9 (also known as growth differentiation factor 2) is one of the potent osteogenic factors that can induce both osteogenic and adipogenic differentiation of MSCs. Nonetheless, the molecular mechanism underlying BMP9 action remains to be fully understood. Leptin is an adipocyte-derived hormone in direct proportion to the amount of body fat, and exerts pleiotropic functions, such as regulating energy metabolism, bone mass, and mineral density. In this study, we investigate the potential effect of leptin signaling on BMP9-induced osteogenic differentiation of MSCs. We found that exogenous leptin potentiated BMP9-induced osteogenic differentiation of MSCs both in vitro and in vivo, while inhibiting BMP9-induced adipogenic differentiation. BMP9 was shown to induce the expression of leptin and leptin receptor in MSCs, while exogenous leptin upregulated BMP9 expression in less differentiated MSCs. Mechanistically, we demonstrated that a blockade of JAK signaling effectively blunted leptin-potentiated osteogenic differentiation induced by BMP9. Taken together, our results strongly suggest that leptin may potentiate BMP9-induced osteogenesis by cross-regulating BMP9 signaling through the JAK/STAT signaling pathway in MSCs. Thus, it is conceivable that a combined use of BMP9 and leptin may be explored as a novel approach to enhancing efficacious bone regeneration and fracture healing.

A simplified system for the effective expression and delivery of functional mature microRNAs in mammalian cells.

MicroRNAs (miRNAs) are ~22 nucleotide noncoding RNAs that are involved in virtually all aspects of cellular process as their deregulations are associated with many pathological conditions. Mature miRNAs (mMIRs) are generated through a series of tightly-regulated nuclear and cytoplasmic processing events of the transcribed primary, precursor and mMIRs. Effective manipulations of miRNA expression enable us to gain insights into miRNA functions and to explore potential therapeutic applications. Currently, overexpression of miRNAs is achieved by using chemically-synthesized miRNA mimics, or shRNA-like stem-loop vectors to express primary or precursor miRNAs, which are limited by low transfection efficacy or rate-limiting miRNA processing. To overcome rate-limiting miRNA processing, we developed a novel strategy to express mMIRs which are driven by converging U6/H1 dual promoters. As a proof-of-concept study, we constructed mMIR expression vectors for hsa-miR-223 and hsa-Let-7a-1, and demonstrated that the expressed mMIRs effectively silenced target gene expression, specifically suppressed miRNA reporter activity, and significantly affected cell proliferation, similar to respective primary and precursor miRNAs. Furthermore, these mMIR expression vectors can be easily converted into retroviral and adenoviral vectors. Collectively, our simplified mMIR expression system should be a valuable tool to study miRNA functions and/or to deliver miRNA-based therapeutics.

lncRNA Rmst acts as an important mediator of BMP9-induced osteogenic differentiation of mesenchymal stem cells (MSCs) by antagonizing Notch-targeting microRNAs.

Understanding the bone and musculoskeletal system is essential to maintain the health and quality of life of our aging society. Mesenchymal stem cells (MSCs) can undergo self-renewal and differentiate into multiple tissue types including bone. We demonstrated that BMP9 is the most potent osteogenic factors although molecular mechanism underlying BMP9 action is not fully understood. Long noncoding RNAs (lncRNAs) play important regulatory roles in many physiological and/or pathologic processes. Here, we investigated the role of lncRNA Rmst in BMP9-induced osteogenic differentiation of MSCs. We found that Rmst was induced by BMP9 through Smad signaling in MSCs. Rmst knockdown diminished BMP9-induced osteogenic, chondrogenic and adipogenic differentiation in vitro, and attenuated BMP9-induced ectopic bone formation. Silencing Rmst decreased the expression of Notch receptors and ligands. Bioinformatic analysis predicted Rmst could directly bind to eight Notch-targeting miRNAs, six of which were downregulated by BMP9. Silencing Rmst restored the expression of four microRNAs (miRNAs). Furthermore, an activating Notch mutant NICD1 effectively rescued the decreased ALP activity caused by Rmst silencing. Collectively, our results strongly suggest that the Rmst-miRNA-Notch regulatory axis may play an important role in mediating BMP9-induced osteogenic differentiation of MSCs.

Developing a Versatile Shotgun Cloning Strategy for Single-Vector-Based Multiplex Expression of Short Interfering RNAs (siRNAs) in Mammalian Cells.

As an important post-transcriptional regulatory machinery mediated by ∼21nt short-interfering double-stranded RNA (siRNA), RNA interference (RNAi) is a powerful tool to delineate gene functions and develop therapeutics. However, effective RNAi-mediated silencing requires multiple siRNAs for given genes, a time-consuming process to accomplish. Here, we developed a user-friendly system for single-vector-based multiplex siRNA expression by exploiting the unique feature of restriction endonuclease BstXI. Specifically, we engineered a BstXI-based shotgun cloning (BSG) system, which consists of three entry vectors with siRNA expression units (SiEUs) flanked with distinct BstXI sites, and a retroviral destination vector for shotgun SiEU assembly. For proof-of-principle studies, we constructed multiplex siRNA vectors silencing ß-catenin and/or Smad4 and assessed their functionalities in mesenchymal stem cells (MSCs). Pooled siRNA cassettes were effectively inserted into respective entry vectors in one-step, and shotgun seamless assembly of pooled BstXI-digested SiEU fragments into a retroviral destination vector followed. We found these multiplex siRNAs effectively silenced ß-catenin and/or Smad4, and inhibited Wnt3A- or BMP9-specific reporters and downstream target expression in MSCs. Furthermore, multiplex silencing of ß-catenin and/or Smad4 diminished Wnt3A and/or BMP9-induced osteogenic differentiation. Collectively, the BSG system is a user-friendly technology for single-vector-based multiplex siRNA expression to study gene functions and develop experimental therapeutics.

PMPCB Silencing Sensitizes HCC Tumor Cells to Sorafenib Therapy.

Hepatocellular carcinoma (HCC) tumors invariably develop resistance to cytotoxic and targeted agents, resulting in failed treatment and tumor recurrence. Previous in vivo short hairpin RNA (shRNA) screening evidence revealed mitochondrial-processing peptidase (PMPC) as a leading gene contributing to tumor cell resistance against sorafenib, a multikinase inhibitor used to treat advanced HCC. Here, we investigated the contributory role of the ß subunit of PMPC (PMPCB) in sorafenib resistance. Silencing PMPCB increased HCC tumor cell susceptibility to sorafenib therapy, decreased liver tumor burden, and improved survival of tumor-bearing mice receiving sorafenib. Moreover, sorafenib + PMPCB shRNA combination therapy led to attenuated liver tumor burden and improved survival outcome for tumor-bearing mice, and it reduced colony formation in murine and human HCC cell lines in vitro. Additionally, PMPCB silencing enhanced PINK1-Parkin signaling and downregulated the anti-apoptotic protein MCL-1 in sorafenib-treated HCC cells, which is indicative of a healthier pro-apoptotic phenotype. Higher pre-treatment MCL-1 expression was associated with inferior survival outcomes in sorafenib-treated HCC patients. Elevated MCL-1 expression was present in sorafenib-resistant murine HCC cells, while MCL-1 knockdown sensitized these cells to sorafenib. In conclusion, our findings advocate combination regimens employing sorafenib with PMPCB knockdown or MCL-1 knockdown to circumvent sorafenib resistance in HCC patients.

A pH-Triggered, Self-Assembled, and Bioprintable Hybrid Hydrogel Scaffold for Mesenchymal Stem Cell Based Bone Tissue Engineering.

Effective bone tissue engineering can restore bone and skeletal functions that are impaired by traumas and/or certain medical conditions. Bone is a complex tissue and functions through orchestrated interactions between cells, biomechanical forces, and biofactors. To identify ideal scaffold materials for effective mesenchymal stem cell (MSC)-based bone tissue regeneration, here we develop and characterize a composite nanoparticle hydrogel by combining carboxymethyl chitosan (CMCh) and amorphous calcium phosphate (ACP) (designated as CMCh-ACP hydrogel). We demonstrate that the CMCh-ACP hydrogel is readily prepared by incorporating glucono δ-lactone (GDL) into an aqueous dispersion or rehydrating the acidic freeze-dried nanoparticles in a pH-triggered controlled-assembly fashion. The CMCh-ACP hydrogel exhibits excellent biocompatibility and effectively supports MSC proliferation and cell adhesion. Moreover, while augmenting BMP9-induced osteogenic differentiation, the CMCh-ACP hydrogel itself is osteoinductive and induces the expression of osteoblastic regulators and bone markers in MSCs in vitro. The CMCh-ACP scaffold markedly enhances the efficiency and maturity of BMP9-induced bone formation in vivo, while suppressing bone resorption occurred in long-term ectopic osteogenesis. Thus, these results suggest that the pH-responsive self-assembled CMCh-ACP injectable and bioprintable hydrogel may be further exploited as a novel scaffold for osteoprogenitor-cell-based bone tissue regeneration.

BMP9-induced osteoblastic differentiation requires functional Notch signaling in mesenchymal stem cells.

Mesenchymal stem cells (MSCs) are multipotent progenitors that can differentiate into multiple lineages including osteoblastic lineage. Osteogenic differentiation of MSCs is a cascade that recapitulates most, if not all, of the molecular events occurring during embryonic skeletal development, which is regulated by numerous signaling pathways including bone morphogenetic proteins (BMPs). Through a comprehensive analysis of the osteogenic activity, we previously demonstrated that BMP9 is the most potent BMP for inducing bone formation from MSCs both in vitro and in vivo. However, as one of the least studied BMPs, the essential mediators of BMP9-induced osteogenic signaling remain elusive. Here we show that BMP9-induced osteogenic signaling in MSCs requires intact Notch signaling. While the expression of Notch receptors and ligands are readily detectable in MSCs, Notch inhibitor and dominant-negative Notch1 effectively inhibit BMP9-induced osteogenic differentiation in vitro and ectopic bone formation in vivo. Genetic disruption of Notch pathway severely impairs BMP9-induced osteogenic differentiation and ectopic bone formation from MSCs. Furthermore, while BMP9-induced expression of early-responsive genes is not affected by defective Notch signaling, BMP9 upregulates the expression of Notch receptors and ligands at the intermediate stage of osteogenic differentiation. Taken together, these results demonstrate that Notch signaling may play an essential role in coordinating BMP9-induced osteogenic differentiation of MSCs.

Transcriptomic landscape regulated by the 14 types of bone morphogenetic proteins (BMPs) in lineage commitment and differentiation of mesenchymal stem cells (MSCs).

Mesenchymal stem cells (MSCs) are ubiquitously-existing multipotent progenitors that can self-renew and differentiate into multiple lineages including osteocytes, chondrocytes, adipocytes, tenocytes and myocytes. MSCs represent one of the most commonly-used adult progenitors and serve as excellent progenitor cell models for investigating lineage-specific differentiation regulated by various cellular signaling pathways, such as bone morphogenetic proteins (BMPs). As members of TGFß superfamily, BMPs play diverse and important roles in development and adult tissues. At least 14 BMPs have been identified in mammals. Different BMPs exert distinct but overlapping biological functions. Through a comprehensive analysis of 14 BMPs in MSCs, we demonstrated that BMP9 is one of the most potent BMPs in inducing osteogenic differentiation of MSCs. Nonetheless, a global mechanistic view of BMP signaling in regulating the proliferation and differentiation of MSCs remains to be fully elucidated. Here, we conducted a comprehensive transcriptomic profiling in the MSCs stimulated by 14 types of BMPs. Hierarchical clustering analysis classifies 14 BMPs into three subclusters: an osteo/chondrogenic/adipogenic cluster, a tenogenic cluster, and BMP3 cluster. We also demonstrate that six BMPs (e.g., BMP2, BMP3, BMP4, BMP7, BMP8, and BMP9) can induce I-Smads effectively, while BMP2, BMP3, BMP4, BMP7, and BMP11 up-regulate Smad-independent MAP kinase pathway. Furthermore, we show that many BMPs can upregulate the expression of the signal mediators of Wnt, Notch and PI3K/AKT/mTOR pathways. While the reported transcriptomic changes need to be further validated, our expression profiling represents the first-of-its-kind to interrogate a comprehensive transcriptomic landscape regulated by the 14 types of BMPs in MSCs.