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.

Reversibly immortalized human umbilical cord-derived mesenchymal stem cells (UC-MSCs) are responsive to BMP9-induced osteogenic and adipogenic differentiation.

Human mesenchymal stem cells (MSCs) are a heterogeneous subset of nonhematopoietic multipotent stromal stem cells and can differentiate into mesodermal lineage, such as adipocytes, osteocytes, and chondrocytes, as well as ectodermal and endodermal lineages. Human umbilical cord (UC) is one of the most promising sources of MSCs. However, the molecular and cellular characteristics of UC-derived MSCs (UC-MSCs) require extensive investigations, which are hampered by the limited lifespan and the diminished potency over passages. Here, we used the piggyBac transposon-based simian virus 40 T antigen (SV40T) immortalization system and effectively immortalized UC-MSCs, yielding the iUC-MSCs. A vast majority of the immortalized lines are positive for MSC markers but not for hematopoietic markers. The immortalization phenotype of the iUC-MSCs can be effectively reversed by flippase recombinase-induced the removal of SV40T antigen. While possessing long-term proliferation capability, the iUC-MSCs are not tumorigenic in vivo. Upon bone morphogenetic protein 9 (BMP9) stimulation, the iUC-MSC cells effectively differentiate into osteogenic, chondrogenic, and adipogenic lineages both in vitro and in vivo, which is indistinguishable from that of primary UC-MSCs, indicating that the immortalized UC-MSCs possess the characteristics similar to that of their primary counterparts and retain trilineage differentiation potential upon BMP9 stimulation. Therefore, the engineered iUC-MSCs should be a valuable alternative cell source for studying UC-MSC biology and their potential utilities in immunotherapies and regenerative medicine.

Niclosamide Exhibits Potent Anticancer Activity and Synergizes with Sorafenib in Human Renal Cell Cancer Cells.

BACKGROUND/AIMS: As the most lethal urological cancers, renal cell carcinoma (RCC) comprises a heterogeneous group of cancer with diverse genetic and molecular alterations. There is an unmet clinical need to develop efficacious therapeutics for advanced, metastatic and/or relapsed RCC. Here, we investigate whether anthelmintic drug Niclosamide exhibits anticancer activity and synergizes with targeted therapy Sorafenib in suppressing RCC cell proliferation. METHODS: Cell proliferation and migration were assessed by Crystal violet staining, WST-1 assay, cell wounding and cell cycle analysis. Gene expression was assessed by qPCR. In vivo anticancer activity was assessed in xenograft tumor model. RESULTS: We find that Niclosamide effectively inhibits cell proliferation, cell migration and cell cycle progression, and induces apoptosis in human renal cancer cells. Mechanistically, Niclosamide inhibits the expression of C-MYC and E2F1 while inducing the expression of PTEN in RCC cells. Niclosamide is further shown to synergize with Sorafenib in suppressing RCC cell proliferation and survival. In the xenograft tumor model, Niclosamide is shown to effectively inhibit tumor growth and suppress RCC cell proliferation. CONCLUSIONS: Niclosamide may be repurposed as a potent anticancer agent, which can potentiate the anticancer activity of the other agents targeting different signaling pathways in the treatment of human RCC.

The Contrasting Effects of Alum-Treated Chicken Manures and KH2PO4 on Phosphorus Behavior in Soils.

Alum [KAl(SO)⋅12HO] is often added to chicken manure to limit P solubility after land application. This is generally ascribed to the formation of Al-PO complexes. However, Al-PO complex formation could be affected by the matrix of chicken manure, which varies with animal diet. Alum was added to KHPO (as a reference material) and two manures from typical chicken farms in China, one from an intensive farm (CMIF) and another from free-ranging chickens (CMFR). These were subsequently incubated with soils for 100 d to investigate P transformations. Alum reduced water-soluble colorimetrically reactive phosphorus (RP) from soils amended with manure more effectively than in soils amended with KHPO. Alum addition lowered Mehlich-3 RP in soils with CMFR but had no influence on Mehlich-3 RP in CMIF- or KHPO-amended soils. A comparison of P in digested Mehlich-3 extracts with RP in undigested samples showed significantly increased P in digests of alum-treated CMFR only. Fractionation data indicated that alum treatment increased P in the NHF-RP (Al-P) fraction only in soils with KHPO, but not in soils with manure treatments. Furthermore, NaOH-extracted nonreactive P was markedly higher in soil with alum-treated CMFR relative to normal CMFR. The CMFR manure was assumed to contain higher concentrations of organic P because these chickens were fed grains only. These results suggest that the formation of alum-organic P complexes may reduce P solubility. By comparing alum-treated KHPO and manures, it appears that organic matter in manure could interfere with the formation of Al-PO complexes.

BMP9 induces osteogenesis and adipogenesis in the immortalized human cranial suture progenitors from the patent sutures of craniosynostosis patients.

The cranial suture complex is a heterogeneous tissue consisting of osteogenic progenitor cells and mesenchymal stem cells (MSCs) from bone marrow and suture mesenchyme. The fusion of cranial sutures is a highly coordinated and tightly regulated process during development. Craniosynostosis is a congenital malformation caused by premature fusion of cranial sutures. While the progenitor cells derived from the cranial suture complex should prove valuable for studying the molecular mechanisms underlying suture development and pathogenic premature suture fusion, primary human cranial suture progenitors (SuPs) have limited life span and gradually lose osteoblastic ability over passages. To overcome technical challenges in maintaining sufficient and long-term culture of SuPs for suture biology studies, we establish and characterize the reversibly immortalized human cranial suture progenitors (iSuPs). Using a reversible immortalization system expressing SV40 T flanked with FRT sites, we demonstrate that primary human suture progenitor cells derived from the patent sutures of craniosynostosis patients can be efficiently immortalized. The iSuPs maintain long-term proliferative activity, express most of the consensus MSC markers and can differentiate into osteogenic and adipogenic lineages upon BMP9 stimulation in vitro and in vivo. The removal of SV40 T antigen by FLP recombinase results in a decrease in cell proliferation and an increase in the endogenous osteogenic and adipogenic capability in the iSuPs. Therefore, the iSuPs should be a valuable resource to study suture development, intramembranous ossification and the pathogenesis of craniosynostosis, as well as to explore cranial bone tissue engineering.

Notch Signaling Augments BMP9-Induced Bone Formation by Promoting the Osteogenesis-Angiogenesis Coupling Process in Mesenchymal Stem Cells (MSCs).

BACKGROUND/AIMS: Mesenchymal stem cells (MSCs) are multipotent progenitors that can differentiate into several lineages including bone. Successful bone formation requires osteogenesis and angiogenesis coupling of MSCs. Here, we investigate if simultaneous activation of BMP9 and Notch signaling yields effective osteogenesis-angiogenesis coupling in MSCs. METHODS: Recently-characterized immortalized mouse adipose-derived progenitors (iMADs) were used as MSC source. Transgenes BMP9, NICD and dnNotch1 were expressed by adenoviral vectors. Gene expression was determined by qPCR and immunohistochem¡stry. Osteogenic activity was assessed by in vitro assays and in vivo ectopic bone formation model. RESULTS: BMP9 upregulated expression of Notch receptors and ligands in iMADs. Constitutively-active form of Notch1 NICD1 enhanced BMP9-induced osteogenic differentiation both in vitro and in vivo, which was effectively inhibited by dominant-negative form of Notch1 dnNotch1. BMP9- and NICD1-transduced MSCs implanted with a biocompatible scaffold yielded highly mature bone with extensive vascularization. NICD1 enhanced BMP9-induced expression of key angiogenic regulators in iMADs and Vegfa in ectopic bone, which was blunted by dnNotch1. CONCLUSION: Notch signaling may play an important role in BMP9-induced osteogenesis and angiogenesis. It's conceivable that simultaneous activation of the BMP9 and Notch pathways should efficiently couple osteogenesis and angiogenesis of MSCs for successful bone tissue engineering.

MiR-362-3p inhibits the proliferation and migration of vascular smooth muscle cells in atherosclerosis by targeting ADAMTS1.

Atherosclerosis is a progressive condition of the large arteries that can cause coronary artery disease (CAD). Growing amounts of evidence have indicated that microRNAs (miRNAs, miRs) can be used as diagnostic biomarkers in many cellular processes associated with CAD. MiR-362-3p has been implicated in many biological cellular functions. However, little is known about the role of miR-362-3p during atherosclerosis. In the present study, significant downregulation of miR-362-3p was observed in 110 atherosclerotic CAD patients and not in the 84 controls. The upregulation of miR-362-3p was demonstrated to inhibit vascular smooth muscle cell (VSMC) proliferation and migration, and impede the G1/S cell cycle transition. Bioinformatics analysis indicated that a disintegrin and metalloproteinase with thrombospondin motifs 1 (ADAMTS1) was a direct target of miR-362-3p. Subsequent experiments demonstrated that miR-362-3p binds to the 3'-untranslated region (UTR) of ADAMTS1 and decreases its levels of mRNA and protein expression. Overexpression of ADAMTS1 partially restored the miR-362-3p-mediated inhibition of VSMC proliferation, cell cycle, and migration. Upregulation of ADAMTS1 in plasma samples was detected in atherosclerotic CAD patients. Taken together, our findings suggested that miR-362-3p inhibits the proliferation and migration of VSMCs by directly targeting ADAMTS1, which might provide novel insight into the molecular mechanisms underlying the action of miR-362-3p in atherosclerosis.

Transient Receptor Potential Vanilloid Subtype 1 Inhibits Inflammation and Apoptosis via the Release of Calcitonin Gene-Related Peptide in the Heart after Myocardial Infarction.

OBJECTIVE: A high mortality rate occurs with silent myocardial infarction (MI), particularly in aging and diabetic populations due to defects in the transient receptor potential vanilloid (TRPV1)-positive sensory nerve function. We have previously shown that TRPV1 deficiency markedly enhances post-MI inflammation and remodeling. However, the mechanisms remain unknown. The objective of this study was to clarify whether calcitonin gene-related peptide (CGRP) release was associated with the protective role of TRPV1 against postmyocardial inflammation and apoptosis. METHODS: TRPV1 gene knockout (TRPV1KO) and wild-type (WT) mice were subjected to left anterior descending ligation or sham operation. The concentration of CGRP in the myocardium was measured at 30 min, 1, 6 and 24 h post-MI. Mice received saline vehicle, CGRP or the CGRP antagonist CGRP8-37 before ligation. Inflammation was evaluated by ELISA assay and histological staining. Apoptosis was assessed by Western blot and TUNEL assay. RESULTS: Post-MI, both TRPV1KO and WT mice displayed elevated CGRP levels in myocardium when compared to sham controls. However, the levels of CGRP were significantly lower in TRPV1KO mice than in WT mice at 30 min after MI. Exogenous CGRP downregulated the levels of tumor necrosis factor-α and interleukin-6 expression in TRPV1KO mice post-MI. Moreover, exogenous CGRP decreased the neutrophil infiltration in TRPV1KO mice, whereas inhibition of CGRP by CGRP8-37 increased the neutrophil infiltration in WT mice. Western blotting data indicated that CGRP attenuated caspase-3 and caspase-9 expression, and enhanced Bcl-2 expression in TRPV1KO mice post-MI. CGRP8-37 upregulated caspase-3 and caspase-9 expression and downregulated Bcl-2 expression in WT mice. CONCLUSION: Our data suggest a protective role of TRPV1 activation against inflammation and apoptosis in mice post-MI, possibly through CGRP release. These findings elucidate a neurogenic mechanism in mice post-MI, which may participate in sensory neurotransmitter-mediated protection in TRPV1 activation.

MicroRNA-101 overexpression by IL-6 and TNF-α inhibits cholesterol efflux by suppressing ATP-binding cassette transporter A1 expression.

BACKGROUND: MicroRNAs play key roles in regulating cholesterol homeostasis. Here, we investigated the role of microRNA-101 (miR-101) in regulating ATP-binding cassette transporter A1 (ABCA1) expression and cholesterol efflux under non-inflammatory and inflammatory conditions in human THP-1-derived macrophages and HepG2 hepatoblastoma cells. METHODS: The cell lines were transfected with one of four lentiviral vectors: miR-101, miR-101 control, anti-miR-101, or anti-miR-101 control. A luciferase reporter assay was used to examine miR-101 binding to the 3' untranslated region (UTR) of ABCA1. Western blotting was conducted to assess ABCA1 protein expression. Cells were loaded with BODIPY-cholesterol and stained with oil red O to assess cholesterol efflux. RESULTS: The luciferase activity assay revealed that wild-type miR-101 binding at site 2 significantly repressed ABCA1 3' UTR activity, suggesting that miR-101 directly targets the ABCA1 mRNA at site 2. In both cell lines, Western blotting revealed that miR-101 expression negatively regulates ABCA1 protein expression and significantly suppresses cholesterol efflux to ApoA1 under both low-density lipoprotein (LDL) and non-LDL conditions, which was confirmed by pronounced lipid inclusions visible by oil red O staining. In HepG2 cells, both IL-6 and TNF-α treatments produced significant miR-101 overexpression; however, in THP-1-derived macrophages, only IL-6 treatment produced significant miR-101 overexpression. Anti-mir-101 transfection under both IL-6 and TNF-α treatment conditions led to ABCA1 upregulation, indicating that miR-101 expression represses ABCA1 expression under inflammatory conditions. CONCLUSIONS: miR-101 promotes intracellular cholesterol retention under inflammatory conditions through suppressing ABCA1 expression and suggests that the miR-101-ABCA1 axis may play an intermediary role in the development of NAFLD and vascular atherosclerosis.

The effectiveness of preoperative trimetazidine on myocardial preservation in coronary artery bypass graft patients: a systematic review and meta-analysis.

BACKGROUND: Coronary artery bypass grafting (CABG) is a key and effective surgical treatment modality for coronary artery disease. Unfortunately, ischemia-reperfusion injury during and after CABG can lead to reversible and irreversible myocardial damage. Trimetazidine [1-(2,3,4-trimethoxybenzyl)piperazine dihydrochloride] is a metabolic anti-ischemic agent with demonstrated cardioprotective effects; however, its effects with respect to myocardial preservation in CABG patients remain unclear. METHODS: We conducted a systematic review and meta-analysis of randomized controlled trials (RCTs) to investigate the effectiveness of myocardial preservation of preoperative trimetazidine therapy in CABG patients by assessing the postoperative levels of several blood-based biochemical markers of myocardial injury, including creatine kinase (CK), creatine kinase-muscle and brain (CK-MB), creatine phosphokinase (CPK), troponin T (TnT) and troponin I (TnI). The RCTs were classified into two subgroup analyses by the timing of sample collection (either ≤12 or >12 h after CABG). RESULTS: Six RCTs were finally included in the meta-analysis. The pooled effect sizes showed significantly lower postoperative levels of CK, CK-MB, TnT and TnI in the trimetazidine-treated CABG patients relative to control CABG patients. However, there were no significant differences in the postoperative CPK levels between trimetazidine-treated CABG patients relative to control CABG patients. In both the ≤12 and >12 h post-CABG subgroup analyses, significant differences in CK, CK-MB, TnT and TnI were detected between the trimetazidine-treated CABG patients relative to control CABG patients. CONCLUSIONS: Preoperative trimetazidine therapy appears to have a positive effect on myocardial preservation in CABG patients.

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.].

Establishment and functional characterization of the reversibly immortalized mouse glomerular podocytes (imPODs).

Glomerular podocytes are highly specialized epithelial cells and play an essential role in establishing the selective permeability of the glomerular filtration barrier of kidney. Maintaining the viability and structural integrity of podocytes is critical to the clinical management of glomerular diseases, which requires a thorough understanding of podocyte cell biology. As mature podocytes lose proliferative capacity, a conditionally SV40 mutant tsA58-immortalized mouse podocyte line (designated as tsPC) was established from the Immortomouse over 20 years ago. However, the utility of the tsPC cells is hampered by the practical inconvenience of culturing these cells. In this study, we establish a user-friendly and reversibly-immortalized mouse podocyte line (designated as imPOD), on the basis of the tsPC cells by stably expressing the wildtype SV40 T-antigen, which is flanked with FRT sites. We show the imPOD cells exhibit long-term high proliferative activity, which can be effectively reversed by FLP recombinase. The imPOD cells express most podocyte-related markers, including WT-1, Nephrin, Tubulin and Vinculin, but not differentiation marker Synaptopodin. The imPOD cells do not form tumor-like masses in vivo. We further demonstrate that TGFß1 induces a podocyte injury-like response in the FLP-reverted imPOD cells by suppressing the expression of slit diaphragm-associated proteins P-Cadherin and ZO-1 and upregulating the expression of mesenchymal markers, α-SMA, Vimentin and Nestin, as well as fibrogenic factors CTGF and Col1a1. Collectively, our results strongly demonstrate that the newly engineered imPOD cells should be a valuable tool to study podocyte biology both under normal and under pathological conditions.

A blockade of PI3Kγ signaling effectively mitigates angiotensin II-induced renal injury and fibrosis in a mouse model.

Chronic kidney disease (CKD) poses a formidable challenge for public healthcare worldwide as vast majority of patients with CKD are also at risk of accelerated cardiovascular disease and death. Renal fibrosis is the common manifestation of CKD that usually leads to end-stage renal disease although the molecular events leading to chronic renal fibrosis and eventually chronic renal failure remain to be fully understood. Nonetheless, emerging evidence suggests that an aberrant activation of PI3Kγ signaling may play an important role in regulating profibrotic phenotypes. Here, we investigate whether a blockade of PI3Kγ signaling exerts any beneficial effect on alleviating kidney injury and renal fibrosis. Using a mouse model of angiotensin II (Ang II)-induced renal damage, we demonstrate that PI3Kγ inhibitor AS605240 effectively mitigates Ang II-induced increases in serum creatinine and blood urea nitrogen, renal interstitial collagen deposition, the accumulation of ECM proteins and the expression of α-Sma and fibrosis-related genes in vivo. Mechanistically, we reveal that AS605240 effectively inhibits Ang II-induced cell proliferation and phosphorylation of Akt in fibroblast cells. Furthermore, we demonstrate that Ang II-upregulated expression of IL-6, Tnf-α, IL-1ß and Tgf-ß1 is significantly attenuated in the mice treated with AS605240. Taken together, our results demonstrate that PI3Kγ may function as a critical mediator of Ang II-induced renal injury and fibrosis. It is thus conceivable that targeted inhibition of PI3Kγ signaling may constitute a novel therapeutic approach to the clinical management of renal fibrosis, renal hypertension and/or CKD.

The development of a sensitive fluorescent protein-based transcript reporter for high throughput screening of negative modulators of lncRNAs.

While the human genome is pervasively transcribed, <2% of the human genome is transcribed into protein-coding mRNAs, leaving most of the transcripts as noncoding RNAs, such as microRNAs and long-noncoding RNAs (lncRNAs), which are critical components of epigenetic regulation. lncRNAs are emerging as critical regulators of gene expression and genomic stability. However, it remains largely unknown about how lncRNAs are regulated. Here, we develop a highly sensitive and dynamic reporter that allows us to identify and/or monitor negative modulators of lncRNA transcript levels in a high throughput fashion. Specifically, we engineer a fluorescent fusion protein by fusing three copies of the PEST destruction domain of mouse ornithine decarboxylase (MODC) to the C-terminal end of the codon-optimized bilirubin-inducible fluorescent protein, designated as dBiFP, and show that the dBiFP protein is highly destabilized, compared with the commonly-used eGFP protein. We further demonstrate that the dBiFP signal is effectively down-regulated when the dBiFP and mouse lncRNA H19 chimeric transcript is silenced by mouse H19-specific siRNAs. Therefore, our results strongly suggest that the dBiFP fusion protein may serve as a sensitive and dynamic transcript reporter to monitor the inhibition of lncRNAs by microRNAs, synthetic regulatory RNA molecules, RNA binding proteins, and/or small molecule inhibitors so that novel and efficacious inhibitors targeting the epigenetic circuit can be discovered to treat human diseases such as cancer and other chronic disorders.

Thermoresponsive Citrate-Based Graphene Oxide Scaffold Enhances Bone Regeneration from BMP9-Stimulated Adipose-Derived Mesenchymal Stem Cells.

Effective bone tissue engineering is important to overcome the unmet clinical challenges as more than 1.6 million bone grafts are done annually in the United States. Successful bone tissue engineering needs minimally three critical constituents: osteoprogenitor cells, osteogenic factors, and osteoinductive/osteoconductive scaffolds. Osteogenic progenitors are derived from multipotent mesenchymal stem cells (MSCs), which can be prepared from numerous tissue sources, including adipose tissue. We previously showed that BMP9 is the most osteogenic BMP and induces robust bone formation of immortalized mouse adipose-derived MSCs entrapped in a citrate-based thermoresponsive hydrogel referred to as PPCNg. As graphene and its derivatives emerge as promising biomaterials, here we develop a novel thermosensitive and injectable hybrid material by combining graphene oxide (GO) with PPCNg (designated as GO-P) and characterize its ability to promote bone formation. We demonstrate that the thermoresponsive behavior of the hybrid material is maintained while effectively supporting MSC survival and proliferation. Furthermore, GO-P induces early bone-forming marker alkaline phosphatase (ALP) and potentiates BMP9-induced expression of osteogenic regulators and bone markers as well as angiogenic factor VEGF in MSCs. In vivo studies show BMP9-transduced MSCs entrapped in the GO-P scaffold form well-mineralized and highly vascularized trabecular bone. Thus, these results indicate that GO-P hybrid material may function as a new biocompatible, injectable scaffold with osteoinductive and osteoconductive activities for bone regeneration.

Adenovirus-Mediated Gene Delivery: Potential Applications for Gene and Cell-Based Therapies in the New Era of Personalized Medicine.

With rapid advances in understanding molecular pathogenesis of human diseases in the era of genome sciences and systems biology, it is anticipated that increasing numbers of therapeutic genes or targets will become available for targeted therapies. Despite numerous setbacks, efficacious gene and/or cell-based therapies still hold the great promise to revolutionize the clinical management of human diseases. It is wildly recognized that poor gene delivery is the limiting factor for most in vivo gene therapies. There has been a long-lasting interest in using viral vectors, especially adenoviral vectors, to deliver therapeutic genes for the past two decades. Among all currently available viral vectors, adenovirus is the most efficient gene delivery system in a broad range of cell and tissue types. The applications of adenoviral vectors in gene delivery have greatly increased in number and efficiency since their initial development. In fact, among over 2,000 gene therapy clinical trials approved worldwide since 1989, a significant portion of the trials have utilized adenoviral vectors. This review aims to provide a comprehensive overview on the characteristics of adenoviral vectors, including adenoviral biology, approaches to engineering adenoviral vectors, and their applications in clinical and pre-clinical studies with an emphasis in the areas of cancer treatment, vaccination and regenerative medicine. Current challenges and future directions regarding the use of adenoviral vectors are also discussed. It is expected that the continued improvements in adenoviral vectors should provide great opportunities for cell and gene therapies to live up to its enormous potential in personalized medicine.

Noncanonical Wnt signaling plays an important role in modulating canonical Wnt-regulated stemness, proliferation and terminal differentiation of hepatic progenitors.

The liver provides vital metabolic, exocrine and endocrine functions in the body as such pathological conditions of the liver lead to high morbidity and mortality. The liver is highly regenerative and contains facultative stem cells that become activated during injury to replicate to fully recover mass and function. Canonical Wnt/ß-catenin signaling plays an important role in regulating the proliferation and differentiation of liver progenitor cells during liver regeneration. However, possible roles of noncanonical Wnts in liver development and regeneration remain undefined. We previously established a reversibly-immortalized hepatic progenitor cell line (iHPx), which retains hepatic differentiation potential. Here, we analyze the expression pattern of the essential components of both canonical and noncanonical Wnt signaling pathways at different postnatal stages of mouse liver tissues and iHPx cells. We find that noncanonical Wnt4, Wnt5a, Wnt9b, Wnt10a and Wnt10b, are highly expressed concordantly with the high levels of canonical Wnts in late stages of liver tissues. Wnt5a, Wnt9b, Wnt10a and Wnt10b are able to antagonize Wnt3a-induced ß-catenin/TCF activity, reduce the stemness of iHPx cells, and promote hepatic differentiation of liver progenitors. Stem cell implantation assay demonstrates that Wnt5a, Wnt9b, Wnt10a and Wnt10b can inhibit cell proliferation and promote hepatic differentiation of the iHPx progenitor cells. Our results strongly suggest that noncanonical Wnts may play an important role in fine-tuning Wnt/ß-catenin functions during liver development and liver regeneration. Thus, understanding regulatory mechanisms governing proliferation and differentiation of liver progenitor cells may hold great promise to facilitate liver regeneration and/or progenitor cell-based therapies for liver diseases.

CRISPR/Cas9-mediated reversibly immortalized mouse bone marrow stromal stem cells (BMSCs) retain multipotent features of mesenchymal stem cells (MSCs).

Mesenchymal stem cells (MSCs) are multipotent non-hematopoietic progenitor cells that can undergo self-renewal and differentiate into multi-lineages. Bone marrow stromal stem cells (BMSCs) represent one of the most commonly-used MSCs. In order to overcome the technical challenge of maintaining primary BMSCs in long-term culture, here we seek to establish reversibly immortalized mouse BMSCs (imBMSCs). By exploiting CRISPR/Cas9-based homology-directed-repair (HDR) mechanism, we target SV40T to mouse Rosa26 locus and efficiently immortalize mouse BMSCs (i.e., imBMSCs). We also immortalize BMSCs with retroviral vector SSR #41 and establish imBMSC41 as a control line. Both imBMSCs and imBMSC41 exhibit long-term proliferative capability although imBMSC41 cells have a higher proliferation rate. SV40T mRNA expression is 130% higher in imBMSC41 than that in imBMSCs. However, FLP expression leads to 86% reduction of SV40T expression in imBMSCs, compared with 63% in imBMSC41 cells. Quantitative genomic PCR analysis indicates that the average copy number of SV40T and hygromycin is 1.05 for imBMSCs and 2.07 for imBMSC41, respectively. Moreover, FLP expression removes 92% of SV40T in imBMSCs at the genome DNA level, compared with 58% of that in imBMSC41 cells, indicating CRISPR/Cas9 HDR-mediated immortalization of BMSCs can be more effectively reversed than that of retrovirus-mediated random integrations. Nonetheless, both imBMSCs and imBMSC41 lines express MSC markers and are highly responsive to BMP9-induced osteogenic, chondrogenic and adipogenic differentiation in vitro and in vivo. Thus, the engineered imBMSCs can be used as a promising alternative source of primary MSCs for basic and translational research in the fields of MSC biology and regenerative medicine.

lncRNA H19 mediates BMP9-induced osteogenic differentiation of mesenchymal stem cells (MSCs) through Notch signaling.

Mesenchymal stem cells (MSCs) are multipotent progenitor cells that can undergo self-renewal and differentiate into multiple lineages. Osteogenic differentiation from MSCs is a well-orchestrated process and regulated by multiple signaling pathways. We previously demonstrated that BMP9 is one of the most potent osteogenic factors. However, molecular mechanism through which BMP9 governs osteoblastic differentiation remains to be fully understood. Increasing evidence indicates noncoding RNAs (ncRNAs) may play important regulatory roles in many physiological and/or pathologic processes. In this study, we investigate the role of lncRNA H19 in BMP9-regulated osteogenic differentiation of MSCs. We demonstrated that H19 was sharply upregulated at the early stage of BMP9 stimulation of MSCs, followed by a rapid decease and gradual return to basal level. This process was correlated with BMP9-induced expression of osteogenic markers. Interestingly, either constitutive H19 expression or silencing H19 expression in MSCs significantly impaired BMP9-induced osteogenic differentiation in vitro and in vivo, which was effectively rescued by the activation of Notch signaling. Either constitutive H19 expression or silencing H19 expression led to the increased expression of a group of miRNAs that are predicted to target Notch ligands and receptors. Thus, these results indicate that lncRNA H19 functions as an important mediator of BMP9 signaling by modulating Notch signaling-targeting miRNAs. Our findings suggest that the well-coordinated biphasic expression of lncRNA H19 may be essential in BMP9-induced osteogenic differentiation of MSCs, and that dysregulated H19 expression may impair normal osteogenesis, leading to pathogenic processes, such as bone tumor development.