Molecular pathogenesis and therapeutic strategies of human osteosarcoma

Osteosarcoma（OS）is a devastating illness with rapid rates of dissemination and a poor overall prognosis,despite aggressive standard-of-care surgical techniques and combination chemotherapy regimens.Identifying the molecular mechanisms involved in disease pathogenesis and progression may offer insight into new therapeutic targets.Defects in mesenchymal stem cell differentiation,abnormal expression of oncogenes and tumor suppressors,and dysregulation within various important signaling pathways have all been implicated in development of various disease phenotypes.As such,a variety of basic science and translational studies have shown promise in identifying novel markers and modulators of these disease-specific aberrancies.Born out of these and similar investigations,a variety of emerging therapies are now undergoing various phases of OS clinical testing.They broadly include angiogenesis inhibitors,drugs that act on the bone microenvironment,receptor tyrosine kinase inhibitors,immune system modulators,and other radio-or chemo-sensitizing agents.As new forms of drug delivery are being developed simultaneously,the possibility of targeting tumors locally while minimizing systemic toxicityis is seemingly more achievable now than ever.In this review,we not only summarize our current understanding of OS disease processes,but also shed light on the multitude of potential therapeutic strategies the scientific community can use to make long-term improvements in patient prognosis.

A simplified noncryogenic strategy to transport mesenchymal stem cells: Potential applications in cell therapy and regenerative medicine

With the rapid advances in stem cell research and po-tential cell-based therapies,there is an urgent need to develop safe and reliable cell transport strategies.Except for autologous stem cell-based therapies,allogeneic stem cell therapies and ex vivo genetically engineered cell therapies would require safe,efficient,and reliable cell preservation and transport methods.

ATDC5来源外泌体包载小分子药物5Z-7-Oxozeaenol治疗骨关节炎

骨关节炎(osteoarthritis,OA)是一种常见的退行性关节疾病。研究表明,TAK1的抑制剂小分子药物5Z-7-Oxozeaenol(5Z-7),用于治疗OA时直接将药物进行频繁关节腔注射,药物的治疗效果有限。本研究选取小鼠胚胎瘤成软骨细胞系(ATDC5),是一种理想的成软骨细胞模型,其增殖速度和培养稳定性均优于间充质干细胞,用于提取外泌体作为药物的载体。本研究提取ATDC5来源的外泌体(ATDC5-Exos),包载药物5Z-7。在炎性细胞因子诱导大鼠软骨细胞模型中,载药外泌体可以促进合成代谢相关基因Col2a1、Sox9的表达,抑制分解代谢相关基因Adamts5、Mmp13的表达。本研究使用8周龄雄性小鼠,行前交叉韧带离断术(ACLT)诱导OA小鼠模型,关节腔注射外泌体或载药外泌体治疗,取膝关节石蜡切片进行组织学评估。结果显示,载药外泌体可缓解创伤后OA模型的病理表型。结合Micro-CT影像学结果显示,治疗能改善ACLT术后膝关节软骨下骨骨小梁的流失和骨赘减少,关节表面更为光滑。本研究证实,ATDC5-Exos包载药物5Z-7在体内和体外实验中均可缓解OA表型。外泌体包载递送5Z-7减少了药物的用量和给药频率,且药物和外泌体可以叠加治疗改善OA的效果。

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 2000 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 preclinical 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.

The wonders of BMP9:From mesenchymal stem cell differentiation,angiogenesis,neurogenesis,tumorigenesis,and metabolism to regenerative medicine

Although bone morphogenetic proteins(BMPs)initially showed effective induction of ectopic bone growth in muscle,it has since been determined that these proteins,as members of the TGF-b superfamily,play a diverse and critical array of biological roles.These roles include regulating skeletal and bone formation,angiogenesis,and development and homeostasis of multiple organ systems.Disruptions of the members of the TGF-b/BMP superfamily result in severe skeletal and extra-skeletal irregularities,suggesting high therapeutic potential from understanding this family of BMP proteins.Although it was once one of the least characterized BMPs,BMP9 has revealed itself to have the highest osteogenic potential across numerous experiments both in vitro and in vivo,with recent studies suggesting that the exceptional potency of BMP9 may result from unique signaling pathways that differentiate it from other BMPs.The effectiveness of BMP9 in inducing bone formation was recently revealed in promising experiments that demonstrated efficacy in the repair of critical sized cranial defects as well as compatibility with bone-inducing bio-implants,revealing the great translational promise of BMP9.Furthermore,emerging evidence indicates that,besides its osteogenic activity,BMP9 exerts a broad range of biological functions,including stem cell differentiation,angiogenesis,neurogenesis,tumorigenesis,and metabolism.This review aims to summarize our current understanding of BMP9 across biology and the body.

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

The authors regret having an image assembly error in Figure 3A,in which the image for "imPOD Synaptopodin DAPl stain"groupwas erroneouslyduplicatedwiththe imagefrom the"tsPOD-33C SynaptopodinDAPIstain"group.We confirm the error is restricted to the image assembly,and the underlying data and conclusions are correct and unchanged.The authors would like to apologize for any inconvenience caused.

Canonical and noncanonical Wnt signaling: Multilayered mediators, signaling mechanisms and major signaling crosstalk

Wnt signaling plays a major role in regulating cell proliferation and differentiation.The Wnt ligands are a family of 19 secreted glycoproteins that mediate their signaling effects via binding to Frizzled receptors and LRP5/6 coreceptors and transducing the signal either throughβ-catenin in the canonical pathway or through a series of other proteins in the nonca-nonical pathway.Many of the individual components of both canonical and noncanonical Wnt signaling have additional functions throughout the body,establishing the complex interplay between Wnt signaling and other signaling pathways.This crosstalk between Wnt signaling and other pathways gives Wnt signaling a vital role in many cellular and organ processes.Dys-regulation of this system has been implicated in many diseases affecting a wide array of organ systems,including cancer and embryological defects,and can even cause embryonic lethality.The complexity of this system and its interacting proteins have made Wnt signaling a target for many therapeutic treatments.However,both stimulatory and inhibitory treatments come with potential risks that need to be addressed.This review synthesized much of the current knowl-edge on the Wnt signaling pathway,beginning with the history of Wnt signaling.It thoroughly described the different variants of Wnt signaling,including canonical,noncanonical Wnt/PCP,and the noncanonical Wnt/Ca2+pathway.Further description involved each of its components and their involvement in other cellular processes.Finally,this review explained the various other pathways and processes that crosstalk with Wnt signaling.

SATB2:A versatile transcriptional regulator of craniofacial and skeleton development,neurogenesis and tumorigenesis,and its applications in regenerative medicine

SATB2(special AT-rich sequence-binding protein 2)is a member of the special AT-rich binding protein family.As a transcription regulator,SATB2 mainly integrates higher-order chromatin organization.SATB2 expression appears to be tissue-and stage-specific,and is governed by several cellular signaling molecules and mediators.Expressed in branchial arches and osteoblast-lineage cells,SATB2 plays a significant role in craniofacial pattern and skeleton development.In addition to regulating osteogenic differentiation,SATB2 also displays versatile functions in neural development and cancer progression.As an osteoinductive factor,SATB2 holds great promise in improving bone regeneration toward bone defect repair.In this review,we have summarized our current understanding of the physiological and pathological functions of SATB2 in craniofacial and skeleton development,neurogenesis,tumorigenesis and regenerative medicine.

Insulin-like growth factor(IGF)signaling in tumorigenesis and the development of cancer drug resistance

One of the greatest obstacles to current cancer treatment efforts is the development of drug resistance by tumors.Despite recent advances in diagnostic practices and surgical interventions,many neoplasms demonstrate poor response to adjuvant or neoadjuvant radiation and chemotherapy.As a result,the prognosis for many patients afflicted with these aggressive cancers remains bleak.The insulin-like growth factor(IGF)signaling axis has been shown to play critical role in the development and progression of various tumors.Many basic science and translational studies have shown that IGF pathway modulators can have promising effects when used to treat various malignancies.There also exists a substantial body of recent evidence implicating IGF signaling dysregulation in the dwindling response of tumors to current standard-of-care therapy.By better understanding both the IGF-dependent and-independent mechanisms by which pathway members can influence drug sensitivity,we can eventually aim to use modulators of IGF signaling to augment the effects of current therapy.This review summarizes and synthesizes numerous recent investigations looking at the role of the IGF pathway in drug resistance.We offer a brief overview of IGF signaling and its general role in neoplasia,and then delve into detail about the many types of human cancer that have been shown to have IGF pathway involvement in resistance and/or sensitization to therapy.Ultimately,our hope is that such a compilation of evidence will compel investigators to carry out much needed studies looking at combination treatment with IGF signaling modulators to overcome current therapy resistance.

The versatile functions of Sox9 in development,stem cells,and human diseases

The transcription factor Sox9 was first discovered in patients with campomelic dysplasia,a haploinsufficiency disorder with skeletal deformities caused by dysregulation of Sox9 expression during chondrogenesis.Since then,its role as a cell fate determiner during embryonic development has been well characterized;Sox9 expression differentiates cells derived from all three germ layers into a large variety of specialized tissues and organs.However,recent data has shown that ectoderm-and endoderm-derived tissues continue to express Sox9 in mature organs and stem cell pools,suggesting its role in cell maintenance and specification during adult life.The versatility of Sox9 may be explained by a combination of posttranscriptional modifications,binding partners,and the tissue type in which it is expressed.Considering its importance during both development and adult life,it follows that dysregulation of Sox9 has been implicated in various congenital and acquired diseases,including fibrosis and cancer.This review provides a summary of the various roles of Sox9 in cell fate specification,stem cell biology,and related human diseases.Ultimately,understanding the mechanisms that regulate Sox9 will be crucial for developing effective therapies to treat disease caused by stem cell dysregulation or even reverse organ damage.

Multifaceted signaling regulators of chondrogenesis:Implications in cartilage regeneration and tissue engineering

Defects of articular cartilage present a unique clinical challenge due to its poor self-healing capacity and avascular nature.Current surgical treatment options do not ensure consistent regeneration of hyaline cartilage in favor of fibrous tissue.Here,we review the current understanding of the most important biological regulators of chondrogenesis and their interactions,to provide insight into potential applications for cartilage tissue engineering.These include various signaling pathways,including fibroblast growth factors(FGFs),transforming growth factor b(TGF-b)/bone morphogenic proteins(BMPs),Wnt/b-catenin,Hedgehog,Notch,hypoxia,and angiogenic signaling pathways.Transcriptional and epigenetic regulation of chondrogenesis will also be discussed.Advances in our understanding of these signaling pathways have led to promising advances in cartilage regeneration and tissue engineering.

Neural EGF-like protein 1(NELL-1):Signaling crosstalk in mesenchymal stem cells and applications in regenerative medicine

Bone tissue regeneration holds the potential to solve both osteoporosis and large skeletal defects,two problems associated with significant morbidity.The differentiation of mesenchymal stem cells into the osteogenic lineage requires a specific microenvironment and certain osteogenic growth factors.Neural EGF Like-Like molecule 1(NELL-1)is a secreted glycoprotein that has proven,both in vitro and in vivo,to be a potent osteo-inductive factor.Furthermore,it has been shown to repress adipogenic differentiation and inflammation.NELL-1 can work synergistically with other osteogenic factors such as Bone Morphogenic Protein(BMP)
2 and
9,and has shown promise for use in tissue engineering and as a systemically administered drug for the treatment of osteoporosis.Here we provide a comprehensive up-to-date review on the molecular signaling cascade of NELL-1 in mesenchymal stem cells and potential applications in bone regenerative engineering.

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 lineagespecific differentiation regulated by various cellular signaling pathways,such as bone morphogenetic proteins(BMPs).As members of TGFb 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 ISmads 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.

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 100e1000 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 e300 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.

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 TGFb1 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,a-SMA,Vimentin and Nestin,as well as fibrogenic factors CTGF and Col1a1.Collectively,our results strongly demonstrate that the newly engineered im-POD cells should be a valuable tool to study podocyte biology both under normal and under pathological conditions.

Stem cell therapy for chronic skin wounds in the era of personalized medicine:From bench to bedside

With the significant financial burden of chronic cutaneous wounds on the healthcare system,not to the personal burden mention on those individuals afflicted,it has become increasingly essential to improve our clinical treatments.This requires the translation of the most recent benchtop approaches to clinical wound repair as our current treatment modalities have proven insufficient.The most promising potential treatment options rely on stem cellbased therapies.Stem cell proliferation and signaling play crucial roles in every phase of the wound healing process and chronic wounds are often associated with impaired stem cell function.Clinical approaches involving stem cells could thus be utilized in some cases to improve a body’s inhibited healing capacity.We aim to present the laboratory research behind the mechanisms and effects of this technology as well as current clinical trials which showcase their therapeutic potential.Given the current problems and complications presented by chronic wounds,we hope to show that developing the clinical applications of stem cell therapies is the rational next step in improving wound care.

Notch signaling:Its essential roles in bone and craniofacial development

Notch is a cellecell 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 noncoding RNA(lncRNA)H19:An essential developmental regulator with expanding roles in cancer,stem cell differentiation,and metabolic diseases

Recent advances in deep sequencing technologies have revealed that,while less than 2%of the human genome is transcribed into mRNA for protein synthesis,over 80%of the genome is transcribed,leading to the production of large amounts of noncoding RNAs(ncRNAs).It has been shown that ncRNAs,especially long non-coding RNAs(lncRNAs),may play crucial regulatory roles in gene expression.As one of the first isolated and reported lncRNAs,H19 has gained much attention due to its essential roles in regulating many physiological and/or pathological processes including embryogenesis,development,tumorigenesis,osteogen-esis,and metabolism.Mechanistically,H19 mediates diverse regulatory functions by serving as competing endogenous RNAs(CeRNAs),Igf2/H19 imprinted tandem gene,modular scaffold,cooperating with H19 antisense,and acting directly with other mRNAs or lncRNAs.Here,we summarized the current understanding of H19 in embryogenesis and development,cancer development and progression,mesenchymal stem cell lineage-specific differentiation,and metabolic diseases.We discussed the potential regulatory mechanisms underlying H19’s func-tions in those processes although more in-depth studies are warranted to delineate the exact molecular,cellular,epigenetic,and genomic regulatory mechanisms underlying the physiolog-ical and pathological roles of H19.Ultimately,these lines of investigation may lead to the development of novel therapeutics for human diseases by exploiting H19 functions.

Damage control:Harnessing prostaglandin E2 as a potential healing factor of tissue injuries

Increasing prostaglandin E2 by knocking out its inhibitor 15-hydroxyprostaglandin dehydrogenase(15-PDGH)or administering a compound that inhibits 15-PDGH was recently found to improve healing in hematopoietic stem cell transplants,colitis recovery,and hepatogenesis after transection in mice.These results are suggestive of pharmacologic therapies or even genetic therapy that could improve patient outcomes,especially since the excess PGE2 and the 15-PDGH inhibitor have proven to be non-toxic.However,elevated levels of PGE2 are associated with increased risk of cancer and blood clotting problems.It would be unacceptable to treat a cancer patient with chemotherapy and replenish the hematopoietic stem cells with the help of PGE2,only to have increased expression of PGE2 and induce another cancer.Therefore,to assess the most therapeutic aspects of PGE2,it is important to consider effects that could induce disease.

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 in-volves disruption of bacteria,removal of membrane lipids,proteins and genomic DNA,purifi-cation 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.