The evolving roles of Wnt signaling in stem cell proliferation and differentiation, the development of human diseases, and therapeutic opportunities

The evolutionarily conserved Wnt signaling pathway plays a central role in develop-ment and adult tissue homeostasis across species.Wnt proteins are secreted,lipid-modified signaling molecules that activate the canonical(β-catenin dependent)and non-canonical(β-catenin independent)Wnt signaling pathways.Cellular behaviors such as proliferation,differ-entiation,maturation,and proper body-axis specification are carried out by the canonical pathway,which is the best characterized of the known Wnt signaling paths.Wnt signaling has emerged as an important factor in stem cell biology and is known to affect the self-renewal of stem cells in various tissues.This includes but is not limited to embryonic,hematopoietic,mesenchymal,gut,neural,and epidermal stem cells.Wnt signaling has also been implicated in tumor cells that exhibit stem cell-like properties.Wnt signaling is crucial for bone formation and presents a potential target for the development of therapeutics for bone disorders.Not surprisingly,aberrant Wnt signaling is also associated with a wide variety of diseases,including cancer.Mutations of Wnt pathway members in cancer can lead to unchecked cell proliferation,epithelial-mesenchymal transition,and metastasis.Altogether,advances in the understand-ing of dysregulated Wnt signaling in disease have paved the way for the development of novel therapeutics that target components of the Wnt pathway.Beginning with a brief overview of the mechanisms of canonical and non-canonical Wnt,this review aims to summarize the cur-rent knowledge of Wnt signaling in stem cells,aberrations to the Wnt pathway associated with diseases,and novel therapeutics targeting the Wnt pathway in preclinical and clinical studies.

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.

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.

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.

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.

Engineered nucleus-free mesenchymal stem cells (MSCs) for the targeted delivery of therapeutics to disease site

Specialized therapeutic delivery, or use of pharmaceuticals and other biomaterials to target specific parts of the body or diseased tissue, has long been sought as an ideal way of treating human diseases. A recent article published in Nature Biomedical Engineering revealed an innovative strategy to engineer nucleus-free human mesenchymal stem cells (MSCs) for targeted delivery of therapeutics to disease site.1 MSCs have emerged as promising vehicles of therapeutic delivery.2,3 MSCs are undifferentiated pluripotent stem cells derived from areas such as bone marrow and adipose tissue.4,5 MSCs are sought after for their chemotaxis, or ability to home towards a chemical stimulus, and capacity for modification with elements such as chemoattractant receptors and adhesion molecules.1 These properties allow for site-specific and minimally-invasive therapeutic administration and treatment.