The Wnt pathway is central to a host of developmental and disease-related processes. The remarkable conservation of this intercellular signaling cascade throughout metazoan lineages indicates that it coevolved with multicellularity to regulate the generation and spatial arrangement of distinct cell types. By regulating cell fate specification, mitotic activity, and cell polarity, Wnt signaling orchestrates development and tissue homeostasis, and its dysregulation is implicated in developmental defects, cancer, and degenerative disorders. We review advances in our understanding of this key pathway, from Wnt protein production and secretion to relay of the signal in the cytoplasm of the receiving cell. We discuss the evolutionary history of this pathway as well as endogenous and synthetic modulators of its activity. Finally, we highlight remaining gaps in our knowledge of Wnt signal transduction and avenues for future research. Expected final online publication date for the Annual Review of Biochemistry, Volume 91 is June 2022. Please see http://www.annualreviews.org/page/journal/pubdates for revised estimates.

The Wnt Pathway: From Signaling Mechanisms to Synthetic Modulators.

Repair of large bone defects remains a significant clinical challenge. Bone marrow stromal cells (BMSCs), a subset of which is known as bone marrow-derived mesenchymal stem cells, show therapeutic potential for bone regeneration. However, their isolation, expansion and implantation will need to be conducted under good manufacturing practices (GMP) at separate locations. An investigation which mimics this clinical scenario where large bone defects shall be regenerated is required before clinical trials can be initiated. Seven batches of 100 million human ex-vivo expanded BMSCs from five donors were transported fresh in syringes from a GMP facility in Germany to France. BMSCs were mixed with biphasic calcium phosphate (BCP) biomaterial prior to subcutaneous implantation in nude mice. The capacity of BMSCs in unison with BCP to regenerate critical sized cranial bone defects was also evaluated. BMSCs expressing luciferase were used to assess the viability and bio-distribution of implanted cells. In situ hybridization, using the human-specific repetitive Alu sequence, was performed for the identification of human cells in explants. Eight weeks after implantation of BMSCs, mineralized bone containing mature bone marrow territories was formed in ectopic sites and in calvaria defects. Significant loss of cell viability was observed by bioluminescence imaging and only 1.5 percent of the initial number of transplanted cells remained after 37 days. After eight weeks, while explants were comprised primarily of host cells, there were also human cells attached along the periphery of BCP and embedded in osteocyte lacunae dispersed throughout the newly formed bone matrix. This study demonstrates the safety and efficacy of BMSC/BCP combinations and provides crucial information for the implementation of BMSC therapy for bone regeneration.

https://www.hal.inserm.fr/inserm-01205389/document

Pre-clinical studies of bone regeneration with human bone marrow stromal cells and biphasic calcium phosphate.

Microfluidic 3D Printing Responsive Scaffolds with Biomimetic Enrichment Channels for Bone Regeneration

In multicellular systems, oriented cell divisions are essential for morphogenesis and homeostasis as they determine the position of daughter cells within the tissue and also, in many cases, their fate. Early studies in invertebrates led to the identification of conserved core mechanisms of mitotic spindle positioning centred on the Gαi-LGN-NuMA-dynein complex. In recent years, much has been learnt about the way this complex functions in vertebrate cells. In particular, studies addressed how the Gαi-LGN-NuMA-dynein complex dynamically crosstalks with astral microtubules and the actin cytoskeleton, and how it is regulated to orient the spindle according to cellular and tissue-wide cues. We have also begun to understand how dynein motors and actin regulators interact with mechanosensitive adhesion molecules sensing extracellular mechanical stimuli, such as cadherins and integrins, and with signalling pathways so as to respond to extracellular cues instructing the orientation of the division axis in vivo. In this Review, with the focus on epithelial tissues, we discuss the molecular mechanisms of mitotic spindle orientation in vertebrate cells, and how this machinery is regulated by epithelial cues and extracellular signals to maintain tissue cohesiveness during mitosis. We also outline recent knowledge of how spindle orientation impacts tissue architecture in epithelia and its emerging links to the regulation of cell fate decisions. Finally, we describe how defective spindle orientation can be corrected or its effects eliminated in tissues under physiological conditions, and the pathological implications associated with spindle misorientation.

Spindle positioning and its impact on vertebrate tissue architecture and cell fate

The maintenance of human skeletal stem cells (hSSCs) and their progeny in bone defects is a major challenge. Here, we report on a transplantable bandage containing a three-dimensional Wnt-induced osteogenic tissue model (WIOTM). This bandage facilitates the long-term viability of hSSCs (8 weeks) and their progeny, and enables bone repair in an in vivo mouse model of critical-sized calvarial defects. The newly forming bone is structurally comparable to mature cortical bone and consists of human and murine cells. Furthermore, we show that the mechanism of WIOTM formation is governed by Wnt-mediated asymmetric cell division of hSSCs. Covalently immobilizing Wnts onto synthetic materials can polarize single dividing hSSCs, orient the spindle and simultaneously generate a Wnt-proximal hSSC and a differentiation-prone Wnt-distal cell. Our results provide insight into the regulation of human osteogenesis and represent a promising approach to deliver human osteogenic constructs that can survive in vivo and contribute to bone repair. Wnt3a protein has been immobilized on a biocompatible bandage and is now shown to induce oriented asymmetric cell division of human skeletal stem cells and can also promote bone tissue repair in vivo.

/pdf/wnt-modified-materials-mediate-asymmetric-stem-cell-division-2ui6pdrnlm.pdf

Wnt-modified materials mediate asymmetric stem cell division to direct human osteogenic tissue formation for bone repair

Spatial cellular organization is fundamental for embryogenesis. Remarkably, coculturing embryonic stem cells (ESCs) and trophoblast stem cells (TSCs) recapitulates this process, forming embryo-like structures. However, mechanisms driving ESC-TSC interaction remain elusive. We describe specialized ESC-generated cytonemes that react to TSC-secreted Wnts. Cytoneme formation and length are controlled by actin, intracellular calcium stores, and components of the Wnt pathway. ESC cytonemes select self-renewal-promoting Wnts via crosstalk between Wnt receptors, activation of ionotropic glutamate receptors (iGluRs), and localized calcium transients. This crosstalk orchestrates Wnt signaling, ESC polarization, ESC-TSC pairing, and consequently synthetic embryogenesis. Our results uncover ESC-TSC contact-mediated signaling, reminiscent of the glutamatergic neuronal synapse, inducing spatial self-organization and embryonic cell specification.

https://www.pnas.org/content/pnas/117/13/7236.full.pdf

Specialized cytonemes induce self-organization of stem cells

To coordinate cell fate with changes in spatial organization, stem cells (SCs) require specific and adaptable systems of signal exchange and cell-to-cell communication. Pluripotent embryonic stem cells (ESCs) use cytonemes to pair with trophoblast stem cells (TSCs) and form synthetic embryonic structures in a Wnt-dependent manner. How these interactions vary with pluripotency states remains elusive. Here we show that ESC transition to an early primed ESC (pESC) state reduces their pairing with TSCs and impairs synthetic embryogenesis. pESCs can activate the Wnt/β-catenin pathway in response to soluble Wnt ligands, but their cytonemes form unspecific and unstable interactions with localized Wnt sources. This is due to an impaired crosstalk between Wnt and glutamate receptor activity and reduced generation of Ca2+ transients on the cytonemes upon Wnt source contact. Induced iGluR activation can partially restore cytoneme function in pESCs, while transient overexpression of E-cadherin improves pESC-TSC pairing. Our results illustrate how changes in pluripotency state alter the mechanisms SCs use to self-organize.

https://rupress.org/jcb/article-pdf/220/4/e202005095/1412445/jcb_202005095.pdf

Pluripotency state regulates cytoneme selectivity and self-organization of embryonic stem cells.

The Wnt-pathway is part of a signalling network that regulates many aspects of cell biology. Recently, we discovered crosstalk between AMPA/Kainate-type ionotropic glutamate receptors (iGluRs) and the Wnt-pathway during the initial Wnt3a-interaction at the cytonemes of mouse embryonic stem cells (ESCs). Here, we demonstrate that this crosstalk persists throughout the Wnt3a-response in ESCs. Both AMPA and Kainate receptors regulate early Wnt3a-recruitment, dynamics on the cell membrane, and orientation of the spindle towards a Wnt3a-source at mitosis. AMPA receptors specifically are required for segregating cell fate components during Wnt3a-mediated asymmetric cell division (ACD). Using Wnt-pathway component knockout lines, we determine that Wnt co-receptor Lrp6 has particular functionality over Lrp5 in cytoneme formation, and in facilitating ACD. Both Lrp5 and 6, alongside pathway effector β-catenin act in concert to mediate the positioning of the dynamic interaction with, and spindle orientation to, a localised Wnt3a-source. Wnt-iGluR crosstalk may prove pervasive throughout embryonic and adult stem cell signalling.

Wnt- and glutamate-receptors orchestrate stem cell dynamics and asymmetric cell division.

Wnt signalling regulates many aspects of cell biology. Wnt-pathway activation and its downstream effects have been extensively studied, but the dynamic analysis of Wnt-ligands on mammalian cellular membranes is obstructed by difficulties of visualization. We overcome this using microbead-tethered Wnts presented to single embryonic stem cells, which undergo Wnt-mediated asymmetric cell division (ACD). Through live imaging and genetic editing, we show that knockout of Wnt co-receptor Lrp5 promotes cytoneme formation and Wnt-recruitment, which requires Lrp6 and β-catenin. Lrp5 facilitates ligand-retention at the membrane, and alongside Lrp6 mediates Wnt-ligand stabilization and positioning. β-catenin or Wnt co-receptor knockout causes misorientation at mitosis, and all but Lrp5 are required for Wnt-orientated ACD. Surprisingly, ionotropic glutamate receptor (iGluR) activity enables initial Wnt-recruitment, positioning, and ultimately oriented ACD. Uniquely, we have scrutinized the early Wnt ligand-membrane interaction, linking roles of Wnt-pathway components and crosstalk with iGluRs in guiding cell fate determination by oriented ACD.

/pdf/wnt-and-glutamate-receptors-orchestrate-stem-cell-dynamics-148uxkjgt6.pdf

Joshua Reeves

Papers

Wnt-modified materials mediate asymmetric stem cell division to direct human osteogenic tissue formation for bone repair

Specialized cytonemes induce self-organization of stem cells

Pluripotency state regulates cytoneme selectivity and self-organization of embryonic stem cells.

Wnt- and glutamate-receptors orchestrate stem cell dynamics and asymmetric cell division.

Wnt- and Glutamate-receptors orchestrate stem cell dynamics and asymmetric cell division