Current approaches in tissue engineering are geared toward generating tissue-specific stem cells. Given the complexity and heterogeneity of tissues, this approach has its limitations. An alternate approach is to induce terminally differentiated cells to dedifferentiate into multipotent proliferative cells with the capacity to regenerate all components of a damaged tissue, a phenomenon used by salamanders to regenerate limbs. 5-Azacytidine (AZA) is a nucleoside analog that is used to treat preleukemic and leukemic blood disorders. AZA is also known to induce cell plasticity. We hypothesized that AZA-induced cell plasticity occurs via a transient multipotent cell state and that concomitant exposure to a receptive growth factor might result in the expansion of a plastic and proliferative population of cells. To this end, we treated lineage-committed cells with AZA and screened a number of different growth factors with known activity in mesenchyme-derived tissues. Here, we report that transient treatment with AZA in combination with platelet-derived growth factor–AB converts primary somatic cells into tissue-regenerative multipotent stem (iMS) cells. iMS cells possess a distinct transcriptome, are immunosuppressive, and demonstrate long-term self-renewal, serial clonogenicity, and multigerm layer differentiation potential. Importantly, unlike mesenchymal stem cells, iMS cells contribute directly to in vivo tissue regeneration in a context-dependent manner and, unlike embryonic or pluripotent stem cells, do not form teratomas. Taken together, this vector-free method of generating iMS cells from primary terminally differentiated cells has significant scope for application in tissue regeneration.

Gastrulation is a stage in embryo development where three germ layers arise to dictate the human body plan. In vitro models of gastrulation have been demonstrated by treating pluripotent stem cells with soluble morphogens to trigger differentiation. However, in vivo gastrulation is a multistage process coordinated through feedback between soluble gradients and biophysical forces, with the multipotent epiblast transforming to the primitive streak followed by germ layer segregation. Here, the authors show how constraining pluripotent stem cells to hydrogel islands triggers morphogenesis that mirrors the stages preceding in vivo gastrulation, without the need for exogenous supplements. Within hours of initial seeding, cells display a contractile phenotype at the boundary, which leads to enhanced proliferation, yes‐associated protein (YAP) translocation, epithelial to mesenchymal transition, and emergence of SRY‐box transcription factor 17 (SOX17)+ T/BRACHYURY+ cells. Molecular profiling and pathway analysis reveals a role for mechanotransduction‐coupled wingless‐type (WNT) signaling in orchestrating differentiation, which bears similarities to processes observed in whole organism models of development. After two days, the colonies form multilayered aggregates, which can be removed for further growth and differentiation. This approach demonstrates how materials alone can initiate gastrulation, thereby providing in vitro models of development and a tool to support organoid bioengineering efforts.

Patients with Waldenström macroglobulinaemia (WM) are at increased risk of severe COVID‐19 infection and have poor immune responses to COVID‐19 vaccination. This study assessed whether a closely monitored pause in Bruton's Tyrosine Kinase inhibitor (BTKi) therapy might result in an improved humoral response to a 3rd COVID‐19 vaccine dose. Improved response was observed in WM patients who paused their BTKi, compared to a group who did not pause their BTKi. However, the response was attenuated after BTKi recommencement. This data contributes to our understanding of vaccination strategies in this patient group and may help inform consensus approaches in the future.

Terminally differentiated murine osteocytes and adipocytes can be reprogrammed using platelet-derived growth factor–AB and 5-azacytidine into multipotent stem cells with stromal cell characteristics. We have now optimized culture conditions to reprogram human adipocytes into induced multipotent stem (iMS) cells and characterized their molecular and functional properties. Although the basal transcriptomes of adipocyte-derived iMS cells and adipose tissue–derived mesenchymal stem cells were similar, there were changes in histone modifications and CpG methylation at cis-regulatory regions consistent with an epigenetic landscape that was primed for tissue development and differentiation. In a non-specific tissue injury xenograft model, iMS cells contributed directly to muscle, bone, cartilage, and blood vessels, with no evidence of teratogenic potential. In a cardiotoxin muscle injury model, iMS cells contributed specifically to satellite cells and myofibers without ectopic tissue formation. Together, human adipocyte–derived iMS cells regenerate tissues in a context-dependent manner without ectopic or neoplastic growth.

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Embryogenesis is orchestrated through local morphogen gradients and endometrial constraints that give rise to the three germ layers in a well-defined assembly. In vitro models of embryogenesis have been demonstrated by treating pluripotent stem cells in adherent or suspension culture with soluble morphogens and small molecules, which leads to tri-lineage differentiation. However, treatment with exogenous agents override the subtle spatiotemporal changes observed in vivo that ultimately underly the human body plan. Here we demonstrate how microconfinement of pluripotent stem cells on hydrogel substrates catalyses gastrulation-like events without the need for supplements. Within six hours of initial seeding, cells at the boundary show elevated cytoskeletal tension and yes-associated protein (YAP) activity, which leads to changes in cell and nuclear morphology, epithelial to mesenchymal transition, and emergence of defined patterns of primitive streak containing SRY-Box Transcription Factor 17 (SOX17)+ T/BRACHYURY+ cells. Immunofluorescence staining, transcript analysis, and the use of pharmacological modulators reveal a role for mechanotransduction-coupled non-canonical wingless-type (WNT) signalling in promoting epithelial to mesenchymal transition and multilayered organization within the colonies. These microscale gastruloids were removed from the substrate and encapsulated in 3D hydrogels, where biomaterials properties correspond to maintenance and spatial positioning of the primitive streak. Together, this approach demonstrates how materials alone can nurture embryonic gastrulation, thereby providing an in vitro model of early development.

Terminally differentiated murine osteocytes and adipocytes can be reprogrammed using platelet-derived growth factor-AB and 5-Azacytidine into multipotent stem cells with stromal cell characteristics. To generate a product that is amenable for therapeutic application, we have modified and optimised culture conditions to reprogram human adipocytes into induced multipotent stem cells (iMS) and expand them in vitro. The basal transcriptomes of adipocytederived iMS cells and matched adipose-tissue-derived mesenchymal stem cells were remarkably similar. However, there were distinct changes in histone modifications and CpG methylation at cis-regulatory regions consistent with an epigenetic landscape that was primed for tissue development and differentiation. In a non-specific tissue injury xenograft model, iMS cells contributed directly to new muscle, bone, cartilage and blood vessels with no evidence of teratogenic potential. In a cardiotoxin muscle injury model, iMS cells contributed specifically to satellite cells and myofibres without ectopic tissue formation. Taken together, human adipocyte derived iMS cells regenerate tissues in a context dependent manner without ectopic or neoplastic growth.

A more efficient and effective adaptive humoral immune response has been proposed as the basis of the usually favourable outcome of paediatric COVID-19. The breadth of virus and vaccine immunogenicity towards the ever-mutating Spike protein amongst variants of concern (VOC) have not yet been compared between children and adults. We utilized molecular cloning and sensitive antibody detection against conformational Spike by flow cytometry to assess Spike antibodies and delineate the immunogenic region in immune naïve children and adults vaccinated by BNT162b2 and ChAdOx1, and naturally infected with Early Clade, Delta, and Omicron variants. Patient sera were analysed against SARS-CoV-2 Spike antigens including naturally occurring VOCs Alpha, Beta, Gamma, Delta, Omicron BA.1, BA.2, and BA.5 variants of interest Epsilon, Kappa, Eta, D.2, and artificial Spike mutants. There was no notable difference between breadth and longevity of antibody responses generated against VOCs in children and adults. Vaccinated individuals displayed similar immunoreactivity profiles across variants to naturally infected individuals. Delta-infected patients had an enhanced immunogenicity toward Delta and some VOCs compared to patients infected by Early Clade SARS-CoV-2. Although Omicron BA.1, BA.2, and BA.5 antibody levels were increased after Omicron infection in both children and adults, immunogenicity against Omicron subvariants was reduced. This decrease was observed across VOC infection, immunization, and age groups. Selected epistatically combined mutations led to an increase of immunogenicity in artificial Spikes, but were unable to compensate overall within Omicron. Our results reveal important molecular features central to the generation of high antibody titers and broad immunoreactivity that should be considered in future vaccine design and global serosurveillance.