We developed a method to produce discrete fibrin microthreads, which can be seeded with human mesenchymal stem cells (hMSCs) and used as a suture to enhance the efficiency and localization of cell delivery. To assess the efficacy of fibrin microthreads to support hMSC attachment, proliferation and survival, microthreads (100 µm diameter per microthread) were bundled together, seeded with 50,000 hMSCs for 2 hours, and cultured for 5 days. Cell density on microthread bundles increased over time in culture, to a maximum average density of 731±101 cells/mm 2 after 5 days. A LIVE/DEAD assay confirmed that the cells were viable and Ki-67 staining verified hMSC proliferation. Additionally, functional differentiation assays demonstrated that hMSCs cultured on microthreads retained their ability to differentiate into adipocytes and osteocytes. The results of this study demonstrate that fibrin microthreads support hMSC viability and proliferation, while maintaining their multipotency. We anticipate that these cell-seeded fibrin microthreads will serve a platform technology to improve localized delivery and engraftment of viable cells to damaged tissue.

No abstract

Our ability to identify genes that participate in cell growth and division is limited because their loss often leads to lethality. A solution to this is to isolate conditional mutants where the phenotype is visible under restrictive conditions. Here, we capitalize on the haploid growth-phase of the moss Physcomitrella patens to identify conditional loss-of-growth (CLoG) mutants with impaired growth at high temperature. We used whole-genome sequencing of pooled segregants to pinpoint the lesion of one of these mutants (clog1) and validated the identified mutation by rescuing the conditional phenotype by homologous recombination. We found that CLoG1 is a novel and ancient gene conserved in plants. At the restrictive temperature, clog1 plants have smaller cells but can complete cell division, indicating an important role of CLoG1 in cell growth, but not an essential role in cell division. Fluorescent protein fusions of CLoG1 indicate it is localized to microtubules with a bias towards depolymerizing microtubule ends. Silencing CLoG1 decreases microtubule dynamics, suggesting that CLoG1 plays a critical role in regulating microtubule dynamics. By discovering a novel gene critical for plant growth, our work demonstrates that P. patens is an excellent genetic system to study genes with a fundamental role in plant cell growth.

The fundamental process of polarised exocytosis requires the interconnected activity of molecular motors trafficking vesicular cargo within a dynamic cytoskeletal network. In plants, few mechanistic details are known about how molecular motors, such as myosin XI, associate with their secretory cargo to support the ubiquitous processes of polarised growth and cell division. Live-cell imaging coupled with targeted gene knockouts and a high-throughput RNAi assay enabled the first characterisation of the loss of Rab-E function. Yeast two-hybrid and subsequent in silico structural prediction uncovered a specific interaction between Rab-E and myosin XI that is conserved between P. patens and A. thaliana. Rab-E co-localises with myosin XI at sites of active exocytosis, and at the growing tip both proteins are spatiotemporally coupled. Rab-E is required for normal plant growth in P. patens and the rab-E and myosin XI phenotypes are rescued by A. thaliana's Rab-E1c and myosin XI-K/E, respectively. Both PpMyoXI and AtMyoXI-K interact with PpRabE14, and the interaction is specifically mediated by PpMyoXI residue V1422. This interaction is required for polarised growth. Our results suggest that the interaction of Rab-E and myosin XI is a conserved feature of polarised growth in plants.

Generation of double-stranded RNA targeting the adenine phosphoribosyltransferase gene in tandem with a target gene enables positive selection of strongly silencing plants.