To exploit the full potential of human pluripotent stem cells for regenerative medicine, developmental biology, and drug discovery, defined culture conditions are needed. Media of known composition that maintain human embryonic stem (hES) cells have been developed, but finding chemically-defined, robust substrata has proved difficult. We employed an array of self-assembled monolayers to identify peptide surfaces that sustain pluripotent stem cell self-renewal. The effective substrates display heparin-binding peptides, which can interact with cell surface glycosaminoglycans, and can be used with a defined medium to culture hES cells for more than 3 months. The resulting cells maintain a normal karyotype and display high levels of pluripotency markers. The peptides are able to support growth of multiple (eight) pluripotent cell lines on a variety of scaffolds. Our results indicate that synthetic substrates that recognize cell surface glycans can facilitate the long-term culture of pluripotent stem cells.

Physical stimuli can act in either a synergistic or antagonistic manner to regulate cell fate decisions, but it is less clear whether insoluble signals alone can direct human pluripotent stem (hPS) cell differentiation into specialized cell types. We previously reported that stiff materials promote nuclear localization of the Yes-associated protein (YAP) transcriptional coactivator and support long-term self-renewal of hPS cells. Here, we show that even in the presence of soluble pluripotency factors, compliant substrata inhibit the nuclear localization of YAP and promote highly efficient differentiation of hPS cells into postmitotic neurons. In the absence of neurogenic factors, the effective substrata produce neurons rapidly (2 wk) and more efficiently (>75%) than conventional differentiation methods. The neurons derived from substrate induction express mature markers and possess action potentials. The hPS differentiation observed on compliant surfaces could be recapitulated on stiff surfaces by adding small-molecule inhibitors of F-actin polymerization or by depleting YAP. These studies reveal that the matrix alone can mediate differentiation of hPS cells into a mature cell type, independent of soluble inductive factors. That mechanical cues can override soluble signals suggests that their contributions to early tissue development and lineage commitment are profound.H uman pluripotent stem (hPS) cells, which include human embryonic (hES) and human induced pluripotent stem cells, possess the remarkable capacity to self-renew indefinitely and differentiate into almost any specialized cell type (1, 2). They represent a potentially unlimited supply of cells for regenerative medicine, drug screening, and studies of human development. These applications require efficient and reproducible conditions to direct hPS cell differentiation into specialized cell types, including neuronal cells. To date, the focus has been on identifying soluble factors, such as growth factors and small molecules, that can influence hPS cell differentiation. The ability of insoluble signals to promote hPS cell-lineage specification remains less clear.Studies in murine ES cells (3, 4) and tissue-specific stem cells (5-10) indicate that the adhesive and mechanical properties of the substratum used can influence cell fate decisions (11). For example, human mesenchymal stem (hMS) cells are sensitive to changes in substrate elasticity and respond by differentiating toward distinct cell lineages depending on the stiffness of the matrix (5). These hMS cells, however, tend to exist in heterogeneous cell populations and lack a specific and unique cell characterization marker (12). Their differentiation capacity is restricted to a few tissues that arise from the mesoderm lineage, such as bone, fat, and cartilage. Indeed, there are questions about whether these cells undergo transdifferentiation to cell types, such as neurons (12)(13)(14). With the unique ability to differentiate into almost any cell type, hPS cells serve as an excellent model for und...

Reaping the promise of human embryonic stem (hES) cells hinges on effective defined culture conditions. Efforts to identify chemically defined environments for hES cell propagation would benefit from understanding the relevant functional properties of the substratum. Biological materials are often employed as substrata, but their complexity obscures a molecular level analysis of their relevant attributes. Because the properties of hydrogels can be tuned and altered systematically, these materials can reveal the impact of substratum features on cell fate decisions. By tailoring the peptide displayed to cells and the substrate mechanical properties, a hydrogel was generated that binds hES cell surface glycosaminoglycans (GAGs) and functions robustly in a defined culture medium to support long-term hES cell self-renewal. A key attribute of the successful GAG-binding hydrogels is their stiffness. Only stiff substrates maintain hES cell proliferation and pluripotency. These findings indicate that cells can respond to mechanical information transmitted via GAG engagement. Additionally, we found the stiff matrices afforded activation of the paralogous proteins YAP/TAZ, which are transcriptional coactivators implicated in mechanosensing and hES cell pluripotency. These results indicate that the substratum mechanics can be tuned to activate specific pathways linked to pluripotency. Because several different hES and induced pluripotent stem cell lines respond similarly, we conclude that stiff substrata are more effective for the long term propagation of human pluripotent stem cells.

Highlights d Biomechanical strain impacts human HSPC formation in vitro d YAP drives endothelial-to-hematopoietic transition downstream of blood flow in vivo d Rho-GTPase stimulation promotes HSPC production in vitro and in vivo via YAP activity

The fate decisions of human pluripotent stem (hPS) cells are governed by soluble and insoluble signals from the microenvironment. Many hPS cell differentiation protocols use Matrigel, a complex and undefined substrate that engages multiple adhesion and signaling receptors. Using defined surfaces programmed to engage specific cell-surface ligands (i.e., glycosaminoglycans and integrins), the contribution of specific matrix signals can be dissected. For ectoderm and motor neuron differentiation, peptide-modified surfaces that can engage both glycosaminoglycans and integrins are effective. In contrast, surfaces that interact selectively with glycosaminoglycans are superior to Matrigel in promoting hPS cell differentiation to definitive endoderm and mesoderm. The modular surfaces were used to elucidate the signaling pathways underlying these differences. Matrigel promotes integrin signaling, which in turn inhibits mesendoderm differentiation. The data indicate that integrin-activating surfaces stimulate Akt signaling via integrinlinked kinase (ILK), which is antagonistic to endoderm differentiation. The ability to attribute cellular responses to specific interactions between the cell and the substrate offers new opportunities for revealing and controlling the pathways governing cell fate.

No abstract

CRISPR/Cas9 has become a powerful tool for genome editing in zebrafish that permits the rapid generation of loss of function mutations and the knock-in of specific alleles using DNA templates and homology directed repair (HDR). We examined the efficiency of synthetic, chemically modified gRNAs and demonstrate induction of indels and large genomic deletions in combination with recombinant Cas9 protein. We developed an in vivo genetic assay to measure HDR efficiency and we utilized this assay to test the effect of altering template design on HDR. Utilizing synthetic gRNAs and linear dsDNA templates, we successfully performed knock-in of fluorophores at multiple genomic loci and demonstrate transmission through the germline at high efficiency. We demonstrate that synthetic HDR templates can be used to knock-in bacterial nitroreductase (ntr) to facilitate lineage ablation of specific cell types. Collectively, our data demonstrate the utility of combining synthetic gRNAs and dsDNA templates to perform homology directed repair and genome editing in vivo.

We review zebrafish models of hepatocellular carcinoma and highlight how the contributions using this model organism are unique. We focus on efforts to model the wide spectrum of genetic mutations found in hepatocellular carcinoma, the biochemical and hormonal changes associated with cirrhosis, the effects of the cancer microenvironment, and the role of metabolic processes such as glutamine and glucose metabolism, autophagy, and oxidative stress.