Pluripotent Cells : Tools


Human Pluripotent Stem Cell-Based Models for Neural Toxicity Screening

UW–Madison researchers have developed 3-D vascularized neuronal tissue models for screening neurotoxic agents. The new constructs are highly uniform and the first to contain every major component of the developing brain: neuronal cells (GABAergic and Glutamatergic), glial cells (astrocytes and oligodendrocytes), interconnected vasculature and mature microglia.

Combined with the modular nature of tunable hydrogels and the power of machine learning tools, the new testing platform enables large-scale, quantitative throughput applications.

Mesenchymal Stem Cell-Educated Macrophages to Treat Radiation Damage and More

UW–Madison researchers have discovered that mesenchymal stem cell-educated macrophages (MEMs) have potent tissue regenerative properties that can minimize tissue damage from radiation and increase survival in clinically significant ways.

They have demonstrated in a relevant preclinical model that their new method is much superior to other forms of cellular therapy, including use of mesenchymal stem cells, for preventing and treating radiation-induced morbidity and mortality, GVHD and other conditions associated with uncontrolled inflammation. They purport that allogenic or autologous MEMs can be administered to exposed or damaged organs to treat acute, subacute or chronic radiation-induced disorder.

Hydrogel Arrays for Screening Cell-Substrate Interactions, Now in Multiwell Format

Building on their previous work, the researchers have now adapted their method to any commercially available, glass or polystyrene-bottom multiwell plate. In the new process, hydrogel is covalently immobilized to the bottom of each well and then selectively polymerized. In this way the spots are completely isolatable, allowing for systemic and independent control of their chemical composition and XYZ physical dimensions.

Once the hydrogel array is formed, each of the spots can be exposed to different soluble factors without risk of diffusion.

Controlling the Formation of Stem Cell Colonies with Tailored SAM Array

Building on their previous work, the researchers have developed a new feature to make SAM arrays an even better tool to control cell aggregation. Specifically, the spots on the array consist of cellular adhesive peptides stuck to the surface by an easy-to-cleave labile bond. The peptides enable layers of cell to form and detach from the array without scraping or other external manipulation.

Any peptide capable of forming such a bond (e.g., a thioester bond) with the SAM surface could be employed.

Hydrogel Arrays for Screening Cell-Substrate Interactions

UW–Madison researchers developed a new method for forming patterned hydrogel arrays featuring any number of test spots possessing different characteristics, such as shape and chemical composition. The arrays can be used to culture a range of cell types and rapidly analyze their behavior (e.g., attachment, spreading, proliferation and differentiation).

The arrays are prepared using a hydrogel precursor solution containing a polymer and crosslinker. The solution is sandwiched between stenciled SAM layers containing hydrophilic (‘water-loving’) and hydrophobic (‘water-hating’) regions, then polymerized and released.

As a result of the process, the array features hydrophilic spots surrounded and isolated by hydrophobic regions, preventing any mixing of contents. The spots can have any desired shape, size and chemical composition.

Controlling Size and Shape of Stem Cell Colonies with SAM Array

UW–Madison researchers have developed a method for generating colonies of stem cells in controlled shapes and sizes.

The method uses self-assembled monolayer (SAM) arrays, which are metal-coated slides patterned with small adherent spots. These tools enable researchers to systematically expose cells to various surface-bound molecules — such as proteins, nucleic acids and polysaccharides — and study how they interact.

The SAM spots can have specified diameters and shapes (e.g., circle, oval or star), and the cells that come in contact with them will adhere accordingly. The cells can be cultured for a sufficient time to form a layer that undergoes a morphogenesis process and then detaches so it can be collected for further analysis.

Regulating Stem Cell Behavior with High Throughput Mineral Coatings

UW–Madison researchers have developed methods of non-viral cell transfection and regulating cell behavior using mineral coatings. The coatings bind polynucleotides and provide a source of calcium and phosphate ions to enhance transfection.

More specifically, a mineral coating is formed by incubating a substrate in a simulated body fluid (SBF). The substrate then is loaded with a polynucleotide (e.g., plasmids, mRNA or proteins), which binds to the coating. Next, a solution of cells is deposited and cultured until a desired level of transfection occurs.

Investigating Cell-Surface Interactions via Chemical Array

UW–Madison researchers have developed an improved approach to SAM-based experiments using simple methods to construct arrays of SAMs that can be used to rapidly investigate how selected types and densities of ligands impact cell behaviors such as adhesion, spreading, proliferation, migration and differentiation.

The substrate comprises a slide coated with metal such as gold onto which a polymer stencil is fitted. Through small holes in the stencil, alkanethiol solutions are deposited, forming micrometer-sized spots (up to 120 or more) of SAM. During this deposition process, a range of biological molecules can be tethered to each SAM spot (at varied densities and mixtures), allowing researchers to examine a wide range of possible biological ligands on a single surface. Furthermore, arrays formed using alkanethiolates that prevent biofouling allow researchers to carefully examine the effects of a particular ligand on the behavior of cells cultured on each array spot. Importantly, array fabrication and subsequent examination of cell behavior can be carried out using tools that are standard in biological laboratories such as pipettes and light/fluorescent microscopes.

Master Human Stem Cell Lines for Gene Expression and Knockdown

UW–Madison researchers have developed a method for inserting transgenes (heterologous gene sequences) into specific ‘hot spots’ in the hESC genome where stable and high gene expression may occur.

Insertion is achieved via the Cre-Lox process—a type of site-specific recombination that has allowed researchers to manipulate a variety of genetic modifications to control transgene expression, delete undesired DNA sequences and modify chromosomes.

The method can be used to create ‘master’ hESC lines that serve as templates for inserting any gene of interest. Once a master hESC line is in place, establishing new transgenic lines becomes a basic laboratory routine.

Multilayer Tissue Regeneration System

A UW-Madison researcher has developed an approach for regenerating natural skeletal tissues that more closely mimics in vivo conditions by localizing and temporally controlling the activity of multiple growth factors. This method for growing tissue is based on a matrix of minerals and growth factors. Engineered protein growth factors are incorporated into the layers of the inorganic matrix. Each layer is designed to dissolve at a separate rate. As the matrix material gradually breaks down, the growth factors are delivered sequentially, enabling the growth of new bone tissue. Alternatively, the growth factors can be engineered to bind to the surface of the inorganic matrix.

Methods of Finding, Selecting and Studying Cells in Heterogeneous Co-Cultures

UW-Madison researchers have developed a method of co-culturing heterogeneous primary cells. The cells are cultured in a very small, convection-free space, such as a microchannel, so they behave more as they would in vivo. Because there is no fluid flow, all movement of components in the environment is by diffusion. The culture contains at least one growth-promoting cell and at least one cell capable of proliferating.

Reprogrammed Stem Cell Line for Research: IISH3i-CB6

UW–Madison researchers have developed a reprogrammed iPSC line called IISH3i-CB6. Their method generates iPSCs free of transgene and vector sequences from human bone marrow and cord blood mononuclear cells using non-integrating episomal vectors.

Reprogrammed Stem Cell Line for Research: IISH4i-CBT4

UW–Madison researchers have developed a reprogrammed iPSC line called IISH4i-CBT4. Their method generates iPSCs free of transgene and vector sequences from human bone marrow and cord blood mononuclear cells using non-integrating episomal vectors.

Thiazovivin was added to improve reprogramming efficiency when creating this line.