Pluripotent Cells : Differentiation


Xeno-Free Protocol for Generating Endothelial Cells from Human Pluripotent Stem Cells

UW–Madison researchers have developed a fully defined and xenogenic material-free method of producing and expanding clinically relevant human ECs for therapeutic and tissue modelling applications. Populations of up to 80 percent CD31+ ECs are generated from both human embryonic and induced pluripotent stem cells.

Like existing protocols, the new method uses factors including Bone Morphogenetic Protein (BMP), Activin A and a TGF-Beta 1 inhibitor.

Derivation of Human Microglia from Pluripotent Stem Cells

UW–Madison researchers have discovered a primitive, macrophage-like cell type of the hematopoietic lineage that has the capability to develop ramified human microglia when added to a neural tissue construct. Accordingly, they have developed the first known protocol of its kind for differentiating and expanding microglia suitable for clinically relevant therapeutic applications.

Albumin-Free Protocol Cuts Cost, Supports Large-Scale Cardiomyocyte Production

UW–Madison researchers have developed a method for generating high yield, high purity cardiomyocytes/progenitors from PSCs under defined, albumin-free conditions. Their discovery that albumin is not necessary, and may even be deleterious, for cardiomyocyte differentiation dramatically reduces the cost of production.

Generating Human Heart Cells via Lineage Reprogramming

UW–Madison researchers have developed a method to reprogram human (or other mammalian) somatic cells to iCPCs by defined factors. The factors include several early cardiac transcription factors and a chromatin remodeling factor. They may be introduced into the cell via standard vector techniques. Successfully reprogrammed cells are separated from the others and cultured in a proliferative state.

Generating Hindbrain and Spinal Cord Cells from Human Stem Cells

UW–Madison researchers have developed a culture medium for producing neural cell populations (caudal lateral epiblasts, posterior neuroectoderm, posterior neuroepithelial cells or motor neurons) with desired positional identity within the hindbrain or spinal cord. This identity can be predicted a priori in a fully defined and scalable way based on HOX gene expression.

The culture medium is composed of standard ingredients and includes a fibroblast growth factor and an activator of β-catenin pathway signaling.

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.

Generating Vasculogenic Cell Populations from Human Stem Cells

UW–Madison researchers have developed a method for generating substantially pure populations of vasculogenic cells (i.e., pericytes and smooth muscle cells) from induced pluripotent stem cells following their differentiation into mesenchymal colony-forming progenitors, called mesenchymoangioblasts (MABs).

The process includes culturing the progenitors in a serum-free medium under conditions that promote differentiation to MABs. Subsequently, the MABs are cultured in medium containing PDGFBB to obtain pericytes, or sphingosylphosphorylcholine (SPC) and transforming growth factor beta (TGFβ) to obtain smooth muscle cells.

Generating Endothelial Cells from Human Pluripotent Stem Cells

UW–Madison researchers have developed a new method for differentiating hPSCs into endothelial cells under chemically defined, growth factor-free conditions. Their method works by activating the Wnt/β signaling pathway for a defined period, e.g., using a Gsk3 inhibitor.

In the new process, hPSCs are contacted with the Wnt/β signaling activator for about two days in suitable culture medium. The cells are cultured in the absence of the activator for up to another 10 days, and undergo at least 14 population doublings. The culture medium is substantially free of exogenous growth factors like VEGF.

The entire process can be embodied in a kit containing (i) a Gsk3 inhibitor, (ii) suitable culture medium and (iii) appropriate instructions.

Xenogen-Free Culture Medium for Stem Cells

UW–Madison researchers have developed a chemically defined, xenogen-free culture system for differentiating human pluripotent stem cells into mesoderm, endothelial and hematopoietic progenitor cells. The new culture system can be used to produce these types of cells from human pluripotent stem cells growing in completely defined E8 medium, thus providing an opportunity to manufacture clinical grade cells.

In the new method, the stem cells (human embryonic or human induced pluripotent stem cells) are seeded as a single cell suspension on a substrate comprising a layer of Tenascin C and IF9S culture medium supplemented with BMP4, Activin A, FGF2 and LiCl. The medium also comprises various hematopoietic cytokines. The stem cells are exposed to the mixture under hypoxic conditions for about two days.

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.

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.

Generating Oligodendrocytes from Human Embryonic Stem Cells

UW–Madison researchers have developed a stepwise, chemically defined protocol for generating oligodendrocytes from hESCs. The process closely mimics that observed in a human embryo.

Firstly, the hESCs are differentiated to uniform neuroepithelial cells, followed by specification to Olig2-expressing neuron/oligodendrocyte precursor cells in the presence of sonic hedgehog protein or purmorphamine.

The precursor cells become mature oligodendrocytes in culture, and can produce myelin sheaths once transplanted into the brains of mice.

Multipotent Lymphohematopoietic Progenitor Cells

UW-Madison researchers have developed a population of cells derived from hES cells that comprise unique, multipotent lymphohematopoietic progenitors. The cells, which were obtained from co-culture of hES cells with OP9 stromal cells, are CD34 and CD43 positive, but CD45 and lin negative. These cells express gene profiles characteristic of definitive, multipotent hematopoietic progenitors and are capable of differentiating into lymphocytes, myeloid cells, erythroid cells and megakaryocytes. They are no longer capable of differentiating into non-hematopoietic lineages. If the cells are cultured until they express CD45, a cell surface marker found in hematopoietic progenitor cells derived from somatic (adult) stem cells, they lose the ability to produce differentiated cells of B lymphoid lineages.

Method of Forming Dendritic Cells from Embryonic Stem Cells

UW-Madison researchers have developed an in vitro method for inducing human embryonic stem cells to differentiate into dendritic cells. Stem cells are cultured successively in three different environments for between seven and 10 days each. The first environment induces them to become hematopoietic cells, the second directs the hematopoietic cells to become myeloid precursor cells, and the third causes the myeloid precursor cells to develop into immature dendritic cells. The resulting dendritic cells are morphologically, phenotypically and functionally similar to dendritic cells in vivo.

Method of Forming Mesenchymal Stem Cells from Embryonic Stem Cells

UW-Madison researchers have developed a method of directing human embryonic stem cells to form mesenchymal stem cells. These cells can then differentiate into a variety of specific cell types, including bone-forming osteoblast cells.

The protocol includes culturing ES cells under conditions that promote the formation of embryoid bodies. The embryoid bodies are then propagated in a mesenchyme-specific medium and digested to form mesodermal cells, which are further cultured in a mesenchyme-specific medium to form a substantially homogenous population of MSCs. These MSCs can then be propagated with 1,25-vitamin D3 to produce a subpopulation of bone-precursor cells, such as pre-osteoblasts and osteoblasts. Ascorbic acid and beta-glycerophosphate may be added to assist the pre-osteoblasts and osteoblasts in mineralizing matrix tissue to form bone. Alternatively, the embryoid bodies may be cultured directly with 1,25-vitamin D3 to produce a relatively homogenous (greater than 90 percent) population of MSCs.

Differentiation of Stem Cells to Endoderm and Pancreatic Cells

UW-Madison researchers have developed methods of directing human embryonic stem cells to differentiate into the lineage of pancreatic islet cells. These methods enable the production of endoderm and pancreatic cells in large numbers.

Three separate techniques, each of which acts independently, are used to increase the percentage of pancreatic progenitor cells in a differentiated cell culture.
  1. Selecting embryoid bodies with greater potential for developing into definitive endoderm cells
  2. Using a medium containing a growth-enhancing factor that promotes the growth of pancreatic cell types
  3. Using both positive and negative selection to eliminate unwanted cells and select for cells of the desired lineage.
In addition, sorting the cells to remove undifferentiated cells eliminates one of the largest barriers to their use in transplantation-the formation of non-malignant tumors, known as teratomas, following transplantation into people.

In Vitro Differentiation of Neural Stem Cells and Neurons from Human Embryonic Stem Cells

UW-Madison researchers have developed a simple and efficient method of differentiating human embryonic stem cells into neural stem cells and neurons for pharmaceutical screening and potential transplant therapy. The system is easily standardized and completely chemically defined. First, hES cells are aggregated and treated with fibroblast growth factors to induce the cells’ development into early neural stem cells. Different combinations of growth factors are then used to direct these naïve neural stem cells to become progenitors of various types of neurons. The neural progenitors organize into neural tube-like rosettes that can be readily enriched and further differentiated into functional spinal motor neurons, midbrain dopaminergic neurons or forebrain dopaminergic neurons.

Method for Generating Primate Trophoblasts

UW-Madison researchers have developed the first method to cause a culture of primate embryonic stem cells to repeatedly, directly and uniformly differentiate into trophoblast cells. Treating primate (non-human and human) embryonic stem cells with a single protein induction factor causes the cells to transform into trophoblast cells. Several protein factors, including bone morphogenic protein (BMP) 4, BMP2, BMP7 and growth and differentiation factor 5, can serve as trophoblast-induction factors. Trophoblast cells are created directly from embryonic stem cells without the intervening step of forming an embryoid body. This method results in a stable and nearly pure culture of primate trophoblast cells.

Endothelial Cells Derived from Human Embryonic Stem Cells

UW-Madison researchers have developed a simple and efficient method of inducing human embryonic stem cells to differentiate into a relatively homogenous population of endothelial cells. The method involves culturing ES cells in a commercially available medium that supports the growth of endothelial cells. The resulting ES-derived endothelial cells have the general morphological characteristics and cell surface markers of endothelial cells. They are capable of inducing and participating in blood vessel formation when transplanted into tissue in vivo.

Differentiation and Purification of Neural Precursors from Human Embryonic Stem Cells

UW-Madison researchers have developed a simple and efficient method of differentiating human embryonic stem cells into neural precursor cells for pharmaceutical screening and potential transplant therapy. This system is easily standardized. First, hES cells are aggregated into embryoid bodies. Next, the embryoid bodies are treated with fibroblast growth factors to induce the cells’ development into a relatively pure culture of neural precursor cells. The resulting neural precursor cells have been shown to differentiate into neurons and glia, both in vitro and after transplantation into neonatal mouse brains.

Method of Making Embryoid Bodies from Primate Embryonic Stem Cells

UW-Madison researchers have developed an improved method of forming embryoid bodies from human and non-human primate ES cells. The method starts with ES cell colonies adhered to a substrate. The colonies are removed from the substrate as clumps through either physical or chemical means. The clumps of ES cells are then incubated under conditions that prevent them from attaching to their container. This allows the ES cells to coalesce into embryoid bodies that can then be differentiated into a variety of desired lineages. In essence, the development of embryoid bodies from primate ES cells depends on maintaining the cells as aggregated clumps, because isolated cells in culture die.

Hematopoietic Differentiation of Human Embryonic Stem Cells

UW-Madison researchers have developed a method for creating hematopoietic cells from human embryonic stem cells. To create hematopoietic cells, a human ES cell culture is exposed to mammalian hematopoietic stromal cells. At least some of the resulting cells are CD34+ (a standard marker for hematopoietic cells), or can form hematopoietic cell colony-forming units in methylcellulose culture, indicating that the cells are capable of further differentiation.
The hematopoietic cells can potentially be transplanted into a patient along with other MHC-compatible human cells, such as pancreatic islets to treat diabetes. Transplanting hematopoietic cells with other MHC-compatible cells improves the acceptance of the other cells.