Drug Discovery : Stem cells


New Target for Diagnosing, Treating Neurodegenerative Diseases

UW–Madison researchers have demonstrated that neurofilament tangles lead to subsequent degeneration and death of motor neurons in ALS patients. They also discovered that these tangles are caused by the reduced expression of a type of neurofilament mRNA. Thus, neurofilament regulation appears to be a promising target for drug screening and gene therapy.

The researchers conducted their studies using motor neurons derived from ALS patients.

Treating Chronic Myeloid Leukemia

With their method, the researchers have discovered a potential gene target to treat chronic myeloid leukemia. The gene, called OLFM4, was identified using iLSCs derived from a patient’s reprogrammed cells. They found that knocking down OLFM4 inhibits leukemia stem cells. Forty other potential gene targets also have been identified.

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.

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.

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.

Culturing Human Embryonic Stem Cells in Well Defined and Controlled Conditions

UW-Madison researchers have developed culture conditions for human ES cells that are entirely free of animal products, feeder cells and conditioned medium. These conditions permit the indefinite culture and proliferation of human ES cells in an undifferentiated state.

The medium includes high levels of a fibroblast growth factor, salts, vitamins, amino acids, glucose, gamma-aminobutyric acid, transforming growth factor beta, pipecholic acid, a lithium salt and lipids. It has been shown to support undifferentiated ES cell proliferation through at least thirteen passages and has proven sufficient to support the derivation of new lines of human ES cells.

The culture conditions also include a biological matrix in the culture vessel. The biological matrix is composed of four human proteins - collagen, laminin, fibronectin, and vitronectin.

No animal products of any kind and no fibroblasts from any species, including humans, are necessary in the culture. Instead, only purified, well characterized components are used to provide a more consistent culture system.

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.