Directed Differentiation

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Collecting human stem cells, whether from an adult or an embryo, is just the first part in a long line of procedures that, hopefully, will lead to a treatment for a medical disorder. Once the cells are collected, they are grown in culture and stimulated in various ways to determine the types of cells they may produce. The procedures used are identical to those established in previous experiments with mouse stem cells. Human stem cells, like mouse stem cells, form embryoid bodies when grown on plates lacking a feeder layer. Although the embryoid bodies vary with regard to cellular composition, they usually include cells that look like neurons and myocytes.

Directed differentiation of human stem cells is always carried out on cells that have been isolated from embryoid bodies and replated to form a monolayer of cells. So far, adult stem cells have been stimulated to produce several cell types, either through exposure to various growth factors or by being injected into mice (see table on page 25). Selecting growth factors for these experiments is an example of educated guesswork. If the intention of the experiment is to produce neurons or epithelial cells, the scientists involved will select growth factors such as epidermal growth factor (EGF) and nerve growth factor (NGF), both of which are known to influence the proliferation of these cells in vivo. Other growth factors, such as transforming growth factor (TGF) or the

DIRECTED DIFFERENTIATION OF HUMAN STEM CELLS

Source

Conditions

Resulting Cell Types

Adult bone marrow

Injection into mice

Hepatocytes Red blood cells White blood cells

Adult bone marrow

Epithelial growth factor (EGF) Neurotrophic growth factor Cultured with fetal rat neurons

Neurons

Adult bone marrow

Transforming growth factor (TGF) Fetal bovine serum Insulin

Adipocyte

Chondrocyte Osteocyte

Cardiomyocyte NGF

Fibroblast growth factor (FGF) Bone morphogenic protein (BMP) Hepatocyte growth factor (HGF) Retinoic acid

Neuron

Blood cell Precursor Liver

Pancreas

Muscle

Embryo (H9 cell line)

Injection into mice Leukemia inhibitory factor

Bone Cartilage Gut epithelia Neural epithelia Smooth muscle Striated muscle

hormone insulin, are known to influence tissues derived from the mesoderm, such as muscle and cartilage. In some cases, the growth factors producing a certain kind of cell are unknown. This occurs when stem cells differentiate in vivo or when they are cultured in the presence of fetal bovine serum (blood serum obtained from a fetal cow), which contains many yet-to-be-identified growth factors.

Directed differentiation of embryonic stem cells is carried out as described for adult stem cells. Retinoic acid, epidermal growth factor (EGF), bone morphogenic protein (BMP), and fibroblast growth factor (FGF) are some of the growth factors that have been used in ES cells. All these factors trigger development of cells that would normally be derived from the ectoderm. Other growth factors, such as activin-A and transforming growth factor (TGF) initiate differentiation of meso-derm-derived cell lines. Hepatocyte growth factor (HGF) and nerve growth factor (NGF) promote differentiation of cells that represent all three germ layers. When all these factors are added individually to cell cultures derived from embryoid bodies, they give rise to 11 different cell types, representing ectoderm, mesoderm, and endoderm (see table on page 25).

Spontaneous differentiation of ES cells in culture will produce several different kinds of cells on a single plate, but stimulating the cultures with any one of the growth factors mentioned above tends to focus the differentiation toward a single cell type. Cultures stimulated with FGF differentiate into epithelial cells that express the marker keratin, a common skin protein. Cultures treated with activin-A produce muscle cells that express a muscle-specific enzyme called enolase. Retinoic acid typically stimulates the production of neurons, but it is also known to initiate development of other cell types.

An important and much sought-after result of directed differentiation is the production of blood cell precursors for the treatment of leukemia. Blood cells are not among the cells produced by human ES cells through spontaneous differentiation, and growth factors so far tested do not initiate the formation of these cells. However, reports indicate some success by growing human ES cells in the presence of Y-irradiated mouse bone marrow cells (Y-irradiated blocks replication of the mouse cells). The mouse cells apparently provide an unknown growth factor that triggers differentiation of the ES cells into blood cells. The differentiated cells express CD34, a marker for blood cell precursors, and under certain conditions, these cells will form erythroid cells, macrophages, and other blood cells.

Growth factor monomer

Growth factor monomer

Activate signaling molecules

Stimulate growth, proliferation, and differentiation

Growth factor receptors. The growth factor forms a dimer (two monomers stuck together) and binds to the receptor, stimulating dimerization of the receptor. This stimulates kinase activity of the receptor, which phosphorylates itself and several signaling molecules. The activated signaling molecules stimulate cell growth, proliferation, and differentiation.

Human ES cells have a greater tendency to differentiate spontaneously when placed in culture than do mouse ES cells. Scientists wishing to produce a culture of human myocytes or neurons through directed differentiation must start with a population of undifferentiated cells, otherwise the product of the experiment might be a curious hybrid cell that could yield unpredictable, and perhaps fatal, results if used in a clinical setting. Markers of the embryonic state, such as stage-specific embryonic antigen (SSEA), are being used, in conjunction with a FACS machine, to isolate and segregate the undifferentiated cells from the rest of the population so they can be used for directed differentiation. In addition, the isolation of the undifferentiated cells will make it possible to study the differences between those cells that remain embryonic and those that become partially or wholly differentiated.

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