What are they and what can we do with them? Stem Cells What are they and what can we do with them?

Stem Cell Characteristics They are undifferentiated cells that can divide indefinitely in culture. They can divide asymmetrically producing one daughter resembling its mother and one that can differentiate into cells of all three germ layers They must originate from an embryonic or adult stem cell (New England Journal of Medicine. 349:267-274. 2003)

Types of Stem Cells Embryonic Umbilical Adult

Embryonic Stem Cells The first human stem cell lines were developed at the Univ. of Wisconsin in 1998. 14 blastocysts were donated by couples in an in vitro fertilization program. The lab established 5 cell lines. The cells were able to develop into various cell types including neural, gut, cartilage, muscle and bone.

Embryonic Stem Cells are derived from fertilized embryos less than a week old, the blastocyst stage of development. (Photo from www.time.com/time/2001/stemcel)l

Developmental Stages (Photo from J. Clinical Investigation

Morula Stage A 32-128 solid ball of cells Each cell is totipotent – it can give rise to all embryonic cell types including extraembryonic tissues necessary for implantation and formation of the placenta and umbilical cord.

Blastocyst Stage The blastocyst (3-5 days in humans) has two regions the inner cells mass contains the undifferentiated stem cells that will form the organism The surrounding cells (trophoblast) help form the placenta and umbilical cord

Inner Cell Mass These cells are called pluripotent and will first form three germ layers (ectoderm, endoderm and mesoderm) through a process called gastrulation The cells in each germ layer are now called multipotent and they follow a path of specialization to become progenitor cells which form specific cell types – e.g. nerve or muscle cell

Pluripotent Cells (Photo from J. Clinical Investigation. 114:1364-1370

Cellular Specialization Cells become specialized as they occupy certain positions in an embryo at a specific time in the life of embryo Specialization occurs as a result of the transcription of a specific set of genes in a cell to cause the translation of proteins that are unique to that cell type – e.g. Dystrophin in muscle cells.

Stem Cells in the Lab Currently, stem cells are grown in association with mouse feeder cells (fibroblasts) and animal proteins which form a matrix on which the stem cells grow Feeder cells keep the stem cells in the undifferentiated state Existing stem cells lines are contaminated with an animal protein

Stem Cells in the Lab Scientists at WiCell Research institute recently published work that eliminates the need for feeder cells, one of the main sources of contamination with pathogens. The research shows a protein called fibroblast growth factor 2 does the same job of the feeder cells: keeping the stem cells undifferentiated. Stem cell cultures still use Matrigel (a matrix of cells from mouse tumors and bovine serum replacement to keep the cells healthy. (Nature Methods. 2:185-190. 2005)

Stem Cell Images (http://www. news. wisc. edu/packages/stemcells/3327

Establishing Stem Cell Lines Inner cell mass cells are transferred to culture dishes where they divide and spread over the dish Subculturing transfers the cells to additional dishes after the first one is filled After 6 months there are millions of cells The cells are evaluated before being called a cell line

How Are ESC Identified (http://stemcells. nih. gov/info/basics/basics3 Growth and subculturing for many months The presence of specific surface protein markers for undifferentiated cells The presence of the protein, Oct-4, a transcription factor unique to ESC Microscopic chromosomal examination Subculturing potency after freezing and thawing and replating Test for pluripotency

Test For Pluripotency (http://stemcells. nih. gov/info/basics/basics3 Allow cells to differentiate spontaneously in cell culture Manipulate cells to differentiate Inject cells into immunosupressed mouse to form a teratoma (benign tumor containing many cell types)

Stem Cell Research Other countries are beginning to take the lead in stem cell research – Korea Private companies in the US can do research with private funds Universities are setting up labs supported solely by private funds- e.g. Univ. Wisconsin, Harvard In 2004, Harvard produced 17 new human ESC lines using private funds

ESC Use in Basic Research (New England Journal of Medicine. 349:267-274. 2003) Study unique aspects of early human development Study origin and mechanisms of chromosomal abnormalities Preclinical testing of candidate therapeutic drugs in many human tissue types

ESC and Treating Diseases Animal experiments have shown the potential therapeutic value of stem cells Mouse ESC have been induced to make dopamine-producing cells in rats with Parkinson’s disease. The symptoms stopped and the rats lived up to 3 months. (Nature. 417:online. June 2002)

ESC and Treating Diseases Mouse ESC have been induced to differentiate into spinal cord motor neurons. The neurons were injected into the spinal cord of a chick embryo and migrated to the proper location in the cord. Some sent out axons to developing limb muscles and formed synapses (J. Clinical Investigation. 114:1364-1370. 2004)

Spinal Cord Experiment (photo from J. Clinical Investigation

Human Trials Considerations How do we control the stem cells – e.g. start and stop differentiation, direct cells to specific tissues Risk of immune rejection Prevent formation of tumors