Stem Cells

1
Stem Cells

• What are they and what can we do with them?

2
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. 349267-274.
  2003)

3
Types of Stem Cells

• Embryonic
• Umbilical
• Adult

4
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.

5
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

6
Developmental Stages(Photo from J. Clinical
Investigation. 1141364-1370. 2004)
7
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.

8
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

9
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

10
Pluripotent Cells(Photo from J. Clinical
Investigation. 1141364-1370. 2004)
11
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.

12
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

13
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. 2185-190. 2005)

14
Stem Cell Images (http//www.news.wisc.edu/package
s/stemcells/3327.html)
15
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

16
How Are ESC Identified(http//stemcells.nih.gov/i
nfo/basics/basics3.asp)

• 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

17
Test For Pluripotency(http//stemcells.nih.gov/in
fo/basics/basics3.asp)

• 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)

18
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

19
ESC Use in Basic Research(New England Journal of
Medicine. 349267-274. 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

20
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 Parkinsons
  disease. The symptoms stopped and the rats lived
  up to 3 months. (Nature. 417online. June 2002)

21
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. 1141364-1370. 2004)

22
Spinal Cord Experiment(photo from J. Clinical
Investigation. 1141364-1370. 2004)
23
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