 Summer 2007 Workshop

 in Biology and Multimedia

 for High School Teachers
Stem Cell Research
Overview
Mountainous Path
Outline
 What are Stem Cells?
 Potential Uses
 Claims Against Using Stem Cells
 Cultivation Process
 Stem Cells and Cloning
 Stem Cell Theory of Cancer
 Worldwide Status
What are stem cells?
 Stem cells are undifferentiated cells that
have many potential scientific uses:
 Cell based therapies
 Often referred to as regenerative or reparative
medicine
 Therapeutic cloning
 Gene therapy
 Cancer research
 Basic research
Two types of stem cells
 Embryonic Stem Cells (ESC): received from:
 Embryos created in vitro fertilization
 Aborted embryos
 Adult Stem Cells (ASC): can be received
from:
 Limited tissues (bone marrow, muscle, brain)
 Discrete populations of adult stem cells generate
replacements for cells that are lost through normal
wear and tear, injury or disease
 Placentalcord
 Baby teeth
Source of ESC
 Blastocyst
 “ballof cells”
 3-5 day old embryo
 Stem cells give rise to multiple specialized cell
types that make up the heart, lung, skin, and
other tissues
 Human ESC were only studied since 1998
 Ittook scientists 20 years to learn how to grow
human ESC following studies with mouse ESC
 How are embryonic stem cells
harvested?
 Human ES cells are derived from 4-5 day old
blastocyst
 Blastocyst structures include:
 Trophoblast: outer layer of cells that surrounds the
blastocyst & forms the placenta
 Blastocoel: (“blastoseel”) the hollow cavity inside the
blastocyst that will form body cavity
 Inner cell mass: a group of approx. 30 cells at one end of
the blastocoel:
 Forms 3 germ layers that form all embryonic tissues (endoderm,
mesoderm, ectoderm)
Blastocyst

http://www.ivf-infertility.com
/infertility/infertility5.php
 Unique characteristics of Stem
Cells
 Stem cells can regenerate
 Unlimited self renewal through cell division
 Stem cells can specialize
 Under certain physiologic or experimental
conditions
 Stem cells then become cells with special
functions such as:
 Beating cells of the heart muscle
 Insulin-producing cells of the pancreas
Unspecialization
 Stem Cells are unspecialized
 They do not have any tissue-specific
structures that allow for specialized function
 Stem cells cannot work with its neighbors to
pump blood through the body (like heart
muscle cells)
 They cannot carry molecules of oxygen
through the bloodstream (like RBCs)
 They cannot fire electrochemical signals to
other cells that allow the body to move or
speak (like nerve cells)
Self - Renewal (Regeneration)
 Stem cells are capable of dividing &
renewing themselves for long periods
 This is unlike muscle, blood or nerve cells –
which do not normally replicate themselves
 In the lab, a starting population of SCs that
proliferate for many months yields millions of
cells that continue to be unspecialized
 These cells are capable of long-term self-renewal
 Specialization of Stem Cells:
Differentiation
 Differentiation: unspecialized stem cells give
rise to specialized (differentiated) cells in
response to external and internal chemical
signals
 Internal signals: turn on specific genes causing
differential gene expression
 External signals include:
 Chemicals secreted by other cells such as growth
factors, cytokines, etc.
 Physical contact with neighboring cells
Differentiation
 Why do your body cells look different
although they all carry the same DNA,
which was derived from one fertilized egg?
 Differentiation example
(http://learn.genetics.utah.edu/units/biotech/microarray/)
Potential of Stem Cells (vocab)
 Totipotent (total):
 Total potential to differentiate into any adult cell type
 Total potential to form specialized tissue needed for
embryonic development
 Pluripotent (plural):
 Potential to form most or all 210 differentiated adult cell
types
 Multipotent (multiple):
 Limitedpotential
 Forms only multiple adult cell types
 Oligodendrocytes
 Neurons
Adult Stem Cells
 Adult or somatic stem cells have unknown
origin in mature tissues
 Unlike embryonic stem cells, which are
defined by their origin (inner cell mass of the
blastocyst)
http://www.stemcellresearch.org/testimony/20040929prentice.htm Reprinted with permission of Do No Harm.
 Adult stem cells continued
 Adult stem cells typically generate the cell
types of the tissue in which they reside
 Stem cells that reside in bone marrow give rise
to RBC, WBC and platelets
 Recent experiments have raised the possibility
that stem cells from one tissue can give rise to
other cell types
 This is known as PLASTICITY
Adult Stem Cell Plasticity Examples
 Blood cells becoming neurons
 Liver cells stimulated to produce insulin
 Hematopoietic (blood cell producing) stem cells
that become heart cells

 CONCLUSION: Exploring the use of adult stem

cells for cell-based therapies has become a very
important (and rapidly increasing) area of
investigation by research scientists!
 Adult stem cells: A brief history
 Adult stem cell research began about 40
years ago
 Stem cell discoveries in 1960s:
 Bone marrow contains 2 populations of stem cells
 Hematopoietic stem cells – forms all blood cell types
 Bone marrow stromal cells – mixed cell population that
generates bone, cartilage, fat and fibrous connective
tissue
 Ratbrain contains two regions of dividing cells,
which become nerve cells
History Cont.
 Stem Cell Discoveries
in the 1990s
 Neural stem cells in
brain are able to
generate the brain’s
three major cell types
 Astrocytes
 Oligodendroglial cells
 Neurons

http://www.alsa.org/images/cms/Research/Topics/cell_targets.jpg
Adult Stem Cell Facts
 Adult stem cells were found in many more
tissues than expected
 Some may be able to differentiate into a number
of different cell types, given the right conditions
 General consensus among scientist:
 Adultstem cells DO NOT have as much potential as
embryonic stem cells
 CLARIFICATION: not all new adult cells arise
from stem cells
 Most arise by MITOSIS of differentiated cells
Potential Uses of Stem Cells
 Basic research – clarification of complex
events that occur during human
development & understanding molecular
basis of cancer
 Molecular mechanisms for gene control
 Role of signals in gene expression &
differentiation of the stem cell
 Stem cell theory of cancer
Potential uses cont.
 Biotechnology(drug discovery &
development) – stem cells can provide
specific cell types to test new drugs
 Safety testing of new drugs on differentiated
cell lines
 Screening of potential drugs
 Cancer cell lines are already being used to screen
potential anti-tumor drugs
 Availability of pluripotent stem cells would allow
drug testing in a wider range of cell types & to
reduce animal testing
Potential uses cont.
 Cell based therapies:
 Regenerative therapy to treat Parkinson’s,
Alzheimer’s, ALS, spinal cord injury, stroke,
severe burns, heart disease, diabetes,
osteoarthritis, and rheumatoid arthritis
 Stem cells in gene therapy
 Stem cells as vehicles after they have been
genetically manipulated
 Stem cells in therapeutic cloning
 Stem cells in cancer
 Embryonic vs Adult Stem Cells
 Totipotent
 Differentiation into ANY
cell type
 Known Source
 Large numbers can be
harvested from embryos
 May cause immune
rejection
 Rejection of ES cells by
recipient has not been
shown yet
 Multi or pluripotent
 Differentiation into some
cell types, limited
outcomes
 Unknown source
 Limited numbers, more
difficult to isolate
 Less likely to cause
immune rejection, since
the patient’s own cells
can be used
 Claims against ESC
(unsubstantiated thus far!)
 Difficult to establish and maintain *
 Difficulty in obtaining pure cultures from dish*
 Potential for tumor formation and tissue*
destruction
 Questions regarding functional differentiation
 Immune rejection
 Genome instability
 Few & modest results in animals, no clinical
treatments
 Ethically contentious * = same problem
with ASC
Cell Culture Techniques for ESC

 Isolate & transfer of inner cell mass into

plastic culture dish that contains culture
medium
 Cells divide and spread over the dish
 Inner surface of culture dish is typically
coated with mouse embryonic skin cells
that have been treated so they will not
divide
 This coating is called a FEEDER LAYER
 Feeder cells provide ES cells with a sticky
surface for attachment
 Feeder cells release nutrients
 Recent discovery: methods for growing
embryonic stem cells without mouse feeder
cells
 Significance
– eliminate infection by viruses or
other mouse molecules
 ES cells are removed gently and plated into
several different culture plates before
crowding occurs
http://www.news.wisc.edu/packages/stemcells/illustration.html Images
depict stem cell research at the University of Wisconsin Madison.
 Cloning of whole organisms
 Purpose:
 Reproductive cloning in animals
 Therapeutic cloning in animals
 Breeding animals or plants with favorable traits
 Producing TRANSGENIC animals that:
 Make a therapeutic product (vaccine, human protein etc)
 Act as animal models for human disease

 Deliver organs that will not be rejected (cells lacking cell

surface markers that cause immune rejection)
 Vaccines in biotech industry: steps in cloning a
gene
 SCNT: Somatic Cell Nuclear
Transfer
 SCNT is a method used for:
 Reproductive cloning such as cloning an embryo
 Regenerative cloning to produce “customized”
stem cells & overcome immune rejection
 Blastula stage cannot continue to develop in
vitro
 Itmust be implanted into surrogate mom
 Surrogate mom is just a container that provides
protection & chemical signals necessary for
development
http://www.kumc.edu/stemcell/early.html Reprinted with permission from the University of Kansas
http://www.stemcellresearch.org/testimony/20040929prentice.htm Reprinted with permission of Do No Harm.
 Challenges of Reproductive
Cloning
 Many animals were cloned after Dolly
 Cats, pigs, mice, goats, cattle, rabbits
 Obstacles:
 Very inefficient process
 Most clones have deleterious effects & die early
 Surviving clones show premature aging signs
 Signs of abnormal embryonic development:
 Clones & their placentas grow much faster than
expected in surrogate mom
 Therapeutic Cloning

 3 goals of therapeutic cloning by SCNT in

humans:
 Use embryo as source for ES cells
 Use ES cells to generate an organ
 In this case the organ generated will carry cells with the
same genetic markers as the patient (recipient)
 Correct genetic error in ESC in blastula stage
Pitfalls of therapeutic cloning (1)

Some immune rejection may occur- WHY?

 About 1% of the DNA in the clone will NOT be
identical to donor cell (patient)
 It will be identical to egg cell used in SCNT
 REASON: mitochonrial DNA in eggs
 Human mitochondria carry about 13 genes, some
of which code for surface proteins
 Pitfalls of therapeutic cloning (2)
 Large number of eggs needed for SCNT
 To harvest large number of eggs:
 Excessive hormone treatment of females to
induce high rate of ovulation
 Surgery to retrieve eggs
 Both can be harmful to female human
 Cow/pig females may be used

 Cow/pig eggs will carry species-specific

mitochondrial genes
 Mixing species is reason for concern!
Common Opinions
 Reproductive cloning is a criminal offense
(it is ILLEGAL worldwide!)
 Therapeutic cloning is acceptable,
however there is still significant
controversy over whether:
the clone is implanted into the uterus of
surrogate mom? OR
the clone is explanted into culture dish to
generate ES cells
 Stem Cell Theory of Cancer
 1855: Rudolf Virchow developed the
Embryonal- Rest Hypothesis
 Microscopic examination of tumor samples
revealed many morphological (structural &
functional) resemblances to ESC in a developing
fetus
 Isolation of teratoma: nonmalignant tumors
 Teratoma represents a ball of almost all cell types
 This indicates that teratoma may originate from
unregulated stem cells that can give rise to almost
all tissues
 Teratoma
 Ovarian Teratoma
 You can see teeth!

http://home.earthlink.net/~radiologist/tf/040802.htm Image courtesy of Leonard J. Tyminski, M.D., Radiologist at

 Current Efforts with SC and Cancer
 Determine difference
between cancer & normal
stem cells
 Identify potential points in
pathways critical for the
survival of cancer SCs
 Develop therapies that
specifically target cancer
SC
Tumor stem cell
 Duke University
Tumor cell
Explanation
Drawn by Christine Rodriguez
 Status of SC research in other
countries
 Great Britain
 Very liberal policies on research
 Therapeutic cloning allowed, use of excess embryos & creation of
embryos allowed
 Stem cell research allowed
 France
 Less liberal politics
 Use of excess embryos from IVF allowed
 Reproductive AND therapeutic cloning banned
 Germany
 Very strict policies
 Use of excess embryos and creation of embryos banned
 Scientists can IMPORT embryos
 Debate in US
 Federal funding available for research using the
Bush lines only:
 ES cell lines that were already in existence by 8/9/01
 Disadvantage of Bush stem cell lines:
 May have lost regenerative ability
 May have accumulated mutations or infections
 Private companies continue to pursue stem cell
research
 Use of human embryos for IVF & therapeutic cloning is
legal in most states
 No federal funding
 Some states are considering banning both
Global Status
 Ongoing debate regarding use of embryos

 United Nations: proposal for a global

policy to ban reproductive cloning only
References
 Stem cells & Cloning Stem cells & Cloning; David A.
Prentice, Benjamin Cummings, 2003
 http://www.pbs.org/wgbh/nova/sciencenow/3302/06.html
 http://www.stemcellresearch.org
 http://www.stemcells.nig.gov/info/nasics/nasics7.asp
 http://www.stemcells.nig.gov/info/scireport/2006report.ht
m
 http://www.whitehouse.gov/news/re;eases/2001/08/2001
0809-2.html
 Stem cells in class; Badran, Shahira; Bunker Hill
Community College, 2007, Boston Museum of Science
Biotechnology Symposium
 Harvard Stem Cell Institute