 Summer 2007 Workshop
 in Biology and Multimedia
 for High School Teachers

 What are Stem Cells?
 Potential Uses
 Claims Against Using Stem Cells
 Cultivation Process
 Stem Cells and Cloning
 Stem Cell Theory of Cancer
 Worldwide Status

 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

 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
 Placental cord
 Baby teeth

 Blastocyst
 “ball of 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
 It took scientists 20 years to learn how to grow human ESC following studies with mouse ESC

 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)

http://www.ivf-infertility.com/infertility/infertility5.php

 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

 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)

 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

 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

 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/)

 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):
 Limited potential
 Forms only multiple adult cell types
 Oligodendrocytes
 Neurons

 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 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

 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 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
 Rat brain contains two regions of dividing cells, which become nerve cells

 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 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:
 Adult stem 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

 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

 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

 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

 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

 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

 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.

 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 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
 It must 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 Medical Center.

http://www.stemcellresearch.org/testimony/20040929prentice.htm
Reprinted with permission of Do No Harm.

 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

 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

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

 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!

 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

 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

 Ovarian Teratoma
 You can see teeth!
http://home.earthlink.net/~radiologist/tf/040802.htm
Image courtesy of Leonard J. Tyminski, M.D., Radiologist at earthlink.net

Tumor stem cell
Tumor cell




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
Duke University
Explanation
Drawn by Christine Rodriguez




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

 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

 Ongoing debate regarding use of embryos
 United Nations: proposal for a global policy to ban reproductive cloning only









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