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

Placental

cord Baby teeth

Source of ESC

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

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
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
This

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):
Limited

potential 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

Rat

brain 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 brains 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:
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

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 Parkinsons, Alzheimers, 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

Multi or pluripotent
Differentiation

into ANY

cell type

Known Source Large numbers can be harvested from embryos May cause immune rejection
Rejection

into some cell types, limited outcomes

of ES cells by recipient has not been shown yet

Unknown source Limited numbers, more difficult to isolate Less likely to cause immune rejection, since the patients 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 * = same problem Ethically contentious
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

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.

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 earthlink.net

Current Efforts with SC and Cancer

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

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 Less liberal politics Use of excess embryos from IVF allowed Reproductive AND therapeutic cloning banned Very strict policies Use of excess embryos and creation of embryos banned Scientists can IMPORT embryos

France

Germany

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