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 cloningGene therapyCancer researchBasic research

Two types of stem cells Embryonic Stem Cells (ESC): received from:

Embryos created in vitro fertilizationAborted 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 cordBaby teeth

Source of ESC

Blastocyst“ball of cells”3-5 day old embryoStem 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 regenerateUnlimited self renewal through cell division

Stem cells can specializeUnder certain physiologic or experimental

conditionsStem 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 periodsThis 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 expressionExternal 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 tissuesUnlike 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 resideStem cells that reside in bone marrow give rise

to RBC, WBC and plateletsRecent 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 brain’s three major cell types

AstrocytesAstrocytes Oligodendroglial cellsOligodendroglial cells NeuronsNeurons

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 cancerMolecular mechanisms for gene controlRole of signals in gene expression &

differentiation of the stem cellStem cell theory of cancer

Potential uses cont.

Biotechnology(drug discovery & development) – stem cells can provide specific cell types to test new drugsSafety testing of new drugs on differentiated

cell linesScreening 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 cloningStem 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 LAYERFeeder cells provide ES cells with a sticky

surface for attachmentFeeder cells release nutrients

Recent discovery: methods for growing embryonic stem cells without mouse feeder cellsSignificance – 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 animalsTherapeutic cloning in animalsBreeding animals or plants with favorable traitsProducing 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 embryoRegenerative cloning to produce “customized”

stem cells & overcome immune rejection Blastula stage cannot continue to develop in

vitro It must be implanted into surrogate momSurrogate 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 processMost clones have deleterious effects & die earlySurviving clones show premature aging signsSigns 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 cellsUse 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 SCNTREASON: 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? ORthe clone is explanted into culture dish to generate ES cells

Stem Cell Theory of Cancer 1855: Rudolf Virchow developed the

Embryonal- Rest HypothesisMicroscopic examination of tumor samples

revealed many morphological (structural & functional) resemblances to ESC in a developing fetus

Isolation of teratoma: nonmalignant tumorsTeratoma represents a ball of almost all cell typesThis 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

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

Tumor stem cell

Tumor cell

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