SC_Adv_research

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 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 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): 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 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: 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 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 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.

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 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 Tumor stem cell Tumor cell

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.htm http://www.whitehouse.gov/news/re;eases/2001/08/20010809-2.html Stem cells in class; Badran, Shahira; Bunker Hill Community College, 2007, Boston Museum of Science Biotechnology Symposium Harvard Stem Cell Institute

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