Summer 2007 Workshopin Biology and Multimedia for High School Teachers

Stem Cell Research OverviewMountainous Path

OutlineWhat are Stem Cells?Potential UsesClaims Against Using Stem CellsCultivation ProcessStem Cells and CloningStem Cell Theory of CancerWorldwide Status

What are stem cells?Stem cells are undifferentiated cells that have many potential scientific uses:Cell based therapiesOften referred to as regenerative or reparative medicineTherapeutic cloningGene therapyCancer researchBasic research

Two types of stem cellsEmbryonic Stem Cells (ESC): received from:Embryos created in vitro fertilizationAborted embryosAdult 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 diseasePlacental cordBaby teeth

Source of ESCBlastocystball of cells3-5 day old embryoStem cells give rise to multiple specialized cell types that make up the heart, lung, skin, and other tissuesHuman ESC were only studied since 1998It 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 blastocystBlastocyst structures include:Trophoblast: outer layer of cells that surrounds the blastocyst & forms the placentaBlastocoel: (blastoseel) the hollow cavity inside the blastocyst that will form body cavityInner 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)

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

Unique characteristics of Stem CellsStem cells can regenerateUnlimited self renewal through cell divisionStem cells can specializeUnder certain physiologic or experimental conditionsStem cells then become cells with special functions such as:Beating cells of the heart muscleInsulin-producing cells of the pancreas

UnspecializationStem Cells are unspecializedThey do not have any tissue-specific structures that allow for specialized functionStem 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 themselvesIn the lab, a starting population of SCs that proliferate for many months yields millions of cells that continue to be unspecializedThese cells are capable of long-term self-renewal

Specialization of Stem Cells: DifferentiationDifferentiation: unspecialized stem cells give rise to specialized (differentiated) cells in response to external and internal chemical signalsInternal 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

DifferentiationWhy 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 typeTotal potential to form specialized tissue needed for embryonic developmentPluripotent (plural):Potential to form most or all 210 differentiated adult cell typesMultipotent (multiple):Limited potentialForms only multiple adult cell typesOligodendrocytesNeurons

Adult Stem CellsAdult 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 continuedAdult 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 typesThis is known as PLASTICITY

Adult Stem Cell Plasticity ExamplesBlood cells becoming neuronsLiver cells stimulated to produce insulinHematopoietic (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 historyAdult stem cell research began about 40 years agoStem cell discoveries in 1960s:Bone marrow contains 2 populations of stem cellsHematopoietic stem cells forms all blood cell typesBone marrow stromal cells mixed cell population that generates bone, cartilage, fat and fibrous connective tissueRat brain contains two regions of dividing cells, which become nerve cells

History Cont.Stem Cell Discoveries in the 1990sNeural stem cells in brain are able to generate the brains three major cell typesAstrocytesOligodendroglial cellsNeuronshttp://www.alsa.org/images/cms/Research/Topics/cell_targets.jpg

Adult Stem Cell FactsAdult stem cells were found in many more tissues than expectedSome may be able to differentiate into a number of different cell types, given the right conditionsGeneral consensus among scientist:Adult stem cells DO NOT have as much potential as embryonic stem cellsCLARIFICATION: not all new adult cells arise from stem cellsMost arise by MITOSIS of differentiated cells

Potential Uses of Stem CellsBasic 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 drugsCancer cell lines are already being used to screen potential anti-tumor drugsAvailability 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 arthritisStem cells in gene therapyStem cells as vehicles after they have been genetically manipulatedStem cells in therapeutic cloningStem cells in cancer

Embryonic vs Adult Stem CellsTotipotentDifferentiation into ANY cell typeKnown SourceLarge numbers can be harvested from embryosMay cause immune rejectionRejection of ES cells by recipient has not been shown yetMulti or pluripotentDifferentiation into some cell types, limited outcomesUnknown sourceLimited numbers, more difficult to isolateLess 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* destructionQuestions regarding functional differentiationImmune rejectionGenome instabilityFew & modest results in animals, no clinical treatmentsEthically contentious* = same problem with ASC

Cell Culture Techniques for ESCIsolate & transfer of inner cell mass into plastic culture dish that contains culture mediumCells divide and spread over the dishInner 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 nutrientsRecent discovery: methods for growing embryonic stem cells without mouse feeder cellsSignificance eliminate infection by viruses or other mouse moleculesES 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 organismsPurpose: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 diseaseDeliver 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 TransferSCNT is a method used for:Reproductive cloning such as cloning an embryoRegenerative cloning to produce customized stem cells & overcome immune rejectionBlastula stage cannot continue to develop in vitroIt 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 CloningMany animals were cloned after Dolly Cats, pigs, mice, goats, cattle, rabbitsObstacles: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 Cloning3 goals of therapeutic cloning by SCNT in humans:Use embryo as source for ES cellsUse ES cells to generate an organIn 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 eggsHuman mitochondria carry about 13 genes, some of which code for surface proteins

Pitfalls of therapeutic cloning (2)Large number of eggs needed for SCNTTo harvest large number of eggs:Excessive hormone treatment of females to induce high rate of ovulationSurgery to retrieve eggsBoth can be harmful to female humanCow/pig females may be usedCow/pig eggs will carry species-specific mitochondrial genesMixing species is reason for concern!

Common OpinionsReproductive 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 Cancer1855: Rudolf Virchow developed the Embryonal- Rest HypothesisMicroscopic examination of tumor samples revealed many morphological (structural & functional) resemblances to ESC in a developing fetusIsolation 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

TeratomaOvarian TeratomaYou 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 CancerDetermine difference between cancer & normal stem cellsIdentify potential points in pathways critical for the survival of cancer SCsDevelop therapies that specifically target cancer SCDuke University ExplanationDrawn by Christine Rodriguez

Status of SC research in other countriesGreat BritainVery liberal policies on researchTherapeutic cloning allowed, use of excess embryos & creation of embryos allowedStem cell research allowedFranceLess liberal politicsUse of excess embryos from IVF allowedReproductive AND therapeutic cloning bannedGermanyVery strict policiesUse of excess embryos and creation of embryos bannedScientists can IMPORT embryos

Debate in USFederal funding available for research using the Bush lines only:ES cell lines that were already in existence by 8/9/01Disadvantage of Bush stem cell lines:May have lost regenerative abilityMay have accumulated mutations or infectionsPrivate companies continue to pursue stem cell researchUse of human embryos for IVF & therapeutic cloning is legal in most statesNo federal fundingSome states are considering banning both

Global StatusOngoing debate regarding use of embryos

United Nations: proposal for a global policy to ban reproductive cloning only

ReferencesStem cells & Cloning Stem cells & Cloning; David A. Prentice, Benjamin Cummings, 2003http://www.pbs.org/wgbh/nova/sciencenow/3302/06.htmlhttp://www.stemcellresearch.orghttp://www.stemcells.nig.gov/info/nasics/nasics7.asphttp://www.stemcells.nig.gov/info/scireport/2006report.htmhttp://www.whitehouse.gov/news/re;eases/2001/08/20010809-2.htmlStem cells in class; Badran, Shahira; Bunker Hill Community College, 2007, Boston Museum of Science Biotechnology SymposiumHarvard Stem Cell Institute

Endoderm = inner organs such as digestive, respiratoryMesoderm = muscles, bone, kidneysEctoderm = skin, nervous system