Molecular Medicine What Are Stem Cells?

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1 Molecular Medicine What Are Stem Cells?
Stem cells are undifferentiated cells that have the potential to differentiate into specific cells. These remarkable building blocks of life can replace or repair damaged tissues and cells. Based on this, scientists insert stem cells into areas of the body to heal or replace tissue and cells effected by disease. Three Classifications of Stem Cells: Totipotent- these are stem cells present in the zygote immediately after fertilization of the egg by the sperm during the first few cell divisions. They can differentiate to not only all cells of the body, but cells of the extraembryonic tissues, such as the placenta. Pluripotent- these are stem cells that can differentiate into any cell of the body. Multipotent- these are stem cells whose potential is limited to differentiation into various cells of their tissue of origin. Researchers see pluripotent stem cells as the holy grail of medicine because of their ability to differentiate into any cell that is needed. The pluripotent are often referred to as "unlimited" stem cells. Turipotent is avoided because extraembryonic tissue is not coveted. Multipotent, while it certainly has great ability and use, is referred to as "limited" stem cells because of their inability to widely differentia 1

2 Molecular Medicine Stem cell therapy Gene therapy
Tumor therapy Therapy Immunotherapy Other therapies Vaccines Genetic diagnostics Diagnostics Medical genomics What Are Stem Cells? Stem cells are undifferentiated cells that have the potential to differentiate into specific cells. These remarkable building blocks of life can replace or repair damaged tissues and cells. Based on this, scientists insert stem cells into areas of the body to heal or replace tissue and cells effected by disease. Three Classifications of Stem Cells: Totipotent- these are stem cells present in the zygote immediately after fertilization of the egg by the sperm during the first few cell divisions. They can differentiate to not only all cells of the body, but cells of the extraembryonic tissues, such as the placenta. Pluripotent- these are stem cells that can differentiate into any cell of the body. Multipotent- these are stem cells whose potential is limited to differentiation into various cells of their tissue of origin. Researchers see pluripotent stem cells as the holy grail of medicine because of their ability to differentiate into any cell that is needed. The pluripotent are often referred to as "unlimited" stem cells. Turipotent is avoided because extraembryonic tissue is not coveted. Multipotent, while it certainly has great ability and use, is referred to as "limited" stem cells because of their inability to widely differentia 2

3 Stem cells Stem cells are defined as cells that can divide into daughter cells that are either identical copies of themselves or are more specialized, such as nerve or muscle cells. In the 10 years since James Thomson of the University of Wisconsin-Madison first grew human embryonic stem (ES) cells in the laboratory, they have fanned a hurricane of hope and hostility that found echoes in the major-party platforms: ES cells may treat grievous conditions like diabetes, Parkinson's and spinal cord injury. This healing potential has sparked interest among patient groups, whose push for faster progress is reminiscent of the early years of the AIDS epidemic. ES cells are ethically tainted because they require the destruction of a human embryo, the tiny ball of cells that can grow into a person. This attitude has spawned laws that restrict federal funding to research on a few lineages of ES cells, forcing researchers to wade through extra paperwork and buy duplicate equipment to do ES work. In response, some states are funding ES-cell research. California's $3-billion stem-cell initiative has transformed the scientific landscape by inducing many researchers, including Thomson, to open labs there to gain state funding.

4 B0 Regeneration medicine: old desire to replace damaged tissues

5 What is a stem cell? B1 reproduce itself or A cell that can….
Differentiate to specialized cells What Are Stem Cells? Stem cells are undifferentiated cells that have the potential to differentiate into specific cells. These remarkable building blocks of life can replace or repair damaged tissues and cells. Based on this, scientists insert stem cells into areas of the body to heal or replace tissue and cells effected by disease. Three Classifications of Stem Cells: Totipotent- these are stem cells present in the zygote immediately after fertilization of the egg by the sperm during the first few cell divisions. They can differentiate to not only all cells of the body, but cells of the extraembryonic tissues, such as the placenta. Pluripotent- these are stem cells that can differentiate into any cell of the body. Multipotent- these are stem cells whose potential is limited to differentiation into various cells of their tissue of origin. Researchers see pluripotent stem cells as the holy grail of medicine because of their ability to differentiate into any cell that is needed. The pluripotent are often referred to as "unlimited" stem cells. Turipotent is avoided because extraembryonic tissue is not coveted. Multipotent, while it certainly has great ability and use, is referred to as "limited" stem cells because of their inability to widely differentia

6 Types of cells B2 Totipotent cells Pluripotent cells Multipotent cells
They can give rise to every cell type  zygote and 8-cell embryo zygote Pluripotent cells Inner cell mass - They an give rise to every cell type, except, trophoblast tissues  blastocyst inner cell mass Trophoblast cells ES cells Multipotent cells - They can give rise a limited type of cells  adult stem cells Cardiac muscle cells Unipotent cells Skin cell - They can divide, but they are able to give rise cells identical with themselves Nondividing cells - neurons, skeletal muscle cells, cardiac muscle cells: Neuron 6

7 Stem cells in our body B3 zygote blastocyst gastrula Unipotent
Non-dividing Zygote is totipotent skin cell neuron pigment cell sperm cells egg cell Germline cells ectoderm zygote blastocyst gastrula brain eye blood liver Bone marrow skin muscle Inner cell mass totipotent pluripotent multipotent Az a lényeg, hogy be kerüljön a vírus genom az ivercellsbe, mert innentől kezdve az utódok minden sejtje tartalmazni fogja az idegen DNS-t mesoderm endoderm cardiac-, skeletal musle cells red blood cells alveolar cells pancreatic cells tubulue cell smooth muscle cell thyroid cells

8 Adult stem cells B4 Brain Umbilical cord blood Multipotent stem cells
What is a mature stem cell? Stem cells are associated with most tissues of the body as part of a tissue/cell renewal mechanism — how the body regenerates its tissues. What is known to date is that mature stem cells are primarily multipotent, meaning they can yield all of the cell types associated with the tissues from which they originate. The mature stem cell is an undifferentiated (unspecialized) cell that is found in a differentiated (specialized) tissue, which can renew itself for a lifetime. What is the role of mature stem cells? Mature stem cells maintain and repair the body’s tissues in which they are found. Can mature stem cells become any type of body cell? Traditionally, mature stem cells have been considered limited in their potential to become any type of body cell. In other words, they only produce cell varieties within their own lineage or type and are considered multipotent. For example, stem cells found in bone marrow can become bone as well as cartilage, fat cells, various kinds of muscle, and the cells that line blood vessels. Can mature stem cells be pluripotent? Unlike early stem cells, there is no evidence to date that any mature stem cells are capable of forming all cells of the body. However, recent studies have demonstrated that mature stem cells may be more flexible than previously thought. More studies are necessary to validate these results. Where can mature stem cells be found? Sources of mature stem cells have been found in areas of the body including bone marrow, blood stream, cornea and retina of the eye, the dental pulp of the tooth, liver, skin, gastrointestinal tract, and pancreas. What is the most common type of mature stem cell used today? The mature stem cells associated with those that form blood in bone marrow are the most common type of stem cell used to treat human diseases today. Multipotent stem cells Neural stem cell Hematopoetic stem cell specialized cells Glial cells neurons Red blood cells mezenchymal cells Platelets White blood cells 8

9 Multipotent stem cells
B5 Adult stem cells Multipotent stem cells Specialized cells What is a mature stem cell? Stem cells are associated with most tissues of the body as part of a tissue/cell renewal mechanism — how the body regenerates its tissues. What is known to date is that mature stem cells are primarily multipotent, meaning they can yield all of the cell types associated with the tissues from which they originate. The mature stem cell is an undifferentiated (unspecialized) cell that is found in a differentiated (specialized) tissue, which can renew itself for a lifetime. What is the role of mature stem cells? Mature stem cells maintain and repair the body’s tissues in which they are found. Can mature stem cells become any type of body cell? Traditionally, mature stem cells have been considered limited in their potential to become any type of body cell. In other words, they only produce cell varieties within their own lineage or type and are considered multipotent. For example, stem cells found in bone marrow can become bone as well as cartilage, fat cells, various kinds of muscle, and the cells that line blood vessels. Can mature stem cells be pluripotent? Unlike early stem cells, there is no evidence to date that any mature stem cells are capable of forming all cells of the body. However, recent studies have demonstrated that mature stem cells may be more flexible than previously thought. More studies are necessary to validate these results. Where can mature stem cells be found? Sources of mature stem cells have been found in areas of the body including bone marrow, blood stream, cornea and retina of the eye, the dental pulp of the tooth, liver, skin, gastrointestinal tract, and pancreas. What is the most common type of mature stem cell used today? The mature stem cells associated with those that form blood in bone marrow are the most common type of stem cell used to treat human diseases today. 9

10 Adult stem cell division
And differentiation B6 A P D A: adult stem cells P: progenitor cells D: differentiated cells

11 Embryonic stem cells B7 (ES cells) Mouse stem cells (1981)
2007 Mouse stem cells (1981) Human stem cells (1998) Martin Evans Gail R. Martin James Thomson Embryonic stem cells (ES cells) were first derived from mouse embryos in 1981 by Martin Evans and Matthew Kaufman and independently by Gail R. Martin. Gail R. Martin is credited with coining the term 'Embryonic Stem Cell'.[3][4]  A breakthrough in human embryonic stem cell research came in November 1998 when a group led by James Thomson at the  University of Wisconsin-Madison first developed a technique to isolate and grow the cells when derived from human blastocysts.[5] Developmental biologist James Thomson, viewing a stem cell culture at the University of Wisconsin-Madison, reported the first isolation of embryonic stem cell lines from a nonhuman primate in 1995, and the first isolation of human ES cells in In 2007, Thomson and a group in Japan reported that they had reprogrammed skin cells to create induced pluripotent stem (iPS) cells, which look and act like ES cells.

12 Generation of ES cells B8 Humán ES cells with cloning Es cells
Tissue compatibility Es cells In vitro fertilization Nuclear transfer 5 day embryo donor nucleus innner cell mass (ICM) enucleated egg egg cell Hwang Woo Suk 2004 Humán ES cells with cloning Adult Stem Cells You might have already guessed where these come from - adult stem cells are taken from adult tissues. You can see in the picture on the right here some of the places in the body that researchers have found adult stem cells. Researchers are still hoping to identify them in many more adult tissues. Shoukhrat Mitalipov 2013

13 Generation of ES cells B9 - from ICM cells feeder cells cultured
morula blastocyst Isolated inner cell mass cells feeder cells cultured ES cells growing colonies 13

14 Generation of ES cells B10 - from ICM cells Blastocyst Inner cell mass
adipocyte Inner cell mass neuron macrophage Cultured ES cells Blastocyst Smooth muscle cell Glial cell 1. In vivo differentiation: implanting directly to the body 2. In vitro differentiation, and then implanting to the body 14

15 Generation of ES cells B11 - from germline cells
zygote totipotent cells blastocyst embryo ICM Isolated ICM cells Inner Cell Mass Isolation from Embryonic Tissue: The inner cell mass of the blastocyst of an embryo constitutes pluripotent cells. With permission from patients, researchers obtain excess embryos from in-vitro fertility clinics to isolate these cells, which are called embryonic stem cells (ES). Primordial Germ Line Isolation from Fetal Tissue: Pluripotent stem cells can be derived from the primitive germ line stem cells that exist from the blastocyst stage until their migration to and conversion within the developing gonads into either sperm or egg stem cells. Researchers obtain these stem cells from terminated pregnancies, where parents independently decide to end the pregnancy and give consent. These cells are called embryonic germ line stem cells (EG) and have very similar properties to ES. Embryonal Germ line stem cells cultivated pluripotent stem cells 15

16 Generation of ES cells B12
- from body cell by nuclear transfer (cloning) Skin cell egg Nuclear reprogramming (epigenetic reprogramming: loose of commitment) nucleus nucleus morula Nuclear Transfer:    [top] The process called nuclear transfer offers another potential way to produce embryonic stem cells. In animals, nuclear transfer has been accomplished by inserting the nucleus of an already differentiated adult cell-for example, a skin cell-into a donated egg that has had its nucleus removed. This egg, which now contains the genetic material of the skin cell, is then stimulated to form a blastocyst from which embryonic stem cells can be derived. The stem cells that are created in this way are therefore copies or "clones" of the original adult cell because their nuclear DNA matches that of the adult cell. As of the summer of 2006, nuclear transfer has not been successful in the production of human embryonic stem cells, but progress in animal research suggests that scientists may be able to use this technique to develop human stem cells in the future. Scientists believe that if they are able to use nuclear transfer to derive human stem cells, it could allow them to study the development and progression of specific diseases by creating stem cells containing the genes responsible for certain disorders. In the future, scientists may also be able to create "personalized" stem cells that contain only the DNA of a specific patient. The embryonic stem cells created by nuclear transfer would be genetically matched to a person needing a transplant, making it far less likely that the patient's body would reject the new cells than it would be with traditional tissue transplant procedures. Although using nuclear transfer to produce stem cells is not the same as reproductive cloning, some are concerned about the potential misapplication of the technique for reproductive cloning purposes. Other ethical considerations include egg donation, which requires informed consent, and the possible destruction of blastocysts. blastocyst ICM Somatic nuclear transfer

17 Generation of ES cells B13
- from germline cells by nuclear transfer (cloning) egg germline cell Reprogramming is simple, since these cells are not differentiated, but their isolation is difficult Nucleus nucleus morula Nuclear Transfer:    [top] The process called nuclear transfer offers another potential way to produce embryonic stem cells. In animals, nuclear transfer has been accomplished by inserting the nucleus of an already differentiated adult cell-for example, a skin cell-into a donated egg that has had its nucleus removed. This egg, which now contains the genetic material of the skin cell, is then stimulated to form a blastocyst from which embryonic stem cells can be derived. The stem cells that are created in this way are therefore copies or "clones" of the original adult cell because their nuclear DNA matches that of the adult cell. As of the summer of 2006, nuclear transfer has not been successful in the production of human embryonic stem cells, but progress in animal research suggests that scientists may be able to use this technique to develop human stem cells in the future. Scientists believe that if they are able to use nuclear transfer to derive human stem cells, it could allow them to study the development and progression of specific diseases by creating stem cells containing the genes responsible for certain disorders. In the future, scientists may also be able to create "personalized" stem cells that contain only the DNA of a specific patient. The embryonic stem cells created by nuclear transfer would be genetically matched to a person needing a transplant, making it far less likely that the patient's body would reject the new cells than it would be with traditional tissue transplant procedures. Although using nuclear transfer to produce stem cells is not the same as reproductive cloning, some are concerned about the potential misapplication of the technique for reproductive cloning purposes. Other ethical considerations include egg donation, which requires informed consent, and the possible destruction of blastocysts. blastocyst ICM Germ line nuclear transfer

18 Cell replacement therapy
B17 ES cells in medicine In vitro fertilization sperm Egg cell zygote morula blastula ES cells Multipotent stem cells What is the primary source of blastocysts for research? The primary sources of blastocysts for use in stem cell research are pre-embryos created by the in vitro fertilization process and donated by consenting adults once they are no longer needed for reproductive purposes. What is Somatic Cell Nuclear Transfer (SCNT)? Somatic cell nuclear transfer (SCNT) is a laboratory procedure that produces a blastocyst from an unfertilized egg and an ordinary adult somatic cell (e.g., from a single skin cell). When were early human stem cells first isolated from IVF-blastocysts? Early human stem cells were first isolated in 1998 by Dr. James Thomson and his research team at the University of Wisconsin. For more information on Dr. Thomson's research please visit his web site at the University of Wisconsin. Between 1998 and 2001, approximately 78 early stem cell lines have been created using IVF-blastocysts globally. For a table with information on the 78 stem cell lines, click here. Differentiated cells Transplantation Cell replacement therapy

19 Cell replacement therapy
B18 ES cells in medicine Egg cell Nuclear transfer Nucleus from a somatic cell of patient zygote morula blastula ES cells Multipotent stem cells How many unwanted pre-embryos are there in the United States? According to a survey conducted in 2003, there are approximately 400,000 unwanted pre-embryos in the United States. (Source: Hoffman, D.I., et al Cryopreserved embryos in the United States and their availability for research. Fertility and Sterility 79: ) These pre-embryos are no longer needed for fertility purposes and remain frozen or will be destroyed. How does SCNT work? SCNT substitutes the nucleus of a somatic cell (which contains all the genetic information of the patient) for the nucleus of a donated egg that has not been fertilized. In cell culture, this customized egg is then coaxed with an electronic or chemical catalyst to develop into a zygote as if it had been fertilized. The zygote begins cell division and develops into a ball of cells called the morula and then into the blastocyst at approximately five days. The inner cell mass of the blastocyst is then removed to generate a pluripotent stem cell line. After the inner cell mass is removed, the blastocyst is no longer capable of further development. When can we expect results from SCNT? The SCNT methodology is still in its infancy. Researchers hypothesize that when the genetic information from the cells of a patient is used, the pluripotent stem cells will be able to make customized tissue that will not be rejected by the patient. SCNT researchers contend that the knowledge gained about developmental biology via the SCNT methodology will allow future researchers to create individualized pluripotent stem cell lines without needing fertilized eggs as sources. Can SCNT be used to clone humans? The purpose of SCNT is to find cures and therapies to treat human disease. SCNT awakens the natural capacity for self-repair that resides in a person's genes. While SCNT has been the technique used to clone animals like "Dolly" the sheep, there is no evidence that it could also successfully clone a human due to the increased complexity of the human organism. Blastocysts produced by a fertilized egg (IVF) and SCNT are considered by many to be fundamentally different, and no SCNT-blastocysts should ever be implanted in a uterus. There is no conception of new life via SCNT. Differentiated cells Transplantation Cell replacement therapy

20 Adult stem cells in medicine
B19 Adult stem cells in medicine - hematopoetikus stem cells Hematopoetic stem cells Cultured stem cells Transplantation Leukemia patient Myeloid progenitor cell In the body of patients The mature stem cells associated with those that form blood in bone marrow are the most common type of stem cell used to treat human diseases today. How do mature stem cells treat diseases like cancer? For more than 30 years, bone marrow stem cells have been used to treat cancer patients with conditions like leukemia and lymphoma. During chemotherapy, most of the leukemia cells are killed as are the bone marrow stem cells needed as a patient recovers. However, if stem cells are removed before chemotherapy, and then re-injected after treatment is completed, the stem cells in the bone marrow are able to produce large amounts of red and white blood cells, to keep the body healthy and to help fight infections in medicine alkalmazott stemsejt-források [szerkesztés] Szöveti cells cells a szervezet számos szövetében megtalálhatók. Ezek az cells cells biztosítják egy életen át a folyamatosan használódó cells újraképződését, annak köszönhetően, hogy az adott szöveti környezetben képesek különböző, a szövetet alkotó speciális funkciót ellátó cellské alakulni. Három jól ismert és terápiában alkalmazott stemsejt-forrás a csontvelő, a perifériás vér és a köldökzsinórvér. Csontvelő: A csontvelő, főként vérképző cells cellset tartalmaz. A vérképző cells cellsből alakulnak ki elsstemorban a vörösvértestek, a fehérvércells, valamint a vérlemezkék. A csontvelői cells cells nyerése altatásban vagy gyakrabban spinális érzéstelenítésben, a hátsó csípőtövisekből, esetleg a szegycsontból történik. Perifériás vér: Nagy dózisú, kolóniastimuláló-faktor (CSF) előkezelés hatására a csontvelőből nagy mennyiségű stemsejt és elkötelezett elődsejt (progenitor sejt) kerül a perifériás vérbe. A transzplantációra alkalmas cells cells gyűjtése a kezelést követően a keringő vérből történik. Köldökzsinórvér: A köldökzsinórvér és az ebből nyerhető cells cells gyűjtése teljesen fájdalom – és kockázatmentesen történik közvetlenül az újszülött születését követően. Továbbá a méhlepényben és a köldökzsinórban visszamaradt vér a szülés után orvosi hulladékként megsemmisítésre kerülne, amennyiben nem rendelkeznek az abból kinyerhető cells cells megőrzéséről. Az cells cells jelenlegi gyakorlati alkalmazása [szerkesztés] A köldökzsinórvér- cells cellset emberben elstemzör a csontvelő-elégtelenségben szenvedők kezelésére használták, mivel a köldökzsinórvér- cells cells könnyen alakulnak át csontvelői cellské. Alkalmazásuk előnyösebb volt a korábban szokásos, más emberből származó csontvelővel végzett transzplantációknál. A köldökzsinórvér- cells cells ilyen célú hasznosítása is ritkábban okozott kilökődést. Másrészt az eddigi tapasztalatok szerint a köldökzsinórvér- cells cells ilyen célú alkalmazása hatékonyabb a adultek csontvelő beültetésénél. Az cells cellset a károsodott területre juttatva képesek arra, hogy a károsodott szöveteket regenerálja, újra életképessé tegye. A csontvelő rosszindulatú daganatos betegségeiben, pl. a fehérvérűség különböző típusaiban, alkalmas a betegek hatékony kezelésére. Az olyan rosszindulatú daganatos betegségekben, amelyek sugárterápiát és/vagy citosztatikus (kemotherapy) kezelést igényelnek, hasznos lenne ezeket minél nagyobb adagban alkalmazni a gyorsan osztódó rákos cells elpusztítására. Ennek azonban határt szab az ugyancsak gyorsan osztódó vérképzstemzervek sejtjeinek nem-kívánt károsodása. Az cells cells rendelkezésre állása azonban megengedhetővé teszi a sugár- és kemotherapy dózisának a növelését, ezáltal pedig a daganatos cells hatékonyabb elpusztítását, mivel a károsodott cells az cells cellsből származó csontvelő beültetésével pótolhatók. Az cells cells jövőbeni felhasználási lehetstemégei [szerkesztés] Az stemsejt-kutatás sokat ígérő eredményei alapján az cells cells azon képességének kihasználásával, hogy különböző szöveti funkcióba léptethetőek, sok más betegségben is hasznosíthatók lesznek. A neves szaklapok, mint a Nature, a Science stb., szinte minden héten beszámolnak új és fontos fejleményekről az stemsejt-kutatásban, az cells cells jövőbeni felhasználásának további sokat ígérő lehetstemégeiről. A jövőben az olyan jelenleg nem, vagy korlátozott hatékonysággal kezelhető betegségek, mint az Alzheimer- és Parkinson-kór, az agyvérzés, bizonyos izomsorvadások stb. esetén is az stemsejt-terápiától várják az előrelépést. Több közlemény jelent már meg, igazolásaként annak, hogy az cells cells laboratóriumi körülmények között képesek például inzulintermelő szigetcellské alakulni. Ezzel remény látszik arra, hogy a cukorbetegség bizonyos fajtáinál az cells cells beültetésével végre gyógyíthatóvá válhat a betegség és kiküszöbölhető lesz a naponkénti injekciós inzulinkezelés. Egy másik példa alapján, laboratóriumi körülmények között köldökzsinórvér- cells cellsből előállított idegcells működőképesek, így a neurológiai betegségek - pl. stroke vagy gerincsérülés – esetén vizsgálják a felhasználhatóságukat. Egy másik fontos alkalmazási területe a szívinfarktuson átesetteknél cells cellset juttatva a károsodott szívizomzatba, képesek voltak szívizomcellset létrehozni és a szövet regenerálódását elstemegíteni. A biotechnológiai ipar szintén ígéretes területe a „tissue engineering”, mégpedig a szövetek és szervek előállítása cells cellsből. Célja olyan szövetek előállítása, melyek segítségével akár szívbillentyűk, ízületek, porckorongok hozhatók létre, hogy aztán beültetve átvegyék a beteg szövetek, szervek funkcióját. Az cells cellsnek szerepük lehet a génterápiában is. Az stemsejtben a hibás gének egészségesre való lecserélésével lehetséges lehet – autológ transzplantáció útján – a betegek gyógyítása a „javított” cells cells beültetésével. Az cells cells hatékonyabb felhasználása érdekében számos kutató-intézetben dolgoznak olyan módszerek kifejlesztésén, melyekkel az cells cells mesterségesen szaporíthatók lesznek. Blood cells Multipotent cells (reduced potency) Hematopoetic cells cell replacement therapy 20

21 Hematopoetic stem cells
B20 Hematopoetic stem cells differentiation NK cell T lymphocyte Neutrophil granulocyte Lymphoid progenitor cell Bazophil granulocyte B lymphocyte hematopoetic Stem cell Eozinophil granulocyte Monocyte Multipotent Stem cell Myeloid progenitor cell Platelets Adult Stem Cell Plasticity It was not until recently that anyone seriously considered the possibility that stem cells in adult tissues could generate the specialized cell types of another type of tissue from which they normally reside—either a tissue derived from the same embryonic germ layer or from a different germ layer (see Table 1.1. Embryonic Germ Layers From Which Differentiated Tissues Develop). For example, studies have shown that blood stem cells (derived from mesoderm) may be able to generate both skeletal muscle (also derived from mesoderm) and neurons (derived from ectoderm). That realization has been triggered by a flurry of papers reporting that stem cells derived from one adult tissue can change their appearance and assume characteristics that resemble those of differentiated cells from other tissues. The term plasticity, as used in this report, means that a stem cell from one adult tissue can generate the differentiated cell types of another tissue. At this time, there is no formally accepted name for this phenomenon in the scientific literature. It is variously referred to as "plasticity" [15, 52], "unorthodox differentiation" [10] or "transdifferentiation" [7, 54]. Approaches for Demonstrating Adult Stem Cell Plasticity To be able to claim that adult stem cells demonstrate plasticity, it is first important to show that a cell population exists in the starting tissue that has the identifying features of stem cells. Then, it is necessary to show that the adult stem cells give rise to cell types that normally occur in a different tissue. Neither of these criteria is easily met. Simply proving the existence of an adult stem cell population in a differentiated tissue is a laborious process. It requires that the candidate stem cells are shown to be self-renewing, and that they can give rise to the differentiated cell types that are characteristic of that tissue. To show that the adult stem cells can generate other cell types requires them to be tracked in their new environment, whether it is in vitro or in vivo. In general, this has been accomplished by obtaining the stem cells from a mouse that has been genetically engineered to express a molecular tag in all its cells. It is then necessary to show that the labeled adult stem cells have adopted key structural and biochemical characteristics of the new tissue they are claimed to have generated. Ultimately—and most importantly—it is necessary to demonstrate that the cells can integrate into their new tissue environment, survive in the tissue, and function like the mature cells of the tissue. In the experiments reported to date, adult stem cells may assume the characteristics of cells that have developed from the same primary germ layer or a different germ layer (see Figure 4.2. Preliminary Evidence of Plasticity Among Nonhuman Adult Stem Cells). For example, many plasticity experiments involve stem cells derived from bone marrow, which is a mesodermal derivative. The bone marrow stem cells may then differentiate into another mesodermally derived tissue such as skeletal muscle [28, 43], cardiac muscle [51, 71] or liver [4, 54, 97]. Red blood cells odipocita osteocytes Stromal cells They protect hematopoetic cells 21

22 Hematopoetic stem cells
B21 Hematopoetic stem cells Use in medicine (1) Bone marrow (from 1950’s): the only successful stem cell therapy - Medication: ◊ leukemia ◊ sickle cell anemia ◊ immune deficiency ◊ bone marrow damages ◊ some metabolic disorders - Unpleasant intervention - Successful if the donor and recipient are genetically similar (2) Umbilical blood and (3) placenta blood (new discovery): - Use: medication of the donor person decades after the isolation or the medication of relatives Bone marrow 22

23 Stem cells in medicine - stromal stem cells Bone marrow B22 stromal
Ex vivo cultivation Bone regeneration requiresex vivo expansion of marrow-derived skeletal stem cells and their attachment to three-dimensional scaffolds, such as particles of a hydroxyapatite/tricalcium phosphate ceramic. This composite can be transplanted into segmental defects and will subsequently regenerate an appropriate three-dimensional structure in vivo. In vivo transplantation 23

24 Stem cells in medicine - cardiomyocyte therapy B23 Blastocyst
Genetic manipulation Smooth- and cardiac muscle cells, endothelial cells ES cells  differentiation in vitro differentiation Blastocyst TRANSPLANTATION TRANSPLANTATION, in vivo differentiation Heart progenitor cells The discovery of dividing cells that resemble immature cardiomyocytes in the zone bordering an infarct border zone suggested that the adult heart contains stem cells, although the origin of these cells was unclear88. Antonio Beltrami and colleagues88 subsequently isolated a lineage-negative (Lin- , a cell population that lacks the immunophenotypic antigens that define mature peripheral blood-cell lineages) Kit-positive cell population from the heart of adult mice that was clonogenic (they are clones of one another), self-renewing and capable of differentiating into cardiomyocytes, vascular smooth muscle cells and endothelial cells both in vitro and in vivo89 (see figure). Later, two other groups reported the isolation of adult cardiac stem cells from mice on the basis of expression of stem-cell antigen 1 (SCA1) or exclusion of Hoechst dye (because of the presence of the ATP-binding cassette transporter ABCG2)90, 91. These dye-excluding cells are also referred to as side population (SP) cells because of their profiles after sorting by flow cytometry. These three populations of cells (Kit+ cells, SCA1+ cells and SP cells) from the adult heart differ phenotypically and in their expression of cell-surface markers92, 93, 94. Their exact relationship to embryonic cardiac progenitor cells that have been found in the fetal heart6, 7, 95 is unknown (see ref. 3 for a review). To determine whether such endogenous cardiac progenitor cells are present in the human heart, scientists isolated a heterogeneous population of cells from biopsied human atrial and ventricular samples by using a mild enzymatic digestion. From the tissue pieces, clonal, multicellular clusters of cells formed in suspension culture, and these are referred to as cardiospheres96. Cardiospheres consist of proliferating Kit+ stem cells in the core and cells expressing cardiac (myosin heavy chain, troponin I and atrial natriuretic peptide) and endothelial-cell (KDR; also known as FLK1) and CD31) markers on the periphery. In addition, cardiospheres express stem-cell markers CD34 and SCA1 (mouse). Cardiospheres can be passaged and frozen, but the cardiomyocytes derived from these will beat only if they are co-cultured with neonatal rat cardiomyocytes. More recently, cardiosphere-derived cells with improved isolation and expansion efficiencies have been generated from biopsied endomyocardial samples from the right ventricle of adult patients; however, these cells were still isolated as a mixed population without cell sorting or antigenic selection97. By contrast, several studies have identified an endogenous Kit+ cell population that could be selected from cardiac tissue by using a Kit-specific antibody98. These cells have been termed human cardiac stem cells. In contrast to human cardiosphere-derived cells, human cardiac stem cells express multidrug resistance protein 1 (MDR1; also known as ABCB1)99, 100. These studies describe another cardiac progenitor-cell population characterized by the presence of the marker MDR1. It is unclear whether these different stem-cell populations are derived from the heart or are associated with it through the circulation. Apart from MDR1, the main difference between human cardiosphere-derived cells and human cardiac stem cells is that cardiac stem cells express CD45 and CD133 (also known as PROM1). CD34, CD45 and CD133 are all expressed by bone-marrow-derived or endothelial progenitor cells, which might indeed populate the heart by way of the circulation. The various cell types that can be isolated from fetal and adult hearts by flow-cytometric separation of cells exposed to antibodies specific for the cell-surface proteins SCA1, Kit and MDR1 are shown in the figure. Cardiac progenitor cells expressing the cardiac transcription factors NKX2-5 and Isl1 may be included within these populations (because they are markers of cardiac progenitor cells). Cardiac progenitor cells can also be isolated from embryonic stem cells (human or mouse) by using flow cytometry, if they have been genetically marked at the NKX2-5 and ISL1 loci. To repair the heart, these cardiac progenitor cells, as isolated by any of the procedures described, could be transplanted directly into a mouse heart, leading to further differentiation of these cells in vivo to become vascular smooth muscle cells, endothelial cells or cardiomyocytes (or a combination of all three cell types). Alternatively, cardiac progenitor cells could be subjected to specific in vitro differentiation protocols for generating these cell types, which could then be transplanted directly into the heart or transplanted after tissue engineering into mice and humans (Box 1). Smooth- and cardiac muscle cells, endothelial cells Heart (embryonic or adult)

25 Stem cells in medicine - skin stem cells B24 Burn injuries:
Traditional treatment – skin transplantation from undamaged body parts, success, since stem cells are located directly under the epidermis. If there were not enough skin, the patient could die. Since 20 years – cultivation of skin stem cells Discovery of new skin stem cells – in deeper layers of skin and in hair bulbs epidermis dermis hypodermis

26 Anterio-lateral chamber subventricular zone (SVZ)
Neural stem cells (NSC) - neurogenesis in adult brain SVZ hippocampus SVZ Olfactory bulb cerebellum NSC NSC New neurons hippocampus Sites of adult neurogenesis (rodent studies) compared with appropriate human brain regions. Neurogenesis has been confirmed in two regions of the adult brain: the subventricular zone (SVZ) of the anterior lateral ventricles (the site of origin for olfactory bulb neurons) and the dentate gyrus of the hippocampus (a brain region involved in learning and memory). In the SVZ, progenitor cells migrate to the olfactory bulb, where they differentiate into neurons. In the dentate gyrus, cells divide along the subgranular zone (also see the figure in the sidebar, below) and migrate into the granule cell layer before terminally differentiating into granule cells. cerebellum hippocampus HUMAN BRAIN RAT BRAIN Anterio-lateral chamber subventricular zone (SVZ)

27 Stem cells in medicine - neural stem cells Parkinson’s disease B26
- Those neurons that are responsible for the elimination of unnecessary movements, are degenerated at the substantia nigra  implantation of stem cells has not been proven to be successful 27

28 Stem cells in medicine - neural stem cells Spinal cord damage B27 28
Broken bone 28

29 Y chromosome in female brain? Y chromosome in neurons:
Éva Mezey New discoveries in stem cell technology are highly promising to treat lots of diseases of different cause. Eva Merey et al. in their recent study revealed that transplanted bone marrow (BMT) generates new neurons in recipients. They have tested four patients, females, of different age, who received BMT, for the presence of Y chromosome positive cells in their brain. It was found that most of Y positive cells found in brain were non neuronal (this is usual). Nevertheless some of them were donor-derived neurons (7 in in the youngest and the longest survived patient). In conclusion this group of scientists resumed that 1 every neurons might derived from the BMT. Such results are promising not only for neurodegenerative disease patients. It is known that different stem cells have the ability to engraft all kind of tissue, what has already been shown on rodents (after BMT in rodents you have 50 engrafted cells per rodent neurons). Using stem cells we can renovate any kind of worn out tissue, as seen here- brain too could be influenced as this is very important. In such a way, making tissues and organism, as efficient as 65 person is a 16-year teen. Unless it is still much work to be done in targeting stem cells where we want them to go and want them to be. Abbreviation: BMT- bone marrow transplantation cells cells Mezey Éva PHD, programvezető, In Situ Hybridization Facility Basic Neuroscience Program, National Institute of Neurological Diseases and Stroke, NIH, Bethesda cells cells: csodatevők vagy csak csodák? "...több vagyok a soknál, mert az stemsejtig vagyok minden stem" József Attila Az utóbbi években mind a tudományos, mind a népszerűsítő irodalomban nap mint nap hallunk az cells cellsről és a velük kapcsolatos reményeinkről. Mi is az stemsejt? A megtermékenyített petesejt osztódása után alakul ki a blasztocita, egy cellskel körülvett üreg, melynek egyik pólusán lévő sejttömegből lesz az embrió. Ez a sejtmassza embryonic cells cellsből áll, melyeket totipotentnek képzelünk. Ez azt jelenti, hogy ezekből az cells cellsből bármilyen szövet kialakulhat. Az embryonic fejlődés során három sejtréteg alakul ki: a külső ektoderma sejtjei a bőrt és az idegrendszert fogják létrehozni; a középső sejtrétegből (mezoderma) képződik majd a csontrendszer, az izmok, és a vérképző rendszer; a belső (endoderma) sejtréteg pedig a gasztrointesztinális rendszert és a tüdőket fogja kialakítani. A három dermalis rétegben lévő cells cells "multipotentek", ami azt jelenti, hogy az adott dermális határokon belül képesek bármilyen sejtté alakulni. Eddig azonban úgy hittük, hogy ezek a cells a "dermális" határokat sosem léphetik át: egy izomsejtből soha nem lehet már bélsejt és fordítva. cells cellset nemcsak a fejlődésben lévő, hanem a adult, kifejlett organizmusokban is találunk. Tekintettel arra, hogy tudjuk, hogy szöveteink regenerálódnak, az cells cells jelenléte adult szervezetben önmagában nem meglepő. Régóta tudjuk, hogy a vércells folyamatosan újraképződnek a csontvelőben lévő differenciálatlan cellsből. Egészen az utóbbi időkig azonban úgy hittük, hogy a adult szervezetben lévő szöveti cells cells csak az adott szövet sejtjeit képesek újratermelni - így differenciálódási lehetstemégük jóval szűkebb a dermális cells cellsénél. Az elmúlt négy évben azonban sok adat látott napvilágot különböző tudományos folyóiratokban, melyek arra mutattak, hogy a természet nem minden esetben követi a fejlődéstanban megtanult szigorú szabályokat. Az új elképzelésnek, hogy adult szöveti cells cells képesek teljesen új irányba differenciálódni és áttörni a dermális gátat, sok támogatója és ellenzője van a szakmában. A jelenséget transzdifferenciálódásnak nevezték el, ami tehát azt jelenti, hogy például egy ektodermális szöveti stemsejt környezeti hatásra képes olyan szöveti sejtté differenciálódni, amely a fejlődés során nem ektodermából (hanem mesodermából vagy endodermából) származott (1. ábra). Az új teóriát ellenzők körébe tartoznak azok, akik az embryonic cells cells terápiás felhasználásán dolgoznak - mivel ha igaznak bizonyul az, hogy szöveti (adult) cells cells használhatók regenerációra, az embryonic stemsejtkutatás politikai és tudományos támogatása jelentstemen csökkenne. Itt mindenekelőtt szeretnénk megjegyezni, hogy az cells cells (bármilyen eredetűek is legyenek) terápiás felhasználása még egyáltalában nem bizonyított. Jelenleg nincs rá megbízható tudományos adat, hogy bármilyen stemsejt képes pótolni sérülés vagy betegség következtében elpusztult szövetet, és így egyetlen fajta stemsejt sem látszik jobbnak a többinél. Az embryonic stemsejtkutatás tehát éppúgy megérdemli a támogatást, mint a szövetspecifikus adult szervezetben található cells cellsé. Az utóbbiakkal kapcsolatos kutatás azonban még gyerekcipőben jár - alapos tanulmányozásuk csupán néhány éve kezdődött meg. Aki a szakmai irodalmat olvassa, nehezen igazodik el az adatokban, melyek a adult cells cells differenciálódási lehetstemégeit vizsgálják. A zavarosság oka részben az, hogy különböző kutatócsoportok különböző oldalról közelítik meg a problémát, és a kép még nem állt össze. A kérdések közül a legfontosabbak egyike, hogy előfordul-e fiziológiásan transzdifferenciálódás. Vajon a vizsgált cells cells átprogramozódnak-e, vagy a bennük lévő genetikus anyag összeolvad egy meglévő (már differenciált) sejt magjával, és ez a magfúzió a magyarázata a sejt karakterváltozásának? Akár a transzdifferenciálódás, akár a fúzió előfordul-e olyan mértékben, aminek terápiás haszna lehet, és ha igen, tudjuk-e a folyamatot irányítani? A fenti kérdések tükrében nézzük meg a csontvelőben található cells cellset. Ezek a cells a legújabb adatok szerint nemcsak a vércellset képezik újra, hanem képesek minden szövet sejtjeihez hozzájárulni - beleértve az agyat is. Ezt úgy bizonyították be, hogy egerekbe kétféle csontvelstemejtet fecskendeztek be: vagy olyan cells cellset, melyekhez genetikusan zöld fluoreszcens festéket kötöttek (Brazelton, 2000); vagy nstemtény állatba hím állatból származó csontvelőt juttattak, és az Y kromoszómát használták nyomkövetésre (Mezey, 2000). A genetikusan jelölt cellskel potenciálisan problematikus lehet, hogy a nyomkövetésre használt zöld fluoreszcens festék expressziója nem stabil (Mezey, 2003). Az Y kromoszóma igen megbízható marker, azonban technikailag nehéz a vizualizálása, valamint az Y kromoszómát tartalmazó cells karakterének egyidejű azonosítása. A nehézség ellenére azonban ez kivitelezhető, és megbízható adatokat szolgáltat. További kérdést vet fel az a tény, hogy sem a fluoreszcens, sem a hím csontvelő nem lett egészséges (kontroll) állatoknak beadva. Ennek oka az, hogy annak érdekében, hogy az új csontvelőcells megtapadjanak és osztódjanak, a fogadó állat saját csontvelejét gyengíteni kell. Ezt általában besugárzással érik el (Brazelton, 2000; Goodell, 2001; Krause, 2001; Nakano, 2001; Theise, 2000; Wagers, 2002), vagy olyan genetikailag előállított egér használatával, mely fehérvércells nélkül születik (Mezey, 2000). Jelenleg még nem tudhatjuk, hogy a besugárzás és/vagy a genetikai manipulálás befolyásolja-e a kapott eredményeket. Amikor a transzplantáció után csontvelőből származó cellset találunk a különböző szövetekben, újabb nehézséget jelent a csontvelőcells markereinek további azonosítása az adott szövetspecifikus cellskel. Az agyban például nem elég kimutatni az Y kromoszómát, hanem idegcellsre jellemző fehérjék kimutatásával azt is be kell bizonyítani, hogy ugyanaz a sejt (vagy sejtmag) tartalmazza az Y kromoszómát, mint a specifikus (idegsejt-specifikus) fehérjét. Ennek egyértelmű kimutatása csak konfokális mikroszkóp segítségével lehetséges, mert ez kizárja, hogy egymás fölött lévő struktúrák átfedése okozná a kolokalizációt. Más szövetekben a feladat könnyebb lehet. A száj nyálkahártyasejtjeit szét lehet kenni egy mikroszkóp tárgylemezére, és a cellset így egyenként lehet megvizsgálni. Ezt a módszert használtuk laboratóriumunkban, amikor szájnyálkahártya cellset gyűjtöttünk olyan, korábban leukémiás nőbetegektől, akik betegségük során férfi csontvelőátültetésben részesültek. Bár hasonló betegek agyában már korábban kimutattuk (Mezey, 2003) igen kis százalékban (0,3%) a csontvelőből származó Y kromoszóma-tartalmú cells jelenlétét, mi is meglepődtünk azon, hogy az Y kromoszómát tartalmazó (azaz a beültetett csontvelőből származó) differenciált szájnyálkahártya-cells száma a betegekben 0,8-12,7 % között mozgott (Tran, 2003). Ezekben a cellsben egyidejűleg meg tudtuk festeni az X és az Y kromoszómákat, és közel tízezer sejt megvizsgálása azt mutatta, hogy csak igen elvétve (két sejt a tízezerből) vannak diploid cells, amiknek a sejtmagjában a normális kromoszómaszám kétszerese van meg, tehát valószínűleg két sejt (egy szájnyálkahártyasejt és egy csontvelstemejt) fúziójából jöttek létre, és nem a csontvelstemejt "átprogramozódásának" a következményei. Ez a kísérlet azt mutatta, hogy emberben a fúzió (legalábbis a szájnyálkahártyában) igen ritka, és azt bizonyította, hogy adult cells cells valóban képesek átváltozni olyan cellské, melyek a fejlődés során más dermális rétegből eredtek. Ez természetesen nem azt jelenti, hogy a sejtmagfúzió jelentstemégével nem kell számolni. Tudjuk, hogy a sejtfúzió kétségtelenül élettani jelenség. A májszövetben például ismert, hogy néha a cells több mint fele diploid - azaz fúzió eredménye. A közelmúltban két kutatócsoport tanulmányozta a genetikailag fumarylacetoacetát-hydroláz enzim hiányában szenvedő egereket (Vassilopoulos, 2003; Wang, 2003). Ezek az egerek kezelés nélkül elpusztulnak. Amikor azonban egészséges (a hiányzó enzimet tartalmazó) csontvelővel transzplantálják őket, képesek egészséges életre. Ezekben a transzplantált egerekben a májcells nagy százaléka az egészséges csontvelőcells és a beteg májcells fúziójának eredményeképpen jött létre. Ezen kísérlet értékelésekor érdemes elgondolkodnunk a máj különleges szerepén. Mivel a máj elsődleges szerepe a méregtelenítés, a májcells folyamatosan károsodásnak vannak kitéve. Amennyiben nem tudják a DNS-üket jó hatásfokkal és gyorsan megjavítani, könnyű elképzelni, hogy nagyszámú mutáció jönne létre, és előbb-utóbb az onkogének mutációjának rákos elfajulás lenne a következménye. Ha azonban feltételezzük, hogy fúzió által egy-egy létszükséges génből nem kettő, hanem négy, nyolc vagy akár tizenhat kópia is lehet egy májsejten belül, akkor már valószínűtlen, hogy ugyanaz a gén ugyanolyan módon mutálódik mindegyik kópiában, tehát így nem jön létre rákos burjánzás. Más szóval a májcellsnél a fúzió az önvédelmi rendszer szerves része lehet. Ennek tükrében azt mondhatjuk, hogy míg ismerten multiploid cells esetében a magfúzió természetes mechanizmus lehet, addig olyan szöveteknél, melyek diploidok maradnak egy életen át (ide tartozik a legtöbb magasabbrendű állati szövet), nem valószínű a fúzió, hanem a cells folyamatos újraképződésében a keringő cells cells transzdifferenciálódása játszhat szerepet. A közelmúltban David Anderson (Anderson, 2001) javasolta, hogy mielőtt transzdifferenciálódásról számolnának be, a kutatók győződjenek meg arról, hogy a kísérletek a következő három feltételt kielégítik-e: (1) a használt cells cells klonálisak, (2) használat előtt nem voltak in vitro körülmények között tenyésztve és (3) az új (például transzdifferenciált) sejttípus teljes mértékben funkcionális az új környezetben. Ezeknek a feltételeknek talán nemcsak elméleti jelentstemégük van. A klonális cells használata valószínűleg nagyban megnövelné az esetleges therapy hatásosságát. Mindenki egyetért azzal, hogy fontos lenne tudni, pontosan melyik fajta csontvelő- cells cellsből származnak neuronok, gliacells, izomcells. Az a feltétel azonban, mely nem engedi a beültetés előtti szövettenyészet használatát, már nem egyértelműen elfogadható. Elképzelhető ugyanis, hogy a szövetekből izolált cellset elstemzör tenyészetben dedifferenciálni kell, vagy esetleg előkészíteni a szükséges irányba való fejődést (például neurális vagy izomsejt) különböző ismert (vagy még nem ismert) anyagok használatával. Erre egy példa Ingvild Mikkola és csoportjának kísérlete (Mikkola, 2002), amikor már teljesen kifejlett B limfocitákat szövettenyészetben kezelve elérték azt, hogy a cells dedifferenciálódtak, majd képesek voltak egy másik sejt (makrofág) irányába fejlődni. A lényeges kérdés nem szükségszerűen az, hogy fiziológiásan mi történik, hanem az, hogy mi lehetséges - esetleg még olyan környezeti és vegyi hatások segítségével is, amiket mesterségesen hozunk létre. A harmadik feltétellel egyet kell értenünk, hiszen a cells funkcionális volta elengedhetetlen ahhoz, hogy terápiásan szöveti regenerációra használhatóak legyenek. Annak bizonyítása azonban, hogy a csontvelőből származó idegcells működőképesek, nem egyszerű feladat. Míg szövettenyészetben lehetséges elektrofiziológia segítségével kimutatni, hogy a cells idegsejtként viselkednek, ezt "in vivo" nem lehet vizsgálni - mivel jelenleg még nem tudjuk a beépült cellset így felismerni. Ha el tudjuk érni, hogy nagyságrendekkel több sejt épüljön be, és váljon neuronná, akkor lehetségessé válna egy-egy rendszer funkciójának vizsgálata. Valószínű, hogy a nehézségek a különböző szövettípustól függően különbözőek. A közeljövő feladata az, hogy kiderítsük, mely szöveteket tudjuk (és mely szöveteket nem tudjuk) cells cells segítségével regenerálni; tudunk-e megfelelő állatmodelleket létrehozni, és tudjuk-e optimalizálni az cells cells kezelését és beadását úgy, hogy sikeres terápiás eszközökké válhassanak. Kulcsszavak: adult stemsejt, csontvelő-stemsejt, transzplantáció, fúzió, transzdifferenciálódás Y chromosome in neurons: in situ hybridization 29

30 A adult stem cell plasticity
B30 A adult stem cell plasticity LIVER CNS stem cells SKELETAL MUSCLE BRAIN BONE MARROW BLOOD CELLS STROMAL marrow cells Adult Stem Cell Plasticity It was not until recently that anyone seriously considered the possibility that stem cells in adult tissues could generate the specialized cell types of another type of tissue from which they normally reside—either a tissue derived from the same embryonic germ layer or from a different germ layer (see Table 1.1. Embryonic Germ Layers From Which Differentiated Tissues Develop). For example, studies have shown that blood stem cells (derived from mesoderm) may be able to generate both skeletal muscle (also derived from mesoderm) and neurons (derived from ectoderm). That realization has been triggered by a flurry of papers reporting that stem cells derived from one adult tissue can change their appearance and assume characteristics that resemble those of differentiated cells from other tissues. The term plasticity, as used in this report, means that a stem cell from one adult tissue can generate the differentiated cell types of another tissue. At this time, there is no formally accepted name for this phenomenon in the scientific literature. It is variously referred to as "plasticity" [15, 52], "unorthodox differentiation" [10] or "transdifferentiation" [7, 54]. Approaches for Demonstrating Adult Stem Cell Plasticity To be able to claim that adult stem cells demonstrate plasticity, it is first important to show that a cell population exists in the starting tissue that has the identifying features of stem cells. Then, it is necessary to show that the adult stem cells give rise to cell types that normally occur in a different tissue. Neither of these criteria is easily met. Simply proving the existence of an adult stem cell population in a differentiated tissue is a laborious process. It requires that the candidate stem cells are shown to be self-renewing, and that they can give rise to the differentiated cell types that are characteristic of that tissue. To show that the adult stem cells can generate other cell types requires them to be tracked in their new environment, whether it is in vitro or in vivo. In general, this has been accomplished by obtaining the stem cells from a mouse that has been genetically engineered to express a molecular tag in all its cells. It is then necessary to show that the labeled adult stem cells have adopted key structural and biochemical characteristics of the new tissue they are claimed to have generated. Ultimately—and most importantly—it is necessary to demonstrate that the cells can integrate into their new tissue environment, survive in the tissue, and function like the mature cells of the tissue. In the experiments reported to date, adult stem cells may assume the characteristics of cells that have developed from the same primary germ layer or a different germ layer (see Figure 4.2. Preliminary Evidence of Plasticity Among Nonhuman Adult Stem Cells). For example, many plasticity experiments involve stem cells derived from bone marrow, which is a mesodermal derivative. The bone marrow stem cells may then differentiate into another mesodermally derived tissue such as skeletal muscle [28, 43], cardiac muscle [51, 71] or liver [4, 54, 97]. ADIPOCYTE EPIHELIAL CELL CARDIAC MUSCLE NEURON GLIAL CELL Stromal (mesenchymal) stem cells in the marrow

31 B32 Embryonic stem cells NO 5 day embryo STOP

32 Adult stem cell plasticity
B31 Stromal cells Mesenchimal stem cells (MSC; stromal stem cells) Not only structural role, but a role in tissue regeneration, too. Sources: - Bone marrow, umbilical cord blood, fat tissue While the terms Mesenchymal Stem Cell and Marrow Stromal Cell have been used interchangeably, neither term is sufficiently descriptive as discussed below: Mesenchyme is embryonic connective tissue that is derived from the mesoderm and that differentiates into hematopoietic and connective tissue, whereas MSCs do not differentiate into hematopoietic cells. Stromal cells are connective tissue cells that form the supportive structure in which the functional cells of the tissue reside. While this is an accurate description for one function of MSCs, the term fails to convey the relatively recently-discovered roles of MSCs in repair of tissue. Because the cells, called MSCs by many labs today, can encompass multipotent cells derived from other non-marrow tissues, such as adult muscle or the dental pulp of deciduous baby teeth, yet do not have the capacity to reconstitute an entire organ, the term Multipotent Stromal Cell has been proposed as a better replacement. Mesenchymal stem cells or MSCs are multipotent stem cells that can differentiate into a variety of cell types. Cell types that MSCs have been shown to differentiate into in vitro or in vivo include osteoblasts, chondrocytes, myocytes, adipocytes, and, as described lately, beta-pancreatic islets cells[1]. However, in vivo results are now thought to be a misinterpretation of spontaneous cell fusion between a damaged neuron and a mesenchymal stem cell placed in the brain Doubts: it is possible that stromal cells only fused with other cell types instead of differentiating to other cell types. MSC: mesenchymal stem cells = marrow stromal cells

33 B33 embryonic stem cells Singapore - Biopolis 33

34 B33 Embryonic stem cells He changed the „stem cell law” at the very first 24 hrs 34

35 What stem cells are good for?
B34 What stem cells are good for? Basic research: generation of knock-out animals Medical research: drug tests Medicine: medication: Real applications: - Testing the cells of sick patients - Leukemia, Parkinson- and disease, neural damage, seizure, cardiac disease, diabetes - Leukemia, skin regeneration, heart attack

36 Genaration of iPS cells
B14 Genaration of iPS cells - by reprogramming body cells Cell reprogramming Mouse iPS cells (2005) Transfer of fibroblast cells to feeder cells Transformed cells Delivery of iPS factors-encoding genes by retrovirus vector Shinya Yamanaka Isolate and culture host cells. e.g. mouse embryonic fibroblasts and adult human dermal fibroblasts. Introduce the ES specific genes (iPS factors) into the cells by using retrovirus vector. Red cells indicate the cells expressing the exogenous genes. Harvest and culture the cells according to the method for ES cell culture using feeder cells (gray). A subset of the cells generates ES-like colonies, that is, iPS cells iPS cells Skin fibroblast cells Some cells are transformed to iPS cells iPSC: induced Pluripotent Stem Cells 36

37 Generation of iPS cells
B15 Generation of iPS cells - By reprogramming body cells Cell reprogramming Body cells Human iPS cells (2007) Oct4, Sox2, Klf4, c-Myc genes delivery James Thomson Reprogrammed cells resemble to the ES cells iPSC: induced Pluripotent Stem Cells 37

38 What and how to deliver? 36 Somatic cells Transcription factors
András Nagy Somatic cells Transcription factors Integrating virus Non-integrating vector Excisable vector Protein Small molecules 38

39 Transcription factors
37 Transcription factors Pluripotent cell Multipotent cell Differentiated cell 39

40 Cell reprogramming and gene therapy in medicine
B28 Genetic improvement by homologous recombination Skin fibroblast cells iPS cells Healthy or sick individual differentiation renewal In 2006, it was shown, by Kazutoshi Takahashi and Shinya Yamanaka83, for the first time that overexpression of only four proteins — OCT4, SOX2, Myc and KLF4 — in mouse embryonic or adult fibroblast cultures could generate pluripotent stem-cell-like cells (see figure). Just a year later, the same group84 and a another group, led by James Thomson85, succeeded independently in generating human induced pluripotent stem (iPS) cells from adult skin fibroblasts. Thomson's group used overexpression of OCT4 and SOX2, in combination with two other proteins, NANOG and Lin28, instead of Myc and KLF4. In both studies, iPS cells showed the essential characteristics of ES cells in terms of their morphology, cell-surface markers, gene-expression profiles and telomerase activity. Furthermore, iPS cell clones could be maintained in culture for several months at least and could be induced to differentiate into derivatives of all three embryonic germ layers both in vitro and in vivo in mouse teratomas. Reactivation of Myc increased tumorigenicity in chimaeric mice derived from mouse iPS cells, and a modified protocol was developed that did not require activation of Myc in either mouse or human cells86. Thus, it became feasible to generate iPS cells from fibroblast cultures from patients (with genetic defects corrected if necessary), and these cells could then, in principle, be induced to differentiate into a variety of patient-specific cell types, allowing transplantation without the risk of immune rejection (see figure). Recently, Jacob Hanna and colleagues87 provided a proof of principle for iPS-cell-based treatment in combination with genetic repair in a mouse model for sickle-cell anaemia, a genetic blood disorder. Tail-tip fibroblasts from these mice were infected with OCT4-, SOX2-, KLF4- and Myc-expressing viruses to generate iPS cells. (Myc copies were then deleted by using the recombinase Cre.) Subsequent homologous recombination in the iPS cells and correction of the genetic defect by the wild-type human variant rescued the phenotype; hematopoietic progenitor cells that were derived from these cells formed aggregates (known as embryoid bodies) in culture and were able to rescue the anaemic mice after transplantation, and the mice were cured. This experiment was the perfect combination of cell and gene therapy. These studies open up exciting prospects for the treatment of various genetic diseases and other abnormalities that might benefit from cell-based therapy. However, the present methods for generating iPS cells require genetic integration by retroviruses or lentiviruses, even though oncogenes, such as Myc, no longer seem to be necessary86. Furthermore, in terms of transplantation, the issues of cell integration, survival and safety still need to be addressed. The more immediate applications of human iPS cells are likely to be the creation of human models of human disease in vitro for studying the underlying molecular mechanisms of disease, for screening drug candidates, and for assessing drug safety and toxicity (see figure). TRANSPLANTATION In vitro testing e.g. selection amon drug candidates 40

41 3D organ printing 41 From adult stem cells or iPSC Problems:
biopolymer biopolymer In 2006, it was shown, by Kazutoshi Takahashi and Shinya Yamanaka, for the first time that overexpression of only four proteins — OCT4, SOX2, Myc and KLF4 — in mouse embryonic or adult fibroblast cultures could generate pluripotent stem-cell-like cells (see figure). Just a year later, the same group84 and a another group, led by James Thomson85, succeeded independently in generating human induced pluripotent stem (iPS) cells from adult skin fibroblasts. Thomson's group used overexpression of OCT4 and SOX2, in combination with two other proteins, NANOG and Lin28, instead of Myc and KLF4. In both studies, iPS cells showed the essential characteristics of ES cells in terms of their morphology, cell-surface markers, gene-expression profiles and telomerase activity. Furthermore, iPS cell clones could be maintained in culture for several months at least and could be induced to differentiate into derivatives of all three embryonic germ layers both in vitro and in vivo in mouse teratomas. Reactivation of Myc increased tumorigenicity in chimaeric mice derived from mouse iPS cells, and a modified protocol was developed that did not require activation of Myc in either mouse or human cells86. Thus, it became feasible to generate iPS cells from fibroblast cultures from patients (with genetic defects corrected if necessary), and these cells could then, in principle, be induced to differentiate into a variety of patient-specific cell types, allowing transplantation without the risk of immune rejection (see figure). Recently, Jacob Hanna and colleagues87 provided a proof of principle for iPS-cell-based treatment in combination with genetic repair in a mouse model for sickle-cell anaemia, a genetic blood disorder. Tail-tip fibroblasts from these mice were infected with OCT4-, SOX2-, KLF4- and Myc-expressing viruses to generate iPS cells. (Myc copies were then deleted by using the recombinase Cre.) Subsequent homologous recombination in the iPS cells and correction of the genetic defect by the wild-type human variant rescued the phenotype; hematopoietic progenitor cells that were derived from these cells formed aggregates (known as embryoid bodies) in culture and were able to rescue the anaemic mice after transplantation, and the mice were cured. This experiment was the perfect combination of cell and gene therapy. These studies open up exciting prospects for the treatment of various genetic diseases and other abnormalities that might benefit from cell-based therapy. However, the present methods for generating iPS cells require genetic integration by retroviruses or lentiviruses, even though oncogenes, such as Myc, no longer seem to be necessary86. Furthermore, in terms of transplantation, the issues of cell integration, survival and safety still need to be addressed. The more immediate applications of human iPS cells are likely to be the creation of human models of human disease in vitro for studying the underlying molecular mechanisms of disease, for screening drug candidates, and for assessing drug safety and toxicity (see figure). Problems: vasculature, innervation Video on ear printing: 41