MBG342 Stem Cell Technologies Fall 2024-2025 PDF

10/14/2024 MBG342 Stem Cell Technologies Fall 2024-2025 Prof.Dr. Sevim Işık Üsküdar University [email protected]...

10/14/2024 MBG342 Stem Cell Technologies Fall 2024-2025 Prof.Dr. Sevim Işık Üsküdar University [email protected] 1 1 Grading: Midterm: % 40 Assignment/Presentation: %10 Final Exam: %50 Attendance: You should attend at least %70 of the classes. Reading: Course Book: Reseach Articles 2 2 1  10/14/2024 Contents ▪ Introduction to Stem Cells ▪ Embryonic Stem Cells ▪ Tissue Stem Cells ▪ Inhduced Pluripotent Stem Cells ▪ Ethical Issues in Stem Cell Research ▪ Stem Cells and Regenerative Medicine ▪ Stem Cells in Cell and Gene Therapy ▪ Stem Cells in Tissue Engineering ▪ Cancer Stem Cells: Basic Concepts and Therapeutic Implications ▪ Stem cell therapeutics for cancer ▪ Stem Cells as Disease Modeling and Drug Screening ▪ Therapeutic Applications of Stem Cell Derived Exosomes 3 Stem Cells 4 2  10/14/2024 A life story… 5 What is a stem cell? A cell that has the ability to continuously divide and differentiate (develop) into various other kind(s) of cells/tissues. The body is made up of about 200 different kinds of specialised cells such as muscle cells, nerve cells, fat cells and skin cells. All cells in the body come from stem cells. A stem cell is a cell that is not specialised yet. The process of specialisation is called differentiation. Once the differentiation pathway of a stem cell has been decided, it can no longer become another type of cell on its own. 6 3  10/14/2024 What is a stem cell? stem cell SELF-RENEWAL DIFFERENTIATION (copying) (specializing) specialized cell stem cell e.g. muscle cell, nerve cell 7 What is a stem cell? Stem cell Stem cell SELF-RENEWAL DIFFERENTIATION (copying) (specializing) Identical stem cells Specialized cells 8 4  10/14/2024 Why self-renew AND differentiate? 1 stem cell 1 stem cell 4 specialized cells Self renewal - maintains Differentiation - replaces dead or damaged the stem cell pool cells throughout your life 9 Where are stem cells found? embryonic stem cells tissue stem cells blastocyst - a very early fetus, baby and throughout life embryo 10 5  10/14/2024 Types of stem cells Stem cell Description Examples type Totipotent Each cell can develop into Cells from early (1-3 days) a new individual embryos Pluripotent Cells can form any (over Some cells of blastocyst (5 to 200) cell types 14 days) Multipotent Cells differentiate, but Fetal tissue, cord blood, and can form a number of adult stem cells other tissues 11 Embryonic Stem Cells What Are Embryonic Stem Cells? Embryonic stem cells are derived from embryos at a developmental stage before the time that implantation would normally occur in the uterus. 12 6  10/14/2024 Tissue-specific stem cells often called “adult” or somatic stem cells but also includes stem cells isolated from fetal tissues and umbilical cord blood involved in tissue homeostasis and repair generally multipotent difficult to isolate and grow in large numbers in the laboratory already used to treat patients (haematological malignancies, diseases of the immune system etc.) 13 Induced pluripotent stem cells 14 7  10/14/2024 This cell Can form the Embryo and placenta This cell Can just form the embryo Multi- potent Fully mature 15 Types of stem cells: 1) Embryonic Stem Cells 16 8  10/14/2024 In late 1998, James Thompson at UW-Madison discovered how to isolate and culture human ES cells. 17 Embryonic Stem Cells The first step in human development occurs when newly fertilized egg , or zygote, begins to devide producing a group of stem cells called an embryo. These early stem cells are totipotent, meaning that they can become any kind of cell in the body. 18 9  10/14/2024 Embryonic Stem Cells Five days after fertilization, the embryo forms a hallow ball-like structure called a balstocyst. Embryos in blastocyst stage contain two types of cells. Embryonic stem cells form the inner cell mass, which ultimately develops into the fetus, or unborn child. Fertilization normally occurs in the oviduct, and during the next few days, a series of cleavage divisions occur as the embryo travels down the oviduct and into the uterus. Trophoblast cells make up the outside of the ball and eventually become the placenta, which supplies nutrients to the fetus. Embryonic stem cells in the blastocyst are pluripotent, meaning that they have the ability to become almost any kind of cell in the body. An embryo in blastocyst stage has not yet developed features characteristics of the human body. 19 Development of Preimplantation Blastocyst in Humans 20 10  10/14/2024 Blastocyst In Vitro After a human oocyte is fertilized in vitro by a sperm cell, the following events occur according to a fairly predictable timeline: Day 1 --- At 18 to 24 hours after in vitro fertilization of the oocyte. By day 2 (24 to 48 hours)--- the zygote (fertilized egg) undergoes the first cleavage to produce a 2-cell embryo. By day 3 (72 hours) --- the embryo reaches the 8-cell stage called a morula. At this stage, the genome of the embryo begins to control its own development. This means that any maternal influences—due to the presence of mRNA and proteins in the oocyte cytoplasm — are significantly reduced. By day 4 --- the cells of the embryo adhere tightly to each other in a process known as compaction. By day 5 --- the cavity of the blastocyst is completed. The inner cell mass begins to separate from the outer cells, which become the trophectoderm that surrounds the blastocyst. 21 Day-5 Blastocysts Are Used To Derive ES Cell Cultures A normal day-5 human embryo in vitro consists of 200 to 250 cells --- Most of the cells constitute the trophectoderm. To derive ES cell cultures, the trophectoderm is removed, either by microsurgery or immunosurgery (in which antibodies against the trophectoderm help break it down, thus freeing the inner cell mass). At this stage, the inner cell mass is composed of only 30 to 34 cells. However, once the inner cell mass is obtained from either mouse or human blastocysts, the techniques for growing ES cells are similar. 22 11  10/14/2024 Derivation of Embryonic Stem Cells The derivation of mouse ES cells was first reported in 1981 The first isolation of embryonic stem cells from human blastocysts was in 1994. Human ES cell lines are derived from embryos produced by in vitro fertilization (IVF), a process in which oocytes and sperm are placed together to allow fertilization to take place in a culture dish. Inner cell mass Trophectoderm 23 Video: Embryo biopsy using acid or laser 24 12  10/14/2024 Immunosurgery of a blastocyst. Antibodies are added that attach to the outermost layer of cells, which on a blastocyst is the trophoblast. After removing any unbound antibodies and adding complement, the trophoblast cells are destroyed, leaving only the inner cell mass. 25 Key Features of Embryonic Stem Cells Embryonic stem (ES) cells are immortal. Many months of growth in culture dishes, form any cell type that makes up the body --- pluripotent. The proliferative and developmental potential of human ES cells promises an essentially unlimited supply of specific cell types for basic research and for transplantation therapies for diseases ranging from heart disease to Parkinson’s disease to leukemia. 26 13  10/14/2024 Sources of Embryonic Stem Cells ▪ Excess embryos from IVF clinics ▪ Embryonic stem cell lines ▪ Embryos created for research by IVF ▪ Therapeutic cloning 27 28 14  10/14/2024 Chemical or electrical activation 29 30 15  10/14/2024 Derivation of (ES) cells: blastocyst cells inside = ‘inner cell mass’ fluid with nutrients culture in the lab embryonic stem cells taken from to grow more cells the inner cell mass outer layer of cells = ‘trophectoderm’ https://www.biointeractive.org/classroom-resources/creating-embryonic-stem-cell-lines 31 Culture of ES Cells The pluripotent cells of the inner cell mass were separated from the surrounding trophectoderm by immunosurgery The inner cell masses were plated in culture dishes containing growth medium supplemented with fetal bovine serum on feeder layers of mouse embryonic fibroblasts that had been gamma-irradiated to prevent their replication. After 9 to 15 days, when inner cell masses had divided and formed clumps of cells, cells from the periphery of the clumps were chemically or mechanically dissociated and replated in the same culture conditions. Colonies of apparently homogeneous cells were selectively removed, mechanically dissociated, and replated. These were expanded and passaged, thus creating a cell line. 32 16  10/14/2024 ES cells in Culture Colonies of human ES cells, line hESM03, grown on a feeder layer of mouse embryonic fibroblasts (×400). This photo was kindly provided by M. A. Lagar’kova (Institute of General Genetics, Russian Academy of Sciences). 33 Embryonic Stem Cells Can Stay ‘Undifferentiated’ in Culture for a Long Time As long as the embryonic stem cells in culture are grown under certain conditions, they can remain undifferentiated (unspecialized) state. Oct4 is an important marker which shows embryonic stem cells are undifferentiated. But if cells are allowed to clump together to form embryoid bodies, they begin to differentiate spontaneously. They can form muscle cells, nerve cells, and many other cell types. Embryonic stem cells Stained embryonic Embryoid bodies in culture medium stem cells 34 17  10/14/2024 A Fig. 5. (I. A. Grivennikov) Embryoid bodies obtained in a culture of mouse ES cells (×200). A) Embryoid bodies on the 3-4th day after attachment to the gelatin substrate; a) embryoid body. B) Onset of cell migration from the embryoid bodies; b) arrow shows cells migrating from the embryoid body after disruption of an external shell. B 35 Directed Differentiation of ES cells differentiation embryonic stem cells PLURIPOTENT all possible types of specialized cells 36 18  10/14/2024 Directed Differentiation of ES cells 37 Directed Differentiation of ES cells Challenges skin neurons embryonic stem cells blood ? liver 38 19  10/14/2024 Criteria for pluripotency ✓ Immortality ✓ Undifferentiation ✓ Clonality ✓ Broad developmental potential 39 Defining Properties of an ES Cell ❑ Growing and subculturing the stem cells for many months. This ensures that the cells are capable of long-term self-renewal (heallthy and remain undifferentiated). ❑ Presence of cell surface markers that are found only on undifferentiated cells. ❑ Presence of transcription factors that are typically produced by undifferentiated cells (such as Nanog and Oct4). Both Oct 4 and Nanog are associated with maintaining the stem cells in an undifferentiated state, capable of self-renewal. ❑ Normal karyotype, no genetic mutations in the cells (observe chromosomes under a microscope; this is a method to assess whether the chromosomes are damaged or if the number of chromosomes has changed. ❑ Testing whether the human embryonic stem cells are pluripotent 1) allowing the ES cells to differentiate spontaneously in cell culture 2) manipulating the cells so they will differentiate to form cells characteristic of the three germ layers 3) injecting the cells into an immunosuppressed mouse to test for the formation of a benign tumor called a teratoma. 40 20  10/14/2024 Testing Whether the Human ES Cells are Pluripotent 1- allowing the ES cells to differentiate spontaneously or to direct their differentiation along specific pathways in cell culture. It is usually accomplished by removing feeder layers and allow embryoid body formation. As long as the embryonic stem cells in culture are grown under certain conditions, they can remain undifferentiated (unspecialized). But if cells are allowed to clump together to form embryoid bodies, they begin to differentiate spontaneously. They can form muscle cells, nerve cells, and many other cell types. Although spontaneous differentiation is a good indication that a culture of embryonic stem cells is healthy, it is not an efficient way to produce cultures of specific cell types. 41 Differentiation of Human Tissues Early embryonic cells unspecialized Three primary tissue layers: – Ectoderm – Mesoderm – Endoderm All tissues develop from ES cells 42 21  10/14/2024 Testing Whether the Human ES Cells are Pluripotent 2. manipulating the cells so they will differentiate to form cells characteristic of the three germ layers: ES cells derived from the inner cell mass of one blastocyst are injected into the cavity of another blastocyst. The “combination” embryos are then transferred to the uterus of a pseudopregnant female mouse, and the progeny that result are chimeras. Chimeras are a mixture of tissues and organs of cells derived from both donor ES cells and the recipient blastocyst. A mouse growing a human ear. Not a chimeric animal ! 43 Testing Whether the Human ES Cells are Pluripotent 3. Teratoma formation Inject the cells into adult mice (under the skin or the kidney capsule) that are either genetically identical or are immune-deficient, so the tissue will not be rejected. In the host animal, the injected ES cells develop into benign tumors called teratomas. Teratomas typically contain a mixture of many differentiated or partly differentiated cell types—an indication that the embryonic stem cells are capable of differentiating into multiple cell types. These tumors contain cell types derived from all three primary germ layers of the embryo. Teratomas typically contain gut-like structures such as layers of epithelial cells and smooth muscle; skeletal or cardiac muscle (which may contract spontaneously); neural tissue; cartilage or bone; and sometimes hair. 44 22  10/14/2024 Potential Use of Pluripotent Stem Cells Basic research in human development – “decision-making genes” (birth defects, cancer) Drug development Toxicity Testing – human cell lines for all cell types Cell therapies – replacement tissue for degenerative conditions (Parkinson’s and Alzheimer’s disease, diabetes, heart disease, stroke, arthritis) Video: ES cells 45 Potential Use of hES Cells Cell Therapy ✓ i.e., to replace or restore tissue that has been damaged by disease or injury. Human ES-derived cells include Parkinson’s disease, diabetes, traumatic spinal cord injury, Purkinje cell degeneration, Duchenne’s muscular dystrophy, heart failure, and osteogenesis imperfecta etc. Advantages: unlimited ability to proliferate in vitro, a broad range of cell types through directed differentiation (pluripotent) 46 23  10/14/2024 Potential Use of hES Cells Disadvantages Tumor formation: undifferentiated ES cells may induce the formation of tumors (teratomas). Immunological rejection of human ES-derived cells. Possible Solutions for immunological rejection ✓ genetically engineering the ES cells to express the MHC antigens of the transplant recipient, ✓ by using nuclear transfer technology to generate ES cells that are genetically identical to the person who receives the transplant. By using somatic cell nuclear transfer technology (so-called therapeutic cloning) in which the nucleus is removed from one of the transplant patient’s cells, such as a skin cell, and injecting the nucleus into an oocyte. The oocyte, thus “fertilized,” could be cultured in vitro to the blastocyst stage. ES cells could subsequently be derived from its inner cell mass, and directed to differentiate into the desired cell type. The result would be differentiated (or partly differentiated) ES-derived cells that match exactly the immunological profile of the person who donated the somatic cell nucleus, and who is also the intended recipient of the transplant 47 Rejection: Possible Solutions Genetic engineering Somatic cell nuclear transfer (SCNT) – develops into a blastocyst Dolly the sheep – cloning (banned by Congress) 48 24  10/14/2024 Somatic Cell Nuclear Transplantation May Provide a Way to Generate Personalized Stem Cells Figure 8-6 Molecular Biology of the Cell (© Garland Science 2008) 49 Personalized Sources of Pluripotent Stem Cells Somatic Nuclear Transfer Dolly (05.07.1996 –14.02.2003) Ian Wilmut, Roslin Institute 50 25  10/14/2024 “Therapeutic” cloning transplantation differentiated donor cells embryonic stem cells early embryo egg skin cells nucleus somatic nuclear transfer 51 “Therapeutic” cloning 52 26  10/14/2024 “Reproductive” cloning Dolly‘s “mother“ Dolly early embryo “reprogramming” skin cells egg nucleus 53 Example of Transplant Therapy: Diabetes Insufficient insulin production in pancreas Insulin needed for glucose uptake “Islet-like” cells derived from stem cells produce insulin Eliminates need for insulin injections 54 27  10/14/2024 Stem Cell Research Worldwide 55 Nations: ES Cell Laws In the European Union, stem cell research using the human embryo is permitted in Sweden, Finland, Belgium, Greece, Britain, Denmark, France and Netherlands, However it is illegal in Germany, Austria, Ireland, Italy and Portugual. United States, several states enforce a complete ban and others giving financial support. Elsewhere, Japan, India, Iran, Israel, South Korea, and China are supportive, Australia is partially supportive (exempting reproductive cloning yet allowing research on embryonic stem cells that are derived from the process of IVF). However New Zealand, most of Africa (excepting South Africa) and most of South America (excepting Brazill) are restrictive. 56 28  10/14/2024 Therapeutic Application of ES Cell Products: Clinical Trials First hES Cell trial: 2009 Second hES Cell trial: 2010 57 Therapeutic Application of ES Cell Products: Clinical Trials https://clinicaltrials.gov/ 58 29  10/14/2024 Therapeutic Application of ES Cell Products: Clinical Trials Ophthalmic diseases 59 Therapeutic Application of ES Cell Products: Clinical Trials Ophthalmic diseases 60 30  10/14/2024 Scientific Challenges Remaining Understanding of cellular events that lead to differentiation and specialization Immunologic rejection - Can stem cells be modified to minimize tissue incompatibility? Time of development and expense of treatments 61 Scientific Challenges Remaining Stem cells need to be differentiated to the appropriate cell type(s) before they can be used clinically. Recently, abnormalities in chromosome number and structure were found in human ESC lines. Stem cell development or proliferation must be controlled once placed into patients. Possibility of rejection of stem cell transplants as foreign tissues is very high. Contamination by viruses, bacteria, fungi, and Mycoplasma possible. The use of mouse “feeder” cells to grow ESC could result in problems due to xenotransplantation. 62 31