Stem Cells And Molecular Embryology Lecture 20 PDF

Unified Lecture 20 STEM CELLS AND MOLECULAR EMBRYOLOGY HIHD-211 TERM-3 Basic Science Department COSHP, KSAU-HS, KSA LEARNING OUTCOMES By the end of the lecture student will be able to: 1. Define stem cells. 2. Describe the different types of stem cells, and where do they come from. 3. Describe the...

Unified Lecture 20 STEM CELLS AND MOLECULAR EMBRYOLOGY HIHD-211 TERM-3 Basic Science Department COSHP, KSAU-HS, KSA LEARNING OUTCOMES By the end of the lecture student will be able to: 1. Define stem cells. 2. Describe the different types of stem cells, and where do they come from. 3. Describe the methods of regulating different activities of stem cells. 4. List the uses of stem cells in regenerative medicine. 5. Discuss key signaling pathway for development 6. Describe molecular regulation of gastrulation 7. Describe molecular regulation of neurulation 8. Describe molecular regulation of neural crest cells 9. Describe molecular regulation of somite formation Dr. Ismail Memon Stem Cells Are: ▪ ▪ Unspecialized cells capable of dividing and renewing themselves Capable of differentiating into specialized cells When stem cells give rise to specialized cells (e.g., nerve cell, blood cells, muscle cells) the process is called differentiation. LO-1 Why can some cells divide and stay as stem cells while others differentiate and can not be stem cells anymore? In stem cells, the continuous expression of certain transcription factors (such as Oct-4, Nanog, and Sox2) will prevent the differentiation of the cell, and thus the cell can maintain its pluripotency characters. How can we identify the stem cells? A stem cell can be identified by the labelling of the several transcription factors which are found only in the cytoplasm of stem cells, and by labelling the cell surface proteins which are specific for stem cells only. Different transcription factors stained in different colors in stem cells. LO-1 What are the possible end results of a stem cell division? When the stem cell divides, the results can be either: 1- two identical daughter stem cells (called symmetric division) 2- two different daughter cells i.e. one stem cell and another progenitor cell (called asymmetric division) What is a progenitor cell? Progenitor cell is a proliferative cell with the capacity to differentiate but with no self-renewal ability. LO-1 Degrees of Stem Cell Potency Cell potency refers to the ability of the stem cells to differentiate into specialized cell types. Cells with high potency can generate more cells types than those with lower potency. LO-2 1- Totipotent Stem Cells.(Zygote) It can differentiate to any of the 220 cell types found in an embryo as well as the placenta. 3- Multipotent Stem Cells (from ectoderm, mesoderm, endoderm, and in adult tissues) It can differentiate into a limited number of cell types and can replace damaged cells in adult tissues. 2- Pluripotent Stem Cells (embryonic stem cells from inner cell mass) can differentiate to all cell types of the body (but not the placenta). 4- Unipotent Stem Cells (e.g. in skin and liver. But not certain If they only differentiate into one type of cells or not. d LO-2 Classification of stem cells Stem cells are classified ,according to their original sources, into three types: 1- embryonic stem cells 2- Adult stem cells 3- Induced pluripotent cells LO-2 adult stem 1- Embryonic stem cells What is the definition of embryonic stem cell? Embryonic stem cells are the stem cells which are derived from the inner cell mass of the blastocyst of the embryos before implantation in the uterus. How can we obtain large number of embryonic stem cells? When the inner cell mass cells are removed from their normal embryonic environment and cultured under appropriate conditions, the inner cell mass cells can continue to divide indefinitely and produce cells that have the potential to form any cell type. LO-2 How to direct the embryonic stem cells to become certain tissue? ▪ 1. 2. 3. The embryonic stem cells are cultured with a mix of growth factors and certain regulatory factors in order to direct these cells towards specific line of differentiation such as: BMP4 ( Bone Morphogenic Protein) molecules induces the formation of mesoderm. Activin/Nodal molecules induces the formation of endoderm FGF (Fibroblast Growth Factor) molecules induces the formation of ectoderm. 10 LO-2,3 Usage Of Human Embryonic Stem Cells (HESCs) 1. HESCs have been programmed to differentiate into type II alveolar lung cells which could enhance the treatment of neonatal lung disease. 2. Insulin-secreting cells have been cultured and can treat diabetes mellitus. Opposition to the use of Human Embryonic Stem Cells (HESCs) o Despite of the great therapeutic promise of human embryonic stem cells in many diseases, there is a great ethical and political opposition to the usage of these cells. The opposition is mainly because the harvesting of HESCs involves the destruction of the human embryo. o This opposition directed the scientific community to research in the field of using adult stem cells in regenerative medicine. LO-2,3 2. ADULT STEM CELLS What is, and how to obtain adult stem cells? 1. They are undifferentiated cells in a tissue, or an organ and they can differentiate to give some or all the specialized cell types of that tissue or organ. 2. The primary function of these adult stem cells is to maintain and repair the tissue in which they are found. 3. They can remain non-dividing until they are activated by the need for new cells to maintain the tissue or due to tissue injury. Adult stem cells have been identified in bone marrow, blood vessels, skeletal muscle, skin, gut , liver, ovarian epithelium and testis. How can the body regulate the number and the differentiation of the adult stem cells? Adult stem cells are in specific areas of each tissue which is called stem cell niche and the microenvironment of every niche will control the stem cells. LO-2,3 What is the definition of a stem-cell niche? A stem-cell niche is an area of a tissue that provides a specific microenvironment, in which stem cells are present in an undifferentiated and self-renewable state. Cells of the stem-cell niche interact with the stem cells to maintain them or promote their differentiation. Different cells which are in the Niche (e.g., endothelium, nerve cells, macrophages ) will have effects on the stem cells and will define the activity and the fate of the stem cells. Several niches have been identified also in many mammalian tissues: 1. hematopoietic system, 2. skin, intestine, 3. brain and 4. muscle 13 LO-2 How the stem cells and the different types of cells in the niche will communicate with each other? Stem cells and niche cells can communicate directly through cell-to-cell contact, or indirectly through secreted factors. What could happen if we change the niche around the stem cells? They may change their differentiation. For example, human neuronal stem cells produced muscle cells when they were implanted in skeletal muscle niche. 14 LO-2,3 Have patients benefited from adult stem cell research? Yes, over 1 million patients worldwide have been treated with adult stem cells and experienced improved health in diseases such as: Diabetes Type I (Juvenile) Liver Cirrhosis Osteoporosis Acute Heart Damage Disadvantages of using adult stem cells in regenerative medicine: 1-Most adult stem cells are restricted in their differentiation = NOT Pluripotent 2- Adult stem cells have a short lifespan in vitro 3- Adult stem cells do not proliferate much in culture. Thus, the search for another source of pluripotent stem cells continued. The question was: how can we force an adult cell to become a stem cell? 15 LO-2 3- Induced pluripotent adult stem cells What is the definition of Induced pluripotent stem cells (iPSCs)? Induced pluripotent stem cells (iPSCs) are adult cells that have been genetically reprogrammed to an embryonic stem cell–like state by being forced to express genes and factors important for maintaining the defining properties of embryonic stem cells. What are the Induced pluripotent stem cells capable of? Human induced pluripotent stem cells shows all the stem cell markers, and they are capable of generating cells from all the three germ layers ( ectoderm, endoderm, mesoderm). What kind of cells could be generated from the human induced pluripotent stem cells? Using iPSC technology scientists have reprogrammed skin cells into active motor neurons, egg and sperm precursors, liver cells, bone precursors, and blood cells. 16 LO-2,3 If all adult cells have the same genetic material, is it possible to take an adult differentiated cell and force it to become stem cell? Yes, this is possible because now we know that certain genes became inactive in these mature differentiated cells and we know that if we can activate these silent genes, then they will produce certain transcription factors that will reprogram the cell to become a stem cell. Who is the first one to succeed in changing an adult cell into a stem cell? This experiment was done by the Nobel-prize-winning Shinya Yamanaka in Japan, who proved in 2006 that the activation of four specific genes (named Myc, Oct3/4, Sox2 and Klf4) will produce specific transcription factors that could reprogram the mature skin fibroblasts to become pluripotent stem cells. These stem cells are called induced pluripotent stem cells = iPSC 17 LO-2,3 Why do we need to study and isolate stem cells? Because in regenerative medicine , the stem cells are needed in repairing the damaged tissues. Regenerative medicine is defined as the process of replacing or "regenerating" human cells, tissues or organs to restore or establish normal function. For example, by using the stem cells to repair some damaged tissues such as bone, cartilage, blood vessels, bladder, or skin. 18 LO-4 Introduction To Molecular Embryology After formation of the zygote as well as during the embryonic development, the dividing cells are directed toward their future specialized cell lineages to form specialized cells and specialized tissues and organs. The precursors of these cells commit to their fate in a step-by-step differentiation process guided by several factors such as: 1-DNA methylation and unequal distribution of cytoplasmic contents 2- Position of the cells within the embryo 3- Signaling between cells Molecular Embryology study the molecular interactions that transform the zygote to a complex embryo that in the end gives rise to a fully-formed human being. LO-5 1- DNA Methylation and DNA Demethylation Methylation is the addition of a methyl group to certain position of cytosine by DNA methyltransferase (DNMT) enzymes. DNA methylation is a major form of DNA modification that plays critical roles in chromatin structure and gene expression during development and cell DNA differentiation. methylation DNA methylation is associated with the silencing of gene expression and thus regulate certain activities of the cells. DNA demethylation of a gene promoter causes transcriptional activation and gene expression. LO-5 DNA demethylation Methylation Epigenetic processes modify gene expression without changing the DNA sequence of the gene. Methylation is considered an important epigenetic process because it can change the activity of a DNA segment without changing its sequence. Methylation Example of global Demethylation followed by methylation of the DNA of the cell can be seen after the formation of the zygote. After fertilization, Demethylation of the DNA occurs in order to remove most methylation marks that were inherited from the parents and thus allows subsequent establishment of the new embryonic characters through a new methylation pattern. LO-5 Demethylation 2- Asymmetric segregation of Cytoplasmic contents This process starts during the earliest stage of embryo development and continue to stimulate the correct specialization of the cells. When the zygote divides and produce separate blastomeres, each of these cells inherit different regions of the cytoplasm of the zygote. Each region of the inherited cytoplasm will contain different cytoplasmic regulatory molecules which will affect its future fate. LO-6,7 zygote asymmetric segregation of cytoplasmic contents in the Zygote Asymmetric division of the cell leads to unequal number of organelles and various regulatory molecules within each cell. Regulatory molecules could be mRNA, proteins, enzymes, several types of small molecules and small granules. The presence of different regulatory factors in each cell of the morula will influence the development of these cells to become inner cell mass or outer trophoblastic cells. LO-5 zygote New cells inherit different regions of the cytoplasm of the original cell How does the body know that the spleen belongs on the left side while the liver is located on the right? Right side In order for the cells to migrate and proliferate in the correct anatomical position, the major body axes need to be established very early (a process called laterality). left side Several molecular mechanisms are needed for the establishment of a left-right body axis. The cells will use two methods to induce laterality: 1- Expression of Specific selective proteins which will stimulate cell division and migration from the current position to another position. 2- Use several Concentration gradients of the signaling molecules. This means that the cells which are close to the source of the secreted signaling proteins will be exposed to higher concentrations of these proteins and this will cause anatomical specialization that is different from the cells which are far from source of secreted signals. A good example for the molecular control of anatomical position can be seen in the primitive streak and the primitive node. Both of them will determine the cranialcaudal and the left-right axes. LO-6,7,8 NODAL Flow Molecular mechanism for establishing right and left axes of the body: 1- The primitive node contains motile cilia which are designed in a unique way so that they move the current of fluid and molecules to the left. NODAL flow 2- This leftward flow (called NODAL flow) causes calcium influx on the left side which in turn induces nodal growth differentiation factor (NODAL) expression. 3- Increasing NODAL levels on the left side induce the secretion of left-right determination factor (LEFTY-2 ) gene expression. 4- Disruptions in the process of left-right body axis determination can result in laterality disorders such as malpositioning of internal organs. LO-6,7 Laterality disorder 3- Signaling Between Cells Paracrine Signaling Tissues and organs are formed by interactions between cells. When one cell causes another cell to change its fate, this process is called induction. The cell which is causing the change is called the inducer, and it can do that by producing molecular signals that will induce the changes in the other cell which is called the responder. Types of cell to cell signaling: 1- Paracrine Signaling 2- Juxtacrine Signaling Juxtacrine Signaling LO-5 Paracrine Signaling Depends on diffusable factors (called paracrine factors or differentiation factors) secreted by a cell. The factors travel a short distance to bind to specific receptors on the surface of the target cells. The binding causes a cascade of interactions that ultimately activates a transcription factor. This transcription factor then activates or inhibits gene expression. Example: Fibroblast Growth factors activate receptors called fibroblast growth factor receptors causing cell division. LO-5 Juxtacrine Signaling (or contact-dependent signalling) Notch as signaling pathway in neurogenesis Does not involve diffusable factors. A protein on one cell surface interacts with a receptor on an adjacent cell. Then it activates a reaction that will activate or inhibit gene expression. Example: Notch signaling pathway which promotes neuronal differentiation. In Notch pathway, cells expressing the Delta proteins in their cell membranes activate neighboring cells that contain the Notch protein in their cell membranes. LO-5 inducer Delta proteins Notch protein Responder (target cell) Cell adhesion molecules in Juxtacrine Signaling Cell adhesion molecules are transmembrane glycoproteins that mediate the connections between cells. They mediate cell-to-cell interactions, and by doing so they trigger intracellular responses affecting intracellular signaling, cytoskeletal organization and gene expression. The cellular adhesion molecules includes members such as integrins, cadherins and selectins. LO-5 Some Important roles for the Cell Adhesion Molecules in implantation: 1- Integrins: play important role in the activation of the genes involved in the implantation process of the blastocyst in the endometrium. 2- Cadherins are a group of calcium-dependent glycoproteins that are responsible for tissue morphogenesis, including cell recognition and boundary formation and coordinated cell movements. cell-to cell-adhesion. E-cadherin, a member of the cadherin family is important for the motility of the trophoblast during implantation. 3- L-selectin is the most relevant during the implantation process. Defects in L-selectin adhesion could explain causes of infertility, early pregnancy loss or insufficient cytotrophoblast invasion which could be related with pregnancy difficulties LO-5 Sonic Hedgehog(SHH): Master Gene for Embryogenesis ▪ The hedgehog gene was named because it coded for a pattern of bristles on the leg of Drosophila that resembled the shape of a Hedgehog ▪ The mutated hedgehog genes often cause birth defects. ▪ Also, if it is activated later in life, certain cancers can be triggered and begin to spread. ▪ Involved in cell growth and differentiation to control organ formation during embryonic development. ▪ Regulates embryonic development, ensuring that tissues reach their correct size and location, maintaining tissue polarity and cellular content. LO-9 Sonic Hedgehog(SHH): Master Gene for Embryogenesis This protein is involved in development of the vasculature Left to right , midline axis formation Development of neural patterning Smooth muscle patterning, and patterning of these following structures -heart, gut, pharynx, lungs, pancreas, kidney, bladder, hair follicles, teeth, thymocytes, inner ear, eyes, and taste buds Induces formation of sclerotome , which is the ventromedial portion of the somite. LO-9