Introduction to Stem Cells PDF

Introduction to Stem cells Dr. Shamsul Azlin Bin Ahmad Shamsuddin Education & work experience 1987-1989 asasi sains Embryologist loc...

Introduction to Stem cells Dr. Shamsul Azlin Bin Ahmad Shamsuddin Education & work experience 1987-1989 asasi sains Embryologist locum, 1989-1993 BSc Zoology Subang jaya medical centre (Sime Darby) 1993-1997 MPhil, UM Metro Hospital, Kelang 1997-2000 Embryologist, Pantai Bangsar Medical Centre Gleaneagles, Penang 2000-2003 Embryologist, Gleaneagles, Ampang Medical Centre Darul Ehsan Medical Centre, Shah Alam 2003-2006 Embryologist, Damansara Sepcialist Hospital Mahkota Medical Centre, Melaka oct 2006- June 2011 PhD, Sheffield University, UK. LPPKN, KL. Embryologist training, Columbia Asia Hospital, Setapak, KL. 1997- Melbourne, Australia (2 Months) Prince Court, Hospital. 2000- Cairns, Queensland, Ausatralia (2 weeks) 2003- NUS, Singapore (3 days)  Regeneration in Nature Outstanding Examples – Planarian – Crayfish – Embryos Inverse Relationship – Increase complexity – Decrease regenerative ability  Regeneration in Humans High Moderate Low  Clinical Needs Cardiovascular – Myocardial infarction – Stroke Bone – Non-union fractures – Tumor resections Nervous – Spinal Cord Injury – Degenerative diseases  Definitions ❑ Stem cells – undifferentiated cells that have the ability to: ❑ divide for indefinite periods in culture (self-renew) ❑ to become specialized cells ❑ Differentiation – ❑ the process whereby cells become specialized ❑ Produces stems cells with decreasing potential (Adult stem cell) ❑ no longer capable of cell division Cardiac muscle cells, neurons produced during embryonic development, differentiate, and are then retained throughout the life of the organism. ❑ Passages- ❑ A passage is the process of removing cells from one culture dish and replating them into fresh culture dishes.  Cell differentiation Self-renew Self-renew Differentiation Self-renew Differentiation Pluripotent Multipotent stem cell stem cells Differentiated cells (muscle, nerve, skin, (Embryonic fibroblast, etc) stem cell) (Adult stem cell) 8  Stem Cells differ by Origin – Embryonic (ES) – Adult stem cells (non-embryonic) (AS) Different “potentials” – Totipotent – Pluripotent – Multipotent – Unipotent  Terminology of potential (plasticity) Prefix Meaning Example Totipotent All Embryonal Pluripotent Many/much Inner cell mass Multipotent Several/much Inner cell mass Pleo- More Hematopoietic, skin Oligo- Few/little Gastrointestinal stem cell Quadri- Four Gastrointestinal stem cell Tri- Three Bronchial lining Bi- Two Bile duct Unipotent One Prostate  Embryonic Stem Cells 1. Embryos are the result of In Vitro Fertilization (IVF) 2. Cells are taken from the Inner Cell Mass (ICM) of a blastocyst 3. ICM cells are nourished in a petri dish in an incubator 4. Cells are given different types of Factors/chemicals These cells can give rise to most types of cells Stem Cell Capacity Human embryonic stem cell lines were first derived in 1998 by Dr. James Thompson. Embryonic Stem Cells  Adult Stem Cells (AS) Adult tissues reported to contain stem cells include: brain, bone marrow, peripheral blood, blood vessels, skeletal muscle, skin, liver and umbilical cord blood but very small number of stem cells in each tissue and difficult to maintain in long term culture. HSCs are found in the bone marrow of adults, with large quantities in the pelvis, femur, and sternum. They are also found in umbilical cord blood and, in small numbers, in peripheral blood.  COMPARISON Adult stem cells Embryonic stem cells From mature body tissues, From inner cell mass of blastocyst umbilical cord and placenta after birth Multipotent- give rise to limited cell Pluripotent- give rise to all cell types types (except the cells of the placenta) First isolated in 1960s First isolated in 1998 at University of Wisconsin Results: Results: 1000+ human therapies Only in animal trial, no human trials to date Embryonic Advantages Disadvantages Stem cell Immortal, produce endless Immune rejection if stem cells were number of cells derived from IVF embryos Easy to extract in laboratory Difficult to control when culture, requires many steps to coax into desired cell types Pluripotent, can make any body cells Derived from IVF embryos donated for research. New technique like SCNT might open up new research potentials Somatic cell nuclear transfer (SCNT) might remove immune rejection problem as patients are using their own cells. Adult Advantages Disadvantages stem cell No immune rejection because Limited longevity- difficult to from patient’s own cells. maintain in culture for long period Mostly specialised, required Difficult to extract from mature less coaxing to differentiate to tissue. Uncommon and more scarce specific cells. with age. Multipotent- limited cell type Questionable quality due to genetic defects; targeted diseases may still present in stem cell genes  Umbilical cord stem cells (UCS cells) Also Known as Wharton’s Jelly Adult stem cells of infant origin Isolated prior to/ immediately following birth Haematopoietic stem cells (Majority) 100,000 stem cells per mL in UCB Alternate to bone marrow stem cells  Umbilical cord stem cells Three important functions of UCS cells: - Plasticity: Potential to change into other cell types like nerve cells - Homing: To travel to the site of tissue damage - Engraftment: To unite with other tissues  Cord blood Vs Bone Marrow Cord Blood Bone Marrow Collection is non-invasive, Collection is invasive and painless, and poses no risk to painful. Must be performed in the donor. a hospital surgical setting. Greater HLA compatibility due Due to the maturity of the to decreased functionality of fetal lymphocytes. stem cells, it requires a greater HLA match to perform Graft versus Host Disease a transplant. (GVHD) is reduced to 10% due to the absence of antibodies Serious GVHD occurs in 60% in the stem cells. of all unrelated Bone Marrow transplants Units are processed and ready for transplant. Bone Marrow is dependent on donor participation. Significantly less expensive Stem Cell Capacity Somatic cell nuclear transfer (SCNT) Somatic cell nuclear transfer can create clones for both reproductive and therapeutic purposes. The diagram depicts the removal of the donor nucleus for schematic purposes; in practice usually the whole donor cell is transferred. Somatic Cell Nuclear Transfer  STEM CELLS RESEARCH IN MALAYSIA haemopaoetic stem cells, UKM mesenchymal stem cells, IMR & UMMC Tooth pulp stem cell –Dental, UMMC An illustration of a rotator cuff tear in the shoulder. Any or all of the white structures (tendon) can be affected. bone marrow stromal stem cells (also called Human tenocytes isolated from semitendinosus tendon. mesenchymal stem cells, or skeletal stem cells) Thank You STEM CELL EPIGENETIC Histone under electron microscopy. http://www.operamedphys. org/OMP_2016_01_0023 Epigenetic differentiation landscape. END INDUCING PLURIPOTENCY By Rohit Satyam END Animal Models of Regeneration  Regeneration Regeneration is the sequence of morphogenetic events that restores the normal structure of an organ after its partial or total amputation. Regeneration in invertebrates: Hydra: Cross amputation of hydra led every part to regenerate to whole hydra species. Planaria: The planaria contains neoblast cells which migrate toward the amputated region and form the lossing parts. Annelids : The blastema of the amputated region formed of ectoderm , mesoderm and neoblast cells which co-ordinated in regeneration of the lossing parts.  Regeneration of vertebrates There are two types of regeneration: 1. Epimorphosis or epimorphic regeneration : This type of regeneration involve the reconstruction of the missing parts by local proliferation from the blastema, or addition of parts to remaining piece. For example: regeneration of tail, limbs and lens in anurans and urodels and other vertebrates. 2. Morpholaxis or morphollactic regeneration: This type of regeneration involving reorganization of the remaining part of the body of an animal.For example: Hydra, planaria and other invertebrates e.g. regeneration of the new individual from body pieces.  Epimorphosis or epimorphic regeneration Regeneration of Tail in amphibians & reptilia : Amphibia : The tail lacks vertebrae and has an unsegmented cartilaginous tube, which contains the regenerated spinal cord which form mainly of the ependymal lining of the central canal. At first very few cells accumulate under the wound epithelium. The ependyma and the various connective tissues dermis, muscle septa, adipose tissues and osteocytes of vertebrae are the sources of cells for the generate. The non-nervous elements proliferate behind the apex, forming both the muscle and cartilage tube ,then, the ependyma proliferate and gradually extend dorsally. Reptilia : For example lizard, the regenerated tail is a quite imperfect tail. It lacks vertebrae and in their place, has an unsegmented cartilaginous tube. This tube contains the regenerated spinal cord, including the extension of the ependymal lining of the central canal of the spinal cord. Steps of regenerated limb  Regeneration of Limb Regeneration begins in 3 phases : 1. Phase of wound healing or pre -blastema stage : Blood clotting and migration of epidermal cells from the basal layer of epidermis toward the centre of the wound. The wound is covered with epithelium which is thicker than the epidermis of the limb. 2. Phase of blastema formation : Cells accumulate beneath the epithelial covering and formed the blastema. Mesenchymal cells accumulate beneath the cap. Mesenchymal – blastemal cells differentiate into myoblasts and muscle cells, early cartilage cells and cartilage. During the dedifferentiate phase Hyaluronate (HA) increases in the distal stump to form blastema. As the blastema forms, the HA will be decrease. The production of HA and break down of collagen represent the establishment of migration from stump tissues. 3. Phase of dedifferentiate and morphogenesis : The blastema begins to restore the part of which the limb was deprived. Specifically, if the fore arm is removed, the blastema differentiated directly into the muscle, bone, cartilage and skin of the fore arm. Reparative Regeneration This is the replacement of lost parts or repair of damaged body organs. In this type of regeneration, wound is repaired or closed by the expansion of the adjoining epidermis over the wound. Example: Regeneration of limbs in salamanders Regeneration of lost tail in lizard Healing of wound Replacement of damaged cells. Autotomy In some animals like starfish, some part of the body is broken off on being threatened by a predator. This phenomenon of self-mutilation of the body is called autotomy Example: Crabs break off their leg on approaching of the enemy Holothurians throw off their internal viscera Starfish breaks off an arm Regenerative capacity in Animal Group The capacity of regeneration varies in its extent in various animal groups. Regenerative capacity is very high among the protozoan, sponges and coelenterates. Invertebrates - In sponges, the entire body can be reconstructed from isolated body cells. The cells rearrange and reorganize to form bilayered sponge body wall. - Regeneration was first discovered in hydra by Tremble (1740). Even 1/1000th part of the body regenerate into new organisms. - In hydra and planaria, small fragments of the body can give rise to a whole animal. When a hydra or a planaria is cut into many pieces, each individual part regenerates into a whole individual. Regeneration in Hydra - Some annelids like earthworms are able to regenerate some segments removed from the anterior and posterior ends of the body. - Some molluscs can regenerate only the eyes and heads while squids can also regenerate their arms. - Many arthropods (e.g., spiders, crustaceans, insect larvae, etc) can regenerate limbs only. Regeneration is faster in the young than in the adults. Regenerated part may not always be similar to the part lost. This type of regeneration is called heteromorphosis. - Echinoderms (like starfish, brittle star, sea lilly) exhibit autotomy. They can regenerate arms and parts of the body. **Regeneration is an usual form of asexual reproduction in several lower groups of animals. Vertebrates Fishes: Lamprey can regenerate its lost tail. Some fishes have the ability to regenerate parts of its fins. Amphibians: The regeneration power is well marked in urodel amphibians like salamanders, newts and their axolotl larvae. They can regenerate limbs, tail, external gills, jaws, parts of eye like lens and retina. Tail and limb regeneration is found in the larval stages of frogs and toads. Reptiles: Lizards exhibit autotomy. When threatened, the lizard detaches its tail near the base to confuse its predator and later regenerates a new tail. The new tail differs from the old one in its shape, absence of vertebrae and the kind of scales covering it. Birds: Regeneration is restricted to parts of the beak. Mammals: Regeneration is restricted to tissues only. External parts are not regenerated. Skin and skeletal tissues possess great power of regeneration. The liver has the maximum capacity of regeneration. If one kidney is damaged or removed, the other enlarges to compensate the lost kidney. This is called as compensatory hypertrophy. Types of Regeneration based on Cellular Mechanism Based on cellular mechanisms regeneration can be of two types: 1) Morphallaxis In this type, regeneration occurs mainly by the remodelling of existing tissues and the reestablishment of boundaries, thus involving very little new growth. As a result, the regenerated individual is much smaller initially. It subsequently increases its size and becomes normal after feeding. This type of regeneration is known as morphallaxis or morphallactic regeneration. Example: Regeneration of hydra from a small fragment of its body 2) Epimorphosis In this type, regeneration involves dedifferentiation of adult structures in order to form an undifferentiated mass of cells. They are highly proliferating and accumulate under the epidermis, which has already expanded. Within two days, bulge transforms into a conical hump. This lump of dedifferentiated cells along with the epidermal covering is called regeneration bud or regeneration blastema. The dedifferential cells continue to proliferate and finally redifferentiate to form a rudiment of the limb. The rudiment eventually transforms into a limb. This type of regeneration is known as epimorphosis or epimorphic regeneration. Example: Limb regeneration in amphibians. 3)Heteromorphosis or heteromorphic regeneration When a different organ develops from the one that has been removed, the phenomenon is called heteromorphosis. Eg. In shrimp Palinurus, eye is regenerated, If it is removed from the eye stalk. But if the eye is removed along with optic ganglion, instead of eye an antenna like organ is regenerated. This type of regeneration is exhibited by lower animals. 4) Super regeneration The development of superfluous number of organs or parts of the body ( eg. Heads, tail limbs) as a result of regeneration is known as super regeneration. When a deep incision is made on the head end of a planaria or earthworm, additional heads will develop. Incisions in the middle part cause the development of both heads and tails. Mechanism of Regeneration Regeneration is a complex process which basically involves histological and physiological events. Regeneration of a Limb of a Newt The mechanism of regeneration in salamander involves the following stages- Wound healing: The epidermal cells from the edges of the wound migrate and spread over the exposed surface. This is known as wound healing. Blastema formation: A few days later, undifferentiated cells accumulate inside the epidermis, resulting in a bulge. This is known as regeneration bud or blastema. Redifferentiation and morphogenesis: The blastema develops rudiments of the lost organ, like the digits which grow into new digits. Growth: The regenerated limb increases and attains the size of a normal limb. In planarians and in Hydra, there are undifferentiated cells called neoblasts which multiply and then migrate from the deeper parts of the body to the cut surface. Growth Factors Wound healing is due to accelerated mitosis. This is mediated by proteins called growth factors which act locally. EGF Epidermal growth factor stimulates the epithelium to undergo mitosis. EGF is also produced in the salivary glands, which is why an animal's licking heals a wound. FGF Fibroblast growth factor stimulates the endothelial cells of the blood vessels to divide and heal the injured blood vessels. Platelet Derived growth factor which stimulates the mitosis of fibroblasts at the site of injury to fill in the damaged areas under the blood clot. Polarity in Regeneration The body segments of Hydra or planarians exhibit distinct polarity during regeneration. Their anterior end always regenerates into head and posterior into the tail.