Stem Cells 101 PDF

Stem Cells 101 Tomar Ghansah, Ph.D. Associate Professor [email protected] MDC 3008-3010 Department of Molecular Medicine Morsani College of Medicine University of So...

Stem Cells 101 Tomar Ghansah, Ph.D. Associate Professor [email protected] MDC 3008-3010 Department of Molecular Medicine Morsani College of Medicine University of South Florida Reading Assignments: Alberts, Johnson, Lewis, Raff, Roberts and Walter, Molecular Salk Institute for Biological Studies Biology of the Cell Chapter 23 (DVD Disc) http://www.technologyreview.com/biomedicine/37787/ Stem Cells Help Regenerate Damaged Heart 2011  2012 British and Japanese Researchers Awarded Nobel Prize For Stem Cell Research The Nobel Prize in Physiology or Medicine 2012 was awarded jointly to Sir John B. Gurdon and Shinya Yamanaka "for the discovery that mature cells can be reprogrammed to become pluripotent"  Objectives Stem Cells Totipotent, Pluripotent, Multipotent and Unipotent Stem Cells Different Origins of Stem Cells Hematopoiesis Immunophenotyping of Stem Cells  Objectives Stem Cell “Niche” Fat Stem Cells Cancer Stem Cells Pre-clinical Studies with Stem Cells Clinical Therapies using Stem Cells Stem Cell: Primitive cell with a very high potential and infinite ability of self renewal and differentiation into other cell types.  What makes a cell a stem cell? Plasticity: A stem cell is plastic, meaning it can develop into another type of cell. Differentiation: The process where a stem cell specializes or develops into another type of cell. Self-Renewal: a stem cell can divide (renew itself) indefinitely (go through mitosis) and  Stem Cell Properties 1) Plasticity: Potential to change into other cell types like nerve cells, heart cells, muscle cells, liver cells, pancreatic cells, brain cells, skin cells, etc. 2) Homing: The ability to travel to the site of tissue, cell or organ damage. 3) Engraftment: To unite with other tissues and regenerate the damage cells, tissue or organ.  Different Types of Stem Cells Totipotent stem cell that has the ability of a single cell to divide and produce all the differentiated cells in an organism, including extra embryonic tissues. Pluripotent stem cell that has the potential to differentiate endoderm (interior stomach lining, gastrointestinal tract, the lungs), mesoderm (muscle, bone, blood, urogenital), or ectoderm (epidermal tissues and nervous system) germ layers. Multipotent stem cell that has the potential to give rise to cells from multiple, but a limited number of lineages. An example is a hematopoietic stem cell that can differentiate into several types of blood cells, only. Unipotent stem cell that has the capacity to differentiate into only one type of tissue/cell type. http://en.wikipedia.org/wiki/Totipotency#Totipotency  Different Types of Stem Cells Embryonic Stem Cells Versatile or Plastic Control of development has not been fully determined Controversial and Restricted Usage Cord Blood Stem Cells Considered as waste product High rate of self renewal Capacity to form bone, muscle, nerve cells, blood vessels Adult Stem Cells Used for over 40 years Treat a plethora of blood malignancies and cancers Through transplant medicine  Different Origin of Stem Cells http://www.pall.com/main/medical/frequently-asked-questions-cell-therapy-38848.page  Embryonic Stem Cells Embryonic stem cells are isolated early in embryonic development. Their derivation requires the use and destruction of the organism at the embryonic stage of development. The interest in these cells is due to their ability to become any cell type in the individual. Therefore, mouse embryonic stem cells have been studied for decades, which have produced an enormous amount of information on how an organism develops and how to manipulate these cells to become different cell types. However, it remains difficult to control exactly what cell types these stem cells can become. A related safety concern associated with the use of embryonic stem cells is that they sometimes form tumors (teratomas). http://content.time.com/time/interactive/ 0,31813,2024008,00.html  Somatic Cell Nuclear Transfer Somatic cell nuclear transfer involves taking the cell from an individual (either a child or an adult donor) and transferring the nucleus, which contains the genetic information in the DNA and chromosomes, and inserting it into an egg. This egg is then stimulated, without fertilization, into developing into a living embryo. It is possible, in principle, to transfer this embryo into a female’s body to initiate a pregnancy that will result in the live birth of an offspring that is nearly genetically identical to the cell donor. http://hypertonicblog.wordpress.com/tag/conservation/  Somatic Cell Nuclear Transfer This technique (sometimes called “reproductive cloning”) was used to clone Dolly the sheep several years ago. It has never been accomplished in primates. Alternatively, the living cloned embryo can be destroyed to isolate the resulting embryonic stem cells. Such cells would be nearly genetically identical to the donor of the somatic cell. This is sometimes called “therapeutic cloning.” No cloned embryonic stem cells have ever been successfully isolated from human beings. http://hypertonicblog.wordpress.com/tag/conservation/  Induced Pluripotent Stem Cells Induced pluripotent stem cells are generated by taking cells, such as skin cells, from a person and then injecting a small number of specific genes or molecules into the cells, which converts the cells into stem cells. Thus, these iPS cells do not result in the death of any embryos or require the use of unfertilized eggs. One concern with iPS cells is that one of the genes that are introduced into the cells is an oncogene (gene that can cause cancer). Additionally, viruses are commonly used to introduce the genes into the cells and these viruses can cause mutations in the cells. Recent studies have reported the generation of iPS cells without using oncogenes or viruses. http://www.eurostemcell.org/factsheet/reprogramming-how-turn- any-cell-body-pluripotent-stem-cell  Specific Transcription Factors Induced Pluripotent Stem Cells http://dx.doi.org/10.1016/j.addr.2011.01.013  Adult Stem Cells Adult stem cells are found in most, if not all, tissues in all living organisms. While these adult stem cells cannot become any cell in the organism, they can potentially become any cell in the organ from where they originated. Because these cells are present in an individual’s body, there is very little chance that the regenerated tissue will be rejected by the immune system. Additionally, these adult stem cells often appear to have the capacity to recognize the location of damage and the dying cell types, such that only those missing cells are regenerated. Thus, adult stem cells do not require the death of any embryos or use unfertilized eggs. www.dimensionsofdentalhygiene.com  Embryonic Stem Cells em cells: Stem cells that are harvested from These cells are pluripotent, meaning they ate into cells from all three germ layers. http://www.allthingsstemcell.com/glossary/#embryonicstemcells  Embryonic Stem Cells Ectoderm: One of the three germ layers. Specifically, this is the outer layer of cells in the embryo and it will develop into skin, the nervous system, sensory organs, tooth enamel, eye lens, and other structures.  Embryonic Stem Cells Mesoderm: One of the three germ layers. Specifically, this is the middle layer of cells in the embryo and it will develop into muscle, bone, blood, kidneys, connective tissue, and related structures.  Embryonic Stem Cells Endoderm: One of the three germ layers. Specifically, this is the inner layer of cells in the embryo and it will develop into lungs, digestive organs, the liver, the pancreas, and other organs. http://www.stemcellclinic.com/main-en/klinika/kletochnaya-terapiya/?lang=en http://www.stemcellsforhope.com/Stem%20Cell%20Therapy.htm  Embryonic Stem Cells Usage of Embryonic Stem Cells  Cord Blood Stem Cells Newborn infants no longer need their umbilical cords, so they have traditionally been discarded as a by-product of the birth process. In recent years, however, the stem-cell–rich blood found in the umbilical cord has proven useful in treating the same types of health problems as those treated using bone marrow stem cells. Umbilical cord blood stem cell transplants are less prone to rejection than either bone marrow or peripheral blood stem cells. This is probably because the cells have not yet developed the features that can be recognized and attacked by the recipient's immune system. Also, because umbilical cord blood lacks well- developed immune cells, there is less chance that the transplanted cells will attack the recipient's body, a problem called graft versus host disease (GvHD). Both the versatility and availability of umbilical cord blood stem cells makes them a potent resource for transplant therapies.  Cord Stem Cells Cord Blood Collection: What is the Process? Cord Stem Cell Transplant Improves Cerebral Palsy Symptoms Cord Blood Stem Cells Cure Child with Sickle Cell Anemia  Adult Stem Cells http://news.bbc.co.uk/2/hi/7929690.stm http://www.stemcellsforhope.com/Stem%20Cell%20Therapy.htm  ADULT STEM CELLS http://science.howstuffworks.com/life/cellular-microscopic/stem-cell3.htm http://en.wikipedia.org/wiki/File:Hematopoesis_EN.svg Pre- and Postnatal Periods http://en.wikipedia.org/wiki/File:Hematopoesis_EN.svg Types of Mature Blood Cells Hematopoietic Growth Factors  Immunophenotyping Cells The cluster of differentiation (cluster of designation) (often abbreviated as CD) is a protocol used for the identification and investigation of cell surface molecules providing targets for immunophenotyping of cells. http://en.wikipedia.org/wiki/Cluster_of_differentiation Medicine » Stem Cell Research » "Innovations in Stem Cell Transplantation", book edited by Taner Demirer, ISBN 978-953-51-0980-8, Published: February 13, 2013 under CC BY 3.0 license Signaling Transduction Pathways of Stem Cells Molecular Mechanisms Regulating Pluripotency of Stem Cells  Immunophenotyping Stem Cells MOUSE vs. HUMAN c-Kit, Thy-1 Low, Sca-1+ CD133, Thy-1 Low, CD34+ Lineage Negative: Lineage Negative: B220 CD38 Mac-1 CD10 Gr1 CD14 CD3 CD15 CD4 CD16 Flow Cytometry  Human CD34+ Hematopoietic Stem Cells (HHSC-CD34+) http://www.lifelinecelltech.com/product-specs/spc-hhsc-cd34.asp  Identification of Hematopoietic Stem Cells in Mouse http://www.ebioscience.com/knowledge-center/cell-type/hematopoietic-stem-cells.htm Stem Cell Microenvironment “Niche” From the following article: No place like home: anatomy and function of the stem cell niche D. Leanne Jones & Amy J. Wagers Nature Reviews Molecular Cell Biology 9, 11-21 (January 2008)  Mobilization of Stem Cells Stromal Derived Factor (SDF-1) binds to chemokine receptor called CXCR4 and activate PI3K and PKC- epsilon signaling events which leads to mobilization and activation, of the stem cell. J Clin Invest. 2005 January 3; 115(1): 27–29. doi: 10.1172/JCI200424013 Stem Cell “Niche”  Adult Stem Cells Adult Stem Cell Success Stories: Dr. Richard Burt Model organism: An organism that is easy to study and manipulate and is similar enough to another organism of interest so that by understanding the model organism, a greater understanding of the other organism may be gained. For example, rats and mice can be used as model organisms to better understand humans.  INTRODUCTION Src Homology Inositol Phosphatase (SHIP-1) SHIP-1, a 145kD protein Inositol 5’ Phosphatase Expressed exclusively in hematopoietic cells. SHIP was first observed as a tyrosine phosphorylated protein associated with Shc (adapter molecule) post growth factor and cytokine stimulation*. Structurally SHIP-1 has a SH2 domain located at the amino-terminal end. - Centrally located is the Inositol 5' phosphatase region -NPXY regions, posterior to enzymatic activity -Located at the carboxyl end are several polyproline rich motifs (PxxP) Rohrschneider et al., 2000 Genes & Development  SHIP KNOCKOUT MICE SHIP KNOCKOUT WILD TYPE Fertile Fertile Reduction of Body Weight Normal Body Weight Life span 2-3 months Life span > 12 months Over production Granulocytes Normal Granulocytes Over production Macrophages Normal Macrophages Myeloid infiltration of the lungs No Myeloid infiltration of lungs Progressive Splenomegaly No Splenomegaly BMMC inflammatory cytokines Normal cytokine production Perturbed Natural Killer (NK) cell Normal NK function development * THE ROLE OF SHIP IN GRAFT versus HOST DISEASES (GvHD) *Wang, Howson, Ghansah et al, 2002 Science (Shared First Co-authors )  Graft versus Host Disease Bone Marrow Transplantation is an essential treatment for a variety of cancers. Therefore, it is critical that patients receive BM from MHC- matched Donor to prevent the BM rejection or GvHD. GvHD is a major complication (side-effect) that can lead to morbidity and mortality post allogeneic BMT. Immune Responses DONOR DONOR T CELL T CELL HOST DONOR APCs T CELL DONOR DONOR DONOR Proliferation T CELL T CELL T CELL Inflammatory Cytokines Dendritic Cell Macrophage Activated B cells Killing of Tissues Post -allogeneic BMT activated donor T lymphocytes produce inflammatory factors that are responsible for the development of acute GvHD which could lead to severe tissue damage, rejection or death.  Allogeneic BM Transplantation Lethal Irradiated DONOR HOST H2d(Balb/c) H2b(C57BL/6) Different Genetic Strains Fully Mis-matched MHC Alleles  SHIPko Mice Accepted and Engrafted Fully Mismatched Bone Marrow 100 SH IP -/ - 75 p = 0.00 7 % 50 SH I P + /+ Survival 0 40 50 60 0 10 20 30 70 Days (Post-BMT) 100 100 100 % 75 Donor 50 50 50 Engraftment 0 0 0 B cells T cells Myeloid cells Wang, Howson, Ghansah et al, 2002 Science (Shared First Co-authors)  Hematopoiesis Bone Marrow Pluripotent Stem Cell Myeloid Lymphoid Progenitor Megakarocyte Progenitor Erythroblast T B Neutrophils Eosinophils Erythrocyte Basophils Monocytes NK DC ? Platelets Mast cell DC Macrophages Immune Dysfunction Immunosuppression Hematological Malignancies and Abnormalities  Immunosuppressive MDSC Microenvironment: Infections (Bacterial or Parasitic) Pro-inflammatory Inflammatory Diseases Cytokines and Growth Graft versus Host Disease Factors Tumor/Cancer Traumatic Stress Activation and Expansion Heterogeneous Population of Myeloid Cells Granulocytes, Macrophages, Dendritic Cells Mice and Humans Early Myeloid Progenitors Natural Suppressor (NS) Immature myeloid cells (IMC) Myeloid Suppressors Cells (MySC) Myeloid Derived Suppressor Cells (MDSC) Phenotypic markers CD11b+GR1+ Phenotypic markers CD33+CD11b+LN- T , B lymphocytes, in vivo and in vitro Immune Responses  Expansion of MDSC in SHIPKO Mice Flow Cytometry Ghansah, T. et al., Journal of Immunology, 2004  Mixed Leukocyte Reaction (MLR) Leukocytes (Blood), Splenocytes (SP), Lymph Node (LN) Cells Irradiated Balb/c SHIP-/- H2Db (C57BL/6) H2Dd SHIP+/+ H2Db (C57BL/6) Add (3H)-thymidine or (incorporated into dividing cells) Radioactivity Proportional to Proliferation Culture 4-5 days Balb/c SHIPko C57BL/6 Responders SHIPwt C57BL/6 Stimulators  SHIPKO MDSC Suppress T cell Immune Responses In Vitro MLR Assay Ghansah, T. et al., Journal of Immunology 2004  Human Bone Marrow Transplantation http://www.makna.org.my/images/ autoglass.gif  OBESTIY Over one third of the U.S. adult population is obese. Obesity increases the risk for other conditions such as cardiovascular disease, metabolic disease type 2 diabetes (T2D) and some cancers. Obesity is characterized by an sity is associated with a state of chronic, low-gra Inflammation in adipose tissue depots.  ADULT STEM CELLS FROM ADIPOSE TISSUE http://nextbigfuture.com/2011/05/stem-cell-therapy-could- reshape.html  HUMAN ADIPOCYTE DERVIED STEM CELLS hADSCs are also known as human adipocyte-derived stem cells. hADSCs are multipotent cells in that they can differentiate into a variety of cell types including, adipocytes, chondrocytes, osteoblasts, neuronal cells and even cardiomyocytes. hADSCs are much more abundant and the process of retrieving adipose tissue is less invasive making them attractive for stem cell therapy use.  Adipogenesis Adipogenesis is the development of White Adipose Tissue http://www.udel.edu/biology/Wags/histopage/colorpage/ http://stemcellscottsdale.com/aboutstemcelltherapy/ http://stemcellscottsdale.com/aboutstemcelltherapy/ Human adipocyte stem cells (HADSC) and Leukocytes from diabetic, normal obese patients Preliminary Data Human fat depots were collected from patients undergoing Bariatric Surgery (USF-IRB Approval #108360). Dr. Michel Murr (Bariatric Surgery, TGH/USF) Dr. Denise Cooper (Diabetic Researcher USF/VA) Dr. Tomar Ghansah (Immunologist, USF)