References  Lecture notes (hyperlink)  Activity notes (hyperlink)  More links… Lecture 2: Introduction to Stem Cells

References  Lecture notes (hyperlink)  Activity notes (hyperlink)  More links… Why do we need stem cells? DEVELOPMENT The development of a multicellular organism begins from a single cell that generates all tissues. Stem cells play a central role in the developing organism. REGENERATION We lose millions of cells every second Without a supply of cells we will lack: – Intestine: 2 days – Skin: 3 weeks – Red blood cells: 4 months 1

References  Lecture notes (hyperlink)  Activity notes (hyperlink)  More links… Functional properties of stem cells 2 Stem cell Daughter stem cell Daughter progenitor cell cardiomyocytes blood cells neuron 1. Self-renew2. Proliferate 3. Progeny can differentiate

References  Lecture notes (hyperlink)  Activity notes (hyperlink)  More links… Introduction to Stem Cells: Embedded Assessment Discuss with a person next to you: Define a stem cell. What makes stem cells different from other cells? Why are these differences important to science and medicine? The Unique Properties of Stem Cells

References  Lecture notes (hyperlink)  Activity notes (hyperlink)  More links… Embryonic stem (ES) cells Present in the inner cell mass (ICM) Pluripotent (can generate all embryonic tissues but NOT extra-embryonic tissues) Proliferate rapidly in culture Give rise to an entire organism when transplanted into a blastocyst Produce tumors when transplanted into the adult 4 Blastocyst ICM Expansion Feeders Hatching Zygote

References  Lecture notes (hyperlink)  Activity notes (hyperlink)  More links… Secreted factors maintain pluripotency of ES cells in culture Secreted factors (proteins): Feeder layer (fibroblasts) secretes proteins that interact with receptors on the ES cell membrane to maintain its pluripotency, either by promoting self-renewal and/or suppressing differentiation. LIF (Leukemia Inhibitory Factor) or basic Fibroblast Growth Factor (bFGF) present in the media binds the LIF receptor on the ES cell plasma membrane, in order to maintain both pluripotency and the rate of cell proliferation. Serum contains BMPs (bone morphogenetic proteins) that maintain pluripotency of mouse ES cells but induce differentiation of human ES cells. 5 ES cellsFeeders Colonies of mouse ES cells in culture

References  Lecture notes (hyperlink)  Activity notes (hyperlink)  More links… Intracellular factors that maintain pluripotency of ES cells Transcription factors (Oct4, Nanog, Sox2) Proteins that are found in the nucleus Bind DNA and regulate gene expression Maintain pluripotency of ES cells by: A) promoting acquisition of ES cell fate and self-renewal ability B) preventing ES cells from acquiring other cell fates (extra-embryonic lineages that are required for embryo implantation into the uterus) ES cells Hypoblast LI F Sox2 Trophoblast 6

References  Lecture notes (hyperlink)  Activity notes (hyperlink)  More links… Adult stem cells Multipotent stem cells found in the adult animal Committed to a particular lineage (e.g. skin, blood, nervous system or gut) Can generate several types of differentiated cells that belong to a particular lineage Divide slowly and are difficult to grow in the lab If the tissue regenerates quickly (blood or gut) or is injured, they divide rapidly They are self-renewing, committed to a tissue and used throughout life 7

References  Lecture notes (hyperlink)  Activity notes (hyperlink)  More links… Tissues maintained by adult stem cells in the body 8 BloodSkin Intestinal epithelium Sperm (Margaret Fuller)

References  Lecture notes (hyperlink)  Activity notes (hyperlink)  More links… Relationship between ES cells and adult stem cells skin brain ES cell Ectodermal cell Mesodermal cell PluripotentMultipotent Endodermal cell Skin stem cell Neural stem cell differentiation Lineage restrictions 9 Multipotent

References  Lecture notes (hyperlink)  Activity notes (hyperlink)  More links… Differences between ES cells and adult stem cells Adult stem cells Quiescent or proliferate very slowly Multipotent cells (committed to a particular lineage such as skin) Found in fetal and fully developed tissues Have partially differentiated into a more mature type of cell Present in small numbers 10 ES cells Proliferate very rapidly in the embryo Pluripotent stem cells (generate all tissues of the embryo) Occur only in the embryo Completely undifferentiated Found in the inner cell mass (ICM) at the blastocyst stage of the embryo

References  Lecture notes (hyperlink)  Activity notes (hyperlink)  More links… Theurapeutic potential of human ES cells versus adult stem cells Adult stem cells Can produce a limited range of differentiated cells Can be harvested from patients and have therapeutic potential Hematopoietic stem cells have been used successfully to treat leukemias and other bone/blood cancers In some cases, are difficult to isolate Limited therapeutic use Difficult to grow in the laboratory 11 ES cells Can produce all tissues of the embryo and have unlimited therapeutic potential Their isolation and use is controversial due to ethical issues Can cause tumors (teratomas) if transplanted into adults. Can be differentiated in culture to make progenitors for various lineages (directed differentiation) and used for therapies. The directed differentiation protocol has been used successfully for many cell types

References  Lecture notes (hyperlink)  Activity notes (hyperlink)  More links… Relationship between adult stem cells and transit amplifying cells 12 skin Transit amplifying progenitors Skin stem cell Differentiation Skin stem cell INJURY REPAIR

References  Lecture notes (hyperlink)  Activity notes (hyperlink)  More links… Stem cell niche 13 stem cell daughter progenitor cell differentiated cells stem cell Loss of contact with the niche Differentiation Local microenvironment where adult stem cells reside in the organism. Influences many properties of stem cells: – Number of stem cells – Stem cell cycle progression and type of division (symmetric versus asymmetric) – Self-renewal properties of stem cells – Differentiation into specific lineages

References  Lecture notes (hyperlink)  Activity notes (hyperlink)  More links… How do niche cells control the properties of stem cell? I. Secreted local factors Stem cell Niche cells Niche cells secrete several factors that are important for stem cell maintenance. Niche cells secrete unique factors for their respective stem cell. Hematopoietic stem cells are maintained by several factors in their niche: a) Stem cell factor (c-Kit receptor) b) Wnt (Frizzled/LRP receptor) c) Angiopoietin-1 (Tie2 receptor) 14

References  Lecture notes (hyperlink)  Activity notes (hyperlink)  More links… How do niche cells control the properties of stem cell? Stem cell Niche cells II. Cell-cell communication Cell-ECM communication Cell-cell communication at the niche occurs through a variety of molecules. Adhesion is important for holding stem cells in the niche. Adherens junctions are specialized intercellular contacts important for cell-cell communication and adhesion. Cadherins and ß-catenin are components of adherens junctions. Integrins are a second class of molecules responsible for adhering stem cells to the extracellular matrix. 15

References  Lecture notes (hyperlink)  Activity notes (hyperlink)  More links… Stem cell niche in response to injury 16 stem cell niche cells Normal state stem cell increased stem cell proliferation differentiation quiescent state: stem cells divide slowly INJURY Disruption of cell contact Upregulation of stem cell factors

References  Lecture notes (hyperlink)  Activity notes (hyperlink)  More links… Somatic-derived stem cells via nuclear transfer Create ES cells that match the donor’s genetic makeup for therapeutic purposes. Currently, no human ES stem cell lines have been derived from this method. ES cells derived from patients can be directed to differentiate into specific lineages (e.g. dopaminergic neurons) to study a particular disease (e.g. Parkinson’s disease). This method may be used for cell-based therapies that would circumvent immune rejection. Not extensively used at present, because: 1) iPS strategies are more feasible, 2) stress to the egg causes a reduced efficiency for ES cell generation. 17 ES cell Neuron Progenitor Enucleated oocyte Fibroblasts from patients Nucleus directed differentiation

References  Lecture notes (hyperlink)  Activity notes (hyperlink)  More links… Induced pluripotent stem cells (iPS cells) A type of pluripotent stem cell artificially derived from an adult somatic cell by "forcing" expression of specific genes. iPS cells are believed to be similar to ES cells with respect to: – A) stem cell gene and protein expression – B) ability to differentiate into all lineages in vitro – C) forming viable chimeras after injection into blastocysts or tumors when transplanted into adult tissues – D) potential to form an entire organism, such as a mouse 18

References  Lecture notes (hyperlink)  Activity notes (hyperlink)  More links… iPS cells - a recent advance IPS cells were first produced in 2006 from mouse tissue, and in 2007 from human. This is an groundbreaking advance in stem cell research, because it allows researchers to obtain pluripotent stem cells, which are important in research and potentially have therapeutic uses, without the controversial use of embryos. Reprogramming adult cells to obtain iPS cells may pose significant risks that could limit their use in humans. If viruses are used to alter the cells’ genome, the expression of cancer-causing genes or oncogenes may potentially be triggered after these cells are introduced into animals. 19

References  Lecture notes (hyperlink)  Activity notes (hyperlink)  More links… Production of iPS cells 20 iPS cell fibroblasts from patients iPS reprogramming factors directed differentiation ectodermal cell mesodermal cell endodermal cell brain heart pancreas

References  Lecture notes (hyperlink)  Activity notes (hyperlink)  More links… iPS reprogramming factors 21 Retroviruses (viruses that contain RNA, and convert RNA into DNA) that infect fibroblast cells are commonly used. Virus encodes four transcription factors: Oct4, Sox2, Klf-4 and c-Myc. C-Myc is a tumor-inducing gene (oncogene). Oct4 and Sox2 are necessary to induce pluripotency of fibroblasts. Transcription factors increase the efficiency of iPS production. Currently, reprogramming is inefficient and slow. Transcription factors modify gene expression in infected cells. Factors turn OFF genes that are part of the differentiated phenotype. Factors turn ON genes that both maintain pluripotency and the ability to self-renew.

References  Lecture notes (hyperlink)  Activity notes (hyperlink)  More links… How do we identify stem cells in the adult organism? Mouse bearing a blue marker in every cell (lacZ) Transplant into recipient mouse Isolate “blue” mammary stem cells by expression of cell surface proteins Stem cells from the donor mouse populate mammary tissue in the recipient mouse and form lactating ducts 22

References  Lecture notes (hyperlink)  Activity notes (hyperlink)  More links… How do we identify stem cells in an adult? 23 Red blood cellwhite blood cell 1. A single cell that resides in the stem cell niche is genetically marked in vivo with green fluorescent protein (GFP). 2. Progenitors derived from the original green cell will be also green. 3. Differentiated cells that are born from green progenitors will also be green. Therefore one can determine that the labeled cell is a stem cell or progenitor.

References  Lecture notes (hyperlink)  Activity notes (hyperlink)  More links… Introduction to Stem Cells: Embedded Assessment Compare and Contrast Stem Cell Types Expected lifespan in tissue culture PotencySourceDevelopmental Stage Human Embryonic Stem Cells (hESCs) Adult Stem Cells (Tissue-specific stem cells: hematopoietic, neural, pancreatic, etc.) Induced Pluripotent Stem Cells (iPSCs)

References  Lecture notes (hyperlink)  Activity notes (hyperlink)  More links… Introduction to Stem Cells: Embedded Assessment Answer Key Compare and Contrast Stem Cell Types Expected lifespan in tissue culture PotencySourceDevelopmental Stage Human Embryonic Stem Cells (hESCs) Immortal: they divide endlessly in culture. Pluripotent, capable of making any cell or tissue in the body. Made from the inner cell mass of a blastocyst. The blastocyst forms very early in development, between 2 and 4 days. Adult Stem Cells (Tissue-specific stem cells: hematopoietic, neural, pancreatic, etc.) Life span is limited, and depends on the type of adult stem cell. Multipotent: They are “lineage restricted”, and make only specific types of cells. Found in organs and tissues of the body, such as heart, bone, fat, brain, and liver. Develop during the fetal stage, and persist throughout adulthood. Induced Pluripotent Stem Cells (iPSCs) Immortal: they appear to divide endlessly in culture. iPS cells are thought to be pluripotent. In theory, any somatic or body cell can be reprogrammed to an embryonic state. Any somatic or body cell can be used, such as a skin cell.

References  Lecture notes (hyperlink)  Activity notes (hyperlink)  More links… Summary TYPES OF STEM CELLS: Embryonic stem (ES) cells Adult stem cells Somatic-derived stem cells via nuclear transfer (SCNT) Induced pluripotent stem (iPS) cells STEM CELL NICHE: Secreted factors Cell-cell interactions 26

References  Lecture notes (hyperlink)  Activity notes (hyperlink)  More links… Introduction to Stem Cells: Concept Mapping Terms Add the key terms/concepts from today’s lecture to your previous concept map. You should include (but are not limited to) the following terms/concepts: E mbryonic stem cell Adult stem cell Induced pluripotent stem cell Nuclear transfer Stem cell niche Transcription factor Self-renewal Lineage restriction Due by Thursday, April 7 27