CHARACTERISTIC FEATURES OF STEM CELLS. CLONING TECHNOLOGIES 1 Science is discovering the unknown Stem cell field is still in its infancy Human embryonic stem cell research is a decade old, adult stem cell research has 30- year head start Holds hope for curing or improving treatments for diseases of old age What are stem cells and why are they so important? Stem cells have three unique features: Remain in undifferentiated state with a high potential to proliferate for a long period of time, increasing its population (mitosis) Can differentiate under the influence of physiological or external signals 4 Stem Cells: definition Self Renewal - undifferentiated cells that can divide repeatedly while maintaining their undifferentiated state Pluripotency ability to differentiate into a variety of different cell types TYPES OF STEM CELLS Types of stem cells. Regenerative potential Totipotent able to generate every cell type including extraembryonic tissues Pluripotent able to generate cells from all three embryonic germ layers Multipotent able to generate a variety of cells from a particular somatic structure Unipotent only generate one cell type Different types of stem cells due to the origin Embryonic stem cells - ESCs, derived from unimplanted embryo at the blastocyst stage Fetal stem cells isolated from buds of tissues emerging between 5-10 weeks of fetal life. These can be obtained after spontaneous abortion or miscarriage. Somatic stem cells - ASCs (Adult Stem Cells), which are obtained from the tissues of adults and mature fetuses or umbilical cord blood at birth. How to distinguish them from each other? Each cell type is characterized by the presence of another set of surface proteins, which allows their isolation and identification EMBRYONIC STEM CELLS (mostly from IVF) isolated from the inner cell mass of blastocyst (4-5-day-old embryos). these cells could be collected from embryos obtained during in vitro fertilization (IVF) procedures Tens of thousands of frozen embryos are routinely destroyed when couples finish their treatment These surplus embryos can be used to produce embryonic stem cells Regenerative medical research aims to develop these cells into new, healthy tissue to heal severe illnesses Main features of ESCs Capable of an infinite number of symmetrical divisions without differentiation Single ESC gives rise to a colony of genetically identical daughter cells having the characteristics of a mother cell They contain and maintain a full set of diploid chromosomes Pluripotent Can be propagated in tissue culture conditions By this method a few dozen lines of embryonic stem cells were obtained and have even been approved by the U.S. government for research within the federal budget ADVANTAGES: pluripotent antigenic homology no recoil after autologous transplantation poorly marked antigenicity - after allogeneic transplantation do not produce a clear response host versus donor do not lead to transplant rejection
DISADVANTAGES: problems with culture pluripotency: unpredictable direction of differentiation (neurons that produce dopamine) transformation into the tumor tissue (teratomas 20%) (protection: thymidine kinase transfection and ganciclovir administration) ethical issues How can we be sure that ESCs, maintained in culture for several months, remain ESCs? As long as we keep cells under controlled conditions, they remain undifferentiated Left alone, they begin to form the so-called embryoid bodies (cell aggregates), which differentiate spontaneously microscopic observations checking karyotype surface markers rating viability after freezing/thawing cells analysis of the differentiation potential: left to themselves stimulated injection of ESCs to nude mice (teratoma). How to make an embryonic stem cell become what we want? We do not know all the factors that allow cells to divide repeatedly and make them remain in the undifferentiated state We do not know the full characterization of cellular signals that control the differentiation of a given cell type There is something we know pluripotency markers Stage-specific antigens: Anti-SSEA 3 and 4 recognize globo-series gangliosides Tra1-60 and Tra1-81: keratin sulfate surface antigens Oct3/4, Sox2, Nanog transcription factors involved in maintaining pluripotency Different culture conditions alter the fate of ES cells in vitro Protocols exist for all three germ layers Many, but not all, protocols involve aggregation of ES cells in embryoid bodies Selection steps can help to remove undesired cell types ADULT STEM CELLS (Somatic SC) Can be found in small amounts in bone marrow, pancreas, liver, epidermis, cornea and retina, where they are responsible for their regeneration Multi-or unipotent, can be transformed into a particular cell or cell line of the tissue from which they originate Include the umbilical cord blood stem cells undifferentiated limited proliferation number decreases with age aging effect found within niches in tissues do not form teratomas Any tissue may contain adult stem cells Their function is involved in organ regeneration after injury or disease There is no complete agreement on how to identify adult stem cells 3
Methods: Labeling cells in vivo and evaluating their regenerative potential Labelling cells ex vivo, in vitro culture to check regenerative potential Labeling cells ex vivo, in vitro culture, differentiation in vitro. Bone Marrow stem cells Hematopoietic SC Can differentiate not only to all blood cell types, but also into: neurons, oligodendrocytes and astrocytes skeletal muscle and myocardium, hepatocytes Mesenchymal SC osteoblasts, chondroblasts, adipocytes, fibroblasts, skeletal muscle and myocardium Factors known to affect stem cells Low stress levels Regular exercise Learning new information Healthy diets: rich in antioxidants Avoid excessive drinking 1 Umbilical cord blood stem cells (Cordial Blood SC) multi-and even pluripotent They can differentiate into many types of mature cells Phenotypic characterization demonstrates that (CB-SCs) display embryonic cell markers (e.g., transcription factors OCT-4 and Nanog, stage-specific embryonic antigen (SSEA)-3, and SSEA-4) and leukocyte common antigen CD45, they are negative for blood cell lineage markers (e.g., CD1a, CD3, CD4, CD8) display very low immunogenicity as indicated by expression of a very low level of major histocompatibility complex (MHC) antigens and failure to stimulate the proliferation of allogeneic lymphocytes. They can give rise to three embryonic layer-derived cells in the presence of different inducers. CB-SCs tightly adhere to culture dishes with a large rounded morphology and are resistant to common detaching methods (trypsin/edta). CB-SCs are the active agent in stem cell educator therapy, which has therapeutic potential against autoimmune diseases like type 1 diabetes Umbilical cord blood is an alternative source of hematopoietic SC (banking, possibility of autogenous and allogeneic grafts (3500 grafts) ADVANTAGES: Availability and time Secure obtaining method Larger pool of available donors Young SC 1 Umbilical cord blood remains in plancental vessels and in the placental part of the umbilical cord after labour easily accessible source of stem cells contains not only hematopoietic cells, but also cells that give rise to other cell lines, such as cells responsible for bone formation, cells of the nervous system
Minimal risk of CMV infection No moral dilemmas 2 DISADVANTAGES: Delayed bone marrow repopulation The risk of genetic load Unknown impact of banking Less SC than in bone marrow and mobilized blood VSELs (Very Small Embryonic Like stem Cells) very small cells having a diameter of 3-5 µm, present in cord blood (BUT also in the bone marrow) researchers are studying the possibilities and directions of VSELs differentiation There are also doubts about VSELs existence STEM CELL TECHNOLOGIES Cloning technologies Reprogramming: ips induced pluripotent stem cells slow and stochastic (until recently) Transdifferentiation conversion of one terminally differentiated cell type into another without de-differentiation to an immature phenotype. Must rule out cell fusion or other explanations. SCNT somatic cell nuclear transfer (reproductive and therapeutic cloning) deterministic and fairly rapid INDUCED PLURIPOTENT STEM CELLS New ways to potentially avoid the use of embryos Disease-specific stem cell lines created The promise and potential pitfalls of this approach Generating ips cells Express transcription factors: Oct3/4, Sox2, Klf4 and c-myc OR Oct3/4, Sox2, Nanog and Lin28 Initial de-differentiation and proliferation (day 1-3, enhanced by Myc); histone modification and chromatin reorganization Pros and Cons to ips cell technology Pros: Cells would be genetically identical to patient or donor of skin cells (no immune rejection!) Do not need to use an embryo Cons: Cells would still have genetic defects One of the pluripotency genes is a cancer gene Viruses might insert genes randomly into the genome (causing mutations)
Transdiferetiation The results of numerous experiments indicate the possibility of transdifferentiation of the second pool of MSC present in cord blood into nerve cells, muscle cells and the liver or pancreas Tissue stem cells (after their concentration), when transplanted into the recipient can differentiate not only into the tissue from which they originate, but also in other tissues that derive from other germ layers (transdifferentiation) The experiments in vitro and in vivo have shown that MSC are very "plastic" and can differentiate into osteoblasts, chondroblasts (cartilage cells) or adipocytes (fat cells) Further research may lead to preparation of cord blood cells capable of repairing various tissues Cloning Gene cloning Gene cloning involves using bacteria to make multiple copies of a gene; Foreign DNA is inserted into a plasmid, and the recombinant plasmid is inserted into a bacterial cell; Reproduction in the bacterial cell results in cloning of the plasmid including the foreign DNA; This results in the production of multiple copies of a single gene. Cloning organisms Cloning organisms produces one or more organisms genetically identical to the parent that donated the single cell. Cloning organisms may lead to other applications (e.g. as models to study genetically inherited diseases therapeutic cloning)
How clone orgnanism? Cloning Plants: Single-Cell Cultures One experimental approach for testing genomic equivalence is to see whether a differentiated cell can generate a whole organism A totipotent cell is one that can generate a complete new organism Plant cloning is used extensively in agriculture Reproductive Cloning of Mammals In 1997, Scottish researchers announced the birth of Dolly, a lamb cloned from an adult sheep by nuclear transplantation from a differentiated mammary cell. Dolly s premature death in 2003, as well as her arthritis, led to speculation that her cells were not as healthy as those of a normal sheep, possibly reflecting incomplete reprogramming of the original transplanted nucleus. Embryo cloning use embryos in pre-implantation stages or primitive embryonic cells Somatic cloning use somatic cells derived from adult tissue or fetal explants Therapeutic cloning - Nuclear transplantation (Somatic Cell Nuclear Transfer) - the nucleus of an unfertilized egg cell or zygote is replaced with the nucleus of a differentiated cell; The nucleus that is extracted from the patient s somatic cell is then inserted into the egg, which had its nucleus previously removed The egg now contains the patient's genetic material It is stimulated to divide and shortly thereafter forms blastocyst
Therapeutic cloning vs. Reproductive cloning Problems Associated with Animal Cloning In most nuclear transplantation studies, only a small percentage of cloned embryos have developed normally to birth, and many cloned animals exhibit defects; Many epigenetic changes, such as acetylation of histones or methylation of DNA, must be reversed in the nucleus from a donor animal in order for genes to be expressed or repressed appropriately for early stages of development. The reprogramming of donor nuclei requires chromatin restructuring, which does not occur fully during cloning procedures.