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Regenerative Medicine Regenerative medicine seeks to revolutionize the ways we improve the length and quality of life by restoring, maintaining, or enhancing tissue and organ function. It involves the merging of several fields, including cell/tissue engineering, biomaterials development, and stem cell biology. Source: NIH, 2003 Stem Cells Stem cells have emerged as the starting material of choice to produce cells and tissues to treat degenerative, diseases. genetic, and immunological Atomic bombings of Hiroshima and Nagasaki 1945 Hallmarks of a Blood-forming Stem Cell McCulloch, E.A., Till, J.E. (1960) The radiation sensitivity of normal mouse bone marrow cells, determined by quantitative marrow transplantation into irradiated mice. Radiation Research 13(1):115-125. Till, J.E., McCulloch, E.A. (1961) A direct measurement of the radiation sensitivity of normal mouse bone marrow cells. Radiation Research 14:213-22 STEM CELLS General definition: special kind of cell that has a unique capacity to renew itself and to give rise to specialized cell types (NIH, 2001). Properties: Self-renewal capacity Multilineage differentiation ability In vivo engraftment ability STEM CELLS What are stem cells? Ability to self-renew by multiplying in an undifferentiated state Ability to differentiate into various cell types that constitute an organism Source: Bongso and Lee in Stem Cells - From Bench to Bedside, World Scientific Publishing Com, 2010 STEM CELLS - Classification Source: www.stemcellresources.org STEM CELLS - Classification Differentiation Potential Totipotent cells: have the capacity to specialize into extraembryonic membranes and tissues, the embryo, and all postembryonic tissues and organs. They have the ability to replicate indefinitely without losing their total potency. Pluripotent cells: capable of giving rise to most tissues of an organism; Multipotent cells: can differentiate into only a limited range of cell types; Unipotent cells: can differentiate into one type of cells. ex: testis stem cells. Totipotent Stem Cells Totipotent stem cell – ex. Zygote (union of male and female germ cells). A single totipotent stem cell has the ability to give rise to the whole organism. Source: Stem Cell Information, NIH The only totipotent cells are the fertilized egg and the cells produced by its cleavage in the first 4 days (as shown by the ability of mammals to produce identical twins, triplets, etc.). Totipotent Stem Cells Mammalian Development Figure 19-5 Molecular Biology of the Cell (© Garland Science 2008) Pluripotent Stem Cells Pluripotent Stem Cells A single pluripotent stem cell has the ability to give rise to all types of cells that develop from the three germ layers (mesoderm, endoderm and ectoderm) from which all cells of the body arise. ESC: derived from a group of cells called the inner cell mass, which is part of the early (5day) embryo, called the blastocyst; EGC: derived from fetal tissue; specifically they are isolated from the primordial germ cells of the gonadal ridge of the 5- to 10-week fetus. Later on, the gonadal ridge develops into the testes or ovaries and the primordial germ cells give rise to eggs or sperm; ECC: isolated from teratocarcinomas, a tumor that occasionally occurs in the gonads. Unlike the other two, they are usually aneuploid; Pluripotent Stem Cells Source: www.nature.com Pluripotent Stem Cells A single pluripotent stem cell has the ability to give rise to all types of cells that develop from the three germ layers of the embryo mesoderm, endoderm and ectoderm from which all cells of the body arise Unlike totipotent cells, pluripotent stem cells fail in its ability to differentiate into extra embryonic membranes, namely the trophectoderm (the outer layer of the developing blastocyst that will give rise to the placenta), and tissues namely the trophoblast (extraembryonic tissue responsible for negotiating implantation, developing into the placenta and controlling the exchange of oxygen and metabolites between the mother and the embryo). PLURIPOTENT STEM CELL TIMELINE 1981: Isolation of mouse embryonic stem cells (mESC) by Evans & Kaufman 1960s-1970s: Isolation of human embryonic carcinoma cells (hECC) 2006: Derivation of induced pluripotent stem cells (iPSC) by Yamanaka and co-workers 1998: Isolation of human embryonic stem cells (hESC) by Thompson and co-workers and human embryonic germ cells (hEGC) by Gearhart and co-workers EMBRYONIC STEM CELLS Undifferentiated cells isolated from the inner cell mass of the blastocyst Inner cell mass Source: NIH, 2001 trophectoderm Source: www.thetechherald.com/ 5-day embryo:200 cells Inner cell mass: 30 cells Embryonic Stem Cells (ESC) Undifferentiated cells isolated from a group of cells called the inner cell mass, which is part of the pre-implantation embryo, called the blastocyst (Source:http://www.stemcellresearchfoundation.org) ESC CHARACTERISTICS Long-term self-renewal capacity Can be cultivated indefinitely (unlimited number of symmetrical divisions) without differentiation Pluripotency Capability to differentiate into all mature cell types that are originated from the three primary germ layers of the embryo even after in vitro long term culture ectoderm (e.g. neuronal cells) endoderm (e.g. pancreatic cells) mesoderm (e.g. cardiomyocytes) ESC PLURIPOTENCY Pluripotent Stem Cells Source: Stem cell Information, NIH hESC – The Pioneers hESC CULTURES Human Embryonic Stem Cells HUMAN EMBRYONIC STEM CELLS - Properties Clonogenic cells, capable of undergoing an unlimited number of symmetrical divisions without differentiating (long-term self renewal) Pluripotent cells capable to give rise to cells from all 3 germ layers of the embryo (ectoderm, mesoderm, endoderm) both in vitro (trough Embryoid Bodies or directed differentiation protocols) Develop into teratomas when injected into SCID mice (immunocompromised mice) Exhibit and maintain a stable, diploid karyotype; Express specific transcription factors such as Oct-4, Sox2 and Nanog, surface markers such as Tra-160, Tra-1-81, SSEA-3 and SSEA-4, and Alkaline Phosphatase (AP) Express high levels of Telomerase Mouse and rat embryonic stem cells should also: contribute to the formation of a healthy chimeric adult when injected into a blastocyst which is then implanted in a female’s uterus (Chimeras: mixture of cells derived from both ESC donor and recipient blastocyst in tissues and organs); enter the germline of these animals, that is, contribute to their pool of gametes; hESC CHARACTERIZATION Surface Markers and Transcription factors hESC CHARACTERIZATION Embryoid Bodies/Directed Differentiation Huangfu D et al., Nature Biotech, 2008 hESC CHARACTERIZATION Telomerase expression On the end of the chromosomes there are noncoding repeating sequences named telomeres; Telomeres can be rebuilt using an enzyme called telomerase. Source: Stem Cell Information, NIH, 2001 hESC lines express high levels of telomerase Source: www.sciencemag.org hESC CHARACTERIZATION Teratoma Formation in SCID mice Mesoderm: Mesoderm: bone cartilage Ectoderm: neural epithelium Mesoderm: striated muscle Mesoderm: fetal glomeruli and renal tubules Endoderm: gut epithelium Source: Odorico et al, Stem Cells, 2001 EMBRYONIC STEM CELLS - Chimera formation Why embryonic stem cells? Feature Embryonic Fetal Adult (ex. Bone marrow) Isolation Proliferation Pluripotency / Differentiation Potential Ethical issues / Safety issues (immunocompatibility, teratoma formation) ? Clinical numbers Sources of human ESC Available hESC lines; Excess embryos from IVF clinics Embryos created for research by IVF Therapeutic cloning (or Somatic Cell Nuclear Transfer, SCNT) Therapeutic Cloning European and International Regulation Source: Elstner et al, SC Research , 2009 on ESC research European and International Regulation Source: Elstner et al, SC Research , 2009 on ESC research Glossary Blastocyst - a preimplantation mammalian embryo, i.e. at the end of cleavage and ready for implantation into the uterine epithelium, made of hundred or more cells depending on species. Consists of a continuous outer layer of cells, the trophectoderm, which give rises to the placenta, a fluid filled cavity , the blastocoel, and a cluster of cells on the interior, the inner cell mass from which ESC are isolated. Cell passage – a round of cell growth and proliferation in culture. Passaging consists of removing cells from one culture plate and replating them into new plates. Embryoid bodies (EBs) – clumps of cellular structures arising from ESC in culture; they contain cells from the 3 germ layers and are not part of normal development, only occurring in vitro. Embryonic stem cells – pluripotent stem cells derived from the ICM upon explantation in culture, which can differentiate in vitro into many different lineages and cell types and, upon injection into blastocysts, can give rise to all tissues including the germline. Embryonic germ cells – pluripotent cell line originating from transformed PGCs. In contrast to pluripotent ESC, PGCs are unipotent in vivo, but become pluripotent in culture. Embryonic carcinoma cells – pluripotent cell line originating from transformed primordial germ cells. These cells are derived from teratocarcinomas. Feeder layer – cells, usually fibroblasts, utilized in co-culture to maintain pluripotent stem cells. These are incapable of division but provide physical support and soluble factors to the other cells. Germline stem cells – unipotent cell line derived from mouse testes, which reconstitutes spermatogenesis when transplanted into sterile recipients. Multipotent germline stem cells have the potential to produce teratomas and chimeric animals. Glossary Inner cell mass – cells of the blastocyst that appear transiently during development and give rise to the 3 germ layers of the developing embryo. Multipotency – ability of a cell to give rise to different cell types of a given cell lineage. These cells include most adult stem cells. Pluripotency - ability of a cell to give rise to all cells of the embryo. Primordial germ cells - give rise to oocytes and sperm in vivo and to embryonic germ cells when explanted in vitro. SCID = severe combined immunodeficiency. These mice lack a functioning immune system (have neither T cells nor B cells) and so cannot reject foreign tissue. Telomerase – an enzyme composed of a catalytic protein component and an RNA template and that synthesizes DNA at the ends of chromosmes and confers replicative “immortality” to cells. Teratoma • A teratoma is a type of tumor; • The word teratoma comes from Greek and means roughly "monstrous tumor".; • A teratoma is a tumor with tissue or organ components resembling normal derivatives of all three germ layers, • Teratomas typically contain gut-like structures such as layers of epithelial cells and smooth muscle; skeletal or cardiac muscle; neural tissue; cartilage or bone; and sometimes hair or teeth. Totipotency – ability of a cell to give rise to all cells of an organism, including embryonic and extraembryonic tissues. Glossary Undifferentiated – not having changed to become a specialized cell type by the generation of structures or production of proteins characteristic of a specialized cell. Unipotency - capacity of a cell to sustain only one cell type or cell lineage. Ex. terminally differentiated cells, certain adult stem cells such as testis stem cells.