Animal Cell and Tissue Culture Lecture 6 PDF

SIO2004 Animal Cell and Tissue Culture Lecture 6 Biotechnology Program Universiti Malaya Instructor: Dr. Nuradilla Mohamad Fauzi Can normal cells divide forever and ever? (in culture) Nope, most normal cells have a finite lifespan! (most of...

SIO2004 Animal Cell and Tissue Culture Lecture 6 Biotechnology Program Universiti Malaya Instructor: Dr. Nuradilla Mohamad Fauzi Can normal cells divide forever and ever? (in culture) Nope, most normal cells have a finite lifespan! (most of them will eventually stop dividing) Replicative cell senescence Multicellular organisms replace worn-out cells through cell division. However, in many cells, proliferation slows down until cell division eventually halts. Cells enter a non-dividing state, known as senescence. In humans this occurs, on average, after 52 divisions, known as the “Hayflick limit”. e.g. Human fibroblasts permanently cease dividing after 25-40 divisions (population doublings) In culture, primary cells have a finite lifespan. Cells stop dividing because the telomeres, protective bits of DNA on the end of chromosomes, shorten with each division, eventually being consumed.  Telomeres A telomere is a region of repetitive nucleotide sequences at each end of a chromosome. Protects the end of the chromosome from deterioration or from fusion with neighboring chromosomes. Disposable buffers at the ends of By U.S. Department of Energy Human Genome Program - chromosomes which are truncated during http://science.nasa.gov/media/medialibrary/2006/03/16/22mar_telomeres_resources/caps.gif, Public Domain, https://commons.wikimedia.org/w/index.php?curid=5234303 cell division; their presence protects the genes before them on the chromosome from being truncated instead. For vertebrates, the sequence of nucleotides in telomeres is TTAGGG.  Telomeres and cellular ageing Each time a cell divides, some of the telomere is lost (usually 25-200 base pairs per division) In humans, average telomere length declines from about 11 kilobases at birth to less than four kilobases in old age During chromosome replication, the enzymes that duplicate DNA cannot continue their duplication all the way to the end of a chromosome, so in each duplication the end of the chromosome is shortened When the telomere becomes too short, the chromosome reaches a "critical length" and can no longer replicate (the cell has become “old”) Cells undergo senescence or apoptosis  Telomerase Telomerase is a reverse transcriptase enzyme that lengthens telomeres in DNA strands Adds telomere repeats Prevents degradation of the chromosomal ends following multiple rounds of replication Telomerase activity is regulated during development. Most post-natal somatic cells are deficient in telomerase.  Cells that express telomerase Which cells express telomerase? Fetal tissues Adult germ cells Stem cells Cancer and transformed cells Telomerase expression can allow senescent cells that would otherwise become post-mitotic and undergo apoptosis to exceed the Hayflick limit, extend the lifespan of the cells, and become potentially immortal (transformation!) Transformation Transformation of cultured cells: a spontaneous or induced permanent phenotypic change resulting from a heritable change in DNA and gene expression, leading to genetic instability. It involves one or all of these phenotypic changes in a cell line: (1) immortalization, the acquisition of an infinite life span, (2) aberrant growth control; the loss of contact inhibition of cell motility, density limitation of cell proliferation, and anchorage dependence, and (3) malignancy, as evidenced by the growth of invasive tumors in vivo. When injected into animals without functional immune system, transformed cell lines cause tumors Note: the acquisition of an infinite life span alone is referred to as immortalization because it can be achieved without grossly aberrant growth control and malignancy, which are usually linked. Transformation and cancer Transformed cell lines are the in vitro equivalent of cancerous cells. Cancer occurs when a somatic cell which normally cannot divide undergoes mutations which cause de-regulation of the normal cell cycle controls leading to uncontrolled proliferation. Immortalized cell lines have undergone similar mutations allowing a cell type which would normally not be able to divide to be proliferated in vitro. Transformation methods Natural/spontaneous Mutations required for immortality can occur naturally, such as in tumor/cancer cells, or randomly arise spontaneously in culture Artificial expression of key proteins required for immortality (e.g. oncogenes) e.g. telomerase! Artificial inactivation of tumor suppressor genes e.g. p53, Rb Chemical mutagens Treating primary cells or nontransformed cell lines with chemical carcinogens, DNA methylation inhibitors Introduction of viral genes Introduction of a viral gene that partially deregulates the cell cycle, such as SV40 T antigen, HPV-16 E6/7 gene TUMOR SUPRESSOR ONCOGENE In normal cells: ON In normal cells: OFF In cancer cells: OFF In cancer cells: ON e.g. p53, Rb e.g. telomerase Example: how SV40 T antigen works The SV40 LT gene is often used to induce immortalization. The product of this gene, T antigen, is known to bind Rb and p53. By doing so, it restricts the DNA surveillance activity p53 and Rb, thereby allowing an increase in genomic instability and an increased chance of generating further mutations favorable to immortalization (e.g. the upregulation of telomerase or the downregulation of one of the telomerase inhibitors). = allows an extended proliferative life span Many cell lines cultured around the world are immortalize/cancer cells! Examples of immortal/cancer cell lines Most immortalized cell lines are classified by the cell type they originated from or are most similar to biologically. HeLa cells – a widely used human cell line isolated from cervical cancer patient, Henrietta Lacks 3T3 cells – a mouse fibroblast cell line derived from a spontaneous mutation in cultured mouse embryo tissue A549 cells – derived from a cancer patient lung tumor Jurkat cells – a human T lymphocyte cell line isolated from a case of leukemia Vero cells – a monkey kidney cell line that arose by spontaneous immortalization F11 cells – a line of neurons from the dorsal root ganglia of rats HEK 293 cells – derived from human fetal cells Wiki Advantages of cancer/immortalized cell lines Immortal! Cells can be grown indefinitely in culture Inexhaustible supply of cells throughout your research project No need to re-establish fresh primary cultures Ease of culture no need complex medium or additives In vitro model for cancer Limitations of cancer/immortalized cell lines Genome instability Abnormal chromosomes Cell lines can change genetically over multiple passages, which leads to accumulation of mutations and variability between stocks Poor model/not representative of normal tissues While immortalized cell lines often originate from a well-known tissue type they have undergone significant mutations to become immortal. This can alter the biology of the cell and must be taken into consideration in any analysis. http://uvmgg.wikia.com/wiki/HeLa_Cells  What if we want both?? Wouldn’t it be nice if we have cells that have long lifespans AND are representative of normal tissues and physiological processes?? Stem cells! Stem cells Definition of stem cells Stem cells are cells that are capable of: Self-renewal: the ability to go through numerous cycles of cell division while maintaining the undifferentiated state. Differentiation the capacity to differentiate into mature, specialized cell types. Alberts et al. (2002) Molecular Biology of the Cell, 4th edition Importance of stem cells in vivo Stem cells are the body's raw materials — cells from which all other cells with specialized functions are generated Cell renewal Produce more cells Replacing dead cells, cells lost through normal wear and tear Repair damaged, diseased tissue Essential for the development of the organism! egg sperm (zygote) (morula)  Blastocoel Trophoblast (cavity) forms extraembryonic tissues, e.g. placenta Inner cell mass (ICM) forms the embryo (all embryonic tissues!) Potency (potential to become Endoderm Mesoderm Ectoderm different cell types) Differentiation Specialization Lineage commitment  egg sperm (zygote) (morula) Totipotent Can differentiate into all embryonic and extraembryonic tissues  Blastocoel Trophoblast (cavity) forms extraembryonic tissues, e.g. placenta Inner cell mass (ICM) forms the embryo Pluripotent (all embryonic tissues!) Can differentiate into all embryonic tissues HSC Multipotent MSC Endoderm Mesoderm Ectoderm Can differentiate into multiple tissue types Oligopotent Can differentiate into a few tissue types Tripotent (3) Bipotent (2) Unipotent (1) (precursor cells) fibroblasts Terminally differentiated cells Development, germ layers and development A germ layer is a primary layer of cells that form during embryogenesis Germ layers eventually give rise to all of an animal’s tissues and organs through the process of organogenesis In vertebrates, three germ layers: Ectoderm is the outer most layer and gives rise to the integument including hair, nails and epidermis, and the epithelial of the nose, mouth and the sense of the eye, the nervous system and inner ear). Mesoderm is the middle layer and it forms the musculoskeletal, circulatory and most of the excretory systems. Mesoderm also develops into gonads and the muscular and connective tissue in the digestive and respiratory system. Endoderm is the inner layer, which will eventually develop into the epithelia linings of the digestive and respiratory tracts including the lungs. It also forms the pancreas, thyroid, bladder and distal urinary tracts. Finally it will also form parts of the liver as well. Development and cell potency The developmental potential, or potency, of a cell describes the range of different cell types it CAN become. The zygote and its very early descendants are totipotent - these cells have the potential to develop into a complete organism. Totipotency is common in plants, but is uncommon in animals after the 2-4 cell stage (still under debate!*). Throughout development; cells undergo progressive restrictions in their developmental potentials. Differentiation occurs when the cell elaborates a cell-specific developmental program. Differentiation results in the presence of cell types that have clear-cut identities, such as muscle cells, nerve cells, and skin cells. As embryonic development proceeds, the potency of cells become progressively more limited. *Stem Cells Dev. 2014 Apr 15; 23(8): 796–812. Q: Are stem cells really immortal? Tbh, scientists are still figuring it out!  Importance of stem cells in research Due to their self-renewal and differentiation abilities, stem cells cultivated in vitro are incredibly useful and important in research! Increase understanding of developmental mechanisms, how diseases occur Stem cell therapy: Regenerative medicine, transplantations Transplantation to regenerate and repair diseased or damaged tissues in people Grow new tissue in vitro Bone marrow transplantations → “old-school” stem cell transplants! Test new drugs for safety and effectiveness Generate specific cell types for testing new drugs Homing and delivery of compounds to target tissues Stem cells can home to tumors, wounds/damaged tissues  Cancer stem cells? CSC Hypothesis—that cancers are perpetuated by a small population of tumor-initiating cells that exhibit numerous stem cell-like properties. Researchers now believe that cancerous stem cells may trigger reproduction and growth of cells within a cancer. These cancerous stem cells lurking within the cancer, under the radar of cancer drugs that target cell proliferation, may underlie the relapse of tumors after surgery of the primary tumor or other cancer treatments. Some breast and prostate cancer cases have fueled the cancer stem cell theory. Often years after the organ or the cancerous lesions are removed and the patient is declared cancer-free, breast or prostate cancer can return in other organs, indicating the cancer had metastasized before it was originally detected. Cancerous stem cells may be the reason for this. The cancer stem cell model suggests that the cells in a tumour are in a strictly organised system with cancer stem cells at the top of the tree, giving rise to all other cancer cells. The stochastic model of cancer growth argues that the cells in a tumour are not in an organised system – any cell has the same intrinsic potential to contribute to tumour growth. Different types of cancer may work in different ways, so it is possible that both of these models apply to different cancers or different stages of tumour development. http://www.eurostemcell.org/factsheet/cancer-disease-stem-cells  Evidence for cancer stem cells There is no definitive proof in favor of either theory of cancer growth. However, an increasing amount of evidence suggests that the cancer stem cell theory holds true in some cases. The first evidence in favor of cancer stem cells came from studies of human leukemia. Researchers found that only a subset of leukemic cells can cause leukemia when transplanted into a healthy body, the key characteristic of cancer stem cells. Since that discovery, many researchers have found cells with cancer stem cell characteristics in a great variety of human and mouse cancers, including breast, brain, skin, prostate and colonic cancers. In some types of tumor the cancer stem cells are rare, for example in colon cancer. In other types of cancer, such as melanoma, a very large number of the tumor cells have cancer stem cell characteristics. http://www.eurostemcell.org/factsheet/cancer-disease-stem-cells How Do Cancer Stem Cells Arise? The molecular pathways that maintain "stem-ness" in stem cells are also active in numerous cancers. This similarity has led scientists to propose that cancers may arise when some event produces a mutation in a stem cell, robbing it of the ability to regulate cell division. This figure illustrates 3 hypotheses of how a cancer stem cell may arise: (1) A stem cell undergoes a mutation, (2) A progenitor cell undergoes two or more mutations, or (3) A fully differentiated cell undergoes several mutations that drive it back to a stem-like state. In all 3 scenarios, the resultant cancer stem cell has lost the ability to regulate its own cell division. https://stemcells.nih.gov/info/Regenerative_Medicine/2006chapter9.htm