Stem Cell and Nuclear Reprogramming

MADE BY APHRODITE PASCOE STEM CELL AND NUCLEAR REPROGRAMMING What are stem cells? - Stem cells have the amazing ability to develop into many different cell types in the body both during early life and growth. - In many adult tissues they can divide, without a limit, to replenish other cells for as long as the organism is alive. - When stem cells divide each cell has the potential to stay as a stem cell or become another cell with a specialized function, such as a muscle cell, brain cells, red blood cell. Characteristics of stem cells 1. They are unspecialized (undifferentiated). 2. They are cells that are capable of renewing themselves through cell division (sometimes even after a very long time of inactivity). 3. Under certain physiological or experimental conditions they can be induced to become tissue or organ specific cells with specialized functions. ▫ In some tissues such as the gut, the bone marrow and the skin, stem cells are constantly dividing to repair or replace damaged or old tissue. ▫ In other tissues such as the heart and pancreas, the stem cells only divide under very special conditions Classes of stem cells ❖ Until recently, scientist only worked with two kinds of stem cells. ▫ Embryonic stem cells ✓ Scientist have figured out how to collect these from mouse embryos in 1981 ✓ Human embryonic stem cells were first obtained in 1998. (where did we get them?) ▫ Non-embryonic (somatic or adult) stem cells ✓ Fetal ✓ Adult ❖ In 2006, scientist have figured out a way to induce specialized cells to take on a stem cell like characteristic. These cells are called induced pluripotent stem cells (iPSCs) Potency of a cell • Potency of a cells is its ability to differentiate into other cell types. Based on this ability we divide the stem cells into different groups. ➢ Totipotent stem cells - Totipotency is the ability of a single cell to divide and produce all of the differentiated cells in an organism. Zygotes are examples of totipotent cells. ➢ Pluripotent stem cells - Pluripotency refers to a stem cell that has the potential to differentiate into any of the three germ layers: endoderm (interior stomach lining, gastrointestinal tract, the lungs), mesoderm (muscle, bone, blood, urogenital), or ectoderm (epidermal tissues and nervous system) ➢ Multipotent stem cells - Multipotency describes progenitor cells which have the gene activation potential to differentiate into discrete cell types. A multipotent blood stem cell is a hematopoietic cell—and this cell type can differentiate itself into several types of blood cell types like lymphocytes, monocytes, neutrophils, etc., but cannot differentiate into brain cells, bone cells or other non-blood cell types. ➢ Oligopotent stem cells -Oligopotency is the ability of progenitor cells to differentiate into a few cell types. Examples of oligopotent stem cells are the lymphoid or myeloid stem cells. ➢ Unipotent stem cells - A unipotent cell is the concept that one stem cell has the capacity to differentiate into only one cell type Some interesting questions ❖ Embryonic stem cells can proliferate for a year in the laboratory without differentiating but adult stem cells cannot. Why? ❖ What are the factors that will normally regulate stem cell renewal and proliferation and differentiation? ❖ Answering these questions will help us better understand embryonic development and even abnormal cell grow conditions such as cancer. ❖ Also, it will enable us to grow and manipulate stem cells more efficiently in the laboratory The stem cell niche and differentiation - Specific factors that allow stem cells to remain undifferentiated is of great importance to us. - Also, factors that prompt the stem cells to turn into specialized cells (differentiate) is also important. - The process of turning unspecialized cells into specialized cells is called differentiation. - The process of differentiation involves several steps and with each step the cells become more and more specialized. The signals can cause the cell to acquire epigenetic marks which can alter its gene expression. ✓ Internal signals of differentiation are controlled by the genes of the cell. ✓ External signals include chemicals produced by other cells, physical contact, molecules in the microenvironment Nuclear reprogramming • It is the process of changing the identity of cells to an embryonic-like state. • It is a like a reset on the cells. ➢ Epigenetic reset (DNA methylation and histone modifications) ➢ DNA replication, nuclear organization, cell division and cell metabolism • There are different types of reprogramming ➢ Revert differentiated cells into a state of increased developmental plasticity (dedifferentiation) ➢ Switch from one cell type to another (Transdifferentiation or direct reprogramming) ➢ Terminally differentiate a cell to a state of totipotency Some examples • Nuclear reprogramming by nuclear transfer to eggs (meiotic metaphase II oocytes) ➢ Somatic nuclear transfer; the oocyte reprograms the nucleus ➢ Low efficacy; 1-2%; reflects the stability of the adult cell • Reprogramming by cell fusion ➢ Fusing the plasma membrane of two cells; The nuclei remains separate • Reprogramming by extract treatment ➢ Protein extracts from pluripotent cells • Reprogramming by transcription factor overexpression ➢ Oct4, Sox2, Klf4 and Myc are virally transfected into somatic cells ➢ iPSCs Transdifferentiation - A number of experiments have shown that some stem cells can differentiate into cell types seen in organs or tissues other than those expected from their lineage. ✓ Brain stem cells differentiate into blood cells ✓ Blood forming cells differentiate into cardiac cells - This issue has been observed in vertebrates but is still under debate in humans. - It is a matter of reprogramming the cells into other cell types using a process of well controlled genetic modification - Programing adult somatic cells into becoming more like embryonic cells (induced pluripotent stem cells) Human Embryonic Stem Cells (hESCs) ❖ Transfer cells from pre implantation-stage embryo to culture dishes with the appropriate growth medium. ❖ The culture dish contains mouse feeder cells, so the stem cells do not die. Now we have figured out a way to grow the cells without the feeder cells. ❖ The cells are grown and re-cultured many times. ❖ Embryonic cells that have grown and multiplied in culture without differentiating for 6 months are pluripotent. At this point they are an embryonic stem cell line. ❖ There are some tests to characterize these cell lines. ▪ Grow and culture them for many months ▪ Presence of specific transcription factors such as Nanog and Oct4 (maintain cells in undifferentiated state) ▪ Surface markers ▪ Chromosome structure ▪ Testing their pluripotency, injecting them into mice to see if they grow teratomas. How do we stimulate ESCs to differentiate? ➢ If we allow the ESCs to clump together they will form embryoid bodies and begins to differentiate spontaneously. ➢ Although the spontaneous differentiation is good it is uncontrolled. ➢ After years of experimentation scientists are now able to control the differentiation to a degree using special growth “recipes” for directed differentiation Adult stem cells • Adults stem cells are thought to a group of undifferentiated cells found among differentiated cells in a issue or organ. • An adult stem cell can renew itself and can differentiate to yield all or some of the major cell types of a tissue or organ. • The primary role is to maintain and repair the organ or tissue they are in. • They live in a special area called the stem cell niche. • Some examples include: - Hematopoietic stem cells - Mesenchymal stem cells - Neural stem cells - Epithelial stem cells - Skin stem cells Comparing between adult stem cells and embryonic stem cell Comparison between stem cells and differentiated cells Potential uses of human stem cells ❖ Help us understand how undifferentiated stem cells become differentiated cells to form organs and tissues. (Why is this important?) ✓ Cancer ✓ Birth defects ✓ Turning genes on or off are at the heart of this process. ❖ Stem cells can be used to test new drugs. ❖ Generation of cells and tissues to use for cell-based therapies. ❖ Creating replacement organs or tissues Some hurdles to overcome in stem cells - Proliferate extensively and generate sufficient quantities of cells for making tissue. - Differentiate into the desired cell type(s). - Survive in the recipient after transplant. - Integrate into the surrounding tissue after transplant. - Function appropriately for the duration of the recipient's life. - Avoid harming the recipient in any way