NPTEL Biotechnology Tissue Engineering. Stem cells
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1 Stem cells S. Swaminathan Director Centre for Nanotechnology & Advanced Biomaterials School of Chemical & Biotechnology SASTRA University Thanjavur Tamil Nadu Joint Initiative of IITs and IISc Funded by MHRD Page 1 of 9
2 Table of Contents 1. STEM CELLS How do stem cells look? Where do we find the stem cells in the tissues? How many different types of stem cells available? What does stem cell plasticity mean? Can stem cells really self-renew? EMBRYONIC STEM CELLS How do you identify the embryonic stem cells? What are the techniques to generate the embryonic stem cells? What is the success rate for the umbilical cord stem cell transplant? What are the major advantages of the cord blood? Where do we store this biological gold (cord stem cells)? ADULT STEM CELLS Differentiation potential of adult stem cell Advantages of using adult stem cells WHAT IS THE DIFFERENCE BETWEEN ADULT AND EMBRYONIC STEM CELLS?...9 Joint Initiative of IITs and IISc Funded by MHRD Page 2 of 9
3 1. Stem cells What are stem cells? Stem cells are undifferentiated or immature cells, which can self-renew, divide and also differentiate into more than one cell types. These cells can produce large number of progeny, which may be unipotent (produce one cell type), pluripotent (produce many cell types) or totipotent (produce all type of cells). The next question is how do we know that the stem cells exist? Suppose when we irradiate a mouse lethally, it is supposed to die due to complete hematopoietic failure. But the mice can be recovered with a very few selected stem cells. They reconstitute the multi-lineage hematopoiesis. In addition, mesenchymal cells (MSCs) in the bone marrow give rise to cells in multiple organs over a period of time in sublethally irradiated mice. Experiments such as implantation of diffusion chamber with 1µm pore size is filled with bone marrow cells develop cartilage and bone after 3 weeks of implantation. They also observed that the hematopoietic markers declined while the marker for bone turnover (alkaline phosphatase) increased. These observations confirm the presence of multilineage cells, which are also known as stem cells. 1.1 How do stem cells look? Hematopoietic stem cells have been isolated using their special surface markers such as CD34 + and CD They are spherical cells of about 6-8 µm in diameter with high nuclear to cytoplasmic ratio. This will express receptors for hematopoietic growth factors and appear to divide rarely. 1.2 Where do we find the stem cells in the tissues? Generally all tissues have their own characteristic time for replacement. Rapidly dividing tissues such as bone marrow, small intestinal epithelium, muscle, and skin have been found to possess stem cells. It is also present in the organs with slow turnover times such as liver, brain, and pancreas. Most cells in the adult tissue are mature and differentiated cells that will die at defined time. Hence, the new cells are derived from a very small population of stem cells i.e. 1 in 100,000 in bone marrow. Joint Initiative of IITs and IISc Funded by MHRD Page 3 of 9
4 1.3 How many different types of stem cells available? Stem cells may be classified based on their source. They are embryonic, fetal, umblical and adult stem cells. Embryonic stem cells are derived from early embryos prior to commitment along three germ layers (endoderm, ectoderm or mesoderm). They are thought to be pluripotent, can produce the progeny from all three germ layers. Fetal stem cells are taken from the aborted fetal tissue. Umbilical stem cells are derived from umbilical cord, which is the rich source of stem cells. These umbilical stem cells are receiving immense attention for storing in a stem cell bank due to its easy availability and lack of well-developed immune cells. Since there are no well developed immune cells in the umbilical cord blood, there is a least chance to graft versus host disease. These stem cells are multipotent. Adult stem cells are present in the adult, which are thought to be tissue specific like neural, skin, skeletal muscle, endothelial progenitor cells. Even bone marrow contains hematopoietic and mesenchymal stem cells which though to be either unipotent or multipotent. 1.4 What does stem cell plasticity mean? It is the capacity of the stem cells to differentiate along the different lineages when kept in the required microenvironment. For example, hematopoietic stem cells have the potential to differentiate into neurons, hepoatocytes or monocytes. 1.5 Can stem cells really self-renew? There are noncoding-repeating sequence in the end of the chromosomes, which are called telomere. Normally all somatic cells reduce its telomere length to around bp/replication. However, when the telomere length reaches to 5-7 kbp, chromosome become unstable, which stops further replication. This phenomenon is called Hay-flick limit. Joint Initiative of IITs and IISc Funded by MHRD Page 4 of 9
5 In case of cancer cells and germ cells, there is a special enzyme called telomerase, which can rebuilt the telomere thereby increase the life span of the cell. Telomerase is a ribonucleoprotein DNA polymerase, which is used to elongate the telomere in eukaryotes. In case of somatic hematopoietic stem cells, telomerase activity is found at a low level. Though there is an evidence for the shortening the telomere length in immature hematopoietic cells during aging, the rate of telomere shortening might be slower as compared to somatic cells. 2. Embryonic stem cells Properties This is derived from the inner cell mass of blastocyst at age of the embryo Pluripotent Long term self renewal It exhibits stable, normal karyotype Capable of integrating fetal tissues during development; clonogenic Express transcription factor oct-4 Lacks G1 checkpoint in cell cycle Factors that affect the differentiation: Microenviroment Cell-cell interaction Growth factors like proteins and nutrients Extracellular matrix Activation and deactivation of genes 2.1 How do you identify the embryonic stem cells? Surface markers that are present only in the undifferentiated cells like oct-4 determine the presence of the stem of cells. Joint Initiative of IITs and IISc Funded by MHRD Page 5 of 9
6 2.2 What are the techniques to generate the embryonic stem cells? Mainly two techniques called in vitro fertilization and somatic cell nuclear transfer are used to prepare embryonic stem cells. However, each technique has its own limitations. In in vitro fertilization, limited number of cell lines are available for federally funded research and risk of developing teratomas i.e tumours from the implantation of undifferentiated stem cells are the two main limitations of this technique. Whereas somatic cell nuclear transfer has not yet achieved with human cells and here also there is potent chance for creating the teratomas. In vitro fertilization: This technique involves the removal of cells from the microscopic inner cell mass of blastocyst and then cultures them to become a cell line. Somatic cell nuclear transfer: This involves removal of nucleus from an egg cell and also from the adult somatic cell. Transfer the nucleus from the somatic cell to egg cell; provide the stimulation to the new cell to grow. Major risk associated with the use of embryonic stem cells are tumour formation; immune rejection and also risk of infection. 2.3 What is the success rate for the umbilical cord stem cell transplant? When these stem cells used for the person from whom it was removed will give 100% success rate. Depending upon the condition, there are 25% chances for perfect match for a sibling. 2.4 What are the major advantages of the cord blood? This is rich in haematopoietic stem cells and progenitor cells used to treat various nonmalignant and malignant haemoatopoietic diseases. The isolation Joint Initiative of IITs and IISc Funded by MHRD Page 6 of 9
7 of such stem cells is through the non-invasive collection, which does not harm the mother or infant. In addition, it is more immune as compared to other stem cells and these young cells possess long telomeres thereby having long self-renewing capacity. 2.5 Where do we store this biological gold (cord stem cells)? There are different cord banks are available to store the cord stem cells namely alpha cord, California Cryobank, Cord Blood Registry, Cordpartners, Cryo-cell, Life bank, UK Cord Blood Bank are some examples. 3. Adult stem cells It is very rare that stem cells, which are known to be undifferentiated cells to be present in the adult or differentiated tissue. It has long-term self-renewal and give rise to differentiated mature cells with specific morphologies. Their primary function is to maintain the steady state functioning of cell, which is also known to maintain homeostasis. Mainly there are two different types of adult stem cells. They are bone marrow derived adult stem cells (hematopoietic stem cell and mesenchymal stem cells) and tissue derived adult stem cells. 3.1 Differentiation potential of adult stem cell Since some are multipotent, they have the ability to differentiate into many different cell types. Precursor cells Neural Oligodendrocyte Bone marrow Derivative Skeletal muscle; epidermis; intestine & liver epithelium; heart tissue; erythroid and myeloid lineage Neurons Neurons ; hepatocytes; skeletal muscle; cardiomyocytes Joint Initiative of IITs and IISc Funded by MHRD Page 7 of 9
8 Muscle Liver Skin Fat Bone marrow; blood Islet cells, hepatocytes, intestine epithelium Smooth muscle, neurons, glial, adipocytes, erythroid & myeloid lineage Adipocytes, cartilage, bone, muscle, neurons Adult stem cell markers Stem cells Markers Human hematopoietic stem cells CD34, Thy-1 Epidermal stem cells β 1 integrin (α2β1, α3β1) Small intestinal stem cells Cytokeratin-15 Retinal stem cells CHX-10, Nestin Neural crest stem cells p75 Liver stem cells AFP, GGT 3.2 Advantages of using adult stem cells Most promising source for treatments Able to generate virtually all adult tissues Can multiply almost indefinitely, providing numbers sufficient for clinical treatments Proven success in laboratory culture Proven success in animal models of disease Proven success in current clinical treatments Ability to home in on damage Avoid problems with tumor formation Avoid problems with transplant rejection Avoid ethical quandary However, limitations such as presence of very minute quantity in adult tissues; difficulty in identification, isolation, maintenance in laboratory; are major issues. Joint Initiative of IITs and IISc Funded by MHRD Page 8 of 9
9 4. What is the difference between adult and embryonic stem cells? Embryonic stem cells are pluripotent, highly proliferative, non-autologous but it has ethical concerns and also tumorigenic whereas, adult stem cells are multipotent having limited proliferative capacity, but autologous, less controversial and also non tumorigenic. Joint Initiative of IITs and IISc Funded by MHRD Page 9 of 9
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