Introduction to Stem Cell Biology (BIO414) Lecture 4: Cord Blood & Stem Cell Banking PDF

Introduction to Stem Cell Biology (BIO414) Lecture 4: Cord Blood & Stem Cell Banking Dr. Shaza Ahmed  Course Plan Differentiation of embryonic stem...

Introduction to Stem Cell Biology (BIO414) Lecture 4: Cord Blood & Stem Cell Banking Dr. Shaza Ahmed  Course Plan Differentiation of embryonic stem Mesenchymal stem cells and Induced Tissue specific stem Basics of Stem Cell cells and Cord Blood & Stem Pluripotent stem cells and cancer Biology Hematopoietic stem cell banking cells and most stem cells cells common methods for generation. Tissue engineering Applications of approaches for stem regenerative cells & challenges of medicine to heal MicroRNA’s and The bioethics of delivering stem cells and repair organs stem cell stem cells for clinical and tissues regulation. research transplantation/diseas through cell es& studies of the 3D replacement culture of cells. therapy. Origins of Stem Cells examples  Cord Blood Umbilical cord blood is rich in potentially life- saving stem cells called hematopoietic progenitor cells (HPCs). In recent years cord blood have been transplanted to more than 10,000 people in the United States alone. Steps:As soon as possible after the delivery and prior to the expulsion of the placenta The needle insertion site should be just above the clamp that remains on the cord. Allow as much blood to flow into the bag as possible. The collection normally takes about 3-5 minutes. Informed consent s a MUST  Advantages of Cord Blood Stem Cells The immune cells are less mature than those from other sources, so their transplantation results in a lower risk of graft-versus-host disease. Cord blood is readily available. Cord blood carries a low potential for infectious disease transmission. It involves minimal risk to the mother or the infant at the time of collection Stem Cells  Stem Cells Banking Banking in the world are divided into transient banking of stem cells in the bone marrow transplant centers for the cases that the stem cells are being harvested prior transplant whether autologus or allogenic , the sample stays in the bank for approximately one month. The second type of banking is the cord blood stem cell banking in which stem cells are stored till needed.  Other types of Banks Public Banks: The public bank, people donates there cord blood in a common pool , in addition to the separation and storage of the stem cells, they perform the Human leukocyte testing (HLA) testing of all the samples thus creating a data base of stem cells with a search engine so that people in need of transplant enroll to find the matched cells. Private Banking: The private banking system is a family banking, where the parent pay money to store their babies stem cells to be preserved for them only The private banks works on three categories of people , The people that have a good knowledge about the benefits of those cells For the people with positive family history The people who wants them for them their family only.  Processing Steps of Cord Blood Manual processing includes the use of Ficol however there is a risk of contamination and Low recovery percentage/ Low viability Automated Processing : Instant thawing of already stored sample in the same rack Instant thawing of already stored sample in the same rack  Storage Steps of Cord Blood (Crypreservation) Ice forms at different rates during the cooling process. During slow cooling, freezing occurs external to the cell before intracellular ice begins to form. As ice forms, water is removed from the extracellular environment and an osmotic imbalance occurs across the cell membrane leading to water migration out of the cell. The increase in solute concentration outside the cell, as well as intracellularly, can be detrimental to cell survival. If too much water remains inside the cell, damage due to ice crystal formation and recrystallization during warming can occur. Cryoprotective agents seem to be most effective when they can penetrate the cell and delay intracellular freezing and minimize the solution effects (Such as DMSO & Glycerol) Sotrage Steps of Cord Blood (Liquid nitrogen) Storage by immersion in liquid nitrogen is not advised. Improperly sealed glass ampoules may have microchannels that lead to liquid nitrogen penetration over time. When these are retrieved from liquid nitrogen to ambient temperature, rapid conversion of the liquid nitrogen to vapor inside the ampoule can result in explosion of the ampoule. Broken ampoules in a liquid nitrogen freezer are a potential source of contamination and contaminants may survive, despite the extremely cold temperatures. When a liquid nitrogen freezer becomes contaminated, the entire unit should be decontaminated after warming to room temperature. The use of liquid nitrogen freezers for long-term specimen cryopreservation becomes most favorable. Ready for Quiz 1 Thank you Any Questions  Introduction to Stem Cell Biology (BIO414) Lecture 5&6: Tissue specific stem cells ,cancer stem cells& role of MircoRNA for Stem Cells regulation Dr. Shaza Ahmed  Course Plan Differentiation of embryonic stem Mesenchymal stem cells and Induced Tissue specific stem Basics of Stem Cell cells and Cord Blood & Stem Pluripotent stem cells and cancer Biology Hematopoietic stem cell banking cells and most stem cells cells common methods for generation. Project: Tissue engineering Applications of approaches for stem regenerative cells & challenges of MicroRNA’s and medicine to heal The bioethics of delivering stem cells stem cell and repair organs stem cells for clinical regulation. and tissues research transplantation/diseas through cell es& studies of the 3D replacement culture of cells. therapy. Definitions Tissue-specific stem cells (also referred to as somatic or adult stem cells) are more specialized than embryonic stem cells. Typically, these stem cells can generate different cell types for the specific tissue or organ in which they live. They are most commonly found in bone marrow. Cancer Stem Cells (CSCs) are a small subpopulation of cells within tumors with capabilities of self-renewal, differentiation, and tumorigenicity when transplanted into an animal host. A number of cell surface markers such as CD44, CD24, and CD133 are often used to identify and enrich CSCs.  What is the importance of studying CSC!! The clinical relevance of CSCs has been strengthened by emerging evidence, demonstrating that CSCs are resistant to conventional chemotherapy and radiation treatment and that CSCs are very likely to be the origin of cancer metastasis. CSCs are believed to be an important target for novel anti-cancer drug discovery. CSCs, which exhibit characteristics of both stem cells and cancer cells. In addition to self-renewal and differentiation capacities, CSCs have the ability to seed tumors when transplanted into an animal host. CSCs can be distinguished from other cells within the tumor by symmetry of their cell division and alterations in their gene expression  What is Cancer Cancer is a multistep process in which normal cells experience genetic changes that progress them through a series of pre-malignant states (initiation) into invasive cancer (progression) that can spread throughout the body (metastasis). The resulting transformed cellular phenotype has several distinct characteristics that enables cells to proliferate excessively in an autonomous manner: 1.Cancer cells are able to proliferate independent of growth signals, unresponsive to inhibitory growth signals, evade programmed cell death (apoptosis) pathways, overcome intrinsic cell replication limits, induce and sustain angiogenesis (form new blood vessels), and form new colonies discontinuous with the primary tumor. 2.The dis-regulation of genes involved in cell proliferation, differentiation and/or apoptosis is associated with cancer initiation and progression.  What is Cancer Cancer is a multistep process in which normal cells experience genetic changes that progress them through a series of pre-malignant states (initiation) into invasive cancer (progression) that can spread throughout the body (metastasis). The resulting transformed cellular phenotype has several distinct characteristics that enables cells to proliferate excessively in an autonomous manner: 3. Oncogenes Proto-oncogenes are genes that normally help cells grow and divide to make new cells, or to help cells stay alive. 4.When a proto-oncogene mutates (changes) or there are too many copies of it, it can become turned on (activated) when it is not supposed to be, at which point it's now called an oncogene. When this happens, the cell can start to grow out of control, which might lead to cancer.  Tumor Suppresor Genes Tumor suppressor genes: Are normal genes that slow down cell division or tell cells to die at the right time (a process known as apoptosis or programmed cell death). They code for negative regulator proteins that when activated can prevent the cell from undergoing uncontrolled division, ex., Rb, p53, and p21. Mutations or deactivation in tumor suppressor genes results in cells that grow out of control. This can lead to cancer.  Tumors are composed of a mixture of cells with different characteristics that fulfill different roles in tumor growth. Cancer Stem Cells (CSCs) are a small proportion of the cells within a tumor. Cancer stem cells (CSCs) are similar to normal stem cells, however the difference is that normal stem cells are usually dormant during adulthood until their regeneration ability is required, whereas CSCs are active. Cancer stem Stemness in Cancer stem cells combines the ability of a cell to perpetuate its lineage, to give rise to differentiated cells, and to cells interact with its environment to maintain a balance between quiescence, proliferation, and regeneration. CSCs are both structurally and functionally distinct from the other cells within a tumor mass and are regarded as playing a central role in tumor progression. They are characterized by their resistance to anticancer therapeutics and are the root of tumor metastasis and recurrence. Current drugs, radio- and chemotherapies kill the bulk of cancer cells but often are not able to eliminate the critical CSCs, which are protected by specific resistance mechanisms. Normal & Cancer Stem Cells  Origin of cancer stem cells Multiple hypotheses concerning the origin of CSCs have emerged: CSCs have emerged from the existing population of non-stem cancer cells (NSCCs) within the tumor mass. NSCCs have only a restricted proliferative capacity but constitute the bulk of the tumor. These non-stem cells, by accumulating various kinds of mutation, might gain stemness potential. The origin of CSCs from somatic / adult stem cells. These stem cells are a compelling target as they survive long enough and accumulate DNA damage which might make them malignant. Somatic stem cells are usually multipotent (or sometimes unipotent), which explains the heterogeneity in most tumors. Although somatic stem cells are generally quiescent, they have remarkable self-renewal potential, which is crucial for tumor expansion. The very small embryonic like stem cells (VSELs), have been recently identified. These cells are a rudimentary /basic germ line-derived VSELs which are deposited in several organs during embryogenesis and persist into adulthood. They have characteristics similar to that of pluripotent stem cells and thus is considered to be another possible origin of the CSCs. Origin of cancer stem cells  Cancer Stem Cells Plasticity Cancer stem cell plasticity is the ability to dynamically switch between CSC and non-CSC states. It is a complex process regulated by both cell intrinsic and extrinsic factors. Plasticity plays an important role in the evolution of therapeutic resistance, tumor relapse and metastasis. EMT: epithelial mesenchymal transition MET: mesenchymal epithelial transition  Cancer Stem Cells Plasticity The role of cancer cell plasticity and cell-cycle quiescence in immune escape: At the site of the primary tumor, differentiated cancer cells and proliferating clusters of CSCs are subjected to immune cells clearance. Instead, CSCs enter in a quiescent state to hide. Hiding quiescent CSCs can enter the bloodstream and, escape / evading the surrounding immune cells, and travel to a new metastatic site. The cells then exit from dormancy and colonize the metastatic site. Metastatic outbreak now occur and the cancer spread to other organs / sites. Like other types of treatments, CSCs are also refractory / resistant to immunotherapies leading to tumor relapse.  Cancer Stem Cells Plasticity  Survival of Cancer Stem Cells SCs cannot survive either outside their environment or in the absence of specific factors and cytokines. The survival of stem cells is ensured at two levels to obtain comparable amount of stem cells and differentiating cells : Cellular asymmetrical division: in which one stem cell gives rise to a stem cell and one differentiating cell. Population asymmetrical renewal: in which one stem cell produces two stem cells or two differentiating cells.  CSC Micoenviornment Solid tumors are regarded as “organs” which are comprised of cancer cells and the tumor microenvironment. The tumor microenvironment is known as “CSC niche”. It regulate CSCs stemness and proliferation and save stem cells from depletion. The CSC niche includes niche cells, such as cancer cells, stromal and endothelial cells extracellular matrix (ECM), signaling molecules, intrinsic factors, blood vessels and other cellular and acellular components such as exosomes. These components together contribute to the CSC renewal and maintain tumor malignancy. The regulation mechanisms in the niche includes intrinsic mechanisms (associated with transcription factors expressed by cells), and extrinsic mechanisms (based on the signaling of the microenvironment and the connection to ECM). CSCs interplay closely with the tumor microenvironment and disrupting this niche microenvironment can lead to phenotypic changes that alter homeostasis of the niche and impairs CSC self-renewal and thereby significantly inhibits the growth of tumors.  CSC Micoenviornment It is known that CSCs not only merely adapt to the existing niche but more aggressively generate such environment. For example; CSCs may promote tumor angiogenesis (the creation of new blood vessels from existing ones), and significant therapeutic advantage can be achieved by treatment with an angiogenesis inhibitor. The niche can contain more than one type of stem cells. The competition between different stem cells within the niche control its function by factors such as E-cadherin. The strength of the stem cell-niche connection can directly affect the fate of stem cells. Understanding of the interaction between CSCs and their niche could be a paradigm shift in the treatment of cancer, thus improving therapeutic outcome. What is the Extracellular Matrix (ECM) in the cancer stem cell niche?? As mentioned, CSCs plasticity, phenotype and function are modulated by the tumor microenvironment. The “CSC niche” must have both anatomic and functional properties that enable CSC reproduction or self-renewal. ECM is secreted molecules, composed of different biochemical components such as protein, glycoprotein, proteoglycan, and polysaccharide. ECM help maintain tissue structure and also performs many other regulatory functions. ECM also takes part in most basic cell behaviors such as cell proliferation, migration, to cell differentiation. ECM is likely to play important roles in tumorangiogenesis and Lymphangiogenesis Tumor angiogenesis; the creation of new blood vessels from existing ones.  Cellular components of the CSC niche Tumor associated fibroblasts (TAFs): Normal fibroblasts are activated into over-proliferative TAFs by the CSCs via the up-regulation of WNT signaling. It is a source of IL-6 (multifunctional cytokine). TAFs induce angiogenesis by secreting various cytokines. TAFs play a role in drug resistance. TAFs induces epithelial to mesenchymal transition (EMT) by secreting TAFs mediated overexpression of BCL-xL (B-cell lymphoma – extra large) protect cancer cells from the cytotoxic effects of tyrosine kinase inhibitors.  Cellular components of the CSC niche Epithelial to mesenchymal transition (EMT): During the development of tissues, some epithelial cells exhibit changes in their characteristics (such as a loss of adhesion contact with other cells and cellular polarity) and transform into a mesenchymal type with improved ability to relocate into different tissues with cancer cells characteristics increasing the risk of cancer metastasis. EMT also have stronger invasiveness and resistance against apoptosis. It is believed that the de-differentiation of cancer cells into CSCs occurs after cancer cells have gone through EMT and migrate to other locations.  Cellular components of the CSC niche Recent research indicates that microRNAs (miRNAs) have an important role in regulating stem cell self-renewal and differentiation by repressing the translation of selected mRNAs in stem cells and differentiating daughter cells. EMT also maintains the CSC population by the activation of NK-kB pathway via secretion of IL-6, IL-8 and over- expression of miR199a. The cytokine-mediated crosstalk between MSCs and cancer cells affects the tumor niche by enhancing the generation of CSCs and their maintenance. MSCs further support the tumor microenvironment by differentiating into tumor associated fibroblasts (TAFs). Thank you Any Questions  Introduction to Stem Cell Biology (BIO414) Lecture 7: Ethical Dilemma’s For Stem Cells Dr. Shaza Ahmed  Course Plan Differentiation of embryonic stem Mesenchymal stem cells and Induced Tissue specific stem Basics of Stem Cell cells and Cord Blood & Stem Pluripotent stem cells and cancer Biology Hematopoietic stem cell banking cells and most stem cells cells common methods for generation. Tissue engineering Applications of approaches for stem regenerative cells & challenges of medicine to heal MicroRNA’s and The bioethics of delivering stem cells and repair organs stem cell stem cells for clinical and tissues regulation. research transplantation/diseas through cell es& studies of the 3D replacement culture of cells. therapy.  Ethical Dilemma’s For SC The use of human embryonic stem cells (hESCs) is particularly contentious, as it involves the destruction of embryos, leading to debates over the moral status of human embryos. Stem cell research offers great promise for understanding basic mechanisms of human development and differentiation, as well as the hope for new treatments for diseases such as diabetes, spinal cord injury, Parkinson’s disease, and myocardial infarction. However, human stem cell (hSC) research also raises sharp ethical and political controversies. The derivation of pluripotent stem cell lines from oocytes and embryos is fraught with disputes about the onset of human personhood. The reprogramming of somatic cells to produce induced pluripotent stem cells avoids the ethical problems specific to embryonic stem cell research.  Ethical issues at different phases of stem cell research In any hSC research, difficult dilemmas arise regarding sensitive downstream research, consent to donate materials for hSC research, early clinical trials of hSC therapies, and oversight of hSC research. These ethical and policy issues need to be discussed along with scientific challenges to ensure that stem cell research is carried out in an ethically appropriate manner.  A. Cord blood stem cells: Hematopoietic stem cells from cord blood can be banked and are widely used Multipotent for allogenic and autologous stem cell Stem Cells transplantation in pediatric hematological diseases as an alternative to bone marrow transplantation.  Multipotent Stem Cells B. Adult stem cells can be isolated through plasmapheresis. They are already used to treat hematological malignancies and to modify the side effects of cancer chemotherapy. Furthermore, autologous stem cells are being used in clinical trials in patients who have suffered myocardial infarction. Their use in several other conditions has not been validated or is experimental, despite some claims to the contrary. Adult stem cells and cord blood stem cells do not raise special ethical concerns and are widely used in research and clinical care. However, these cells cannot be expanded in vitro and have not been definitively shown to be pluripotent.  Embryonic Stem Cell Research Ethical Dilemma’s Human embryonic stem cell (hESC) research is ethically and politically controversial because it involves the destruction of human embryos. In the United States, the question of when human life begins has been highly controversial and closely linked to debates over abortion. It is not disputed that embryos have the potential to become human beings; if implanted into a woman’s uterus at the appropriate hormonal phase, an embryo could implant, develop into a fetus, and become a live-born child.  Embryonic Stem Cell Research Ethical Dilemma’s Some people, however, believe that an embryo is a person with the same moral status as an adult or a live-born child. As a matter of religious faith and moral conviction, they believe that “human life begins at conception” and that an embryo is therefore a person. According to this view, an embryo has interests and rights that must be respected. From this perspective, taking a blastocyst and removing the inner cell mass to derive an embryonic stem cell line is tantamount to murder Many other people have a different view of the moral status of the embryo, for example that the embryo becomes a person in a moral sense at a later stage of development than fertilization. Few people, however, believe that the embryo or blastocyst is just a clump of cells that can be used for research without restriction.  In 2001, President Bush, allowed federal National Institutes of Health (NIH) funding for stem cell research using embryonic stem cell lines already in existence at the time, while prohibiting NIH funding for the derivation or use of additional embryonic stem cell lines. This policy was a response to a growing sense that hESC Existing research held great promise for understanding and treating degenerative diseases, while still opposing further destruction of human embryos. embryonic stem President Bush’s rationale for this policy was that the cell lines embryos from which these lines were produced had already been destroyed. Allowing research to be carried out on the stem cell lines might allow some good to come out of their destruction. However, using only existing embryonic stem cell lines is scientifically problematic. Originally, the NIH announced that over 60 hESC lines would be acceptable for NIH funding. However, the majority of these lines were not suitable for research; for example, they were not truly pluripotent, had become contaminated, or were not available for shipping.  Existing embryonic stem cell lines However, these lines may not be safe for transplantation into humans, and long-standing lines have been shown to accumulate mutations, including several known to predispose to cancer. Currently, federal funds may not be used to derive new embryonic stem cell lines or to work with hESC lines not on the approved NIH list. NIH-funded equipment and laboratory space may not be used for research on nonapproved hESC lines. Both the derivation of new hESC lines and research with hESC lines not approved by NIH may be carried out under nonfederal funding. Thank you Any Questions Introduction to Stem Cell Biology (BIO414) Lecture1: Basics of Stem Cell Biology Dr. Shaza Ahmed  Course Plan Differentiation of embryonic stem Mesenchymal stem cells and Induced Stem cell banking & Tissue specific stem Basics of Stem Cell cells and Pluripotent stem Bone marrow cells and cancer Biology Hematopoietic stem cells and most transplantation stem cells cells common methods for generation. Tissue engineering Applications of approaches for stem regenerative cells & challenges of medicine to heal MicroRNA’s and The bioethics of delivering stem cells and repair organs stem cell stem cells for clinical and tissues regulation. research transplantation/diseas through cell es& studies of the 3D replacement culture of cells. therapy.  Diffrentiation: Is a process of a cell becoming specialized by selective expression of certain genes Specialized Cell: A specialized cell is a type of cell that have a particular structure to serve its specific function Terminologies Unspecialized Cell: A type of cell that is not diffrentiated/Originated from meiosis or mitosis Stem Cell: A type of cell that is not specialized and can diffrentiate into any type of cell. Potency: Ability of diffrentiation Regenerative medice: To use stem cells for replacing damaged body tissues  Stem Cells The study of stem cell proliferation, differentiation, migration, and signal transduction can contribute to the trauma repair and regeneration of body tissues, leading to the discovery of new ways to promote the self-repair and renewal of patients. What factors initiate the regeneration of tissues? What factors inhibit regeneration? Where do stem cells come from? And what are the types of stem cells How do stem cells proliferate, migrate to a specified location, and differentiate into specific tissue cells?  Embryonic Stem Cells: ESC are the most basic cells and highly Types of undifferentiated Adult stem cells: Adult Stem stem cells multipotent and have are limited renewal abilities Cells Hematopoietic stem sells: derived from the blood.  Origin of Stem Cells Stem Cells have self-renewal capability and can proliferate and differentiate into a variety of functionally active cells that can serve in various tissues and organs. Stem cells exist in: Embryonic tissues (ESCs) Adult such as bone marrow (bone marrow stem cells, BMSCs) Fat (adipose-derived stem cells, ADSCs) Dental pulp (dental pulp stem cells, DPSCs) Blood (hematopoietic stem cells, HSCs) Amniotic fluid (amniotic fluid stem cells, AFSCs) Umbilical cord (umbilical cord stem cells, UCSCs)  Different levels of stem cell potency Totipotent stem cells: The highest potency of a stem cell is totipotent and can only be found in the first four to eight cells of a developing fetus (e.g. Zygot formed at egg fertilization) Pluripotent stem cells: Pluripotent found in embryonic stem cells. Embryonic stem cells come from the inner cell mass. Only at this stage of early development can embryonic stem cells be harvested and cultured in vitro. Multipotent stem cells: During early development the inner cell mass forms three germ layers and the cells reach the next level of potency. Multipotent stem cells have more restricted function based on the tissue they are to form and play an important role in driving organogenesis (formation of organs). Oligopotent: Able to differentiate into a few different cells types (e.g. Lymphoid or myeloid stem cells) Unipotent: Able to produce only their own type of cells (e.g. Muscle stem cells)  Characteristics of Stem Cells Second is that under certain First, they are physiologic or experimental unspecialized cells that conditions, they can be induced renew themselves for to become cells with special functions such as the beating long periods through cells of the heart muscle or the cell division. insulin producing cells of the pancreas.  A stem cell cannot work with its neighbors to pump blood through the body (like a heart muscle cell); it cannot carry molecules of oxygen through the bloodstream (like a red blood cell) However, unspecialized stem cells can give rise to specialized cells, including heart muscle cells, blood cells, or nerve cells. Stem cells are capable of dividing and renewing themselves for long periods. Characteristics of Stem Cells Unlike muscle cells, blood cells which do not normally replicate themselves stem cells may replicate many times. When cells replicate themselves many times over it is called proliferation. A starting population of stem cells that proliferates for many months in the laboratory can yield millions of cells. If the resulting cells continue to be unspecialized, like the parent stem cells, the cells are said to be capable of long-term self-renewal.  Scientists are just beginning to understand the signals inside and outside cells that trigger stem cell differentiation. The internal signals are controlled by a cell's genes, which are interspersed across Stem Cells long strands of DNA, and carry coded instructions for all the structures and Research functions of a cell. The external signals for cell differentiation include chemicals secreted by other cells, physical contact with neighboring cells, and certain molecules in the microenvironment.  When the blastocyst is used for stem cell research, scientists remove the inner cell mass and place these cells in a culture dish with a nutrient-rich liquid where they give rise to embryonic stem cells. Embryonic stem cells seem to be more flexible than stem cells found in adults, because they have the potential to produce every cell type in the human body. Stem Cells However, such undifferentiated stem cells could not be used directly for tissue transplants because they can Research cause a type of tumor called a teratoma. To be used for therapies, embryonic stem cells would first need to be differentiated into specialized cell types. Some find embryonic stem cell research to be morally objectionable, because when scientists remove the inner cell mass, the blastocyst no longer has the potential to become a fully developed human being.  Adults Stem Cells Adult stem cells, also called somatic stem cells, are the cells found in specific tissues that function to repair and form cells of only the tissues they are found on. These cells are considered less potent than embryonic stem cells as they cannot differentiate to different cell types. Adult stem cells exist in niches or areas created by other cells which secrete fluids and nutrient for the stem cells to remain alive on. Adult stem cells are found in both children and adults and mostly localized in tissue like the epidermis, bone marrow, and lining of the intestine.  Adult stem cells can be found from various sources including: 1.Bone marrow: A rich source of Mesenchymal stem cells (MSCs) and hematopoietic stem cells (HSCs). MSCs can Sources of differentiate into diverse cell types, while HSCs give rise to all blood cell types. Adult Stem 2.Adipose tissue: Adipose-derived stem cells (ADSCs) found in fat tissue are capable of differentiating into cell Cells types like adipocytes, cartilage cells, and bone cells. 3.Menstrual blood: Another source of MSCs, with potential therapeutic applications. 4.Nervous system: Neural stem cells (NSCs) located in specific brain areas can generate nerve cells, astrocytes, and oligodendrocytes. 5.Nucleus pulposus: These cells, found in spinal discs, have the potential to differentiate into disc tissue cells. 6.Salivary glands: Salivary gland stem cells are being explored for treating salivary gland dysfunction.  Adult Stem Cells Research There are a very small number of stem cells in each tissue. Stem cells are thought to reside in a specific area of each tissue where they may remain quiescent (non-dividing) for many years until they are activated by disease or tissue injury. The adult tissues reported to contain stem cells include brain, bone marrow, peripheral blood, blood vessels, skeletal muscle, skin and liver. Scientists in many laboratories are trying to find ways to grow adult stem cells in cell culture and manipulate them to generate specific cell types so they can be used to treat injury or disease. Some examples of potential treatments include replacing the dopamine-producing cells in the brains of Parkinson's patients, developing insulin-producing cells for type I diabetes and repairing damaged heart muscle following a heart attack with cardiac muscle cells. B. What tests are used for identifying adult stem  Adult and embryonic stem cells differ in the number and type of differentiated cells types they can become. All cell types of the body because they are Embryonic stem cells pluripotent. Adult stem cells are generally limited to differentiating into different cell types of their tissue of origin. Adult Stem This is an important distinction, as large numbers of cells are needed for stem cell replacement therapies. Cells A potential advantage of using stem cells from an adult is that the patient's own cells could be expanded in culture and then Research reintroduced into the patient. The use of the patient's own adult stem cells would mean that the cells would not be rejected by the immune system. This represents a significant advantage as immune rejection is a difficult problem that can only be circumvented with immunosuppressive drugs. Embryonic stem cells from a donor introduced into a patient could cause transplant rejection. However, whether the recipient would reject donor embryonic stem cells has not been determined in human experiments. Thank you Any Questions Introduction to Stem Cell Biology (BIO414) Lecture 2: Differentiation of Embryonic stem cells Dr. Shaza Ahmed  Course Plan Differentiation of embryonic stem Mesenchymal stem cells and Induced Stem cell banking & Tissue specific stem Basics of Stem Cell cells and Pluripotent stem Bone marrow cells and cancer Biology Hematopoietic stem cells and most transplantation stem cells cells common methods for generation. Tissue engineering Applications of approaches for stem regenerative cells & challenges of medicine to heal MicroRNA’s and The bioethics of delivering stem cells and repair organs stem cell stem cells for clinical and tissues regulation. research transplantation/diseas through cell es& studies of the 3D replacement culture of cells. therapy.  Human Embryonic Stem Cells Types of Adult stem cells: Stem 1) Mesenchymal Stem Cells 2) Hematopoietic Stem Cells 3) Neural Stem Cells Cells Induced Pluripotent Stem Cells  Embryonic stem cells Stem Cells Adult stem cells 1) Hematopietic Stem Cells( harvesting 2)Mesenchymal Stem Ceells( 3) Neural Stem Cells Cord blood What stages of early embryonic development are important for generating embryonic stem cells? Embryonic stem cells, as their name suggests, are derived from embryos. Specifically, embryonic stem cells are derived from embryos that develop from eggs that have been fertilized then donated for research purposes with informed consent. They are not derived from eggs fertilized in a woman's body. The embryos from which human embryonic stem cells are derived are typically four or five days old and are a hollow microscopic ball of cells called the blastocyst. The blastocyst includes three structures: the trophoblast, which is the layer of cells that surrounds the blastocyst; the blastocoel, which is the hollow cavity inside the blastocyst; and the inner cell mass, which is a group of approximately 30 cells at one end of the blastocoel.  Human Embryonic Stem Cells  Culturing Cell Lines and Stimulating Stem Cells to Differentiate Human embryonic stem cells are isolated by transferring the inner cell mass into a plastic laboratory culture dish that contains a nutrient broth known as culture medium. The inner surface of the culture dish is typically coated with mouse embryonic skin cells that have been treated so they will not divide. This coating layer of cells is called a feeder layer. The reason for having the mouse cells in the bottom of the culture dish is to give the inner cell mass cells a sticky surface to which they can attach. Also, the feeder cells release nutrients into the culture medium. Recently, scientists have begun to devise ways of growing embryonic stem cells without the mouse feeder cells, because of the risk that viruses or other macromolecules in the mouse cells may be transmitted to the human cells. The cells of the inner cell mass proliferate and begin to crowd the culture dish. When this occurs, they are removed gently and plated into several fresh culture dishes. The process of replating the cells is repeated many times and for many months, and is called subculturing. Each cycle of subculturing the cells is referred to as a passage. The original 30 cells of the inner cell mass yield millions of embryonic stem cells. Embryonic stem cells that have proliferated in cell culture for six or more months without differentiating, are pluripotent, and appear genetically normal are referred to as an embryonic stem cell line.  What laboratory tests are used to identify embryonic stem cells? At various points during the process of generating embryonic stem cell lines, scientists test the cells to see whether they exhibit the fundamental properties that make them embryonic stem cells. This process is called characterization. A) Growing and subculturing the stem cells for many months. This ensures that the cells are capable of long-term self-renewal. 1)Scientists inspect the cultures through a microscope to see that the cells look healthy and remain undifferentiated. 2)Using specific techniques to determine the presence of surface markers that are found only on undifferentiated cells. 3)Another important test is for the presence of a protein called Oct-4(Octamer- binding transcription factor 4 ), which undifferentiated cells typically make. Oct-4 is a transcription factor, meaning that it helps turn genes on and off at the right time, which is an important part of the processes of cell differentiation and embryonic development. 4)Examining the chromosomes under a microscope. This is a method to assess whether the chromosomes are damaged. B)Testing whether the human embryonic stem cells are pluripotent by: 1) Allowing the cells to differentiate spontaneously in cell culture 2)Manipulating the cells so they will differentiate to form specific cell types. 3) Injecting the cells into an immunosuppressed mouse to test for the formation of a benign tumor called a teratoma.  ESCs grow as a cluster of cells, which can be Microscopic image of seen in the middle of the figure. Around this human ESCs cluster are darker cells supporting the growth of the ESCs, which are called support cells.  Embryonic stem (ES) cells, are controlled by 13 sequence-specific Transcription factors(TF) (Nanog, Oct4, STAT3, Smad1, Sox2, Zfx, c-Myc, n-Myc, Klf4, Esrrb, Stem Cells Tcfcp2l1, E2f1, and CTCF) and 2 transcription regulators (p300 and Suz12). Singaling During embryonic development, Oct4 is expressed initially in all blastomeres. pathways At maturity, Oct4 expression becomes confined exclusively to the developing germ cells Oct4 controls the pluripotency of stem cells in a quantitative fashion. Specifically, they determined that high level of Oct4 expression drives ES cells to endoderm and mesoderm lineages.  Effect of Growth factors on Stem Cells Once growth factors are added to pluripotent stem cells under certain conditions, they are able to direct differentiation into three different germ layers 1)endoderm 2)mesoderm 3)ectoderm from which various cell types are derived. Neural Differentiation (Ectoderm): Major neural cell types of the central nervous system, neurons, astrocytes. Hematopoietic Differentiation (Mesoderm): Mesoderm cell lineages, including blood, heart, fat tissue, bone, and muscle cells. Insulin-Producing Pancreatic Cells (Endoderm): Pancreatic B cells and liver cells.  Advantages & Disdavantages of Embryonic Stem Cells Disadvantages: 1)Ethical issue donating embryos for research is ethically problematic because it involves intentional destruction of a blastocyst that will never develop into a human being. Alternatives to this the blastocyst can be adopted by infertile couples, giving it a fair chance of life 2)Embryonic stem cells can replicate themselves in an undifferentiated state for very long periods of time before stimulating them to create specialized cells. However, such undifferentiated stem cells could not be used directly for tissue transplants because they can cause a type of tumor called a teratoma. Advantages: 1)Embryonic stem cells are very flexible , more flexible than stem cells found in adults, because they have the potential to produce every cell type in the human body. 2)They are also generally easier to collect, purify and maintain in the laboratory than adult stem cells.  The creation of Embryonic Stem Cells through a Nuclear Transfer Nuclear Transfer: The process called nuclear transfer offers another potential way to produce embryonic stem cells. In animals, nuclear transfer has been accomplished by inserting the nucleus of an already differentiated adult cell-for example, a skin cell-into a donated egg that has had its nucleus removed. This egg, which now contains the genetic material of the skin cell, is then stimulated to form a blastocyst from which embryonic stem cells can be derived. The stem cells that are created in this way are therefore copies or "clones" of the original adult cell because their nuclear DNA matches that of the adult cell.  The creation of Embryonic Stem Cells through alternative Nuclear Transfer (ANT) In this variation of the nuclear transfer technique, scientists create a blastocyst whose genetic material has been changed so that further development and implantation into the uterus is not possible. It aims to create embryo-like entities that are not truly embryos but that can be a source of pluripotent stem cells. ANT, so far only tested with mouse blastocysts, could allow the creation of embryonic stem cells without destroying a viable human blastocyst. Some who object to embryonic stem cell research support ANT because the resulting blastocyst could never develop into a full human being and therefore would not have the moral status of a human embryo. However, this procedure is objectionable to some because they believe that it involves the creation of an imperfect blastocyst that is designed to be destroyed. Thank you Any Questions Introduction to Stem Cell Biology (BIO414) Lecture 3: Adult Stem Cells Dr. Shaza Ahmed  Course Plan Differentiation of embryonic stem Mesenchymal stem cells and Induced Stem cell banking & Tissue specific stem Basics of Stem Cell cells and Pluripotent stem Bone marrow cells and cancer Biology Hematopoietic stem cells and most transplantation stem cells cells common methods for generation. Tissue engineering Applications of approaches for stem regenerative cells & challenges of medicine to heal MicroRNA’s and The bioethics of delivering stem cells and repair organs stem cell stem cells for clinical and tissues regulation. research transplantation/diseas through cell es& studies of the 3D replacement culture of cells. therapy. Types of Adult Stem Cells Adult stem cells: 1) Mesenchymal Stem Cells 2) Hematopoietic Stem Cells 3) Neural Stem Cells 4) Intestinal Stem Cells 5)Stem Cells of the Epidermis & Hair Follicle  Adult Stem Cells Adult stem cells, also called somatic stem cells, are undifferentiated cells that are found in many different tissues throughout the body of nearly all organisms, including humans. Unlike embryonic stem cells, which can become any cell in the body (called pluripotent), adult stem cells, which have been found in a wide range of tissues including skin, heart, brain, liver, and bone marrow are usually restricted to become any type of cell in the tissue or organ that they reside (called multipotent). These adult stem cells, which exist in the tissue for decades, serve to replace cells that are lost in the tissue as needed, such as the growth of new skin every day in humans.  Adult Stem Cells They are also termed tissue progenitor cells and are usually multipotent. Adult stem cell or progenitor cell populations are present in many adult tissues such as bone marrow/peripheral blood (hematopoietic stem cells),gastrointestinal tract, brain, skin, muscle, nerve cells, liver, eye, pancreas, and dental pulp. They are usually tissue-specific and can only differentiate into cell types associated with the tissue they reside in. They are controlled by their microenvironment or niche. Stem Cells  Differential Characteristics of Adult Stem Cells During normal cell division, the two daughter cells produced are equivalent and the same as the mother cell from which they are derived (symmetric division). After this, the descendant cells can evolve along different routes, either following certain programmes of differentiation or retaining the potential of their initial state. Maintaining a suitable balance between the rates of proliferation and differentiation enables most tissues to establish homeostatic control of their shape and size, avoiding uncontrolled growth, associated with, for example, tumour formation. On the other hand, SC seem to be regulated by a conservative division mechanism (asymmetric), in such way that the division produces one cell identical to the mother cell and the other is in charge of the rest of the differentiation programme. In theory, this mechanism should allow strict regulation of the number of SC existing in any given organ  Hematopoietic Stem Cells (HSC) Are blood-forming stem cells, including a red blood cell, white blood cells, and platelets. Often used after bone marrow transplants to help people with cancer make new blood cells after their own hematopoietic progenitor cells have been killed by chemotherapy treatment. Are the only stem cells approved for use by the FDA in the US and can also be sourced from umbilical cord blood cells. Of all the various cell types, hematopoietic stems cells which are CD34+, are extensively studied and used in therapeutic applications. Exceptions to the tissue-specific characteristic of adult stem cells are the multipotent mesenchymal stem cells (MSCs), which are derived from the bone marrow. They are referred to as multipotent adult progenitor cells and are able to differentiate into a variety of tissues such as neuronal, adipose, muscle, gut, liver, lungs, and spleen, but not bone marrow or gonad cells. Adult stem cells can differentiate into another type of tissue under suitable growth conditions. This property has been termed as “plasticity”.  Types of Adult Stem Cells and Their Markers Hematopoietic Stem Cells Hematopoietic stem cells (HSCs :Because mature blood cells are predominantly short lived, stem cells are required throughout life to replenish multi-lineage progenitors and the precursors committed to individual hematopoietic lineages. Hematopoietic stem cells (HSCs) are rare cells of mesodermal origin residing in the adult mammalian bone marrow (one HSC can be found in approximately 104 of bone marrow nucleated cells) which sit atop a hierarchy of progenitors that become progressively restricted to several or single lineages. These progenitors yield blood precursors devoted to unilineage differentiation and the production of mature blood cells.  Types of Adult Stem Cells and Their Markers: Hematopoietic Stem Cells True HSCs are long-term cells capable of unlimited self-renewal. They remain mostly in the quiescent state in the adult tissue, and give rise to short-term HSCs which have limited self-renewing capacity (6-8 weeks). When the short-term HSC leaves the undifferentiated self- renewing state it first chooses between two fates it can become either a common myeloid progenitor (CMP) or a common lymphoid progenitor (CLP). In terms of potency degree ,CMPs and CLPs are oligopotent cells that can produce multiple cell types but not the complete spectra of the tissue specific cells. The myeloid lineage further gives rise to erythrocytes, monocytes and macrophages, neutrophils, basophils, eosinophiles, megakaryocytes/platelets, and dendritic cells.  Types of Adult Stem Cells and Their Markers: Hematopoietic Stem Cells Generally, murine HSCs are the best experimentally described model of HSCs. Despite many similarities, the murine and human HSCs differ in the presence of different antigenic markers. As these long-term HSCs begin to develop as distinct cell lineages the cell surface markers are no longer identified. For example, murine long-term and short-term HSCs can be distinguished by the presence of CD34: long-term HSCs are CD34− and short-term HSCs are CD34+. Other types of hematopoietic progenitors can be defined by different combinations of these markers. Nevertheless, the cells sorted by the presence of these surface markers are not exclusively the long-term HSCs, but represent a heterogeneous population consisting of other types of hematopoietic progenitors as well. For that reason the invention of other ways of HSCs discernment is the subject of high scientific interest. One of these alternative methods is the SLAM code, which uses the SLAM (signaling lymphocyte activation molecule) family of surface molecules (CD150,CD48,CD244).  Mesenchymal Stem Cells (MSCs) Mesenchymal stem cells (MSCs) are a very diverse adult multipotent cell population. The majority of these reside in bone marrow stroma. The MSC’s from bone marrow possess the natural ability to differentiate into mesodermal tissues such as muscle, tendon, adipocyte lineages. However, it is possible to find minor cell populations in nearly all other organs, where they are able to produce a healing environment in case of damage, injury or inflammation. Another important medical source of MSCs are neonatal tissues such as placenta cord blood, and Wharton’s jelly. MSCs are incorporated in the regenerative processes of adult tissues, for example in the heart after infarction, but the precise mechanism of regulation is still unknown. MSCs may directly differentiate into particular cells from damaged tissues, but may also serve as paracrine regulators of healing processes. The MSCs possess a large variety of diffusible cytokines directly regulating immune cells. Types of Adult Stem Cells and Their Markers: Mesenchymal Stem Cells (MSCs) Multipotent adult fibroblast like stem cells. Have self-renewal capacity and differentiation potential into several mesenchymal lineages including osteoblasts (bone cells), chondrocytes (cartilage cells), myocytes (muscle cells) and adipocytes (fat cells which give rise to marrow adipose tissue). MSCs are characterized by expression of cell surface markers of MSCs (CD105, CD73, CD90) and the absence of hematopoietic markers (CD45, CD34, CD14 or CD11b, CD79α or CD19 and HLA- DR). MSCs have been applied clinically in patients with inflammatory bowel diseases, liver disorders and cardiac diseases with very encouraging results. Culturing of MSC & drawbacks Issues with Mesenchymal Stem Cells (MSCs) Serious adverse events noticed in some of MSC-treated patients could be explained by the fact that MSCs either suppress or promote inflammation in dependence of inflammatory environment to which they are exposed to. The primary concerns for clinical application of MSCs (labeled with question marks) are unwanted differentiation of the transplanted MSCs and their potential to suppress anti-tumor immune response and generate new blood vessels that may promote tumor growth and metastasis.  Neural Stem Cells There are several types of “specialized” cells in the brain such as neurons, oligodendrocytes, and astrocytes. Neurons, with their projections called “dendrites” and “axons,” enable the different regions of our brain to communicate with one another and allow the brain to talk to (and control) the rest of the body, which enables us to move about and sense changes in our environment. Neurons transmit and receive information. Oligodendrocytes wrap around the neurons, providing support that enables the neurons to transmit this information quickly. Astrocytes (star cells) support the nervous system by providing nutrition and regulating what can pass into the brain from the rest of the body. Stem cells can keep dividing as long as they are alive (“self- renewing”), and they have two important features: They can create other stem cells. They can become multiple types of more specialized cells. In the brain, we have neural stem cells. That means that these neural stem cells can give rise to neurons, astrocytes, or oligodendrocytes. There are many stem cells in the brain of an embryo because the neural stem cells give rise to all the cell types of the brain. The majority of brain cells are born in the embryo stage. Neural stem cells persist in the brain even into adulthood, where they are located in specific parts of the brain.  Neural Stem Cells to Treat Injury or Diseases of the Brain? Scientists are actively studying how neural stem cells (either those already existing in the brain or those grown in a laboratory or taken from another brain) can help to treat things such as stroke (when normal blood flow to the brain stops and therefore cells cannot get enough nutrients and oxygen), spinal cord injury, and Parkinson’s disease (a disease in which cells that contribute to control body movements progressively stop working and die). Neural stem cells are affected in some brain diseases, such as Parkinson’s and Alzheimer’s (a disease of the brain that leads to memory loss and mental impairments).. In these diseases, neural stem cells seem to have lower proliferation rates and are less likely to become fully developed and healthy neurons. The mechanisms explaining how and why stem cells help recovery from brain injury are an important and active area of research.  Reference for reading https://www.sciencedirect.com/topics/ biochemistry-genetics-and-molecular- biology/adult-stem-cell Thank you Any Questions