SIO2004 Animal Cell and Tissue Culture Lecture 7 PDF

SIO2004 Animal Cell and Tissue Culture Lecture 7 Biotechnology Program Universiti Malaya Instructor: Dr. Nuradilla Mohamad Fauzi Definition of stem cells Stem cells are cells that are capable of: Self-renewal: the ability to go through numerous cycles of c...

SIO2004 Animal Cell and Tissue Culture Lecture 7 Biotechnology Program Universiti Malaya Instructor: Dr. Nuradilla Mohamad Fauzi 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. Identification of stem cells Morphology and behavior Cell surface markers (e.g. “CD” proteins) Flow cytometry Ability to self-renew Clonogenic assay Ability to differentiate Differentiation assays Morphology Cell staining (cytochemical) Gene expression of markers of differentiation Types of stem cells commonly cultured Unipotent stem cells / precursor cells Fibroblasts Osteoblasts Multipotent stem cells Mesenchymal stem cells (MSCs) Hematopoeitic stem cells (HSCs) Neural stem cells (NSCs) Pluripotent stem cells Embryonic stem cells (ES cells/ESCs) Induced pluripotent stem cells (iPS cells/iPSCs) Primordial germ cells (PGCs, especially in chickens) http://www.nature.com/nrcardio/journal/v10/n7/fig_tab/nrcardio.2013.81_F1.html Hematopoietic stem cells (HSCs) Multipotent stem cells that give rise to all blood cells through the process of hematopoiesis (Greek: “to make blood”) In a healthy adult person, approximately 1011–1012 new blood cells are produced daily in order to maintain steady state levels in the peripheral circulation HSC research began in the 1950s with the demonstration that intravenously injected bone marrow cells can rescue irradiated mice from lethality by reestablishing blood cell production. HSC are rare, with estimated frequencies of 1 in 10,000 bone marrow cells and 1 in every 100,000 blood cells. Figure 22-35 A tentative scheme of hemopoiesis The multipotent stem cell normally divides infrequently to generate either more multipotent stem cells, which are self-renewing, or committed progenitor cells, which are limited in the number of times that they can divide before differentiating to form mature blood cells. As they go through their divisions, the progenitors become progressively more specialized in the range of cell types that they can give rise to, as indicated by the branching of the cell-lineage diagram in the region enclosed in the gray box. Many of the details of this part of the lineage diagram are still controversial, however. In adult mammals, all of the cells shown develop mainly in the bone marrow—except for T lymphocytes, which develop in the thymus, and macrophages and osteoclasts, which develop from blood monocytes. Some dendritic cells may also derive from monocytes. Mast cells (not shown) are thought to develop from circulating basophils. By User:Mikael Häggström and A. Rad - File:Hematopoiesis (human) diagram.png, by A. Rad., CC BY-SA 3.0, https://commons.wikimedia.org/w/index.php?curid=11107853 Diagram including some of the important cytokines that determine which type of blood cell will be created. SCF= Stem cell factor Tpo= Thrombopoietin IL= Interleukin GM-CSF= Granulocyte Macrophage-colony stimulating factor Epo= Erythropoietin M-CSF= Macrophage-colony stimulating factor G-CSF= Granulocyte-colony stimulating factor SDF-1= Stromal cell-derived factor-1 FLT-3 ligand= FMS-like tyrosine kinase 3 ligand TNF-a = Tumour necrosis factor-alpha TGFβ = Transforming growth factor beta  Isolation of HSCs HSCs are found in the bone marrow. They are HSC also found in umbilical cord blood and, in small numbers, in peripheral blood. To harvest HSCs from the circulating peripheral blood, blood donors are injected with the cytokine granulocyte-colony stimulating factor (G-CSF), which induces HSCs to leave the bone marrow and circulate in the blood vessels. CD34 The classical marker of human HSCs is CD34. They do not express cell surface markers that are found on mature blood cells. If we tag HSCs with fluorescent antibodies that bind to CD34, flow cytometry can select for these cells!  Flow cytometry Flow cytometers are automated instruments that quantitate properties of single cells, one cell at a time. Flow cytometers take in a suspension of single (unclumped) cells and run them one at a time past a laser beam. As each cell passes through the laser beam, scattered and fluorescent light are quantitated. The machine sorts and counts the cells that have been labelled or tagged using fluorescent antibodies or dyes. They can count cells, measure cell size, cell granularity, amounts of cell components such as total DNA, mRNA, amounts of intracellular proteins, or amounts of specific surface receptors. Resulting data show the proportion different sub-populations of cells with different properties within the sample. Fluorescence-activated cell sorting (FACS) A specialized type of flow cytometry that sorts a heterogeneous mixture of biological cells into two or more containers, one cell at a time, based upon the specific light scattering and fluorescent characteristics of each cell. Provides fast, objective and quantitative recording of fluorescent signals from individual cells as well as physical separation of cells of particular interest. Can be used to select for the sub-population of cells (e.g. stem cells) that you are interested in! In the case of HSCs: cells that are CD34+ ! http://www.unifr.ch/pathology/assets/images/FACS%20Schema.jpg e.g. antibody against CD34 By SariSabban - Sabban, Sari (2011) Development of an in vitro model system for studying the interaction of Equus caballus IgE with its high- affinity FcεRI receptor (PhD thesis), The University of Sheffield, CC BY-SA 3.0, CD34- cells CD34+ cells https://commons.wikimedi a.org/w/index.php?curid=1 8139883 https://www.youtube.com/watch?v=CeC2cFIsA0U Clinical applications of HSC The application of most classes of adult stem cells is either currently untested or is in the earliest phases of clinical testing. The only exception is HSCs, which have been used clinically since 1959 and are used increasingly routinely for transplantations, albeit almost exclusively in a non-pure form. To date, close to 1.5 million hematopoietic cell transplants have been performed in more than 1,500 transplantation centers worldwide Virtually all HSC transplants are carried out with either non-purified, mixed cell populations (mobilized peripheral blood, cord blood, or bone marrow) or cell populations that have been enriched for HSCs (e.g. by selection for CD34+ cells), for treatment of: hematopoietic cancers (leukemias and lymphomas), the use of high-dose chemotherapy for non-hematopoietic malignancies (cancers in other organs) Diseases that involve genetic or acquired bone marrow failure, such as aplastic anemia, thalassemia, sickle cell anemia, and increasingly, autoimmune diseases.  HSC Risk of graft versus host disease GVHD: donor immune cells attacks the host  Banking of UCB samples: if expansion of fully functional HSCs in tissue culture becomes a reality, HSC transplants may be possible by starting with small collections of HSCs from UCB banks Collections of HSCs from volunteer donors or umbilical cords could be theoretically converted into storable, expandable stem cell banks useful on demand for clinical transplantation and/or for protection against radiation accidents. https://www.forbes.com/sites/tylerroush/2024/07/18/hiv-stem-cell-treatment-likely-cures-7th-person-in-history-of-virus-doctors-say/ http://www.nature.com/nrcardio/journal/v10/n7/fig_tab/nrcardio.2013.81_F1.html Mesenchymal stem cells: history Also known as such as “mesenchymal stromal cells” or “multipotent stromal cells” In 1970, Friedenstein et al. discovered a rare population of precursor cells from the bone marrow that were able to form fibroblastic colonies and were capable of bone formation. These progenitor cells isolated from marrow tissue were then dubbed “mesenchymal stem cells” by Arnold Caplan in 1991, referring to their mesenchymal tissue of origin and ability to self-renew and differentiate into either osteoblasts or chondrocytes. In 1999, Pittenger et al. reported the isolation of these cells from human bone marrow tissue and showed that these cells possessed multipotent differentiation capacities.  Clonogenic assay Cells that have the capability to self- renew are able to proliferate from a single cell into a colony – MSCs can! Each colony is a ‘clone’ Can estimate % of stem cells within a sample (colony-forming units, CFU) J Anim Sci Biotechnol. 2015 Jan 11;6(1):1. doi: 10.1186/2049-1891-6-1. eCollection 2015. http://www.nvrb.org/2016/10/31/radiation/ MSCs are multipotent adult/somatic stem cells Have been shown to differentiate into mesodermal tissue types: bone (osteocytes) cartilage (chondrocytes) fat (adipocytes) smooth muscle (smooth myocytes) skeletal muscle (skeletal myocytes) cardiac muscle (cardiomyocytes) tendon (tenocytes) and ligament Front. Immunol., 04 September 2013 | http://dx.doi.org/10.3389/fimmu.2013.00201 *only applicable to human MSCs? Differentiation = manipulating culture conditions to stimulate the process of differentiation Mimic cell-to-cell contact necessary for differentiation (e.g. confluency) Adding compounds (growth factors, hormones, cytokines, etc.) to the media Induce expression of genes important for differentiation into the specific tissue type In vitro differentiation: osteogenic Standard procedure: treatment of a confluent monolayer with a cocktail of dexamethasone, ascorbic acid and β-glycerophosphate. These substances on intracellular signaling cascades that lead to osteogenic differentiation Dexamethasone induces expression of RUNX2 (earliest marker of osteogenesis; a transcription factor that is essential for osteogenic differentiation) Journal of Cell Science 2008 121: 1002-1013; doi: 10.1242/jcs.019315  Cytochemical staining Following fixation, cells and tissues could be stained with dyes that target specific compounds. These stains are used to selectively stain cells and cellular components used to characterize tissues. Alizarin Red S – stains calcium (for bone) Mohamad-Fauzi et al. Journal of Animal Science and Biotechnology 2015, 6:1 / Mohamad-Fauzi personal collection  Dexamethasone (Dex) induces the osteogenic differentiation of stem cells by increasing the transcription of FHL-2. Binding of FHL-2 to β-catenin potentiates the transport of β-catenin to the nucleus, where it binds TCF/LEF-1 (T-cell factor/lymphoid enhancer factor) and leads to the transcription of Runx2. Dex also contributes to osteogenic differentiation by increasing the expression of the Runx2 co-activator TAZ. Additionally, Dex treatment induces the expression of the gene encoding MKP-1 (a component of the mitogen-activated protein kinase (MAPK) signaling pathway), which dephosphorylates and thereby activates the key transcription factor Runx2 via extracellular related kinase (ERK) signaling. The addition of ascorbic acid (Asc) facilitates osteogenic differentiation by increasing secretion of collagen type I (Col1), resulting in increased binding of α2β1 integrins to Col1. This leads to the phosphorylation of ERK1/2 in the MAPK signaling pathway, and a subsequent translocation of P-ERK1/2 to the nucleus where it activates Runx2 by phosphorylation. Abbreviations: ECM, extracellular matrix; +OH, hydroxylation; MEK, Stem Cell Res Ther. 2013; 4(5): 117. MAPK/ERK Kinase; FAK, Focal Adhesion Kinase; P, phosphate. In vitro differentiation: chondrogenic Similar to osteogenic media: inductive media contains dexamethasone and ascorbic acid, but with the addition of insulin-transferrin-selenium (ITS) and TGF-β3 or TGF-β1 Done in pellet cultures, or by differentiating cells in a micromass culture system Culturing in these systems mimics the cellular condensation phase that occurs at the initiation of mesenchymal chondrogenesis Cell-to-cell contact and cell shape appear to promote chondrogenesis Stem Cell Res.10(3): 464-76, 2013  Cytochemical staining Following fixation, cells and tissues could be stained with dyes that target specific compounds. These stains are used to selectively stain cells and cellular components used to characterize tissues. Alcian Blue – stains proteoglycans Alizarin Red S – stains calcium (for cartilage) (for bone) Mohamad-Fauzi et al. Journal of Animal Science and Biotechnology 2015, 6:1 / Mohamad-Fauzi personal collection In vitro differentiation: adipogenic Addition of dexamethasone, isobutyl methylxanthine (IBMX), indomethacin and insulin to the media Cell-to-cell contact is necessary cells that do not eventually reach full confluence fail to differentiate  Cytochemical staining Following fixation, cells and tissues could be stained with dyes that target specific compounds. These stains are used to selectively stain cells and cellular components used to characterize tissues. Alcian Blue – stains proteoglycans Alizarin Red S – stains calcium Oil Red O – stains triglycerides (for cartilage) (for bone) (for fat) Mohamad-Fauzi et al. Journal of Animal Science and Biotechnology 2015, 6:1 / Mohamad-Fauzi personal collection Tissues of origin MSCs can be isolated from: BM-MSCs Bone marrow UC-MSCs Adipose tissue Wharton’s Jelly Umbilical cord blood AD-MSCs Even though MSCs isolated from different tissues appear similar/identical in morphology, they have been shown to demonstrate phenotypical differences, such as in expansion rates, cell surface marker profile, and differentiation capacities. MSCs have been isolated from a multitude of species to date!  MSCs are one of the most studied classes of adult stem cells Ease of culture and expansion in vitro in medium with no or minimal additives Possible isolation from multiple tissue sources isolation from adult tissue allows for autologous transplantation Ability to differentiate into bone and cartilage, hence serving as a potential source of cells for osteochondoral regeneration such as repairing segmental bone defects and restoring cartilage in the context of injury or diseases such as osteoarthritis and osteoporosis in one clinical trial, as patients who received autologous BM-MSC transplantations for articular cartilage showed no signs of tumors or abnormalities after 11 years Wilson SM et al. Journal of Oral and Maxillofacial Surgery [01 Mar 2012, 70(3):e193-203]  Low immunogenicity due to their immunosuppressive abilities heterologous (allogeneic) transplantation of MSCs is feasible treat autoimmune diseases, such as rheumatoid arthritis and systemic lupus modulate inflammation MSC MSC  Critically ill COVID-19 patients treated with non- altered stem cells from umbilical cord connective tissue were >2x as likely to survive as those who did not have the treatment. The trial used stem cells obtained through explants from actual umbilical cord tissue. https://www.eurekalert.org/news-releases/827321  Remember that MSCs have … Low immunogenicity due to their immunosuppressive abilities heterologous (allogeneic) transplantation of MSCs is feasible treat autoimmune diseases, such as rheumatoid arthritis and systemic lupus support allografts by suppressing host immune rejection: infusion of MSCs also treat existing graft-versus-host disease (GVHD) in patients who received heterologous HSC transplants Donor immune cells against host tissues Reduce risk of graft versus host disease MSC MSC HSC  Ability to migrate and promote healing to damaged tissue/sites of injury, direct repair and support tissue regeneration to sites of inflammation, and reduce inflammation and recruit immune cells to tumors Secretes signaling factors to nearby cells (paracrine) growth factors, cytokines, and other bioactive factors produced by MSCs may be contained in exosomes and microvesicles that function in a paracrine manner these exosomes can be administered as cell-free therapy https://www.nature.com/articles/s41536-019-0083-6 https://doi.org/10.1016/j.ymthe.2018.05.009  Ability to migrate and promote healing to damaged tissue/sites of injury, direct repair and support tissue regeneration to sites of inflammation, and reduce inflammation and recruit immune cells to tumors Potential of using modified MSCs as delivery vehicles deliver therapeutics to tumors https://doi.org/10.1016/j.jconrel.2020.10.037  Typical immunotherapies work by harnessing the power of the immune system. In CAR T-cell therapy, for example, patients receive a transfusion of their own T cells that have been modified to recognize a specific protein on the surface of cancer cells and then destroy the cancer. “But if you reprogram immune cells to kill cancer by fiddling around with your endogenous immune system, [there could be] some side effects,” says coauthor and bioengineer Martin Fussenegger of ETH Zurich. In a study published [November 13, 2017] in Nature Chemical Biology, researchers have generated nonimmune cells with the ability to kill cancer cells on contact.... the research team built a cancer-detecting sensor in … human mesenchymal stem cells.... When the receptors come into contact with a cancer cell presenting the antigens they recognize... [this interaction] launches a falling-domino cascade of reactions—some endogenous and some that Fussenegger and colleagues engineered into the cell lines—ultimately resulting in the release of a drug-activating enzyme. The enzyme is then trafficked into nearby cells and, in the presence of a prodrug, converts the medicine into its active form, killing the https://www.the-scientist.com/?articles.view/articleNo/50933/title/Researchers- cells. Build-a-Cancer-Immunotherapy-Without-Immune-Cells/ https://doi.org/10.1016/j.ymthe.2018.05.009  Multipotent stem cells are cool, but their differentiation capacity is limited … What if we can work with stem cells that can potentially divide into any tissue type?