SIO2004: Stem Cells and Techniques

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Questions and Answers

What is the primary function of hematopoietic stem cells (HSCs)?

  • Give rise to all blood cells (correct)
  • Differentiate into muscle cells
  • Generate nerve cells
  • Produce fat cells

Which method is NOT typically used to identify stem cells?

  • Flow cytometry
  • Cytochemical staining
  • Gene expression analysis
  • Protein crystallization (correct)

Which type of stem cell is known to differentiate into multiple specialized cell types but not all?

  • Pluripotent stem cells
  • Totipotent stem cells
  • Unipotent stem cells
  • Multipotent stem cells (correct)

What is the significance of self-renewal in stem cells?

<p>It helps maintain the stem cell population. (D)</p> Signup and view all the answers

Which cell type is an example of unipotent stem cells?

<p>Osteoblasts (A)</p> Signup and view all the answers

What is the estimated frequency of hematopoietic stem cells in bone marrow?

<p>1 in 10,000 (B)</p> Signup and view all the answers

Which of the following is a characteristic of pluripotent stem cells?

<p>They can differentiate into any cell type of the body. (D)</p> Signup and view all the answers

What happens to committed progenitor cells derived from multipotent stem cells?

<p>They become specialized and limit further divisions. (A)</p> Signup and view all the answers

What primary purpose does a flow cytometer serve in cell analysis?

<p>It quantifies and sorts individual cells based on their properties. (A)</p> Signup and view all the answers

Which of the following components can flow cytometers measure?

<p>Cell size and intracellular proteins. (A)</p> Signup and view all the answers

What characteristic is specifically used to sort hematopoietic stem cells (HSCs) in flow cytometry?

<p>Presence of CD34+ marker (A)</p> Signup and view all the answers

What does fluorescence-activated cell sorting (FACS) allow researchers to do?

<p>Sort cells into multiple containers based on their properties. (D)</p> Signup and view all the answers

What type of light is utilized by flow cytometers for measuring cell properties?

<p>Laser light (D)</p> Signup and view all the answers

What challenge exists regarding the clinical application of adult stem cells?

<p>Most adult stem cell applications are currently untested or in early testing. (B)</p> Signup and view all the answers

What information does flow cytometry provide when analyzing cell populations?

<p>Shows proportions of different subpopulations within a sample. (A)</p> Signup and view all the answers

Which of the following statements regarding fluorescent antibodies or dyes is accurate for flow cytometry?

<p>They are essential for accurately tagging specific cell populations. (D)</p> Signup and view all the answers

What is the primary purpose of modifying T cells in CAR T-cell therapy?

<p>To enable T cells to recognize and destroy cancer cells. (A)</p> Signup and view all the answers

What potential issue is raised regarding the reprogramming of immune cells?

<p>It may cause the immune system to attack healthy cells. (A)</p> Signup and view all the answers

What is the main benefit of the cancer-detecting sensor developed by the research team?

<p>It allows nonimmune cells to kill cancer cells on contact. (B)</p> Signup and view all the answers

What triggers the release of the drug-activating enzyme in the engineered cells?

<p>Interaction with cancer cells presenting specific antigens. (B)</p> Signup and view all the answers

What role does FHL-2 play in relation to β-catenin's function?

<p>It enhances the transport of β-catenin to the nucleus. (D)</p> Signup and view all the answers

Which type of stem cells were used to create the cancer-detecting sensor?

<p>Human mesenchymal stem cells. (B)</p> Signup and view all the answers

What is a possible consequence of modifying endogenous immune cells as noted by Martin Fussenegger?

<p>Unintended side effects on the immune system. (A)</p> Signup and view all the answers

How does Dex influence osteogenic differentiation?

<p>By inducing the expression of MKP-1. (A)</p> Signup and view all the answers

What term describes the process by which the engineered interactions in the sensor lead to action against cancer cells?

<p>Signal transduction. (A)</p> Signup and view all the answers

Which of the following factors enhances osteogenic differentiation alongside ascorbic acid?

<p>Insulin-transferrin-selenium (ITS) (D)</p> Signup and view all the answers

What effect does ascorbic acid have on collagen type I?

<p>It increases the secretion of collagen type I. (A)</p> Signup and view all the answers

What is one of the roles of the drug-activating enzyme released by the engineered cells?

<p>To induce apoptosis in cancer cells. (D)</p> Signup and view all the answers

Which process is facilitated by the phosphorylation of ERK1/2 in the MAPK pathway?

<p>Translocation of P-ERK1/2 to the nucleus. (A)</p> Signup and view all the answers

What is the purpose of using Alcian Blue dye in cytochemical staining?

<p>To stain proteoglycans in cartilage. (C)</p> Signup and view all the answers

Which factor does NOT enhance the differentiation process in chondrogenesis?

<p>High oxygen availability (C)</p> Signup and view all the answers

What happens during the cellular condensation phase in mesenchymal chondrogenesis?

<p>Increased intracellular signaling (B)</p> Signup and view all the answers

Which of the following cells primarily develop in the bone marrow?

<p>Macrophages (C), Osteoclasts (D)</p> Signup and view all the answers

What is the primary function of granulocyte-colony stimulating factor (G-CSF) in hematopoiesis?

<p>It induces hematopoietic stem cells (HSCs) to circulate in the blood. (A)</p> Signup and view all the answers

Which surface marker is commonly used to identify human hematopoietic stem cells (HSCs)?

<p>CD34 (A)</p> Signup and view all the answers

How are dendritic cells primarily derived in adult mammals?

<p>From blood monocytes (C)</p> Signup and view all the answers

Where can hematopoietic stem cells (HSCs) be found in the human body?

<p>In umbilical cord blood and peripheral blood (A)</p> Signup and view all the answers

What role does flow cytometry play in identifying HSCs in the laboratory?

<p>It selects for cells expressing CD34. (A)</p> Signup and view all the answers

Which cytokine is involved in the stimulation of platelet production?

<p>Thrombopoietin (A)</p> Signup and view all the answers

Which of the following cytokines is associated with the regulation of granulocyte development?

<p>Granulocyte colony-stimulating factor (C)</p> Signup and view all the answers

What does a clonogenic assay estimate?

<p>The percentage of stem cells within a sample (B)</p> Signup and view all the answers

Which tissue types can multipotent adult stem cells differentiate into?

<p>Mesodermal tissue types including bone and cartilage (A)</p> Signup and view all the answers

What is the role of dexamethasone in osteogenic differentiation?

<p>It induces expression of RUNX2 (D)</p> Signup and view all the answers

Which compounds are generally included in the cocktail for osteogenic differentiation?

<p>Dexamethasone, ascorbic acid, and β-glycerophosphate (D)</p> Signup and view all the answers

What does cytochemical staining help to characterize?

<p>Cells and cellular components in tissues (C)</p> Signup and view all the answers

How does manipulation of culture conditions affect stem cell differentiation?

<p>It can mimic necessary cell-to-cell contact (D)</p> Signup and view all the answers

What is Alizarin Red S used to stain?

<p>Calcium for bone identification (C)</p> Signup and view all the answers

Which factor is NOT typically involved in inducing differentiation in stem cells?

<p>Antioxidants (C)</p> Signup and view all the answers

Flashcards

Stem Cells

Cells capable of self-renewal and differentiation into specialized cell types.

Self-renewal

Ability of stem cells to divide and create more stem cells while maintaining an undifferentiated state.

Differentiation

Process where stem cells become specialized cell types, such as blood cells or nerve cells.

Hematopoietic Stem Cells (HSCs)

Multipotent stem cells that generate all blood cells through hematopoiesis.

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Multipotent Stem Cells

Stem cells capable of differentiating into multiple, but limited, specialized cell types.

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Unipotent Stem Cells

Stem cells that can only differentiate into one specific type of cell.

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Hematopoiesis

The process of blood cell formation.

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Clonogenic Assay

A method to measure the ability of stem cells to form colonies.

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Hematopoiesis

The process of blood cell formation in mammals.

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Bone marrow

Primary location for blood cell development (most cells), except T lymphocytes and some others.

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T lymphocytes

Develop in the thymus, a specialized organ.

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HSCs

Hematopoietic Stem Cells; immature blood cells that can become different types of blood cells.

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Cytokines

Proteins that regulate the differentiation and development of blood cells.

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G-CSF

Granulocyte Colony-Stimulating Factor. A cytokine that mobilizes HSCs into the bloodstream.

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CD34

A cell surface marker that identifies human Hematopoietic Stem Cells (HSCs).

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Flow Cytometry

A technique used to analyze and quantify cells based on their characteristics.

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Flow Cytometry

A technique that analyzes cells by passing them one at a time through a laser beam, measuring light scatter and fluorescence.

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FACS (Fluorescence-Activated Cell Sorting)

A specialized form of flow cytometry that physically separates cells based on their fluorescence and light-scattering properties.

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Light Scatter

The amount of light deflected by a cell as it passes through a laser beam, used to measure cell size and granularity.

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Fluorescence

A cell's emission of light when illuminated with a specific wavelength of light, often used to identify specific cell types or components.

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Antibody

A protein that binds to a specific target molecule (e.g., a cell surface receptor), allowing its detection or isolation.

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Cell Sorting

The process of separating different types of cells from a mixture based on their unique characteristics.

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CD34+

A marker/characteristic used to identify specific types of cells (e.g stem cells).

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Stem Cells

Cells with the potential to develop into various cell types in the body. They are important for repair.

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Clonogenic Assay

A test to measure stem cell colony formation from a single cell.

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MSCs

Multipotent adult stem cells that form colonies.

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Osteogenic Differentiation

Process to stimulate MSCs to make bone.

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Dexamethasone

A compound used to induce osteogenic differentiation.

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RUNX2

A protein crucial in bone development.

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Alizarin Red S

Dye that stains calcium, helping to identify bone.

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In vitro Differentiation

Differentiation of cells in a lab, controlled.

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Colony-forming Units

Estimate stem cells in a sample using colony formation.

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β-catenin transport

FHL-2 binds β-catenin, moving it to the nucleus to bind TCF/LEF-1, initiating Runx2 transcription.

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Dexamethasone's effect

Dex increases Runx2 co-activator TAZ and MKP-1 (MAPK pathway component) expression, activating Runx2.

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Ascorbic Acid's role

Asc increases collagen type I (Col1) secretion, promoting α2β1 integrin binding and ERK1/2 phosphorylation for Runx2 activation.

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Chondrogenic differentiation

Inductive media similar to osteogenic, but with ITS and TGF-β, used in pellet/micromass cultures to mimic cellular condensation.

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Cytochemical staining

Dyes target specific compounds (e.g., Alcian Blue stains proteoglycans, Alizarin Red S stains calcium) to characterize tissues.

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Runx2 Activation

Runx2 is activated by ERK signaling, phosphorylation by P-ERK1/2.

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Inductive media

Similar to osteogenic media; includes dexamethasone and ascorbic acid, with added ITS and TGFβ3/1.

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Micromass cultures

Cell culture method that uses a small amount of cells to form compact structures mimicking early tissue development.

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CAR T-cell therapy

A cancer treatment using modified patient T-cells to target and destroy cancer cells.

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Non-immune cells for cancer treatment

Cells engineered to detect and kill cancer cells upon contact.

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Cancer-detecting sensor

A component built into cells to identify cancer cells.

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Human mesenchymal stem cells

Cells used to create a cancer-detecting system.

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Falling-domino cascade

A chain reaction triggered by the interaction with cancer cells.

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Drug-activating enzyme

An enzyme released to trigger cancer cell destruction after specific signals.

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Endogenous pathway

An existing pathway within cells that creates reactions.

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Engineered pathway

A programmed process within cells that contributes to a cascade of reactions.

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Study Notes

SIO2004: Animal Cell and Tissue Culture, Lecture 7

  • Course: Biotechnology Program, Universiti Malaya
  • Instructor: Dr. Nuradilla Mohamad Fauzi

Definition of Stem Cells

  • Stem cells are cells able to:
    • Self-renewal: Divide repeatedly while maintaining an undifferentiated state.
    • Differentiation: Develop into mature, specialized cell types.

Identification of Stem Cells

  • Morphology and behavior: Visual characteristics and growth patterns
  • Cell surface markers (e.g., "CD" proteins): Specific proteins on the cell surface
  • Flow cytometry: A technique to measure and sort cells based on their physical and fluorescent properties
  • Ability to self-renew: Tested via clonogenic assays
  • Clonogenic assay: Tests the ability of a single cell to form a colony
  • Differentiation assays: Evaluates the potential of stem cells to differentiate into various cell types:
    • Morphology: Observe changes in cell shape and structure
    • Cell staining (cytochemical): Use specific stains to visualize specific products or structures
    • Gene expression of markers of differentiation: Evaluate gene expression pertinent to specific differentiation pathways.

Types of Stem Cells Commonly Cultured

  • Unipotent stem cells/precursor cells:
    • Fibroblasts
    • Osteoblasts
  • Multipotent stem cells:
    • Mesenchymal stem cells (MSCs)
    • Hematopoietic 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 found in chickens

Hematopoietic Stem Cells (HSCs)

  • Multipotent stem cells giving rise to all blood cells through hematopoiesis
  • In healthy adults, approximately 1011-1012 new blood cells are produced daily
  • HSCs have been researched since the 1950s, demonstrating their ability to rescue irradiated mice
  • HSCs are rare (~1 in 10,000 bone marrow cells, 1 in 100,000 blood cells)

Isolation of HSCs

  • HSCs are found in bone marrow, umbilical cord blood, and peripheral blood
  • Cytokine granulocyte-colony stimulating factor (G-CSF) can be administered to mobilize HSCs from bone marrow into peripheral blood
  • The classical marker for human HSCs is CD34
  • Using fluorescent markers and flow cytometry, cells can be sorted based on cell surface markers

Flow Cytometry

  • Automated instruments quantifying single cell properties
  • Suspensions of single cells pass through a laser beam, enabling measurement of scattered and fluorescent light
  • Cells tagged with fluorescent antibodies can be sorted and counted
  • Data can reveal cell size, granularity, amounts of cell components (e.g., DNA, mRNA, proteins) and specific surface receptors

Fluorescence-Activated Cell Sorting (FACS)

  • Specialized flow cytometry to sort diverse cell populations into different containers based on fluorescent characteristics
  • Facilitates fast, objective and quantitative recording of fluorescent signals and physical separation of specific cell populations

Clinical Applications of HSCs

  • Most classes of adult stem cells are not widely tested in clinical settings
  • HSCs have been utilized clinically since 1959 for a wide variety of applications
  • Used to treat hematopoietic cancers, non-hematopoietic malignancies, and various diseases involving bone marrow failure, including autoimmune diseases

Banking of UCB Samples

  • Expanding fully functional HSCs in tissue cultures may rely on the collection of small amounts of HSCs from umbilical cord banks

In Vitro Differentiation of Stem Cells

  • Osteogenic Differentiation: Stimulates bone formation; utilizes dexamethasone, ascorbic acid, and β-glycerophosphate
  • Chondrogenic Differentiation: Promotes cartilage development, uses dexamethasone, ascorbic acid and insulin-transferrin-selenium (ITS) and TGF-β3 or TGF-β1
  • Adipogenic Differentiation: Induces fat cell production; involves dexamethasone, isobutyl methylxanthine (IBMX), indomethacin, and insulin
  • Cytochemical staining: Techniques used to identify specific elements or components within tissues and cells (e.g. Alizarin Red S for calcium, Alcian Blue for proteoglycans)

MSCs are Multipotent Adult/Somatic Stem Cells

  • Differentiate into mesodermal tissues:
    • Bone (osteocytes)
    • Cartilage (chondrocytes)
    • Adipose (adipocytes)
    • Smooth muscle
    • Skeletal muscle
    • Cardiac muscle
    • Tendon (tenocytes) and ligament

Clonogenic Assay

  • A method to estimate the proportion of stem cells in a sample
  • Measures the ability of a single cell to form a colony

Mesenchymal Stem Cells: History

  • Friedenstein et al. (1970) discovered precursor cells in bone marrow that form fibroblastic colonies and contribute to bone formation
  • Arnold Caplan (1991) coined the term "mesenchymal stem cells"
  • Pittenger et al. (1999) isolated these cells from human bone marrow and demonstrated their multipotent differentiation capabilities

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