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?

It helps maintain the stem cell population. Signup and view all the answers

Which cell type is an example of unipotent stem cells?

Osteoblasts Signup and view all the answers

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

1 in 10,000 Signup and view all the answers

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

They can differentiate into any cell type of the body. Signup and view all the answers

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

They become specialized and limit further divisions. Signup and view all the answers

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

It quantifies and sorts individual cells based on their properties. Signup and view all the answers

Which of the following components can flow cytometers measure?

Cell size and intracellular proteins. Signup and view all the answers

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

Presence of CD34+ marker Signup and view all the answers

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

Sort cells into multiple containers based on their properties. Signup and view all the answers

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

Laser light Signup and view all the answers

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

Most adult stem cell applications are currently untested or in early testing. Signup and view all the answers

What information does flow cytometry provide when analyzing cell populations?

Shows proportions of different subpopulations within a sample. Signup and view all the answers

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

They are essential for accurately tagging specific cell populations. Signup and view all the answers

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

To enable T cells to recognize and destroy cancer cells. Signup and view all the answers

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

It may cause the immune system to attack healthy cells. Signup and view all the answers

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

It allows nonimmune cells to kill cancer cells on contact. Signup and view all the answers

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

Interaction with cancer cells presenting specific antigens. Signup and view all the answers

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

It enhances the transport of β-catenin to the nucleus. Signup and view all the answers

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

Human mesenchymal stem cells. Signup and view all the answers

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

Unintended side effects on the immune system. Signup and view all the answers

How does Dex influence osteogenic differentiation?

By inducing the expression of MKP-1. 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?

Signal transduction. Signup and view all the answers

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

Insulin-transferrin-selenium (ITS) Signup and view all the answers

What effect does ascorbic acid have on collagen type I?

It increases the secretion of collagen type I. Signup and view all the answers

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

To induce apoptosis in cancer cells. Signup and view all the answers

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

Translocation of P-ERK1/2 to the nucleus. Signup and view all the answers

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

To stain proteoglycans in cartilage. Signup and view all the answers

Which factor does NOT enhance the differentiation process in chondrogenesis?

High oxygen availability Signup and view all the answers

What happens during the cellular condensation phase in mesenchymal chondrogenesis?

Increased intracellular signaling Signup and view all the answers

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

Macrophages Signup and view all the answers

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

It induces hematopoietic stem cells (HSCs) to circulate in the blood. Signup and view all the answers

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

CD34 Signup and view all the answers

How are dendritic cells primarily derived in adult mammals?

From blood monocytes Signup and view all the answers

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

In umbilical cord blood and peripheral blood Signup and view all the answers

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

It selects for cells expressing CD34. Signup and view all the answers

Which cytokine is involved in the stimulation of platelet production?

Thrombopoietin Signup and view all the answers

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

Granulocyte colony-stimulating factor Signup and view all the answers

What does a clonogenic assay estimate?

The percentage of stem cells within a sample Signup and view all the answers

Which tissue types can multipotent adult stem cells differentiate into?

Mesodermal tissue types including bone and cartilage Signup and view all the answers

What is the role of dexamethasone in osteogenic differentiation?

It induces expression of RUNX2 Signup and view all the answers

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

Dexamethasone, ascorbic acid, and β-glycerophosphate Signup and view all the answers

What does cytochemical staining help to characterize?

Cells and cellular components in tissues Signup and view all the answers

How does manipulation of culture conditions affect stem cell differentiation?

It can mimic necessary cell-to-cell contact Signup and view all the answers

What is Alizarin Red S used to stain?

Calcium for bone identification Signup and view all the answers

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

Antioxidants Signup and view all the answers

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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Description

Explore the fundamentals of stem cells, including their definitions, identification methods, and differentiation potential. This quiz delves into techniques such as flow cytometry and clonogenic assays, vital for understanding stem cell biology in the context of biotechnology.