Deriving and Characterizing Mouse Embryonic Stem Cells Flashcards

Comprehensive practice questions covering the derivation, identification, and characterization of mouse embryonic stem cells based on the lecture transcript.

According to Czechanski et al. (2014) in Nature Protocols, what is the full citation for the primary source of these lecture notes?

Czechanski et al. Nature Protocols. 2014. 9; 3, 559-574.

What are the two defining characteristics of stem cell behavior regarding reproduction and progeny?

Stem cells reproduce themselves (self-renewal) and generate progeny destined to differentiate into functional cell types.

How long do stem cells typically persist according to the classical definition?

Stem cells persist for a long time, potentially for the individual's whole life.

What regulates stem cell behavior according to the immediate micro-environment?

The niche.

What is the primary difference between pluripotent stem cells and tissue-specific stem cells?

Pluripotent stem cells (ESCs or iPSCs) can grow in vitro and produce any cell type, while tissue-specific stem cells generally produce only their own tissue types.

Name three examples of tissues with characteristic architecture maintained by self-renewing cell populations.

The epithelial lining of the gut, the epidermis, and the bone marrow (haematopoietic stem cells).

What is another name for progenitor cells that divide a limited number of times before terminal differentiation?

Transit-amplifying cells.

Define 'Induced Pluripotent Stem Cells' (iPSCs).

Somatic (regular body) cells taken from a patient and reprogrammed backward into a pluripotent state.

What are the components of the haematopoietic stem cell niche in the bone marrow mentioned in the text?

Secreted factors (paracrine signals, chemokines), extracellular matrix components, physical conditions (hypoxia/metabolic states), and neighboring cellular components.

What are the primary proteins that build the Extracellular Matrix (ECM) in the stem cell niche?

Collagen, fibronectin, and laminin.

How do stem cells anchor themselves to the Extracellular Matrix (ECM)?

Via integrins.

In the CHIMPS acronym for niche features, what does the 'C' stand for?

Cell-cell interactions: Heterologous (different cell types) interactions providing complex, bidirectional signaling.

In the CHIMPS acronym, what does the 'H' stand for and why is it important?

Hypoxia (low oxygen): Requires tight metabolic regulation to keep stem cells quiescent and capable of long-term self-renewal.

In the CHIMPS acronym, what does the 'I' stand for regarding the niche?

Immunological cells provide dynamic regulation, often under strict 'immune privilege'.

In the CHIMPS acronym, what does the 'M' stand for and what is its role?

Matrix (ECM proteins): Provides physical scaffolding, instructive signals via integrin binding, and acts as a reservoir for soluble factors.

In the CHIMPS acronym, what does 'P' represent?

Physical parameters: Mechanical forces like tissue shape, stiffness, elasticity, and blood flow.

In the CHIMPS acronym, what does 'S' represent and what are some examples?

Secreted factors: Soluble and membrane-bound molecules like Wnt, Notch, SCF, and chemokines.

What are five historical attributes used to identify and study stem cells?

Possession of specific markers, label retaining (slow dividing), clonogenic in vitro/transplantable in vivo, asymmetric division, and producing more than one type of differentiated cell.

Name three common pluripotency markers mentioned in the notes.

$Oct4$, $Sox2$, and $Nanog$.

Is there a universal stem cell marker?

No, there is no universal stem cell marker; some are essential to function while others are not.

Which marker is essential for stem cell function, and which is provided as an example of a marker that is not always essential?

$Oct4$ is essential; $CD34$ is an example of a marker that is not always essential.

Where is the marker $CD34$ expressed in the body according to the text?

In epidermal stem cells (bulge region of the hair follicle) and hematopoietic (blood) stem cells.

What is the 'label retaining' property of stem cells?

Stem cells divide slowly and infrequently, so they retain a DNA label over a long period while fast-cycling progenitors dilute it through rapid divisions.

What are two specific DNA labels used to identify slow-dividing stem cells?

$BrdU$ and $EdU$.

What does the term 'quiescent' mean in the context of stem cells?

In a state or period of inactivity or dormancy; stem cells that do not divide very much.

Name two systems where truly quiescent stem cells exist.

The hematopoietic system and skeletal muscle (satellite cells).

What is one limitation of using label retention to identify stem cells in culture?

Some types of stem cells (like iPSCs or human epidermal SCs) can be kept in constant proliferation in a dish, which dilutes the label.

What is 'clonogenicity'?

The ability of a single cell to clone itself and grow into a full colony of cloned cells.

What are the specific colonies in the epidermis that contain solely proliferative, clonogenic cells?

Holoclones.

Define 'asymmetric division'.

A process where one daughter cell remains a stem cell and the other becomes terminally differentiated.

According to lineage tracing experiments, is stem cell division strictly asymmetric at the individual cell level in solid tissues?

No, it is often random or stochastic ($50:50$ chance), and properties belong to the population rather than individual cells.

What is an example of a stem cell that produces only one type of differentiated cell?

A subset of spermatogonia, which only produce sperm.

What is the simplified operational definition of adult tissue stem cells provided by Clevers et al. (2019)?

The process by which a given tissue replaces its lost cells through cell division.

From which embryonic stage and day range are mouse embryonic stem cells (mESCs) derived?

Pre-implantation blastocysts at stage $E3.5$ to $E4.5$.

Why are pre-implantation blastocysts used for embryo collection in mESC derivation?

They are free-living and unattached, allowing them to be flushed out of the uteri using a syringe and media.

What tool is used to wash embryos through media droplets and assess their quality?

A mouth pipette.

What are 'MEFs' and what is their role in mESC derivation?

Mouse Embryonic Fibroblasts; they serve as 'feeder cells' providing growth factors and ECM components to support ESCs.

Why must MEFs be inactivated before plating ESCs?

So they do not proliferate and outcompete the stem cells while still providing support.

What is the 'zona pellucida'?

The outer layer that the blastocyst must hatch out of before attaching to the feeder layer.

In mESC derivation, what part of the blastocyst outgrows after attachment?

The Inner Cell Mass (ICM).

Around what day of the derivation protocol is the ICM outgrowth disaggregated?

Around Day 10.

Describe the visual appearance of proper mESC colonies around Day 12.

Bright, small, compact clusters with smooth edges, appearing as 'big white blobs'.

What are the two major properties researchers must prove to characterize a cell as an ESC?

That they are self-replicating and pluripotent.

What are the two categories of assays used to characterize newly derived mESCs?

Descriptive assays and functional assays.

List four pluripotency markers used in descriptive assays for mESCs.

$NANOG$, $OCT4$, $SOX2$, and $REX1$ (or $SSEA-1$).

List four specific cell fate markers used to detect spontaneous differentiation in suboptimal cultures.

$GATA6$, $SOX17$, $GATA4$, and $SOX7$.其余的包括 $CDX2$ 和 $GATA3$.

What methods are used in descriptive assays to detect mRNA and protein?

Immunostaining and PCR.

What does a 'patchy' expression of pluripotency markers in a colony indicate?

Suboptimal culture and spontaneous differentiation.

What enzyme is a reliable, quick, and cheap marker of high-quality, undifferentiated ESCs?

Alkaline phosphatase.

What color is seen in a positive alkaline phosphatase staining?

Pink.

What does a colony-forming assay test for?

It tests if the cells are self-replicating (one cell yielding one colony) and evaluates their morphology.

What morphological features characterize 'good' vs 'poor' colonies in a colony-forming assay?

Good colonies are tightly compacted; poor colonies are sparse, loose, and have cells projecting out from the edges.

Describe the process of Embryoid Body (EB) formation.

mESCs are seeded in small aggregates and cultured in hanging drops, methylcellulose media, or suspension without pluripotency-maintaining factors.

What is the goal of an Embryoid Body (EB) formation assay?

To prove the cells can spontaneously differentiate into various cell types representing all three germ layers.

What are the three germ layers represented in embryoid bodies?

Mesoderm, ectoderm, and endoderm.

What is a teratoma in the context of ESC characterization?

A tumor containing tissues from all three germ layers, formed when ESCs are injected into a host.

Name three types of tissue structures that might be found in a teratoma to prove pluripotency.

Respiratory epithelium, squamous cells with keratin pearls, columnar glands, neuroectodermal rosettes, or osteoid (bone).

Which characterization technique is considered the 'Gold Standard' for proving ESC function?

Chimera formation.

How is a chimera formed in ESC research?

ESCs (e.g., from a black-coated mouse) are injected into the blastocyst of a different strain (e.g., albino) and implanted into a surrogate.

What confirms a successful chimera in the resulting offspring?

A mixed black-and-white coat, proving the cultured ESCs contributed to normal in vivo development.

What is 'stem cell tourism'?

The advertising and pursuit of bogus, unproven, and often profitable stem cell therapies that lack controlled trials.

What usually happens to the cells in stem cell allografts used in 'tourism' therapies?

It is generally agreed that all cells die soon after grafting.

If a slight effect is seen in bogus stem cell therapy trials, what is it usually ascribed to?

Substances released by the dying cells.

What allows ESCs and iPSCs to be used for therapy despite needing massive quantities?

They can be grown in massive batches while culture conditions are carefully maintained to keep them strictly pluripotent.

How can researchers 'direct' pluripotent stem cells to differentiate into specialized cell types?

By mimicking the natural developmental signaling pathways (growth factors) known from in vivo embryonic development.

What is a 'holoclone'?

A type of colony in human epidermal cell culture that contains solely proliferative, clonogenic stem cells.

What can happen to holoclones in culture to make them useful for therapy?

They can form 3D stratified epithelial sheets that can be grafted back into a patient.

Why is asymmetric division considered to provide 'flexibility' at a population level?

It allows the balance between stem cells and differentiated cells to adjust, which is useful following tissue damage.

What is the function of the Inner Cell Mass (ICM)?

It is the part of the blastocyst that gives rise to the embryo and is the source for deriving ESCs.

What is the purpose of the mouth pipette in the derivation protocol?

To wash embryos through series of media droplets to remove debris and assess quality.

What does a 'tightly compacted' colony with smooth edges indicate in ESC culture?

High quality, undifferentiated embryonic stem cells.

What happens to the DNA label in fast-cycling progenitor cells?

It is quickly diluted and erased through numerous rapid divisions by the time they terminally differentiate.

Identify the cell fate marker for endoderm mentioned in the descriptive assays.

$SOX17$ (or $GATA6$/$GATA4$).

Why is the teratoma formation assay described as 'invasive'?

It involves injecting cells into a living host to form a tumor, which wouldn't be done every time unless changing a protocol.

Is CD34 a pluripotency marker?

No, it is a marker for certain tissue-resident stem cells like hematopoietic and epidermal stem cells, but not for pluripotency.