Lecture 2: Embryonic stem cells
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Questions and Answers

What key characteristic indicates the pluripotency of stem cells when observing teratomas formed in immunocompromised mice?

  • Rapid proliferation of undifferentiated cells
  • Formation of benign tumors with differentiated cells from all three germ layers (correct)
  • High nucleus-to-cytoplasm ratio
  • Presence of a single type of differentiated cell

Which method is specifically unable to be applied to human cells due to ethical concerns?

  • Tetraploid embryo complementation (correct)
  • Morphological observation of stem cell colonies
  • Gene expression analysis in vitro
  • Teratoma formation in mice

What morphological feature distinguishes pluripotent stem cells in culture?

  • Large cell size with irregular edges
  • Low nucleus-to-cytoplasm ratio
  • Tightly packed colonies with smooth edges (correct)
  • Rapidly forming large aggregates

Which of the following best describes the chromosomal composition of a tetraploid embryo used in the assessment of pluripotency?

<p>Twice the usual number of chromosomes (B)</p> Signup and view all the answers

What role do genes expressed in pluripotent stem cells primarily have?

<p>Coding for proteins crucial for embryonic development (A)</p> Signup and view all the answers

What is the primary characteristic that defines stem cells?

<p>Ability to divide and produce identical daughter cells (A)</p> Signup and view all the answers

What type of cell is no longer able to proliferate and has defined specialized functions?

<p>Terminally differentiated cell (A)</p> Signup and view all the answers

What effect do external signals from the niche have on stem cells?

<p>They influence whether a stem cell self-renews or differentiates (B)</p> Signup and view all the answers

What is meant by 'asymmetric division' in stem cells?

<p>Daughter cells exhibit different fates due to uneven internal determinants (D)</p> Signup and view all the answers

Which statement correctly describes totipotent cells?

<p>They have the highest potential for differentiation (A)</p> Signup and view all the answers

What is the significance of divisional asymmetry in stem cell function?

<p>It allows for a balance between self-renewal and differentiation (B)</p> Signup and view all the answers

What outcome occurs when a stem cell undergoes terminal differentiation?

<p>It loses its capacity for further divisions (C)</p> Signup and view all the answers

What is the primary purpose of transferring a somatic cell nucleus into an enucleated oocyte?

<p>To produce pluripotent stem cells for research (A)</p> Signup and view all the answers

Which of the following is NOT a positive aspect of using SCNT-derived stem cells?

<p>Completely eliminates the need for human oocytes (D)</p> Signup and view all the answers

What is the status of the blastocyst from which embryonic stem cells can be harvested?

<p>It should be between 5-7 days old for legal and ethical compliance (D)</p> Signup and view all the answers

How does SCNT potentially benefit the study of mitochondrial disorders?

<p>By allowing the creation of '3-parent embryos' (C)</p> Signup and view all the answers

Which process involves transfer of a nucleus from a somatic cell to an enucleated egg cell?

<p>Somatic Cell Nuclear Transfer (SCNT) (A)</p> Signup and view all the answers

What is a significant challenge associated with obtaining human oocytes for SCNT?

<p>Human oocytes are difficult to obtain due to ethical concerns (A)</p> Signup and view all the answers

What is the developmental stage called when embryonic stem cells are harvested?

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

What advantage do SCNT-derived stem cells offer in terms of immunology?

<p>They reduce the risk of immune rejection as they are genetically identical to the donor (A)</p> Signup and view all the answers

What does the process of embryonic stem cell harvesting contribute to?

<p>Potential treatments for various diseases through disease modeling (A)</p> Signup and view all the answers

Which negative aspect is associated with the SCNT process?

<p>It requires human oocytes which are rare and challenging to procure (D)</p> Signup and view all the answers

What is the primary characteristic of human embryonic stem cells (hES) in terms of their karyotype?

<p>They maintain a normal diploid karyotype. (B)</p> Signup and view all the answers

What is the purpose of using MEF (mouse embryonic fibroblast) feeder layers in hES cell culture?

<p>To provide necessary support while preventing overgrowth. (C)</p> Signup and view all the answers

What is the significance of using a teratoma assay for assessing stem cell capabilities?

<p>It assesses the ability to form tumors representing embryonic development. (C)</p> Signup and view all the answers

What is the recommended culture technique for human embryonic stem cells to maintain their undifferentiated state?

<p>Subculture weekly as clumps of cells. (C)</p> Signup and view all the answers

What does the term 'pluripotency' refer to in the context of stem cells?

<p>The capacity to contribute to all tissue types of an organism. (C)</p> Signup and view all the answers

What is a significant outcome of introducing hES cells into immunodeficient mice?

<p>Tumors composed of various embryonic cell types. (D)</p> Signup and view all the answers

During the process of assessing the pluripotency of stem cells through differentiation, what signifies successful differentiation?

<p>Contribution to somatic and extraembryonic tissues. (D)</p> Signup and view all the answers

Why is mechanical dissection used during the differentiation process of hES cells?

<p>To separate differentiated cells from undifferentiated ones. (A)</p> Signup and view all the answers

What is a common requirement for the culture medium used in hES cells?

<p>Inclusion of fetal bovine serum. (B)</p> Signup and view all the answers

What does the term 'chimera' refer to in the context of stem cell injection into a blastocyst?

<p>An organism formed from multiple genetic sources. (B)</p> Signup and view all the answers

What characteristic differentiates pluripotent cells from totipotent cells?

<p>Pluripotent cells can form nearly any cell type but not extra-embryonic tissues. (A)</p> Signup and view all the answers

Which type of stem cells are associated with neural potential?

<p>Multipotent neural stem cells (D)</p> Signup and view all the answers

What best describes unipotent stem cells?

<p>They differentiate into only one specific cell type. (C)</p> Signup and view all the answers

Which of the following is a source of multipotent cells?

<p>Adult brain tissue (B)</p> Signup and view all the answers

From which source are embryonic carcinoma (EC) cells derived?

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

How are embryonic stem cells characterized in comparison to multipotent cells?

<p>They can differentiate into almost any cell type, unlike multipotent cells. (C)</p> Signup and view all the answers

Which type of cell can arise from regions of the brain?

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

Which of the following is NOT a characteristic of embryonic stem cells?

<p>They can only develop into one specific cell type. (D)</p> Signup and view all the answers

Which option best describes aneuploidy in embryonic carcinoma cells?

<p>They possess an abnormal number of chromosomes. (C)</p> Signup and view all the answers

Flashcards

Stem Cell

A cell capable of dividing indefinitely and producing identical daughter cells (self-renewal) and specialized cells (differentiation).

Self-Renewal

The process by which a stem cell divides to create two identical daughter cells.

Differentiation

The process by which a stem cell develops into a specialized cell type with a specific function.

Terminally Differentiated Cell

A cell that has irreversibly lost its ability to proliferate and become a specialized cell type.

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Progenitor Cell

A non-stem cell daughter produced from a stem cell division that is committed to becoming a specific cell type, but still has a limited number of divisions.

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Environmental Asymmetry

External signals and environmental factors that influence a stem cell's fate (self-renewal or differentiation).

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Divisional Asymmetry

Internal differences in the distribution of cellular components and determinants within a stem cell.

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Totipotent Cells

Cells that can differentiate into any cell type in the body, including extra-embryonic tissues like the placenta.

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What is the source of totipotent cells?

Cells derived from fertilized eggs, capable of giving rise to all cell types of an organism.

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Pluripotent Cells

Cells capable of differentiating into almost any cell type within the body, excluding extra-embryonic tissues.

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What's the source of pluripotent cells?

Embryonic stem cells derived from the inner cell mass of a blastocyst, a stage in early embryonic development.

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Multipotent Cells

Cells that can differentiate into a limited range of cell types within a specific tissue or organ.

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Give an example of a multipotent cell.

Neural stem cells are a type of multipotent cell found in the brain that can differentiate into neurons, astrocytes, and oligodendrocytes.

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Unipotent Cells

Cells capable of differentiating into only one specific cell type.

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What is the source of unipotent cells?

Unipotent cells are found in specific regions of the brain and are responsible for generating specific types of neurons.

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What are Embryonic Carcinoma (EC) cells?

Embryonic carcinoma (EC) cells are derived from teratocarcinomas, a type of tumor that often forms in the testes or ovaries.

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Somatic Cell Nuclear Transfer (SCNT)

A method of generating stem cells that are genetically identical to the donor, potentially for therapeutic cloning.

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SCNT-derived stem cells

Pluripotent stem cells created through SCNT, genetically identical to the donor.

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Therapeutic Cloning

The process of using a somatic cell nucleus to create a blastocyst, which can then be used for research or therapy.

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Blastocyst

A 5-7 day old embryo that contains a pluripotent inner cell mass.

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Pluripotent stem cells

Stem cells that can differentiate into any cell type of the body.

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Self-renewal of stem cells

The ability of a stem cell to divide and produce two identical daughter cells.

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Differentiation of stem cells

The process by which a stem cell develops into a specialized cell type with a specific function.

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Disease modeling with stem cells

The use of stem cells to create in vitro models of diseases and explore potential treatments.

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3-parent embryos

Using SCNT to potentially correct mitochondrial disorders by replacing the mitochondria of an egg with a healthy donor.

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Environmental influence on stem cell differentiation

Stem cells can differentiate into specific cell types based on external signals and environmental factors.

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Blastocyst Injection (Mouse ES Only)

A gold standard method for testing pluripotency in mice, involving the injection of stem cells into a developing blastocyst. The resulting chimeric embryo provides evidence of pluripotency if the injected cells contribute to various tissues in the offspring.

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Tetraploid Embryo Complementation (Mouse ES Only)

A technique for assessing pluripotency in mice where stem cells are injected into a tetraploid embryo (which cannot develop alone). If the stem cells contribute to the development of a complete organism, it signifies their pluripotency.

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Teratoma Formation

A method to check for pluripotency in both mouse and human stem cells. Stem cells injected into immunocompromised mice develop into teratomas, which are benign tumors containing cells from all three germ layers.

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Morphology of Pluripotent Stem Cells

A characteristic of pluripotent stem cells, they usually have a larger nucleus compared to the cytoplasm, forming tightly packed colonies with smooth edges.

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Gene Expression in Pluripotent Stem Cells

Pluripotent stem cells express specific genes that code for proteins involved in embryonic development. Analyzing these transcripts is a key method to characterize their pluripotency.

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What is a critical characteristic of hES cells?

Maintaining a normal diploid karyotype (correct and stable number of chromosomes like 46 in humans) is essential for proper development.

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How does the 'pluripotent' nature of hES cells affect their differentiation?

They have the potential to differentiate into various cell types found throughout the body and extraembryonic tissues like the placenta.

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Why are hES cells considered 'immunologically matched'?

They are immunologically compatible with the embryo they originated from, meaning they won't be rejected by the embryo's immune system.

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What is a key test for hES cell pluripotency?

When injected into immunodeficient mice, hES cells can form tumors containing various cell types representative of the developing embryo.

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What is 'subculturing' or 'passaging' in hES cell culture?

The process of transferring a small portion of a cell culture to a new dish to allow continued growth. This process is crucial to prevent overcrowding and sustain the culture.

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What is the role of 'MEF feeder layers' in hES cell culture?

A layer of inactivated mouse embryonic fibroblasts (MEF) that provide essential support and growth factors for hES cell culture.

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What is the typical culture medium composition for hES cell culture?

DMEM, 20% fetal calf serum (FCS), 0.1 mM β-mercaptoethanol, 1% non-essential amino acids, 2 mM glutamine, and penicillin/streptomycin.

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Why are hES cells cultured as clumps instead of single cells?

hES cells are cultured as clumps rather than as single cells to maintain their undifferentiated state.

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What is the primary goal of assessing the pluripotency of hES cells?

The assessment of the pluripotency of stem cells involves evaluating their ability to differentiate into various cell types and contribute to all tissues of an organism.

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How does the 'chimera generation' test assess hES cell pluripotency?

Injected hES cells contribute to various tissues in the offspring, creating a 'chimera'—an organism composed of cells from different genetic origins.

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Study Notes

Embryonic Stem Cells

  • Stem cells possess two key properties: self-renewal and differentiation.
  • Self-renewal means a stem cell divides to create identical daughter cells.
  • Differentiation allows a stem cell to develop into various specialized cell types.
  • Stem cells are not terminally differentiated; they have the capacity to proliferate.
  • Stem cells have limitless divisions, meaning they can replicate numerous times.
  • Asymmetric division produces either stem cells (renewal) or cells that undergo terminal differentiation (progenitor, or transit amplifying cells).
  • Environmental asymmetry is when external signals influence a stem cell's fate.
  • Divisional asymmetry results from unevenly distributed components and determinants in stem cells during division.
  • Daughter cells receive different internal determinants upon division.

Hierarchy of Potency

  • Totipotent cells have the highest potency, capable of differentiating into any cell type in the body and extraembryonic tissues (e.g., the placenta).
  • A zygote is an example of a totipotent cell.
  • Pluripotent cells can differentiate into almost any cell within the body, but not extraembryonic tissues (e.g., embryonic stem cells).
  • Embryonic stem cells are derived from the inner cell mass of a blastocyst.
  • Multipotent cells have a more limited differentiation potential, able to produce many cell types within a specific tissue or organ (e.g., neural stem cells).

Different Types of Pluripotent Stem Cells

  • Embryonic carcinoma (EC) cells originate from teratocarcinomas.
  • EC cells are typically aneuploid (abnormal chromosome number).
  • Teratomas are tumours derived from parthenogenetically activated oocytes.
  • They display a variety of tissue types (e.g., hair, muscle, bone).
  • Teratomas in the ovary are usually benign, while those in the testes can be malignant (teratocarcinomas).

Embryonic Germ (EG) Cells

  • EG cells are isolated from cultured mouse primordial germ cells (PGCs).
  • PGCs develop into sperm or eggs.
  • EG cells are pluripotent.
  • EG cells can differentiate into various types of cells within the three germ layers.
  • EG cells can retain the characteristics of germ cells, including the ability to erase imprinted genes.
  • Mouse EG cells are cultured on feeder cell layers, similar to ES cells.
  • Leukemia inhibitory factor (LIF) and fibroblast growth factor 2 (FGF-2) assist EG cells to maintain their undifferentiated state.

Somatic Cell Nuclear Transfer (SCNT)

  • SCNT involves transferring the nucleus of a somatic cell into an enucleated oocyte.
  • The resulting cells are genetically identical to the donor.
  • Cloned human embryos are a source for ES cells.

Isolation of ICM from Human Embryos

  • Zona pellucida is a glycoprotein layer surrounding the blastocyst.
  • Immunosurgery removes the trophectoderm to access the inner cell mass (ICM).
  • Pronase (an enzyme) digests the zona pellucida.
  • ICM cells are cultured on mitotically inactivated mouse embryonic fibroblast (MEF) cells.

Characteristics of Human Embryonic Stem (hES) Cells

  • hES cells can be grown indefinitely.
  • hES cells maintain a normal diploid karyotype (chromosomes).
  • hES cells are capable of differentiating into multiple somatic and extraembryonic tissues, both in vitro and in vivo.
  • hES cells are immunologically matched to the embryo of origin.

Establishment of ES Cells in Culture

  • Subculturing/passaging involves transferring a portion of the culture to a new dish.
  • Establishing hES cells involves incubating them on a layer of mouse embryonic fibroblast (MEF) cells.
  • DMEM media, with 20% FCS, is used.

Key Assessments for Pluripotency

  • Evaluating a cell's ability to differentiate into various cell types and contribute to all tissues.
  • Cloned cell lines and differentiation are key indicators.
  • Chimera formation is a gold standard.
  • Tetraploid embryo complementation identifies cells that rescue a tetraploid embryo where transplanted hES cells form a full organism.
  • Teratoma formation is crucial, indicating cells differentiate into cells of all three germ layers thus confirming pluripotency.

Differences between Human and Mouse ESCs

  • In vivo differentiation of human ES cells, ability of a human ES cell line to form teratomas is the best test of pluripotency.
  • Xenografts in SCID mice allows implantation for testing beneath the testis capsule.

Hurdles for Stem Cell Therapies

  • Maintaining hES cell growth in clinically acceptable conditions (no exposure to non-human serum proteins).

Ethical and Legislative Implications of hPSC Use

  • The use of hPSCs raises ethical and legal issues.
  • Regulations and guidelines dictate cell culture techniques (e.g., GMP guidelines).

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Lecture 2: Embryonic Stem Cells PDF

Description

Test your knowledge on the characteristics of stem cells and their pluripotency. This quiz covers key concepts such as teratomas, asymmetric division, and the effects of external signals on stem cell behavior. Challenge yourself to identify the differences between stem cell types and their roles in development.

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