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BMB 402 Final > BMB 460 Final > Flashcards

BMB 460 Final Flashcards

1
Q

What are stem cells?

A

Cell types that can divide, renew, and can be eventually differentiated into different cell lineages

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2
Q

What are the properties of stem cells?

A

They can make copies of themselves and they can become two or more other cell types

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3
Q

What differentiates one stem cell type from another?

A

How many cell types they can become

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4
Q

What are totipotent cells?

A

Cells that can form all the cell types in a body, plus the extraembryonic, or placental, cells. Embryonic cells within the first couple of cell divisions after fertilization are the only cells that are totipotent

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5
Q

What are pluripotent cells?

A

Cells that give rise to all of the cell types that make up the body. Embryonic stem cells are considered pluripotent

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6
Q

What are multipotent cells?

A

Cells that can develop into more than one cell type, but are more limited than pluripotent cells. Adult stem cells and cord blood stem cells are considered multipotent

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7
Q

Up to what stage in embryogenesis are cells considered totipotent (can produce placenta and all cells of the organism)?

A

Up to the 8-cell stage

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8
Q

Cells beyond the 8-cell stage form what kind of cells?

A

These cells are pluripotent and can produce all cells of the adult organism but not the placenta. Cells of the inner cell mass are pluripotent

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9
Q

What are blastocysts and are they totipotent?

A

They are a type of blastula and are pluripotent

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10
Q

What does the inner cell mass and the trophoblast become?

A

The inner cell mass becomes the fetus while the trophoblast becomes the placenta

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11
Q

Where are embryonic stem cells isolated from?

A

From cells in a blastocyst (a very early stage embryo)

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12
Q

What happens to embryonic stem cells once they are isolated from the blastocyst?

A

They form colonies in culture (closely packed groups of cells)

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13
Q

Are embryonic stem cells pluripotent? What can they differentiate into?

A

ESCs are pluripotent and they can differentiate into cells from all three germ layers (mesoderm, ectoderm, and endoderm) which later make up the adult body

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14
Q

When do embryonic stem cells lose their pluripotency?

A

At gastrulation (cell movements lead to the formation of 3 layers of cells called germ layers)

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15
Q

What is one source of embryonic stem cells?

A

In vitro fertilization (IVF): all donated eggs are fertilized but not all eggs are implanted, illegal to use since donors were not asked for permission

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16
Q

What are the benefits of human embryonic stem cells?

A

They are pluripotent which provides maximum possibilities for developing therapeutic uses and are a valuable system to study differentiation and development

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17
Q

What are the drawbacks of human ESCs?

A

Ethical issues, no federal funding, difficult to grow and manipulate

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18
Q

Where are adult stem cells found?

A

In several organs that need a constant supply of cells. They replace “worn out” cells in the body

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19
Q

Can stem cells no longer proliferate?

A

Yes, if an undifferentiated cell produces daughter cells that undergo terminal differentiation into particular cell types, the cells can no longer proliferate

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20
Q

Hematopoietic stem cells are what type of stem cells?

A

Tissue-specific stem cells

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21
Q

What are unipotent stem cells?

A

Cells that give rise a single cell type (myoblasts into muscle)

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22
Q

What is the differentiation potential of human adult stem cells?

A

Mutltipotent

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23
Q

Describe the process of nuclear transfer.

A

Nucleus of already differentiated adult stem is inserted into a (ex: skin cell) donated egg that had its nucleus removed.

Egg is stimulated to form a blastocyst where embryonic stem cells can be derived.

Stem cells created this way are “clones” of the original adult cell because the nuclear DNA matches the adult cell, not the skin cell.

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24
Q

What is the motivating hypothesis in the first paper (Bone marrow cells adopt the phenotype of other cells by spontaneous cell fusion)?

A

Maybe ES cells can “reprogram” other adult stem cells into a pluripotent state. “Reprogramming” might be due to cell-cell interaction or production of a soluble factor

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25
Q

What is the prediction in the first paper (Bone marrow cells adopt the phenotype of other cells by spontaneous cell fusion)?

A

If bone marrow cells are cultured with ES cells, the ES cells may “reprogram” the stem cells to a pluripotent type of cell that will then differentiate into many cell types

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26
Q

What is the experimental strategy in the first paper (Bone marrow cells adopt the phenotype of other cells by spontaneous cell fusion)?

A

1) Isolate bone marrow (BM) cells from GFP+, puromycin resistant female mice

2) Incubate female GFP+, puromycin resistant BM cells with male, puromycin-sensitive ES cells (1 week)

3) Submit to positive and negative selection: Maintain LIF to support growth of any new ES-like cells, add IL3 to maintain growth of hematopoietic cells (days 0-7), and add puromycin to kill male ES cells and remove IL3 to terminate hematopoietic cells (days 7-14, maintain LIF to sustain “induced” ES cells

27
Q

Why are the stem cells grown in the presence of LIF and IL3?

A

LIF (leukemia inhibitory factor) - allows growth of mouse ES cells without feeder cell layer. Feeder cells consist in a layer of cells unable to divide, which provides extracellular secretions to help another cell to proliferate

IL3 - supports growth of hematopoietic cells

28
Q

What is the purpose of puromycin in the culture?

A

Antibiotic that inhibits protein synthesis, allows kills puromycin-sensitive parental cells

29
Q

What is the purpose of leukemia inhibiting factor (LIF) in the culture?

A

LIF is a secreted cytokine that is necessary to maintain ES cells in an undifferentiated state. It is also necessary to maintain mouse embryonic stem cell pluripotency, regulates embryonic stem cell self-renewal, and without it, LIF ES lose their stem cell character

30
Q

What is the purpose of interleukin-3 (IL-3) in the culture?

A

It is a potent growth promoting cytokine. By removing IL-3 at day 7 from the mixed culture of cells, bone marrow cells that had not been altered to the ES-like state would stop proliferating

31
Q

What does the GFP+ colonies in figure 1 of the first paper (Bone marrow cells adopt the phenotype of other cells by spontaneous cell fusion) indicate? What do the flattened colonies show?

A

Shows GFP+ colonies are growing and indicated the cells are derived from bone marrow. The flattened colonies have tight cell-cell junctions which look like ES colonies. These cells also proliferate at a rate similar to ES cells for more than 6 months

32
Q

How do we know that BMESL cells appear to be pluripotent?

A

When LIF is withdrawn from cultured ES cells, they differentiate into a variety of cell types. When LIF is withdrawn from BMESL cells, a sphere of aggregated cells of various morphologies forms (similar to ES cells)

33
Q

What do undifferentiated pluripotent ES cells specifically express?

A

Transcription factors Oct 3/4 and UTF1

34
Q

What is the significance of the expression of Oct3/4 and UTF decreasing? What analysis shows these results?

A

Undifferentiated ES cells express Oct3/4 and UTF. If the expression is decreasing, it means that cells are differentiating (expression of differentiation genes goes up). RT-PCR indicates BMESL cells behave similarly and pluripotency is indicated by expression of genes from all 3 germ lineages

35
Q

What additional evidence is there for the pluripotency of BMESL cells?

A

When ES cells are injected into mice, they form teratocarcinomas. BMESL cells behave similarly - a GFP+ tumor is formed at injection site and tissue staining shows various cell types, including striated muscle

36
Q

Why was genetic analysis necessary for the first paper?

A

The results so far indicated that an ES cell-like pluripotent cell line was formed from bone marrow cells by incubating them with ES cells. This must be verified by genetic analysis

37
Q

What is the genetic analysis workflow of the first paper?

A

The number of DNA copies (DNA ploidy) is examined by fluorescence-activated cell sorting (FACS analysis). How? Stain ES with propidium iodide (small fluorescent molecule that stains the nucleus) which intercalates into the major groove of DNA. Fluorescence is quantified in a flow cytometer with a lase

38
Q

What is FACS and how is it used in the first paper?

A

It is fluorescence-activated cell sorting and is commonly used in cell-cycle analysis. Flow cytometry is a verified method to determine ploidy. ES are kind of unstable and tend to gain or lose chromosomes.

FACs analysis was used to examine chromosome copy number of BMESL. Found that all 13 clones were over-diploid, 11 were tetraploid, and 2 were hexaploid

39
Q

What do the peaks in the FACs analysis of the first paper show?

A

One peak is for G1 and the other is G2 (G1 cells will have one copy of DNA intensity, G2/M phase will have two copies.

G1 peak of BMESL cells have twice as much DNA as the G1 peak of ES cells (same for G2 peaks). This means BMESL cells have more DNA

40
Q

What does PCR analysis of genomic DNA from BMESEL cells further indicate?

A

It indicates cell fusion. PCR detected the length of a specific chromosomal locus and the length of this region varies in the two strains. But the BMESL strains have chromosomes of each type

41
Q

To indicate that BMESL cells were generated by cell fusion of embryonic stem and bone marrow cells, what sequences did PCR amplify?

A

Amplified simple sequence repeats (SSRs/micro satellites) which are tandemly repeated short DNA sequences

42
Q

Which cells are actually fusing in the first paper (Bone marrow cells adopt the phenotype of other cells by spontaneous cell fusion)?

A

Maybe macrophages since they can fuse and IL3 is known to promote their fusion. BM cells that were purified to contain a higher percentage of HSC did not produce BMESL more efficiently and suggests that HSC are not the cells that are fusing

43
Q

What the hypothesis of the second paper (Induction of pluripotent stem cells from adult human fibroblasts by defined factors)?

A

The same four factors that can induce a pluripotent state in adult mouse cells will induce a pluripotent state in adult human cells

44
Q

What is transdifferentiation?

A

Cell regress to a point when they can switch lineages

45
Q

What is reprogramming?

A

Induce differentiated cells into reverting to pluripotency. From here, they can differentiate into almost any cell type

46
Q

What is transduction?

A

The process of stably introducing genetic material into cells (genes into genome)

47
Q

How did the authors of the second paper identify genes that produce “induced pluripotent stem” (IPS) cells? Just a general overview

A

1) Generated a transgenic mouse that had a transgene designed to select ES-like cells: Fbx15 promoter only active in early embryos and ES cells, beta-galactosidase generates blue color with X-gal substrate, neomycin resistant protein (geo) renders cells resistant to neomycin, beta-geo encodes a beta-galactosidase-Neomycin resistant fusion protein

2) Infect transgenic embryonic or adult fibroblasts simultaneously with 24 different retroviruses each engineered to express a transcription factor that is specifically expressed in ES cells or implicated in self-renewal

3) Select neomycin resistant, beta-gal positive cells in culture (yielded ES-like cells)

4) Systematically infect with subsets of viruses until the minimal set is identified

5) Four genes found to induce pluripotency: Oct 3/4, Sox2, c-Myc, and Klf4 (Yamanaka factors)

48
Q

In the second paper, how were the identified transcription factors applied to human cells?

A

1) Human dermal fibroblasts (HDF), which can be harvested from just below the skin, were purchased for this work

2) Oct3/4, Sox2, c-Myc, and Klf4 were transduced into cells via 4 retroviruses

3) Plate cells on mitomycin C-treated feeder layer of cells (mitomycin C damages DNA and prevents DNA replication in the feeder cells)

4) Cells were cultured in primate ES media with basic fibroblast growth factor (bFGF)

5) Cells that looked like hES cells were picked 30 days later

6) Cell population was expanded and tested for ES-like properties

49
Q

What is a retrovirus and how do they deliver genes to cells?

A

Virus that uses RNA as its genomic material. Upon infection with a retrovirus, a cell converts the retroviral RNA into DNA, which in turn is inserted into the DNA of the host cell

50
Q

What is retroviral transduction?

A

Retroviruses have the ability to transform their single-stranded RNA genome into a double-stranded DNA molecule. This allows retrovirus RNA to be stably integrated into the genome of dividing target cells

51
Q

What makes retroviruses a powerful gene delivery tool?

A

They have a number of features that make them unique as a gene delivery vehicle, genetic stability-life cycle includes an integrated state in the DNA of the host chromosome, have high expression, and have a flexible genome that can accommodate changes to its configuration

52
Q

How do you engineer a retrovirus?

A

In the retroviral genome, substitute gag (polyprotein that produces capsid protein), pol (reverse transcriptase), and env (envelope protein) with a sequence of interest and moving gag, pol, and env somewhere else in the genome.

The transcript initiated at the LTR (contains promoter for transcription, sequences for recombination into the genome, and sequences for packing the RNA into a virus particle) will be packaged into a virus particle containing the proteins encoded by gag, pol, and env but not the genetic information for making gag, pol and env

53
Q

Describe the retroviral packaging system.

A

Virus packaging cell lines provides the packaging proteins gag-pol-env. A transfer plasmid (recombinant viral vector) provides RNA encoding genes of interest

54
Q

What is the difference between an ecotropic envelope and an amphotrophic envelope?

A

Ecotropic envelope, Phonenix-ECO (for infection of mouse and rat cells

Amphotropic envelope, Phoenic-AMPHO (for the infection of mammalian cells

They both are capable of infecting human dermal fibroblasts but not producing more virus (efficient means of introducing foreign DNA into a cell)

Transduction efficiency in the study was 20% for amphotropic particles and 80% for mouse cells

55
Q

How and why was retroviral transduction efficiency optimized?

A

Why: 4 different viruses into one cell requires efficient transduction

Proof of concept experiment: Introduce GFP into adult human dermal fibroblasts (HDF) with amphotropic retrovirus produced in PLAT-A packaging cells

Issue: Transduction efficiency with HDFs was low when tested with a GFP-retrovirus

Solution: Introduce into the HDFs a gene for the mouse receptor for retroviruses called Slc7a1 (lead to mouse cells transduced more efficiently than HDFs because of Slc7a1). Resulting HDF-Slc7a1 cells have high transduction efficiency

56
Q

In the protocol for human iPS cell induction, what is the purpose of FBS?

A

FBS (fetal bovine serum) - provides the growth factors that nuture the adult dermal cells

57
Q

In the protocol for human iPS cell induction, what is the purpose of feeder cells?

A

Feeder cells: layer of cells unable to divide, which provides extracellular secretions to help another cell to proliferate while keeping them undifferentiated

58
Q

In the protocol for human iPS cell induction, what is the purpose of ES medium + bFGF?

A

ES medium is embryonic cell culture medium and bFGF is a multifunctional cell growth factor that stimulates the growth of cells derived from mesodermal and neuroectodermal cells

59
Q

What are the ES cell specific markers?

A

SSEA-3 and SSEA-4 (hES cell-specific surface antigens)

60
Q

Is SSEA-1 a ES cell specific marker?

A

NO

61
Q

What are TRA-1-60, TRA-1-81, and TRA-2-49/6E?

A

Tumor-related antigens

62
Q

What is NANOG?

A

A DNA binding homeobox transcription factor involved in embryonic stem (ES) cell proliferation, renewal, and pluripotency

63
Q

In the figure that shows that human iPS express ES cell surface markers, what marker is show and what color is it?

A

Signal is green or red

SSEA-3, SSEA-4, all the TRAs, and NANOG

64
Q

What conclusions can be made with the RT-PCR analysis of human iPS cells?

A

Human iPS cells expressed many un-differentiated ES cell-marker genes (confirmed by RT PCR and Western Blot) with levels at least as high as ES cells

BMB 402 Final (6 decks)

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