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BB20149 > Stem cells > Flashcards

Stem cells Flashcards

1
Q

What are stem cells?

A

Undifferentiated cells that have an unlimited capacity to proliferate. They are immortal and have the capacity to self renew and give rise to other stem cells. They can differentiate into one or more specialised cell types.

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2
Q

What are the types of stem cells?

A

Adult stem cells, embryonic stem cells and induced pluripotent stem cells (iPS).

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3
Q

Where are stem cells found?

A

In early embryos and in adult tissue.

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4
Q

How can stem cells be isolated from early embryos?

A

From the intracellular matrix of the blastocyst.

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5
Q

Where did the first evidence of stem cells come from?

A

From the haematopoietic system using in vivo grafting studies.

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6
Q

Give details of the study where stem cells were discovered.

A

Grafting of haematopoietic tissue from normal mice to irradiated recipient mice repopulated the recipients bone marrow. These experiments were completed by James Till and Ernest McCulloch. There was the development of the clonogenic assay to show that bone marrow gradting produced splenic nodules containing cells of myeloid and erythroid lineages coming from a single cell.

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7
Q

What is haematopoietic tissue?

A

Tissue that gives rise to blood cells.

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8
Q

What were the nodules formed in Till and McCulloch’s experiments called?

A

Spleen colony forming units/ CFU-S.

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9
Q

What are the features of the spleens from Till and McCulloch’s experiments?

A

Each colony/nodule is derived from a single cell, the colony consists of different types of blood cells and injection of cells from the colony to another mouse forms new colonies.

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10
Q

What are the characteristic features of stem cells?

A

They are self renewing, they make up a small percentage of the total cells of an organ, undifferentiated, slow cycling, a large nucleus to cytoplasmic ratio, clonogenic, can be pluripotent, multipotent or unipotent.

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11
Q

What is the order of potency of stem cells, starting with the highest potency?

A

Toti/pluripotent, multipotent, unipotent.

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12
Q

What does totipotent mean?

A

Zygote - able to form the embryo and extraembryonic membranes.

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13
Q

What does multipotent mean?

A

Progenitor cells that are capable of giving rise to a limited range of differentiated lineages e.g. gut and CNS.

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14
Q

What are unipotent cells?

A

Cells only capable of giving rise to one type of cell e.g. epidermis.

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15
Q

What is the ICM?

A

The inner cell mass in the embryo - it gives rise to the embryo and some membranes.

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16
Q

What are the steps in the isolation of embryonic stem cells?

A

Pregnany mice are ovariectomised and injected with progesterone to delay implantation. Blastocysts are then isolated and placed in drops of media on top of feeder fibroblasts (fibroblasts treated with mitomycin C to prevent division) and the ICMs grow and divide. The ICM colony is trypsinised and plated on fresh feeders and give rise to ES cell colonies.

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17
Q

What is essential for stem cells to maintain their pluripotency?

A

Need the presence of growth factors such as LIF and BMP. These inhibit signalling pathways that lead to cell differentiation.

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18
Q

What are features of the improved method for derivation and maintenance of ES cells?

A

Using leukemia inhibitory factor (LIF) to activate different proteins. This prevents the differentiation of mouse embryonic SC in vitro. It was shown that with inhibition of MEK/ERK and GSK3 was sufficient (with activation of STAT3 by LIF) to promote stem cell derivation.

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19
Q

When are the first ES colonies present in cell derivation from a nanog-GFP reporter expressing mouse?

A

Day 13.

20
Q

Who pioneered the technique of blastocyst injection with ICM cells to generate chimeras?

A

Richard Gardner.

21
Q

What is a chimeras?

A

An organism with two sets of DNA.

22
Q

What potency do embryonic stem cells have?

A

Pluripotent - can give rise to all 3 germ layers.

23
Q

What factors do embryonic stem cells express?

A

Oct4, Nanog, Sox2, Klf4 and surface antigens such as SSEA.

24
Q

What are the physical features of embryonic stem cells?

A

They have a large nucleus and prominent nucleoli (the nucleolus). There is little cytoplasm.

25
Q

What are the characteristics of the ES cell growth cycle?

A

It is a short cell cycle with minimal growth phases.

26
Q

What do embryonic stem cells require in order to remain as stem cells?

A

Feeder fibroblasts or LIF (the growth factor).

27
Q

What is a fibroblast?

A

A cell in connective tissue that produces collagen and other fibres.

28
Q

What are embryoid bodies?

A

Aggregations of pluripotent cells that are induced to differentiate by a combination of factors.

29
Q

What can induce specific lineage differentiation in embryonic stem cells?

A

Dimethylsulfoxide and hexamethylene bis acetamide.

30
Q

What else can induce differentiation in embryonic stem cells?

A

Sonic hedgehog, FGF, Wnt.

31
Q

How can neurons be formed artificially from embryonic stem cells?

A

ES cells grown into embryoid bodies that are then plated onto PA6 feeder cells with retinoic acid will form neurons.

32
Q

What are induced pluripotent stem cells?

A

Embryonic stem cell-like cells that are derived by reprogramming from differentiated somatic cells.

33
Q

How do iPS cells resemble embryonic stem cells?

A

Morphology, proliferation, gene expression and teratoma formation.

34
Q

What is a teratoma?

A

A tumour composed of tissues not normally present at the site.

35
Q

What are early approaches to derived iPS cells?

A

Nuclear transfer and cell fusion.

36
Q

What is a recent and commonly used approach to derive iPS cells?

A

Exogeneous expression of 4-5 factors that are important for self renewal of embryonic stem cells: OCt4, Sox2, Klf4, Myc, Lin28

37
Q

What are the vectors used for iPS generation?

A

Retroviral or lentivirus based.

38
Q

What are problems with the vectors used for iPS generation?

A

Oncogenicity and mutagenesis, deleterious consequences of expression of the reprogramming factors, retroviral insertions may alter expression of nearby genes.

39
Q

How can iPS cells be generated from fully differentiated cells?

A

Expressing pluripotency factors.

40
Q

When were mouse iPS cells derived?

A

2006.

41
Q

When were human iPS cells derived?

A

2007.

42
Q

What can human iPS cells be used for?

A

To generate neurons and motor neurons.

43
Q

How can human iPS cells be used to generate neurons and motor neurons?

A

They can be treated with dual Smad inhibitor to generate neuroectoderm which is then converted to neural progenitors by treatment with RA. These are then converted to motor neuron progenitors by co-culture of PA6 feeder cells and RA treatment. Eventually to post-mitotic motor neurons by the treatment with RA and Shh agonist. THey are functionally active as seen by calcium flux.

44
Q

What is the main advantage of human iPS cells?

A

There is no rejection by the immune system, which might be a problem with other stem cells.

45
Q

How can human iPS cells be used to treat disease?

A

In diseases caused by mutations, gene targetting by homologous recombination and then generate replacement tissue.

46
Q

How can iPS cells be used to study disease?

A

Cells from patients can be used to study the disease in vitro and to screen for drugs to treat the disease.

BB20149 (16 decks)

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