Tissues And Stem Cells 1 Flashcards

Question

Cell turnover: carbon-14/carbon-12 ratios

Answer 1

~0.4 trillion cells (~1%) die each day and are replaced by mitosis. But which cells are being replaced? Atmospheric nuclear weapons testing between 1955 and 1963 resulted in an increase in the atmospheric concentration of a number of radioisotopes, includng carbon-14, followed by exponential decay. (a) Carbon-14 in tree rings from 1950-2000. (b) Carbon-14 in DNA of autopsy samples from a person born in 1967 and who died in 2003. Levels in the brain were similar to atmospheric levels close to the time of birth, while those of the intestine are closer to atmospheric levels at the time of death

Answer 2

Cells of the epihelium of the small intestine turn over rapidly. They are “born” in the intestinal crypts, which contain a population of proliferating stem cells. These can be seen as a green fluorescence in the the figure on the right, where DNA synthesis was labelled with bromodeoxyuridine. Differentiating cells move up the villi before apoptosing and sloughing off from the tip. All the cells of the villi are replaced every 6-7 days.

Answer 3

Undifferentiated cells that retain plasticity Have the potential for unlimited cell proliferation. They are capable of self renewal, producing more stem cells, or differentiation as specialized cells. As stem cells differentiate they often produce a population of transit amplifying cells that are only capable of dividing a few times, but their division can be regulated to meet the demand for new differentiated cells

Answer 4

1. Self renewal + differentiation: daughter cells form one of each cell-type. 2. Self renewal: daughter cells identical to the parent cell. 3. Differentiation: daughter cells form more specialised cell types. Stem cells can maintain themselves either (1) by repeated asymmetrical cell division or (2,3) generating stem cell and transit amplifying daughter cells with equal frequency but at separate divisions

Answer 5

Stem cells require continuous exposure to signals from surrounding cells in the “niche” to maintain their stem cell behaviour. This was first demonstrated in the ovary of Drosophila, where germ line stem cells must be in contact with somatic cap cells. These cells secrete Dpp, a BMP-like signalling protein that maintains the stem cells in mitosis. If the stem cells lose contact with the cap cells then they are exposed to less Dpp and begin to differentiate.

Answer 6

Pluripotent Multipotent Unipotent

Answer 7

Can differentiate into cell types from all three germ layers

Answer 8

Cells differentiate into closely related cell types e.g. from a single germ layer

Answer 9

Cells can only differentiate into a single type

Answer 10

ESCs Tissue SC Neural stem cell (Multipotent/unipotent) iPSCs Check slides

Answer 11

Multipotent

Answer 12

Stem cells of the mouse small intestine were genetically labelled and then analyzed. (A) One mutant stem cell may come to occupy an entire crypt, with its progeny forming streams up to the tip of adjacent villi. (B and C) Histological sections through mouse small intestine in which stem cells have been genetically labelled with ß-Gal. (B) After 1 day just a few stem cells are labelled in the crypts. (C) After 5 days a “ribbon” of labelled descendants can be clearly seen in the epithelium of villi.

Answer 13

Crypts appear after birth and contain all the proliferative cells of the epithelium. All the epithelial cells are produced by mitotic stem cells at the base of the crypts, which are intermingled with Paneth cells. These stem cells give rise to a zone of rapidly dividing, undifferentiated, transit amplifying cells. New cells migrate out of the crypt and along the villi, differentiating along the way. At the tip of the villi they undergo apoptosis and are shed into the intestinal lumen. In this way, the epithelium is replaced every 5-7 days.

Answer 14

Notch signalling directly targets the intestinal stem cells (ISC) to maintain proliferation and promote cell survival, Paneth) but also acts to promote specification of the absorptive lineage and repress specification of secretory lineage. The specification of these different lineages is based on small variations in the expression of the Notch ligand Delta, which activates Notch in adjacent cells. Activated Notch promotes development of absorptive progenitors (AP) and represses expression of Math1, a transcription factor that activates expression of Delta and promotes development of secretory progenitors (SP).

Answer 15

Stem cells in the crypts of the small intestines are initially polyclonal but over time become monoclonal. This was first shown in chimeric mice in which two embryos with different molecular markers were aggregated. The crypts of newborn mice contained both molecular markers but after a few weeks one of the markers is lost (randomly). More recently this has been shown using “confetti mice”, in which different stem cells are labelled with differently coloured fluorescent proteins. Initially, newborn mice contain crypts with multiple colours but eventually only a single colour is found. This is because stem cells that only produce differentiating cells will be lost from the stem cell pool

Answer 16

The results with “confetti mice” are consistent with the model that not all stem cell divisions are asymmetric. A stem cell that only produces transit amplifying cells will be lost from the stem cell pool while a stem cell that only produces stem cells will give rise to a larger clone. Over a number of generations, these stochastic events will see a polyclonal stem cell pool replaced by one that is monoclonal

Answer 17

Haematopoietic stem cells in the bone marrow are self renewing but will also form stem cells of both the lymphoid and myeloid lineages. Lymphoid cells form lymphocytes and natural killer cells, while the myeloid lineage forms erythrocytes, platelets, monocytes (macrophages), eosinophils, basophils and neutrophils. The signals regulating the formation of all of these cells are now largely understood.

Answer 18

1. Bone marrow removed or blood is drawn 2. Stem cells are collected 3. Stem cells frozen and stoned 4. Stem cells returned to bloodstream Autologous bone marrow transplant Bone marrow transplants were pioneered in the 1960’s by Donnall Thomas. More than 50,000 transplants are now performed (worldwide) each year. Most (57%) are autologous, using the patients own bone marrow. Bone marrow transplantation is a risky procedure and is only used when a patients condition is critical (it is mainly used to treat acute leukaemia and neoplastic lymphoproliferative disorders).

Answer 19

Pluripotent ESCs were first produced in 1981 by Evans and Kaufman and independently by Martin in mice They removed inner cell mass (ICM) cells from mouse blastocysts and cultured them in vitro Under appropriate conditions they will divide indefinitely but can also form aggregates called embryoid bodies that differentiate into a range of specialised cell types By altering the conditions, differentiation can be sent in different directions in which particular cell types from any of the three germ layers predominate

Answer 20

Sir Martin Evans - Italian geneticist Matthew Kaufman - British stem cell biologist (1942-2013) Oliver Smithies - British-American geneticist (1925-2017) They were awarded the 2007 Nobel Prize in Physiology or Medicine “for their discoveries of principles for introducing specific gene modifications in mice by the use of ESCs”

Answer 21

Mutant genes can replace a copy of the normal gene by homologous recombination in ESCs. Heterozygous ESCs are introduced back into a blastocyst and some mutant cells may be incorporated into the germline. Breeding allows for the production of mice that are homozygous for the mutation

Answer 22

James Thomson and colleagues (1998) removed ICM from humans blastocysts donated from IVF treatments. They cultured them in a layer of irradiated mouse fibroblasts to form established cultures Even after several months of culture they were able to differentiate tissues from all three germ layers including gut (endoderm), muscle (mesoderm) and neurons (ectoderm)

Answer 23

Pluripotent

Answer 24

• Knew that the transcription factors Oct4, Sox2 and Nanog are expressed in the inner cell mass and are required to maintain its pluripotent state. • Demonstrated that forced expression of these genes in mouse fibroblasts transform them to embryonic stem cells. These cells are pluripotent and can be induced to form a range of differentiated cells types

Answer 25

From mouse iPSCs derived from a model of sickle cell anaemia they corrected the haemoglobin mutation and then induced the cells to differentiate as blood stem cell. These cells rescue sickle cell animal when transplanted back into the mutant mice demonstrating the enormous potential of iPSCs

Answer 26

One of the first organoids to be described was that for the retina. Here GFP has been linked to the Retinal Homeobox gene (Rx), one of the first genes to be expressed in the developing retina. Rx exression (GFP fluorescence) can be detected after 5 days of culture and an optic cup is evident after 9 days of culture. Photoreceptors can be isolated from these organoids and transplanted into the retina where they restore some function in macular degeneration.

Answer 27

The hope is that these retinal organoids can produce cells required to repair retinal damage, in conditions such as macular degeneration (where photoreceptors in the centre of the visual field degenerate). There have been promising results in mouse experiments and clinical trials have commenced on humans, which involve UCL scientists.

Answer 28

Culture adaptation of human pluripotent stem cells. During prolonged culture many cells either die or differentiate, leading to selection for cells carrying mutations that permit them to grow (self renewal). Culture adaptation leads to cells with enhanced growth characteristics and sometimes to embryonal carcinoma cells (malignant ES cells) that have lost the ability to differentiate.

Answer 29

120 human ES cell lines were analysed for karyotype abnormalities and grouped according to whether abnormalities were seen at early or late passage. Most (66%) were normal but the study identified a tendency to acquire chromosomal abnormalities with prolonged culture. Chromosomes 1, 12, 17, and 20 were most commonly affected. Presumably they contain a gene, or genes, that confer growth advantage in culture.