Stem cell and regenerative biology

what do you need to research before you can completely characterize a stem cell?

-It's ability to self renew (the stemness hallmark) -the ability to differentiate into certain cell type(s). -Prospective isolation (how can you tell this cell apart from other cells of the tissue? essential to show that it's this specific cell that show stem cell characteristics. this can be done using cell surface markers and FACS sorting). -Functional assay (one HSCs ability to reconstitute the entire blood system). -Characterization of the niche. (it's important to identify the stem cells normal microenviroment in order to maintain it in culture).

list some reasons that can help explain the inefficiency of cloning? (somatic cell nuclear transfer to Oocyte in order to produce ESCs)

-laboratory variation -Oocyte source and quality -Genomic imprinting (a specific kind of epigenetic change) -Failure to reprogram the transferred nucleus -Artificial methods for membrane activation without fertilization. -methods of embryo culturing - Doner cells type and cell cycle stage.

what have been identified as the major challanges in performing somatic cell nuclei transfer (SCNT)

-premature exit from meiosis (egg) -suboptimal oocyte activation. Caffeine (protein phosphatase inhibitor) appear to be the solution. Can't remember exactly what it does.

what are some of the unanswered questeion on the cancer stem cell field?

-where do tumor initatiation cells come from? -are cancer stem cells rare? -are cancer stem cells dormant? -are cancer stem cell drug resistent? -are the some bone fide cancer stem cell markers? or molecular divers?

What are the limitations to cadavric pancreatic islet transplantation?

1) 2 whole pancreas's are needed per patient. 2) the pancreas is the first organ to degrade after death (within an hour), due to necrotic cell death, leading to release og the acidic content of the lysosomes which activates the digestive enzymes produced in the exocrine pancreas. 3) no time to coordinate histocompatability between donor and receipient

As a rule of thumb, what is the cloning succes rate for mammels with somatic cell nuclei transfer?

1-2%

how many erythrocyts are removed from the circulation every day by the spleen?

10^11

Every time you clone a new species it poses challanges. very simplified you need to fit 3 step in your protocol to the given species. what are these steps?

3 steps that often vary between species -how to remove the "old" nuclei from the doner egg (micro needle, irradiation, etc) -how to obtain the new nuclei (cell cycle stage, nuclei isolation, etc) -how to fuse the nuclei and egg (injection, electroporation).

what is a CpG island (why is it called C-p-G island)?

A CpG island is a base sequence of cytosin and guanine read from 5' to 3' orientation, with a phosphodiester linkage between them, illustrated by the "p". 5'- C-p-G-3' The C can be methylated(in the 5 posistion of the pyrimidin) which, when a lot of methylated C's are close together, will bind proteins with a methyl binding domain, and thus make the DNA less accessible to the transcription machinery. A CG sequence is a palindromic sequence (will be identical on the complimentary strand 5'- C-p-G-3') which allows for conservation of the cytosine methylation upon replication.

What are the fractions of misregulated pan genes and misregulated imprinted genes according to humphrey et al (2002) study (not one we have read, but is referenced in the lecture).

A study by Humphrey et al (2002) have showed that 4% of genes are misregulated in cloned embryos compared to normal control embryos. But the fraction of misregulated imprented genes is a staggering 50%.

what are the "phases" an activated stem cell go through, on the path to a differentiated cell.

Activation proliferation maturation differentiation

what is Alkaline phosphatase activity normally a marker for?

Alkaline phosphatase activity is normally a marker for Pluripotency. However, AP activity can also be found in spermatogonial cells which are unipotent. however, spermatogonial cells can form cell types of all germ layer in vitro, if some factors are removed.

what is an IRES?

An internal ribosome entry site, abbreviated IRES, is an RNA element that allows for translation initiation in a cap-independent manner, as part of the greater process of protein synthesis. In eukaryotic translation, initiation typically occurs at the 5' end of mRNA molecules, since 5' cap recognition is required for the assembly of the initiation complex. The location for IRES elements is often in the 5'UTR, but can also occur elsewhere in mRNAs.

How can you use germline transmission to check wether pluripotent stem cell(PSC) injected into the blastocysts got incorporation into the germ line.

By crossing a chimeric animal(male) with a WT(female) you can check whether traits from the injected pluripotent stem cell (like fur color) are found in the offspring. If yes, the PSCs will have contributed to the establishment of germ line cells in the chimeric parent.

How can you experimentally test for the Hematopoietic stem cell criteria?

By irradiating a mouse with a lethal dose of radiation, and subsequently give it a bone marrow transplant the mouse is able to survive. But it will take time for the newly transplanted HSC to reconstitute all the cells of the blood system, so you will need to transplant other cells of the BM as well (some of the progenitor cells). by labelling the HSC, but not the remaining transplanted cells, you are able to track the a few months later all the cells of the blood will have formed from the labelled HSC.

List some different methods the can be used to trace cell division?

By supplying cells with labelled Thymidine analog, you can look for their incorporation into DNA, the analogs will only be found in the cells that replicated while the thymidine analog was present. some examples are: -3H-thymidine (used to look for regeneration in canary birds - and their song generation pattern that includes new neurons in their circut). -brdU (remember that thymidine is not found in RNA).

how can you design a pulse-chase experiment to determine wether a cell population is renewed by a stem cell population?

By using a pulse that is specific for the differentiated cell type (like temporally inducing (Cre-ER with addition of TAM) the expression of GFP under the control of the insulin promoter(if you wanna pulse beta-cells), using low dose of TAM to label approx 50% of the cells) followed by a chase period that is sufficiently long for the cells to turn over a few times. three outcomes are imaginable: 1) most of the labelled cells are gone, replaced by unlabelled cells (indicative of cell population being maintained by a stem cell). 2) the labelled cell population retain the lebel, but newly formed cell populations(islets) are label free. this indicated that a stem cell give rise to the new forming cell populations, but that the old cell population os self maintaining. 3) the degree of label is the same as when the cell were initially pulsed. The cell population is maintained by lateral cell divisions. no stem cell population.

what are the terms used to describe cell rich, normal and cell poor bone marrow cellularity? and when can the different stages be seen?

Cell rich bone marrow: Hypercellular (seen in leukemia) normal bone marrow: normal Cell poor bone marrow: Aplastic bone marrow (can be seen after a lethal dose of radiation)

what is meant by "Label retaining cells"?

Cells that will remain "tagged" for a long time, after the labelling pulse has been removed. An example could be a pulse-chase experiment with brdU, which is a thymidine analog that gets incorporated into DNA. If the cells are actively dividing during the chase phase, this label gets "washed" out quickly. Whereas slowly cycling cells will keep the label for longer i.e. label retaining. This is seen for the stem cell located at the hair follicle. these stem cells rarely divide, instead they form daughter cells that are transiently amplifying. This is a cleaver tactic, since mutations most often happens during replication, thus having the stem cell in a rather dormant stage decrease the chance of mutations in this cell population. Whereas the mutation probability is higher for the transient amplifying cells, which is okay, since this cell only gives rise to a limited number of cells for a limited period of time.

How does the oocyte "curve" for a mammalian female look like from conception to menopause?

Contrary to the male continuous germ cells production, the female is born with all the eggs she will ever have. Actually the highest number of oocytes (6-7 mio) will be found in the prenatally, with most cells dying off before birth(1-2mio), and and continuous decrease throughout life, starting puberty with approx 300.000 oocytes, until menopause where there's basically no oocytes left. Throughout a life time, a human female ovulates approx 500 oocytes.

which enzymes add and remove methyl to/from the DNA?

DNA methyl transferases and demethylases

list some mature cell type that arise from the different germ layers?

Endoderm: -alveolar cells of the lungs -pancreatic cells -thyroid cells -epithelial lining of the gastrointestinal tract (The endoderm forms: the pharynx, the esophagus, the stomach, the small intestine, the colon, the liver, the pancreas, the bladder, the epithelial parts of the trachea and bronchi, the lungs, the thyroid, and the parathyroid.) Mesoderm: -muscle cells (skeletal, cardiac, and smooth) -blood cells -tubule cells of the kidney (The mesoderm forms: muscle (smooth and striated), bone, cartilage, connective tissue, adipose tissue, circulatory system, lymphatic system, dermis, genitourinary system, serous membranes, and notochord.) Ectoderm: -neurons -epidermis -pigment cells (The ectoderm generates the outer layer of the embryo, and it forms from the embryo's epiblast.[9] The ectoderm develops into the surface ectoderm, neural crest, and the neural tube.[10] -The surface ectoderm develops into: epidermis, hair, nails, lens of the eye, sebaceous glands, cornea, tooth enamel, the epithelium of the mouth and nose. -The neural crest of the ectoderm develops into: peripheral nervous system, adrenal medulla, melanocytes, facial cartilage, dentin of teeth. -The neural tube of the ectoderm develops into: brain, spinal cord, posterior pituitary, motor neurons, retina.) germ cells: -sperm -Oocyts

How does FACS work, and what does it stand for?

FACS: Fluorescence activated cell sorting It works by mixing a heterogenous cell population with fluorescence conjugated antibodies. the cells are moved, in a single file, past a laser that detects the fluorescence. the cells are then seperated into droplets, to which a charge is added, base on the fluorescence signal detected. The charge can be used to sort the cells into different colletion chambers.

which factor is required for spermatogonial self renewal in vivo and in vitro?

GDNF: Glial cell-derived neurotrophic factor expressed by basement membrane sertoli cells.

Define genomic imprinting

Genomic imprinting is an epigenetic phenomenon that causes genes to be expressed in a parent-of-origin-specific manner. Forms of genomic imprinting have been demonstrated in fungi, plants and animals. As of 2014, there are about 150 imprinted genes known in the mouse and about half that in humans.

What was Hans Spemanns embryo-splitting experiment about?

He did two experiments (according to the lecture): 1) He split a 2-cell stage embryo into two individual cells, and observed that they both developed into newts(salamanders). 2) he tied up the blastula with a baby hair, having all the genomic material in one of the two "blobs", and later releasing the knot, letting both sides develop. Hi noticed that the side containing the Genomic material developed normally, while the other side had abarrent and slow development. According to the web: the cytoplasmic factors(in the gray cresent) were the important factors for development.

how does histone modifications contribute to altered gene expression patterns?

Histones, mainly their "tails", undergo different forms of post translational modifications(PTMs). however, unlike DNA methylations, the different modifications do not have a unique effect on transcription; methylations in some posistion can either promote DNA condensation while in other posisiton the same form of PTM can promote DNA relaxation. Acetylations of Lysins are generally accepted to promote euchromatin formation (relaxed state) since the neutralize the positive charge on lysins which interact with the negatively charged DNA. Lysin methylations, on the other hand, preserve the positive charge, and is considered to promote heterochromatin condensation. serine phosphorylation add a negative charge which "repel" the negative DNA, again promoting DNA relaxation. Lysin methylations preserve the positive charge, which promotes DNA condensation. This is not an exhaustive list, other histone modifications exist.

What types of cues might regulate stem cells (contribute to the stem cell niche)

Humoral factors (blood, nutrient, oxygen supply etc). structural factors Paracrine (and juxtacrine) signals Metabolic factors physical factors neural control

How is transcription prevented from genes with hypermethylated promoters?

Hyper methylated promoters (and other hyper methylated DNA sites) will bind proteins that contain Methyl binding domains (MBD) and will prevent the transcription machinery from assembling and in initiating transcription at the given site.

How can you form chimeric animals?

If you inject pluripotent stem cells into the blastocyst, without prior removal of the existing cell mass, then the newly injected cells will contribute to the developing organism along site the preexisting pluripotent cells.

how can you use homologous recombination to perform trageted mutagenesis?

If you wish to mutate (introduce insertion/deletions) into the specific site in the genome, you can exploit the cells own homologous recombination(HR) machinery. HR is usually known from meiosis (germline cells) but can also be used by somatic cells as a DNA repair mechanism. when you use the HR machinery for mutagenesis it is during the mitotic cell devision. You will need a DNA construct containing homology arms (which are sequences complimentary to the region in which you want to insert/delete a sequence). In between the homology arms, you have your sequence of interest. Usually your sequence of interest will contain a positive selection gene (like antibiotic resistance, potentially flanked by restriction enzyme clevage site) and next to the homology arm you will have a negative selection gene, allowing you to select for clones where only the sequence of interest have been added by HR and deselect for clones where the entir construc have been randomly integrated and the negative selection marker is present.

How can you isolate the embryo inner cell mass using immunosurgery?

Immunosurgery is a method of selectively removing the external cell layer (trophoblast) of a blastocyst through a cytotoxicity procedure. Immunosurgery of a blastocyst. Antibodies are added that attach to the outermost layer of cells, which on a blastocyst is the trophoblast. After removing any unbound antibodies and adding complement, the trophoblast cells are destroyed, leaving only the inner cell mass

HSCs are found in the bone marrow and sometimes in the circulation. What does HSC interact with in the BM and how are they able to find their way back from the circulation to the BM?

In the bone marrow HSC are always found in contact with osteoblasts (boneprecursor cells). HSC are attracted to calcium, and that is how they find their way back to the BM.

how can you perform liniage tracing of intestinal stem cells?

Instestinal stem cells express lgr5. In the lecture bill went through the following construct: --Lgr5 promoter [EGFP]-[IRES]-[CRE-ER]--- --Rosa26 promoter [STOP]-[LACZ]--- The IRES is an internal ribosomal entry site, allows for translation of 2 different proteins from the same mRNA transcript. the Cre will only be expressed in lgr5+ positive cells, and by adding tamoxifen, you can get these cells to express lacZ. using only a low dose of Tam, allows you to label only a single or a few cells. Over time you can follow the fate, and the progeny cell types, formed from the originally labelled cells. can be considered a pulse chase experiment, where the pulse is tamoxifen induced expression of lacZ, by the excision of the stop sequence. and the chase is the wait. these results shows that these lgr5+ cells are not label retaining(quiescent), had the label been something that could be watered down like brdU and not constitutively expressed like the lacZ gene on the rosa26 promoter.

which question can you ask yourself to figure out which genetic manipulation system to use?

Is this a genetic problem? (1) (1) no: it's probably a biochemical problem (1) yes: does the experiment need to be tissue specific (spatial control)? (2) (2) no: use homologous recombination or a transgenic mouse (2) yes: Do you need temporal contol of gene expression? (3) (3) no: Use Cre-LoxP (3) yes: Does it need to be reversible? (4) (4) no: use Cre-ER-Tamoxifen (4) yes: use Tet-on or tet-Off

blood cancers can potentially form at 2 different stages, which? and which is therapeutically favorable?

It can form at the level of the hematopoietic stem cell or at the common progenitor (CMP or CLP). The common progenitor level is therapeutically favorable, since you can isolate un-transformed HSCs from the patient, before irradiating the BM, and thus the patient can serve is his/her own bone marrow donor (i.e. 100% histocompatibility). this is not possible if tumourigenic transformation happened in the HSC.

What is the function of adult neurogenesis?

It is unknown, but studies have shown that; -most adult born neurons do not survive. -that exercise and enriched enviroment boosts adult neurogenesis. -the depression and neuropsychiatric disorders are inversely correlated with adult neurogenesis. some question, that are yet to be answered in regards to adult neurogenesis is: -is adult neurogenesis a mechanism for learning? -how much of adult neurogenesis is experience dependent? -how does the degree of adult neurogenesis differ between individuals?

what is cytosine-beta-D-arabinofuranoside (araC)?

It's an anti mitotic drug, that do not kill of the cells but prevent cell division. It was used in an example provided in the lecture where it was used to assess the relationship between A, B, C and E cells in the sub-ventricular zone. Addition of araC caused elimination of cell population A and C, but B and E remained. 2 days after araC treatment the C cells reappeared, 4 days after the A cells came back. conclusion: the A and C cells are turning over, and can't be replenished due to the AraC treatment. when the drug is removed C cells can form, and they will later form A cells. They did another experiment(with brdU staning) to elucidate wether it was the B or the E cells that had stem cell properties, and found that it's the B cell. the E cells (ependymal cells) are have a long life span, which is why they ware not affected by the araC treatment.

When cloning a sheep (Dolly) the Donor Nuclei need to be in the G1 cell cycle stage. how do you transfer a Nuclei in G1 stage where the chromosomes are not condensed?

It's difficult, if not to say immposible, to isolate a nucleus with un-condensed DNA. So what they did when they cloned Dolly, was that they transferred the entire somatic cell and placed it next to the enucleated Oocyte and then fused the two cells with electroshock.

what is ki67 a marker for?

Ki67 is an antigen expressed by dividing cells, and can be used to label dividing cells.

how can you engraft intestinal stem cell in the colon of a mouse?

Like for the hematopoietic stem cells, the instestinal stem cells won't engraft if the niche is "full". In order to get the stem cells to engraft, you can use a mouse model with acute collitis (cells can engraft at the ulcers).

What is lineage tracing and what can you use it for?

Lineage tracing can be used to track all the different cells that arise from a single population of cells (like a stem cell). The method described was use to track the cells arising from the astrocyte (type B cell/ neural stem cell). This was done by driving the expression the avian virus receptor (Tva) under the expression of a promoter specific to astrocytes (GFAP). the expression of this receptor then allowed for infection of the cells with avian virus (RCAS-AP), whose genome got incorporated into the genome of the host cell. This permanent genomic change is then passed on to all progeny of the astrocyte, and they will all express alkaline phosphatase. RCAS: replication competent avian leukosis virus AP: alkaline phosphatase (allows for detection)

belgian blue are a type of cattle with excessive muscle mass, what gene is causeing this phenotype?

Myostatin (also known as growth differentiation factor 8, abbreviated GDF-8) is a myokine, a protein produced and released by myocytes that acts on muscle cells' autocrine function to inhibit myogenesis:

list some human diseases that are amenable to regenerative therapy

Neurudegenerative diseases - parkinsons -Alz -luo gehring CVDs Diseases of the blood system Diabetes

What is tetraploid complementation?

Normal mammalian somatic cells are diploid. The assay starts with producing a tetraploid cell, by taking an embryo at the two-cell stage and fusing the two cells by applying an electrical current. The resulting tetraploid cell will continue to divide, and all daughter cells will also be tetraploid. Such a tetraploid embryo can develop normally to the blastocyst stage and will implant in the wall of the uterus. The tetraploid cells can form the extra-embryonic tissue (placenta etc.), however the tetraploid inner cell mass cannot give rise to a viable fetus. In the tetraploid complementation assay, you can inject your cloned pluripotent cells into the tetraploid blastocyts, and the injected diplod cells will be the ones that gives rise to the embryo. The embryo will then develop somewhat normally; the fetus is exclusively derived from the injected pluripotent cells, while the extra-embryonic tissues are exclusively derived from the tetraploid cells.

Explain the relationship between satellite cells and notch signalling.

Notch signalling is necessary for satellite cell maintenance and self renewal. Notch1 is expressed by the satellite cells (and the ligand is probably expressed by the neighbouring muscle cells. Notch signalling is juxtacrine form of signaling, where the ligand and receptor are both surface bound.

What 4 factors are needed to reprogram skin fibroblasts to iPSCs?

Oct4, Sox2, Klf4, hc-Myc (yamanaka factors)

What's experiment was performed i order to isolate the HSC?

Once the HSC had be hypothesized to exist, one needed means of isolating this specific cell from the remaining cells, in order to test it experimentally. what weissman and colleagues did was to raise monoclonal antibodies against all surface antigens on bone marrow cells. Next, by testing these antibodies one at the time on a heterogenous BM cell population, he could subdivided cells into groups. Then, by transplanting these subgroups into mice irradiated with a lethal dose of radiation, he could identify in which group the HSC was to be found. (based on which mice that were able to survive i.e. restore the cells of the blood system) for mouse he found that HSC was: Sca-1+, Thy-1 low, and negative for lineage specific markers. for humans: CD34+, Thy-1+, negative for lineage specific markers. (basically the same fundamental setup as modern day FACS sorting).

what is parabiosis?

Parabiosis: "living beside", In the field of experimental physiology, parabiosis is a class of techniques in which two living organisms are joined together surgically and develop single, shared physiological systems, such as a shared circulatory system.

which transcription factor is important for satelite cells (used as a marker for satelitte cells)

Pax7

what's the name of the organism the can regenerate itself, no matter which way you cut it?

Planarian (flatworm).

How does the cre-lox system work?

See illustration for Cre mediated excision of a flox'ed gene. The system can also be used for induction of gene expression, by either inversion of the gene, or removal of a transcription termination (polyadenylation site) located between the promoter region and the coding region for the gene you wish expressed. after cre excision, one loxP site will remain.

what cell types can you find in the testis?

Spermatogenesis: -spermatogonium (Type A1, A2, and B) -primary spematocyte -secondary spermatocyte (haploid) -spermatids (haploid) -sperm (haploid) Other: -sertoli cells (suport cells)

What are Teratocarcinomas?

Stem cell tumors. The derive from a stem cells and can form organised and well differentiated cell type of all germlayers (endo-, meso- and ectoderm).

what did the study with a parabiotic young and old mouse show in regards to muscle regeneration?

Supplementation of systemic GDF11 levels, which normally decline with age, by heterochronic parabiosis or systemic delivery of recombinant protein, reversed functional impairments and restored genomic integrity in aged muscle stem cells (satellite cells). Increased GDF11 levels in aged mice also improved muscle structural and functional features and increased strength and endurance exercise capacity.

How does the "2 stem cell populations" theory work?

The 2 stem cell populations theory is the idea of a tissue having two distinct stem cells populations, one that is quiescent and one that is actively dividing. for the intestinal stem cell population, the quiescent stem cell population is found in an inhibitory zone where the Wnt signal is OFF but BMP signal is ON. These cell can give rise to the active stem cell. The active stem cell, performs asymmetric cell divisions, where it self renew and forms a progenitor that can further proliferate and differentiate. The active stem cell is found in an active zone, where BMP signals are OFF and Wnt is ON.

The hematepoietic stem cells(HSC) is a multipotent stem cells, that can give rise to all cell types of the blood. When the HSC devides it can self renew, form a commen myeloid progenitor (CMP) or a commen lymphoid progenitor (CLP). What are the CMP and CLP?

The CMP can form alle the cells of the myeloid lineage (erythrocytes, granulocytes, macrophages, etc) and the lymphoid progenitor can form the lymphocytes (NKT, T and B cells/plasma cell). these cells are progenitor, but not stem cells, that have self renewal capacities for a short time span until they eventually differentiate.

How does notch signaling work?

The Notch protein spans the cell membrane, with part of it inside and part outside. Ligand proteins binding to the extracellular domain induce proteolytic cleavage and release of the intracellular domain, which enters the cell nucleus to modify gene expression. The receptor is normally triggered via direct cell-to-cell contact, in which the transmembrane proteins of the cells in direct contact form the ligands that bind the notch receptor. The Notch binding allows groups of cells to organize themselves such that, if one cell expresses a given trait, this may be switched off in neighbouring cells by the intercellular notch signal. Pax7 is a nodal transcription factor that is essential for regulating the maintenance, expansion, and myogenic identity of satellite cells during both neonatal and adult myogenesis. Deletion of Pax7 results in loss of satellite cells and impaired muscle regeneration. Here we show that ectopic expression of the constitutively active intracellular domain of Notch1 (NICD1) rescues the loss of Pax7-deficient satellite cells and restores their proliferative potential. Strikingly NICD1-expressing satellite cells do not undergo myogenic differentiation and instead acquire a brown adipogenic fate both in vivo and in vitro. NICD-expressing Pax7-/- satellite cells fail to upregulate MyoD and instead express the brown adipogenic marker PRDM16. Overall these results show that Notch1 activation compensates for the loss of Pax7 in the quiescent state and acts as a molecular switch to promote brown adipogenesis in adult skeletal muscle.

During development the primordial germ cell migrate from their original location at the posterior side, along the gut to the genital ridges. How does this migration take place.

The PGC are migrating towards a chemoattractant. The distance the cells needs to migrate, are too long to the chemoattractant to diffuse from the genital ridges to the PGC cell origin. Instead, several cells along the migration path express the chemoattractant, once one chemoattractant expressing cells is reached, its production of chemoattractant will be suppressed, so the migrating PGC is attracted to the next chemoattractant cells along the path. Migration depend on steel (the ligand) and c-kit (the receptor. This is a survival signal. If the cells migrate off path, they die from the lack of steel ligand. If the cells due not die off, for some reason, when they migrate off path, they can form teratomas.

list and describe the different classes of stem cells

The full classification includes: Totipotent: These stem cells can differentiate into all possible cell types. The first few cells that appear as the zygote starts to divide are totipotent. (can form extra embryonic cell types, such as the placenta, yolksac, etc) Pluripotent: These cells can turn into almost any cell. Cells from the early embryo are pluripotent. Multipotent: These cells can differentiate into a closely related family of cells. Adult hematopoietic stem cells, for example, can become red and white blood cells or platelets. Oligopotent: These can differentiate into a few different cell types. Adult lymphoid or myeloid stem cells can do this. Unipotent: These can only produce cells of one kind, which is their own type. However, they are still stem cells because they can renew themselves. Examples include adult muscle stem cells(i.e. satellite cells). Embryonic stem cells are considered pluripotent instead of totipotent because they cannot become part of the extra-embryonic membranes or the placenta. (spermatogonial stem cells can only give rise to sperm)

what's the "conveyor belt" model for cell renewal of the intestinal epithelial cell layer.

The inteastinal (actively dividing) stem cells are found at the bottom of the instestinal crypts, in close contact with panath cells (Sato et al. 2011). these cell replenish all cell types of the villus, by dividing at the bottom of the crypt and having the cell move upwards towards the tip of the villus like a conveyor belt. Cells are sloughed off at the tip. the intestinal epithelium is replaced every 1-3 days. Accoding to Bill, another intestinal stem cell that is dormant, is located further up the crypt. this cell is bypassed when the cell in the conveyor belt moved upwards.

Explain "large offspring" syndrome?

The large offspring syndrome refers the higher than normal birth weight, and correspondingly larger placentas, of cloned animals at birth. It does not, however, refer to the offspring of the cloned animals, which are normal at birth. This phenotype could potetially be explained by abarrent imprinting of the H19 gene (which under normal conditions are paternally imprinted, leading to preferential expression of the more upstream Igf2 gene). Higher igf2 levels, would serve as a resonable explaination for the large offspring phenotype since the same phenomenon is observed in humans offspring of mother suffering from untreated gastational diabetes. However, according to the Humphrey et al (2001) paper this phenotype cannot be explained by the imprinting pattern of a single gene, since they cannot show any consistent pattern between imprinting pattern (mRNA level) and birth/placental weight. Gastational diabetes: Suger, but not insulin, can cross the placenta. If the mother suffers from untreated gastational diabetes, which resembles Type II diabetes with hyperglycemia, large amount of suger is transfered to the fetus which will produce large amounts of fetal insulin which is a potent growth promoter.

How does the tet-off system work?

The tet off system can be used to turn off gene expression by addition of doxycyclin-tetracyclin. doxy-/tetracyclin will bind to the transcription factor tTA and prevent its association with the tet operator(tetO), turning off the expression of the gene controlled by the tetO. In the figure the TetO is refered to as the tet response element (TRE).

how does the tet-on system work?

The tet-on system work oppositely the tet-off system: when doxy-/tetracyclin is present it can bind to rtTA which then can bind to the tet operator (tetO) leading to transcription. (the gene is Turned on in the presence of Tet)

over a 6 month period, the beta cells are turned over approximately 3 times. how?

There are no stem cells of the pancreas, the cell turnover happens by "horizontal" cell division, where an existing beta cells divides to give rise to two new beta cells.

Which marker can you use to stain for germ cells?

Transcription factor Oct4 are expressed in germ cells. and they have alkaline phosphatase activity. Migrating primordial germ cells, oocytes and spermatogonial cells. (Do not express Nanog, and in the presence of steel

the stem cell found in the subventriculat zone (SVZ) are called type B cells, what are the stem cells in the subgranular zone(SGZ) called ?

Type 1 cells

What is Waddington's developmental/epigenetic landscape?

Waddington's epigenetic landscape is a metaphor for how gene regulation modulates development. Among other metaphors, Waddington asks us to imagine a number of marbles rolling down a hill. The marbles will sample the grooves on the slope, and come to rest at the lowest points. These points represent the eventual cell fates, that is, tissue types.

Pronuclear injection of your gene of interest, can form transgenic animal - but only if integrated into the genome; otherwise it will not be passed on to progeny during cell devision. (This is the method used in the "Hayashi & McMahon: Efficient Recombination in Diverse Tissues by a Tamoxifen-Inducible Form of Cre: A Tool for Temporally Regulated Gene Activation/Inactivation in the Mouse" paper). Explain why this is very inefficient and do not for revers genetics.

When the construct is injected into the pronucleus, it can either integrate or not. In case of integration, the construct will integrate at random. This means that the integration can cause mutation of other genes, integrate in regions that will be condensed into heterochromatin or converse integrate into a region that is actively transcribed all with a different effect on transcript-ability. This method do not allow for revers genetics (targeted mutagenesis) since the DNA fragment will integrate at random.

how the the Wnt signalling pathway work?

Wnt is an important signalling pathway in the intestines. Without Wnt: Dishevelled cannot bind to the receptor frizzled, and is thus inactive. the Axin:APC:GSK3:CK1 can form and target beta-catenin for degradation. With Wnt: Wnt can bind to the Frizzled receptor, that activates dishevelled. This sequesters Axin at the membran, and the beta-catenine degrading complex cannot form (GSK3 that phosphorylates beta-catenine is inactive). Beta-catenine concentration can increase, and translocated to the nucleus and promote transcription of downstream target genes (like lgr5 expressed in intestinal stem cells).

What does a beta-catenine staining of intestinal epithelium look like?

beta-catenine is also a cytoskeletal protein that bind to the inner surface of the plasma membrane. this protein is present in all cell of the small intestines. to look for active Wnt signalling, one should look for beta-catenine stain in the nucleus.

How can spatially and temporally control the expression of Cre?

by having Cre expressed under the control of a tissue specific promoter, you can get spatially control where the recombinase is being expressed. by fusing the Cre to a modified version of the estrogen receptor (ER-TM) you can sequester the protein in the cytosol (via binding to Hsp90) in the absence of Tamoxifen. (the modified version of the estrogen receptor cannot bind endogenous estrogen, only exogenous Tamoxifen)

If a child(boy) has to undergo chemotherapy, the germ cells might be affected. Since the child cannot yet form sperm, it's impossible to cryopreserve a sperm sample for later use. An alternative approach can be used to insure that the child, when he reached adulthood, can father children. How does this work?

by taking a testis biopsy from the child, containing germ stem cells (spermatogonial cells) and cryopreserving it, the cell can later (after ended chemo treatment) be transplanted back into the child's older self, where the cells will reinhabitate the seminiferous tubules and give rise to sperm cells.

what does the niche hypothesis say?

"stem cells persist and their activity is regulated through interaction with a specialized microenviroment or niche"

under which conditions does new beta-cell islets form?

during growth, pregnancy and in obesity.

What are the different structure found associated with the hair follicle?

follicle structure: dermal papilla inner root sheath outer root sheath sebaceous gland hair-shaft cell compartments: downmost you have the hair bulb above that you have the "bulge" which is where the skin/hair stem cells reside.

what is "gating" (context: FACS)

gating is the process of (somewhat arbitrarily) dividing FACS results into subcategories. It's the lines drawn on the FACS result sheet, tha subdivides the cells into groups.

what does cardiotoxin do?

it's a myonecrotic drug that induces muscle injury. muscle injury triggers muscle regeneration. muscle regeneration is used to study satellite cells.

How can you isolate the embryo inner cell mass using laser surgery?

laser is used to kill/destroy the trophoblast cells, leaving the inner cell mass (ICM) intact and easily accessible.

What was the conclusion from the sherwood et al paper from 2004 on muscle stem cells?

that the cells that contribute to muscle regeneration do not come from the hematoipoetic liniage, but the some other cells from the bone marrow exhibited some degree of myogenic potential.

What kind of experiment was used to show that the GFAP+ cells is responsible for generation of all new neurons in the SVZ and SGZ?

the HSV-tk (herpes simplex virus gene thymidine kinase) was inserted in the genome, under the control of the GFAP promoter. when adding glanciclovir to these cells, the HSV-tk gene will comnvert it to a cytotoxic product that kills of the cells. An absence of new neurons were observed, by the lack of DCX staining in histological sections of the SVZ and SGZ. Doublecortin (DCX) is a microtubule-associated protein expressed by neuronal precursor cells and immature neurons in embryonic and adult cortical structures. Neuronal precursor cells begin to express DCX while actively dividing, and their neuronal daughter cells continue to express DCX for 2-3 weeks as the cells mature into neurons. Downregulation of DCX begins after 2 weeks, and occurs at the same time that these cells begin to express NeuN, a marker for mature neurons

what is Rosa26?

the Rosa26 promoter is ubiquitously expressed in all tissues of the mouse, but the RNA transcribed does not code for anything. Knocking out this gene does not manifest in a phenotype. Can be cloned to ubiquitously express exogenous genes in mice. This is gods gift to scientist working with gene modifications in mouse.

Describe how CRISPR-Cas9 and dead-Cas9 works

the bacterial derived Cas9 protein has endonuclease activity. By providing a exogenous singlestranded guide RNA strand (sgRNA) you can target the Cas9 protein at a specific nucleotide sequence. If you want to introduce a mutation, you can provide a "repair template" (dsDNA with homology arms) which can be used as a template for homologous recombination. dead-cas9 (dCas9) on the other hand has no enzymatic activity. But it can still be recruited to a specific sequence in the DNA when provided with the guide strand. by tethering other proteins such activators, repressors, as methyl transferases, demethylases, cytidine deaminases(C->T), which can be used for a multitude of purposes. THe major concern with using CRISPER is potential off target effects. CRISPR: Clustered Regularly Interspaced Short Palindromic Repeats

how is the posterior / anterior orientation of the embryo established?

the extra embryonic ectoderm (2) secrete BMP (1) the diffuses down to the epiblast (3). The epiblast cells are surrounded by the visceral endoderm (4). The viceral endoderm asymmetrically (toward the soon to be anterior side) express signalling molecules that determine anterior/posterior orientation of the embryo. The signalling molecules includes BMP inhibitors, i.e. only the soon-to-be posterior axis see BMP. The primordial germ cell (PCG) forms along the posterior axis. 1. BMP 2. extra-embryonic ectoderm 3. Epiblast: later give rise to the embryo 4. visceral endoderm: Forms yolk sac and early hematopoiesis.

Tumors are heterogeneous. Two different models, explaining the origin of this heterogeneity, has been proposed. What are the two models?

the first model is that; 1) "clonal expansion model": tumors cells are heterogeneous and most cells within the tumor can proliferate extensively and give rise to new tumors. --> tumor initiating cells are common. 2) "cancer stem cell model": tumors cells are heterogeneous,however there is a subset of cells, the tumor stem cells, which are the only cells that have the ability to proliferate extensively and form new tumors. --> tumor initiating cells are rare.

which layers make up the skin?

the outmost part is the epidermis which is an ectodermal derivative, which is separated from the mesodermal dermis by a basal lamina. the epidermis has a basal cell layer, and granular cell layer, squamous cells and dead keratinocytes. In addition, hair follicles, sebaceous- and sweat glands belong to the epidermis. The basal cell have stem like potential and can repair skin, in areas where the hair follicle bulge cells are absent.

which embronic structure/cells give rise to the germ cells?

the primordial germ cells (PGCs)

Define Epigenetics

the study of changes in organisms caused by modification of gene expression rather than alteration of the genetic code itself. Epigentic changes can be DNA modifications (CpG methylations) or histone modifications (methylations, acetylations, etc). In class she used the text/sentence analogy, where the letter where the genetic code, and the punctuations and spaces are the epigenetic modifications. the punctuations can change the interpretation of a sentence. example: 1) A woman without her man is nothing or 2) A woman. Without her, man is nothing.

What can you use the xenograft assay for, and is it reliable?

the xenograft assay is injection of tumor cells into an immunodeficient mouse and observe the rate of tumor formation. previously the NOD/SCID mouse were used, but the paper by Quintana et al (2008) show that the NOD/SCID Il2rg-/- was a better model since it is more immuno compromised, and allow for a high degree of tumors to form. neither of them are ideal. NOD= non-obese diabetic SCID= severe combined immunodeficiency Il2rg: Interleukin 2 receptor gamma.

"Transplant-frindly" pigs can be the future of organ transplantation. How does these pigs vary from normal pigs?

they are KO for the Alpha-1,3-gal transferase, that adds alpha-1,3-gal to the mambrane surface of pig somatic cells. humans do not have alpha-1,3-gal, so if the protein is present on transplanted organs, it will provoke an immune reaction.

satellite cells are muscle stem cells. Where are these cells found (the location of their niche)

underneath the basal lamina, next to the sarcolemma of the muscle fiber.

what are the difference between forward and reverse genetics?

very simplified: In forward genetics(phenotype --> genotype) you generate mutations at random, find a phenotype of interest and go back and examine the genotype causing the phenotype to arise. In reverse genetics (genotype--> phenotype) you do introduces mutation in your gene of interest (targeted mutagenesis) and then you observe the phenotype

under which conditions can lacZ form a blue substrate?

when X-gal, an otherwise colorless substrate, is added. The enzymatic activity of lacZ converts this into a blue product.

Are there stem cells in the adult human brain?

yes, in some areas of the brain (sub ventricular and the subgranular zone). Some cells, when isolated from the human brain, has the capacity to form neurospheres. The cells with this renewing capacity are called neural stem cells/ astrocytes / type B cells and can give rise to a transient amplifying cell (progenitor) / type C cells, that next form the migrating neuroblast cells (type A cells). These cells reside just below the ependymal cell layer that lines the ventricle. In the 14C method paper by Spalding et al (2013) they also showed turnover of hippocampal neurons throughout life.

How can you test for pluripotency and what are the limitations to the different techniques?

you can do: -in vitro differentiation and look for makers of all the three germ layers. (limitations: marker expression do not test for functionallity, and can simply be a cellular stress response). -Teratoma formation. By injecting the cells into a histocompatible or immuno suppressed mouse to can form a teratoma and look at the cells types present. these cell well have morphologies and functionalities resembling those of all three germlayers. (limitation: does not test for the cells ability to promote normal development). -formation of chimera animals: by injecting the cells into a blastocyts you can asses their contribution to normal development. (limitation: host derived cells may compliment cell non-autonomous defects). -germline contribution (mating of chimeric animals) will test for the pluripotents cells ability to form functional germ cells. (limitation: this exclude genetic, but not epigenetic defects that could interfere with development). -tetraploid complimentation: the most stringent test, the formed animal will consist exclusively of your pluripotent cells. (limitations: will not thest for the ability to form trophectoderm (placental) lininage ("totipotency").

How can you test for methylated cytosines?

you can use the bisulfite sequencing, where you compare the (sanger) sequencing result from the untreated DNA with the (sanger) sequencing result from the bisulfite treated DNA. The cytosines indentified in the bisulfite treated DNA, where the ones that were methylated originally.

In broad terms, what are the developmental steps from zygote to beta cell.

zygote -> endoderm -> pancreatic precursor -> endocrine precursor -> beta cell.