User:Kinkreet/SCRA/sandbox

The genomes of both humans and zebrafish are diploid. However, zebrafish is known to harbour many gene-duplication evenets. As a result, the zebrafish genome contains many paralogs in which a gene is duplicated and the function of each gene diverge through mutation, until eventually the two genes provide distinct but related functions; often one paralog retains one part of the function (subfunction) of the original gene, while the other paralog retains the other subfunction, thus ultimately resulting in the division of labour. This is known as subfunctionalization; another possibility is that the paralog takes on a completely new function (neofunctionalization).

Growth and regeneration
Both human and zebrafish contain the vertebrate body plan, and both grow to a definite adult size. Human infants and zebrafish exhibits saltatory growth pattern where there are episodic growth for 1 day to 1 week or growth and 2 days to 2 months of null-growth in humans However, the regenerative potential in zebrafish is significantly higher than that of humans - zebrafish is able to regenerate many tissues and organs, such as heart, fin and retina, whereas humans can only limitedly regenerate the fingertips.

Rainbow zebrafish
The rainbow zebrafish follows in the step of the brainbow mice

A rainbow zebrafish contains repeats of RFP, CFP GFP and YFP genes under the regulation of the constitutively active promoter β-act2; between each gene is a loxP site. This zebrafish is then crossed with another zebrafish containing the Cre combinase, expressed under a cell-type specific marker. The resulting progeny will express Cre recombinase, which will excise between loxP sites; however, because Cre is not efficient, recombination will not be complete for all loxP sites. Instead, by chance, the Cre recombinase will excise between two 'pseudorandom' loxP site. Thus, individual stem cell will express different levels of each fluorescent protein, and so simply by probability, it is very likely that cells adjacent to each other will exhibit a different colour; thus we are able to trace the progeny of each cell individually.

Rainbow zebrafish is ideal for lineage tracing, as it is able to trace the progeny of each individual stem cell or progenitor.

From study of the heart using the rainbow zebrafish, it was found that the surface of the heart originally contains many stem cells, which gave many clonal populations; however, as the zebrafish matures, the number of clonal populations, and thus presumably the number of stem cell population on the surface of the heart, decreases. However, the inner linings of the heart still exhibited many colours. In the same study, they were also able to track individual neuronal connection to see how the neurons at the heart interact with each other.

Drosophila melanogaster
Genetics approaches to study somatic stem cells

The most well-characterized population of stem cells in Drosophila has been the germ line cells. However, more research is being conducted on the adult stem cell populations of Drosophila. Drosophila melanogaster is small, have a short life span (thus good for aging studies as you don't have to wait too long for the fly to age). Because of the fruit fly is highly prolific, they are widely used in population studies.

It has been long known that heat shock can be used to control the expression of certain genes, which sprung the whole study of heat shock proteins (HSP)

In Drosophila, haematopoiesis occurs in the lymph gland. Progenitors (prohaemocytes) at the lymph gland can give rise to three types of circulating haematocyte populations: plasmatocytes, crystal cells and lamellocytes. Plasmatocytes are phagocytes similar to monocytes, and constitute the majority of the myeloid cell population; crystal cells are immune effector cells and lamellocytes are cells that encapsulate invading organisms and large foreign bodies; the latter two cell types are not found in mammals. In contrast, there are no lymphocytes or red blood cells in Drosophila. Lamellocytes are not found in abundance as it only becomes differentiated after a stimuli such as parasitization by wasps.

The posterior signalling centre (PSC) is a cluster of signalling cells which controls the balance between maintaining the multipotent prohaemocyte populations, and the proliferation and differentiation of the prohaemocytes into different lineages. The PSC control the haematopoietic progenitors by expressing the COE transcription factor Collier, a gene only expressed in the PSC, which maintain JAK/STAT signalling in prohaemocytes and prevents differentiation. The role of Collier is similar to that of mammalian early B-cell factor, but has also been shown to mediate Hh-induced patterning and muscle formation during development.

Feeder cells
Feeder cells provide leukemia inhibitory factor (LIF, an interleukin 6 class cytokine) for mouse embryonic stem cells which leads to an increase in STAT3, which in turn leads to increased cell renewal. Thus, feeder cells maintain the undifferentiated state of stem cells using LIF, although this does not work for human ESCs.

Induced pluripotent stem cells
Embryonic stem cells have the ability to divide indefinitely while maintaining pluripotency, this is a highly desirable trait in therapy as it can be used to replenish and repair cells after injury. However, because embryonic stem cells are derived from the inner cell mass of mammamlian blastocysts, the use of human embryos poses ethical issues. Furthermore, ES cells derived this way have unique surface antigens which may be rejected by the recipient. Induce pluripotent stem cells (iPS) overcome this issue by reprogramming differentiated cells, derived from the patient itself, back to the pluripotent state. iPS cells can also be derived from disease-state cells, to allow for drug screening and toxicological tests.

Four transcription factors - Oct3/4, c-Myc, Sox2, Klf4 - were introduced into mouse somatic cells (embryonic fibroblasts and tail-tip fibroblasts) through retrovirus infection, and was sufficient to transform adult fibroblast into embryonic stem cells. c-Myc and Sox2 are not absolutely required for this process, but makes it much more efficient.

First, GFP was introduced using amphotropic retrovirus derived from PLAT-A packaging cells; this is to test the transduction efficiency, which was no more than 20%. To improve the transduction rate, Yamanaka first introduced Slc7a1 (a mouse receptor for retrovirus) into HDF using lentivirus, and then introduced GFP using ecotropic retrovirus produced in PLAT-E packaging cells. The ecotropic retrovirus binds to Slc7a1 and gave a transduction efficiency of 60%. Once this system is optimized, four transcription factors - Oct3/4, c-Myc, Sox2, Klf4 - were introduced into human dermal fibroblasts, derived from the facial dermis of 36-year-old Caucasian female, using the ecotropic retrovirus and incubate for 6 days to allow for optimal transduction. After transduction, the cells are plated onto mitomycin C-treated SNL (Immortalized cell line derived from mouse fibroblast STO cell transformed with murine LIF and neomycin resistance genes) feeder cells. Mitomycin C is a chemotherapy drug which glues DNA together so it cannot divide for grow, meaning that the feeder cells are no longer mitotically active and will not contribute to the apparent stem cell population. The medium was also changed the next day into one designed for primate ES cell culture supplemented with bFGF.

Around two weeks later, granulated colonies, which are not characteristic of hES appears, and colonies which resembles hES (flat and tightly-packed) appears ~25 days post-treatment. More hES-like colonies was observed in low density (starting with 5×104 fibroblasts) compared to high density (starting with 5×105 fibroblasts) Thee hES-like cells have a large nuclei and a sparse cytoplasm, and are the proposed iPS cells.

iPS cells express hES markers
To test its similarity to hES cells, Yamanaka next tested for markers of differentiated cells and hES cells. The iPS cells expressed hES-specific surface markers such as SSEA-3, SSEA-4, tumor-related antigen (TRA)-1-60, TRA-1-81 and TRA-2-49/6E (alkaline phosphatase), and NANOG protein, and cell-markers genes such as Oct3/4, Sox2, Nanog, growth and differentiation factor 3 (GDF3), reduced expression 1 (REX1), fibroblast growth factor 4 (FGF4), embryonic cell-specific gene 1 (ESG1), developmental pluripotency-associated 2 (DPPA2), DPPA4, and teomerase reverse transcriptase (hTERT) were expresses at equivalent or higher levels than those found in hES. In contrast, it did not express stage-specific embryonic antigen SSEA-1. Global gene expression shows similar but not identical profiles, with 1267 genes (out of 32266 analysed) showing greater than 5-fold difference between iPS cells and hES cells.

Promoters of ES cell-specific genes are active in human iPS cells
Activity of a gene can be determined through epigenetic analysis of its promoter, looking at epigenetic marks such as the methylation state of CpG islands and the histone modifications and variants. iPS cells shows low levels of methylation of CpG dinulceotides in the promoter regions of pluripotent-associated genes, such as OCT3/4.REX1, and NANOG; this is in contrast with HDFs, which shows a high level of methylation on these genes. Methylation of DNA can only occur on the carbon-5 position of cytosine of a CpG dinucleotide, and methylation of DNA at promoter regions is associated with gene silencing, probably because transcription factors can no longer bind to methylated DNA sequences to activate transcription. Bisulfite sequencing treats DNA with bisulfite to convert cytosine residues to uracil, but 5-methylcytosine is not affected. The bisulfite-treated DNA can then be directly sequenced and compared to the untreated DNA to identify the methylation sites. The level of transcription of pluripotent-associated genes such as OCT3/4 and REX1 was high in human iPS cells compared to HDF, whereas ubiquitously-expressed genes such as PolII shows similar levels in both iPS cells and HDF.

Immunoprecipitation of the promoter regions of pluripotency-associated genes (Oct3/4, Sox2 and Nanog) of iPS cells shows high levels of H3K4 trimethylation (associated with gene activation), and low levels of H3K27 trimethylation (associated with gene repression), compared to HDF cells. In iPS cells, development-related genes, such as Gata6, Msx2, Pax6, and Hand1, shows similar levels of methylation on both H3K4 and H3K27, which is characteristic of hES cells, as hES must maintain pluripotency by not expressing cell-type specific genes.

iPS cells are able to differentiate into different cell types
iPS cells were cultivated in floating culture into embryoid bodies (EBs) to test for their ability to differentiate into different cell types. EBs were observed after 8 days, at which point they were transferred onto gelatin-coated plates and cultivated for a further 8 days. The resulting cell plate showed a variety of cell types, representing all three germ layers - high levels of βIII-tubulin, glial fibrillary acidic protein (GFAP) and the high levels of expression of MAP2 and PAX6 represents the ectoderm; presence of α-smooth muscle actin (α-SMA) and desmin plus the high levels of expression of BRACHYURY and MSX1 illustrates mesoderm cells; and presence of α-fetoprotein (AFP) and high level of expression of FOXA2, AFP, cytokeratin 8 and 18, SOX17 represents the endoderm. The dfferentiated cells also show depletion of OCT3/4, SOX2 and NANOG expression.

It has also been shown that the differentiation process can be directed, at least for neural and cardiac cells, using the same methodology as used for hES cells.

In mouse cells at least, these differentiated cells derived from iPS cells can be reprogrammed unlimited times.

Other hES-like traits
Human iPS cells shows a high level of telomerase activity through its high expression of hTERT. When the iPS cells were transplanted into dorsal flanks of immunodeficient (SCID) mice, teratoma was observed after 9 weeks, and confirmed by histology to contain cells of all three germ layers.

Mechanism
Oct3/4 and Sox2 are transcription factors which activates genes associated with stemness, while suppressing differentiation genes. They lose their function in differentiated cells because of methylation, histone modification, or binding of other factors which prevents the action of Oct3/4 and Sox2. Klf4 is known to interact with p300 histone acetyltransferase and promotes acetylation associated with gene activation; thus Klf4 might modify the structure of chromatin to allow Oct3/4 and Sox2 to operate again, again transcribing stemness genes. This is supported by the fact that inhibiting HDACs (which deacetylates histones) increased iPS generation efficiency. c-Myc has been shown to induce differentiation and apoptosis, and might not be involved in the self-renewal properties of stem cells, but instead to promote differentiation when it is signaled to do so. It has since been shown that c-Myc is not required for reprogramming. The results show less background cells and the iPS cells generated were of higher quality and did not develop tumours.

These four factors are needed initially but is later silenced when endogenous Oct4, Myc, Sox2 and Klf4 are re-activated after a certain time. From profiles of expression of 48 genes in single cells shows a marked variation in gene expression in early stages of reprogramming compared to late stages. This means that the four endogenous factors may signal through some mechanism to activate another mechanism which maintains the stemness. Gene expression early on in reprogramming shows a stochastic characteristic whereas later on it is more hierarchical with Sox2 directing the hierarchy.

Implications and Limitations
Human iPS cells were similar to human embryonic stem cells in morphology, proliferation, surface antigens, gene expression, epigenetic status of pluripotent cell-specific genes, and telomerase activity. The iPS cells are able to form all three germ layers and can form teratomas.

The iPS cells derived were derived from the HDF tissue. PCR of genomic DNA of the HDF and the clones reveal that each clone has the transgene incorporated three to six times, and each clone has an unique pattern of integration. The patterns of 16 short tandem repeats also identity with the original HDF, which is highly unlikely to occur by chance. However, the possibility of those cells originating from undifferentiated stem or progenitor cells coexisting in the fibroblast culture cannot be ruled out.

Such high levels of retroviral integration increases the chance of a negative mutation which might lead to tumorigenesis. Other methods might be used for introducing the factors, such as adenoviruses (which do not integrate into the genome) and the use of cell-permeable recombinant proteins, as the factors only need to be present for a short period of time before the cell produce these stem cell factors endogenously.

Comparing the level of transduction compared to the proportion of iPS cell colonies, shows that not every transduction event results in induced pluripotency. This might mean that the genes must be inserted into specific loci, or loci which do not interfere with other genes, in order for pluripotency to be induced.

Yamanaka has shown that pluripotency can be induced into human somatic cells using the same four factors as mouse, but the maintenance of pluripotency requires different factors - bFGF is essential to maintain pluripotency in hES cells, and LIF/Stat3 pathway is essential for pluripotency in mES cells; the same is true for differentiation signals, where BMP induces differentiation in hES cells but maintains self-renewal in mES cells. This means that the fundamental transcriptional network governing pluripotency is common in human and mice, but there are differences in the extrinsic factors which signals for maintenance of pluripotency as opposed to differentiation.

However similar, iPS cells are not identical to ES cells, and several iPS clones have shown abnormalities in mRNA and miRNAs, especially in imprinted genes.

Cancer stem cells
Stem cells are long-lived and so throughout its lifetime will accumulate many mutations. Mutations in non-stem somatic cells are tolerated, because the cells will soon die off; however mutations in the stem cell population will affect all its progeny, and thus more likely to cause cancer. Luckily, stem cells do not divide very often, some stem cells are only known to replicate less than ten times; since most mutations arise during DNA replication, at least in this respect mutations in stem cells are limited.

Cancer stem cells are derived from mutations in cells which gives the mutated cell the ability to proliferate indefinitely. This mutation can occur at different stages. If it occurs at the stem cell stage, all of the cancer stem cell's progeny will gain the function to proliferate indefinitely and causes a tumour; if it occurs at the nearly-differentiated cell stage, the nearly-differentiated cell will gain the ability to proliferate indefinitely, and so by definition becomes a cancer stem cell. Thus, cancer stem cells can be derived from almost any cell type, and might not be identical or even similar to the stem cells of the tissue the cancer derives from.

The generation of cancer stem cells from normal tissues are not likely to be due to a single mutation event, but rather an accumulation of many events, leading a normal tissue into clonal cells, then into abnormal clonal cell (not persistent), then into a neoplastic cell (persistent), malignant cell, and finally to cancer stem cell.

Cancer stem cells have self-renewal properties by dividing asymmetrically, interacts with the CSC niche to maintain its stemness, and escape chemotheray by locating themselves around stromal cells, which absorbs most of the chemotherapeutic agents.

Within a tumor, the majority of tumor cells have limited ability to proliferate. a small population of cells, cancer stem cells (CSCs), within some tumors possess the ability to self-renew and proliferate and are thus able to maintain the tumor. The terms 'cancer initiating cells' and 'cancer stem cells' are often used interchangably; however, non-cancerous stem cells and progenitors are also cancer initiating cells. Progenitors are already proliferating heavily and so can be easily upregulated to induce uncontrolled proliferation, leading to cancer; acutemyeloid leukemia most likely to be originated from progenitor cells. Therefore, to eradicate a tumour, the CSCs must be eradicated. In normal tissues, cells replicate and differentiate towards the terminally differentiated state; however, some cancer cells are thought to be able to reverse differentiation.

Epithelial-mesenchymal transition (EMT) are transdifferentiation programs that are required for tissue morphogenesis during embryonic development, regulated by a diverse array of cytokines and growth factors, such as transforming growth factor (TGF)-beta. When these factors are dysregulated, epithelial tumour cells can undergo EMT to become vascularized and metastasize, changing from a flat shape to spindle shaped so it becomes more invasive. Once it has metastasized, it will undergo mesenchymal-epithelial transition (MET) to return back to epithelial tumour cells. Cells which have undergone EMT generate cancer more efficiently, maybe due to the fact that the epithelial cells have reverse differentiation to become a more potent cancer stem cell, able to replicate and differentiate more efficiently, causes increased metastasis, and contributes to drug resistance. Furthermore, it has been shown that activation of the Ras-MAPK pathway was also able to generate CSCs from human mammary epithelial cells. Thus, even eradicating the CSCs alone might not be enough to eradicate the tumour - the whole tumour has to go or else the chance of relapse cannot be abolished.

Relapses are probably caused by cancer stem cells. When we use chemotherapy, we only kill the progeny of the stem cell, and so the parental stem cell population can still divide and differentiate after chemotherapy treatment.

How cancer cells can originate from differentiated and stem cells.

Therapies - pros and cons

Wnt signalling elongates telomeres of cancer cells and allow them to not age. Telomerase-targetting drugs such as GRN163L have already been in clinical trial.