Stem Cells

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Stem cells

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have the ability to divide by which they can form a copy of themselves and a cell that differentiates. How many and which kind of cell depends on the type of stem cell → embryonic stem cells, somatic stem cells, iPS induced pluripotent stem cells.

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Embryonic stem cells

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A fertilized egg cell has the potential to develop in all cell types of a certain species. Therefore this is a totipotent cell. Embryonic stem (ES) cells are pluripotent which means that they have the potential to form all cell types (>200 different cell types in a mouse body) except the placenta. This are the cells of the inner mass of an early (mouse) embryo (blastocyst stage).

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Somatic stem cells

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stem cells that occur in specific niches, they are multipotent and can develop in a few cell types. Stem cells and cells directly derived from them are mitotically active. Stem cells divide but slower than the cells of the proliferative region → divide a few times and then differentiate (transiently amplifying cells). The Paneth cells next to the stem cells form an organizer, secreting signals to keep the stem cells identity.

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Renewal of the small intestine

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dividing stem cells lie in protected regions deep in the crypts, they generate absorptive cells an secretion cells.

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Molecular signals that organize the whole stem-cell system

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tumours arise from stem cells that have lost both copies of Apc. It codes for a protein that prevents inappropriate activation of the Wnt signaling pathway. Loss of Apc means a continuous activation of Wnt. Wnt normally keeps the crypt cells in a proliferative state, this needs to stop when they leave the crypt. Normal APC increases the affinity of the degradation complex for B -catenin, which in excess can enter the nucleus and promote transcription of key target genes for cell proliferation.

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Delta-notch signaling

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within the crypt population mediates lateral inhibition, which forces the cells to diversify, in such a way that secretory cells become surrounded by absorptive cells. Notch signaling is a competitive interaction that drive neighboring cells to different fates. Wnt signaling switches on the expression of all components necessary for Notch signaling. Without Notch signaling all cells stop dividing and differentiate into secretory cells. On the other hand when Notch signaling is activated in all cells, no secretory cells are formed. Wnt signaling promotes cell proliferation and confers competence for the full range of modes of differentiation, while preventing differentiation from occurring immediately.

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Lateral inhibition by notch signaling

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when a precursor cell commits to becoming a secretory cell, it
signals to its immediate neighbors not to do the same; the inhibited cells develop into epithelial cells instead. This is activated by a transmembrane protein called Delta on the secretory cell which bind to a Notch receptor on neighbouring cells to signal it should not become secretory. Notch is proteolytically processed to function. The tail translocates to the nucleus to bind a DNA-binding protein which will activate genes.

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In vitro embryonic stem cells

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when grown on a feeder layer of fibroblasts they remain their pluripotent nature. They can form embryoid bodies like organoids, the cells of these bodies can be induced to differentiate.

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Signals of fibroblast feeder layer

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the molecules secreted by these cells are sufficient to maintain stemness. Cytokine LIF, BMP4 and Wnt play a role.

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Genes involved in pluripotency

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Oct 4 (Octomer, POU domain transcriptional regulator). Nanog (Nan Og = homeodomain transcription factor). Sox2 (a high mobility group box transcription factor). Without Oct4 and Nanog the cells immediately differentiate.

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Chip on chip technology has been used to identify which genes are targets of these transcription factors

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Transcription factors are cross linked to chromatin and subsequently by immune precipitation the regions interacting with a certain TF are precipitated, which genes interact with the specific TF can be identified for example by micro array analysis. The TFs interact with themselves (positive feedback) and genes involved in early differentiation (repressed).

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Fibroblasts can be reprogrammed to create induced pluripotent stem cells (iPS)

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by introducing Oct4, Sox2, Klf4 and Myc into fibroblasts they are reprogrammed permanently into cells similar to ES cells. This means ES cells can be derived from adult human cells. The conversion is slow and inefficient however

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Cascade of changes in iPS cells

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the conversion is induced by a long cascade of changes like the expression of individual genes and changes in chromatin state. Myc loosens the chromatin to promote binding of the other master regulators in concert → leads to positive feedback so the signal is sustained. They also repress and activate a whole lot other genes and change the epigenetics. At the end the introduced factors are no longer necessary and the iPS is self-sustaining and making the necessary proteins from its own endogenous genes.

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ES and iPS cells can be guided to generate specific adult cell types

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in culture they need to be exposed to an appropriate sequence of signal proteins and growth factors delivered in the right time. They can even form embryoid bodies.

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Organoid formation from somatic stem cells

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Lgr5 (target gene of Wnt) is only expressed in the crypts. More specifically the cells expressing Lgr5 are the stem cells of the small intestine and colon. Noggin could be used to form organoids from crypt cells.