SCs different tissues Flashcards
How much time does the intestinal epithelium need to regenerate completely?
Three to five days.
What are the intestinal stem cells and what are the niche cells?
The crypt base cells (CBC) are the intestinal SCs and the Paneth cells the niche cells.
How was the proliferation potential of the intestine discovered?
Proliferation can be detected using BrdU (DNA base analogon) which can be incorporated in the DNA when there is cell replication. The incorporation can be detected with anti-BrdU antibodies. With this assay was found that proliferation in the intestine happens in the crypt.
Explain lineage tracing.
With lineage tracing, an inheritable and inducible marker is used to track the fate of cells and their daughter cells. A cell is extracted, genetically modified with the marker, and transplanted in a recipient. If a stem cell is labeled, the whole tissue unit will eventually be labeled.
How was lineage tracing used to prove that crypt base cells are the intestinal SCs?
The Cre-ERT2 system was used in cells expressing Lgr5 (only expressed in CBCs), by placing the Cre-ERT2 gene under the control of a Lgr5 promotor. ERT2 is inducible by tamoxifen (4-OHT). When cells are induced, the Cre-ERT2 complex migrates to the nucleus where Cre cuts out the stop codon before the Lac-Z gene, making the cells turn blue. The daughter cells inherit this genomic change, making it possible to track the lineage.
A low concentration of tamoxifen was used to induce several crypts, which turned all daughter cells and eventually the whole villi blue. This proved that Lgr5+ cells (CBCs) are the intestinal SCs.
How was proven that intestinal SCs compete and outgrow each other?
Multiple lineage tracing was used to prove that intestinal SCs outgrow other SCs in the same crypt. Lineage tracing of several SCs was done with different colors. The different colors were obtained by inverting the IoxP sequence, leading to expression of different fluorescent markers. After several weeks the crypts only show one color.
What is the function of Lgr5 in CBCs?
Lgr5 is a receptor which is co-expressed with Lgr4. A double knockout of Lgr4 and Lgr5 leads to cell death. Lgr4/5 are receptors for Rspondin, which is a cytokine produced by the Paneth cells. Binding of Rspondin prevents degradation of Wnt receptors, preventing the degradation of ß-catenin, leading to stronger and longer Wnt signaling.
Where are the skin SCs found and what is their niche?
Different parts of the skin are maintained by their own SCs and their exact roles are analyzed. The epidermal SCs are found in the basal layer of the interfollicular epidermis. They are organized into proliferative units and differentiate while migrating upwards.
Another population of SCs in the skin are the hair follicle SCs. They are found in the bulge of the follicle and their niche is the mesenchymal cells of the dermal papilla.
How many divisions can HSCs and epidermal SCs undergo when culturing and expanding them?
Epidermal SCs can divide more than 180 times in culture. HSCs are challenging to expand in culture.
How was demonstrated that bulge cells are SCs?
The bulge cells are the SCs in the hair follicle. This was demonstrated by transplantation assays, where single bulge cells were cultured and implanted in the skin of nude mice. Different cell types were marked using lineage tracing, showing the multipotency. The assay was repeated over multiple lineages of mice (serial transplantation), showing the long-term proliferation potential.
A second test was done to prove the multipotency of the bulge cells. The bulge region was removed from a non-marked follicle from a wild-type mouse and the bulge region of a marked donor follicle was inserted. This chimeric follicle turned out to be completely marked, proving multipotency of bulge SCs.
How does skin SC treatment work and what are the challenges?
Epidermal SC treatment is used for treating severe burn wounds. 1-5 cm2 healthy skin is needed. The cells are expanded on a feeder layer of mouse fibroblasts or fibrin matrix for 2-3 weeks and transplanted onto the patient with surgical glue. The treatment leads to long-term survival of many patients, but the quality of life is low. This is because only the epidermis is regenerated, leading to absence of hair, sweat glands and sebaceous glands.
Where are the corneal SCs found and what is their niche?
The SCs are located in the limbus of the cornea. Their niche is not defined.
How does cornea transplantation work?
The treatment is used for patients with loss of limbus SCs due to injury or disease. Isolate limbus SCs of a healthy part of the eye and expand them on fibrin substrate to generate in vivo cornea. Remove the injured area in the eye and transplant on the corneal region. The treatment can lead to full recovery of visual activity.
Where are the breast SCs found and what is their niche?
The SCs are present everywhere in the tissue and are called mammary gland stem cells. Their niche is not defined.
How was demonstrated that there are SCs in the mammary gland?
Different breast cell type populations were identified and separated. These populations were tested with different methods:
- Generation of colonies in adherent cultures
- Generations of mammospheres in non-adherent cultures
- Serial transplantation assay of breast epithelium into fat pad
