[Lecture Notes] Fundamentals of Biotechnology - L18: Cloning and stem-cells

Stem cells

Stem cells are undifferentiated cells with the ability to become other types of cells depending on their environment. While undifferentiated they are able to keep dividing, whereas specialised cells mostly lose that ability.

The ultimate stem-cell is a zygote which is totipotent, it divides to form every single cell in your body. Taking cells from the inner cell mass of a blastula yields embryonic stem cells, which are able to differentiate into any type of cell. Stem cells can divide indefinitely, produce unique stem cell transcription factors and they produce daughter cells which can differentiate into specialised cells. Stem cell lines are routinely tested for these characteristics to ensure that they truly remain stem-cells, as changes in their environment may cause them to differentiate.

Different growth factors can induce the stem cells to start differentiating into certain types of cells; they form embryoid bodies and start becoming muscle or neurons etc.

Somatic stem cells can still differentiate, produce offspring which can differentiate, and can divide indefinitely, and can repopulate tissue, but we call them multipotent. The type of somatic stem cell governs the extent of its differentiation. Muscle stem cells can only differentiate into different types of muscle tissue for example.

Fate mapping to identify adult stem cells.

Stem cell types in adults are unique, and there is no one marker which can identify any stem cell. Fate mapping labels a suspected stem cell and then tracks its progeny over subsequent generations. Normal cells do not divide by mitosis and most do not divide at all (postmitotic cells), and so if it is a true stem-cell, the marker will be passed down to the daughter cells, and those daughter cells will pass down the marker as well. Markers used are typically GFP or beta-galactosidase (visualised with X-GAL media).

Transplantation

One can mark stem cells with GFP and transplant them into a developing embryo. As the tissue grows one can track where the stem-cells go and differentiate and can keep identifying the stem-cells throughout their lives.

Stem cell niche

The stem cell niche is a micro-environment that protects the stem cells from differentiating and essentially keeps them growing. Niche cells maintain the micro-environment, secreting chemicals which stop the stem-cells from differentiating. Stem cells themselves can secrete autocrine signals which stop themselves differentiating. Maintaining the environment is important as over-proliferation of stem-cells leads to tumours, but loss of stem cells means no more tissue regeneration can occur.

Asymmetric cell division

Stem cells divide to produce a differentiated daughter cell and another stem cell. Intrinsic asymmetry is due to signals between daughter cells, one receives a signalling molecule and the other does not, and extrinsic asymmetry, where one daughter cell may be physically pushed out of the niche and into an environment causing it to differentiate. Symmetric renewal produces two stem cells and symmetric differentiation is the opposite, producing two differentiated somatic cells.

A stem cell is kept in its niche because it adheres to the extracellular matrix which anchors the cell within the niche.

Hematopoietic stem cells

These are stem cells found in the bone marrow. The interface between bone marrow and bone is called the endosteum, and contains endosteal cells. This area is an endosteal niche. HSCs are important because they replenish the red and white blood cell supply of our body. There are two HSC populations: one constantly self-renews and the other stays dormant and maintains itself. The renewing HSCs can become either lymphoid or myeloid progenitors. Myeloid progenitors form erythroid lines which produce RBCs and macrophates. The lymphoid progenitors produce T cells and B cells. (fig 18.13)

Induced pluripotent stem cells

It has been found that differentiated tissue can be converted back into pluripotent stem cells. These are called induced pluripotent stem cells (IPSCs). They have the characteristics of stem cells, dividing indefinitely and differentiating when exposed to various hormones/factors. Fibroblasts can be undifferentiated and then re-differentiated into muscles or neurons for example.

To make an IPSC one needs to induce the expression of four factors: OCT4, SOX2, NANOG and LIN28. Upon expression the cell will become undifferentiated.

Stem cell therapy

HSC transplants can be used to save people with radiation sickness, conferring upon them the ability to make blood again after their hematopoietic have been destroyed. White blood cell cancers can be treated with HSC transplants. The donor of the HSCs can be the patient themselves, if the cells are harvested from the body before undergoing chemotherapy. Once chemotherapy is over, the cells can be put back into the bone marrow. This is autologous bone marrow.

If this is not possible then allogenic bone marrow needs to be used. A match between the donor and patient need to be found. In some cases people who are not related can match with one another but most likely it will be a family member. The match needs to have the same blood type and similar human leukocyte types.

Other sources of HSCs may come from blood of the umbilical cord. One can pre-emptively freeze and store these cells upon birth should the person ever need them. Because these blood cells are even less mature than bone marrow cells, they do not need to be as closely related to the recipient’s cells.

If the patient is injected with granulocyte-colony stimulating factor (GCSF) then some of their HSCs will migrate out of the bone marrow and into the blood stream. A large enough dose of this cytokine will yield enough HSCs in the blood stream (up to 20% of cells) to harvest, and this method is less invasive and painful.

In general terms (more info in the book): some types leukaemia can be cured with bone marrow transplants. Read the textbook

Cloning: Dolly the sheep

Udder cells from a donor animal were taken and starved so that the DNA stopped dividing. G0 nuclei are resting, and so when one of these nuclei is taken and put into an egg cell (fused with electroporation) which has had its own nucleus removed, this new hybrid cell starts dividing again. This egg cell is then put back into the foster mother and an embryo can develop. This is the principle used to clone Dolly the sheep. (fig 18.17). Dolly was not a perfect clone because the mitochondrial DNA was left in the egg cell, so she had the mitochondrial DNA of one sheep, and the nuclear DNA of another.

Practical applications of this technology: standardisation of quality among livestock products can be achieved, however epidemics become more of an issue due to there being no variability. Cloning can keep endangered species alive without diluting their genes with crossbreeding. Transgenic animals can be cloned to produce more product faster.

References: my notes are made from, and follow the structure of my course textbook which is Biotechnology 2nd edition by David P. Clark, which can be found for purchase here.