Stem cell bones are getting closer to the clinic

Bone grafts can be obtained from reprogrammed human cells

Yulia Kondratenko, "Elements"

For the first time, American researchers have gone all the way to transform specialized human cells (skin fibroblasts and bone marrow cells) into bone tissue cells. To change the specialization of cells, they were first turned into induced pluripotent cells – cells that could potentially acquire different specializations. Bone tissue cells were obtained from induced pluripotent cells, which were grown in a special bioreactor to obtain artificial bones. The grown bones had the properties of natural human bones – they had a similar cell density, contained the necessary proteins, and isolated the necessary extracellular substances. 12 weeks after implantation to mice, the resulting bones retained their properties.

To replace the damaged organs and tissues of the patient – transplantation – donor organs, artificial transplants, as well as autologous transplantation (transplantation of the patient's own cells) are now used. All these methods have obvious drawbacks: donor organs can be attacked by the body's immune system, artificial transplants are not in all respects similar to natural organs, and autologous transplantation is not applicable if the patient has pathologies of tissues necessary for transplantation, and besides, it is not always convenient due to damage to areas of the body, from which cells are taken from.

That is why there was a dream of creating the required organs from their own cells, minimally injuring the patient himself. Such organs, as well as autografts, will not cause immune rejection and will have all the properties of natural organs. Work in this direction is already underway: for example, an article was published in 2010 (see: Grayson et al., 2010. Engineering anatomically shaped human bone grafts), the authors of which managed to grow a fragment of the mandibular bone from the mesenchymal stem cells of the patient isolated from the bone marrow (Fig. 1).

Fig. 1. The process of obtaining a fragment of the mandibular bone from the mesenchymal cells of the patient's bone marrow.
A, B – shows the fragment, a copy of which was required to be obtained.
C is the basis for growing bone obtained from calf bone.
D is the type of the required fragment in different projections, E is the scheme of the bioreactor,
F is the bioreactor chamber in which the bone growth base is placed.
A nutrient medium is fed into the holes, stimulating the transformation of mesenchymal cells into bone tissue cells.
Illustration from Grayson et al., 2010.Mesenchymal cells are progenitor cells capable of developing into bone tissue cells, cartilage cells and fat cells.

To obtain bone, it was only necessary to start the process of converting these cells into bone tissue cells and grow them on a special basis of anatomical shape.

Unfortunately, the isolation of mesenchymal cells is a rather laborious process, and therefore the next step was to obtain bones from those cells that are easier to get. However, such cells are not natural precursors of bone tissue cells, therefore, in order to obtain artificial bone, the isolated cells must be "reprogrammed".

Theoretically, nothing is impossible in this, because every cell of our body carries all the information necessary for the functioning of any cell types. However, in specialized cells, most of this information is not used: for example, there are genes whose work makes a nerve cell a nerve cell, but all these genes are silent in liver cells, in retinal cells, in heart muscle cells and in all other cells with a different specialization. To change the specialization of a cell, you first need to "turn off" its current specialization – to transfer it from a differentiated (specialized) state to a pluripotent one (from Latin pluralis – multiple, potentia – strength, power, opportunity, in a broad sense can be translated as "the possibility of development under different scenarios"). This is done by introducing DNA molecules encoding four pluripotency factors into a differentiated cell – it turned out that only four factors are enough to disable the current cell specialization and transfer it to a state from which it can turn into a different type of cell. The cells obtained in this way are called induced pluripotent cells. From induced pluripotent cells (Induced pluripotent stem cell, iPS cells, iPSCs), by adding special factors, cells of the required type can be obtained.

The authors of the work recently published in the journal PNAS (2013. V. 10. P. 8680-8685) (see its popular retelling in the article "Full–fledged bones from stem cells" - VM) managed to carry out all this sequence of actions and get an "artificial" bone. They used fibroblasts and bone marrow cells as starting cells, converted these cells into pluripotent cells, then transferred them to mesenchymal cells capable of being precursors of bone tissue cells, cartilage cells and fat cells. Bone tissue cells were obtained from mesenchymal cells by adding a special medium. In order to form a bone of the required shape and structure, the cells need to be grown on a special basis, which is a fragment of calf bone completely cleared of cells.

For the formation of bone tissue similar to natural, a small amount of mesenchymal cells is applied to the base of calf bone. After that, the future bone is in a special bioreactor for five weeks, where the growing cells are regularly washed with a nutrient medium. The medium should also contain factors contributing to the transformation of mesenchymal cells into bone tissue cells. The characteristics of the medium flow affect the properties of the resulting bone, and by changing the flow of the nutrient medium through different parts of the resulting bone, it is possible to obtain, for example, different cell densities in different parts of it. A detailed study of the influence of the properties of the medium flow on cell growth is important when growing anatomically shaped bones.

It turned out that artificially obtained human mesenchymal cells can successfully populate such a base and, when grown in an appropriate environment, can turn into bone tissue cells. The resulting specialized bone cells will secrete the necessary extracellular components, completing the formation of artificial bone.

To check the immutability of the properties of the obtained bones, they were implanted under the skin of mice, extracted after 12 weeks and checked whether the properties of the bones remained the same. Since the bones were grown using human cells, and experiments with implantation were carried out on mice, so that the transplanted bone was not attacked by the immune system, a line of immunodeficient mice was used in the experiments. It turned out that in a living organism, artificially grown bones remain stable – that is, the cells remain viable, and the same genes work in them as before implantation. Interestingly, in 12 weeks, the bone began to be perceived by the body as its own: vessels sprouted into it and host cells-osteoclasts, engaged in the restructuring of bone tissue, were found in it. Capsules of loose connective tissue were formed around the bones (Fig. 2).

Fig. 2. Micrographs of bones after twelve weeks of implantation.
Arrows indicate vessels, asterisks mark osteoclasts (bone tissue cells).
Photos from additional materials to the discussed article in PNASDuring implantation, the mineralization of the implanted bones also increased (Fig. 3).

Fig. 3. 3D models of artificially created bones obtained by computed tomography.
The names of the cell lines from which induced pluripotent cells were obtained are marked on top.
The upper row shows the bones before implantation, in the lower row — after twelve weeks of implantation.
It can be seen that during implantation, the bone substance has become more dense, that is, the formation of bone tissue
it continued after implantation. Figure from the article under discussion in PNASThe authors also studied which cells are better to take as a basis to grow artificial bones.

The work compared the properties of bones obtained from bone marrow cells, as well as from skin fibroblasts. In addition, when obtaining pluripotent cells, different methods of delivering DNA fragments encoding pluripotency factors were used – different vector molecules.

To deliver foreign DNA into the cell, viral DNA molecules are often used, in which all the genes responsible for the reproduction of the virus are removed and some other gene is inserted instead. Such a virus (viral vector) is non-infectious, but it is able to introduce a new gene into the cell, included in the viral DNA.

The set of factors itself also differed in different experiments. It turned out that the induced pluripotent cells obtained by different methods result in bone tissue with different properties – the activity of genes important for the work of bone tissue cells slightly differed in the resulting cells, the amount of calcium deposited and the rate of cell growth in the bioreactor differed. Bone tissue cells obtained from fibroblasts with the introduction of pluripotency factors OCT4, SOX2, KLF4 and C-MYC using a vector based on the human-safe "mouse" Sendai virus had the best indicators.

Unfortunately, it is not yet clear which types of cells are most suitable for growing bones of real patients: so far, all experiments have been conducted only on mice and on model cultures of human cells, not to mention that serious comparative studies are required to select optimal cells. In addition, the mechanisms by which cells of different types are reprogrammed into bone cells in different ways are unknown. The situation is similar with pluripotency factors and vector molecules – scientists are still only selecting their optimal combinations, but it is still difficult to talk about why such a combination is good, and what are the mechanisms of its influence on the future of the cell.

Nevertheless, the work has taken an important step towards obtaining the organs required by the patient from his own cells, which are easy to isolate. Now it is clear that at least for bones, this technology is quite applicable. It is still difficult to say whether it will be possible to obtain other organs by this method – for example, those with complex innervation or the structure of blood vessels. Nerve cells and vascular endothelial cells develop along special paths, and various types of progenitor cells will need to be added to grow them. At the same time, it will be necessary to somehow force them to form the correct structure inside the growing organ. However, it seems that these tasks, although difficult, are solvable, and that we have every reason to believe in the rapid development of a new type of medicine.

Source: de Peppo et al., Engineering bone tissue substitutes from human induced pluripotent stem cells.

Portal "Eternal youth" http://vechnayamolodost.ru12.07.2013