20 October 2015

Safe induced pluripotent stem cells

IPSK fuse

Dmitry Dzhagarov, "Biomolecule" 

Biomolecula has repeatedly published materials on the revolutionary technology of reprogramming somatic cells into rejuvenated stem cells called induced pluripotent stem cells (iPSCs). It would seem that humanity has received a fountain of eternal youth and health. However, doctors are in no hurry to use these wonderful cells to heal patients, as treatment with their help turned out to be associated with the risk of getting cancer. How to reliably protect patients who need therapy with cells derived from IPSC? This mini-review is dedicated to the latest developments and achievements in solving this problem.

For a long time, the painful isolation of stem cells from the human body and the lack of high-quality methods of their reproduction in culture held back the breakthrough in cell therapy. However, not so long ago, a revolutionary technology for reprogramming somatic cells into rejuvenated stem cells, called induced pluripotent stem cells (iPSCs), was introduced to the world. Its main features are the "reproduction" of stem cells and their transformation into various specialized cells, and besides, along the way (if necessary) the implementation of gene therapy to correct mutant genes [1].

Moreover, it turned out that with the help of reprogramming in IPSC, it is possible to obtain completely updated stem cells from the cells of old and even super-old (centennial) patients, capable of differentiating into young somatic cells [2]. Therefore, the technology of obtaining IPSC should also be considered as a method of radical rejuvenation [3]. It is assumed that with the help of these cells it will be possible to fight diseases previously considered incurable [4]. For example, the rejuvenated mesenchymal cells obtained from IPSC can be used to treat the heart, kidneys, nervous tissue, joints, for bone regeneration and treatment of inflammatory diseases, as well as to suppress the rejection reaction during transplantation [5], since they differ little from stem cells from bone marrow tissue [6].

Despite these and many other evidences of the beneficial (therapeutic) effects of cells obtained from IPSC, they have not yet been "adopted" in the clinic, since there are serious obstacles to their use for therapeutic purposes – the danger of tumor formation and the immunogenicity of pluripotent cells [7]. Injecting a patient with his own iPSCs usually leads to an immune reaction that prevents their engraftment, or even to the formation of teratoma-type tumors. This ability of iPSCs to form tumors is associated with the failure of the mechanism of epigenetic rearrangement, and not with irreversible genetic mutations [8], and can be predicted by the activation of certain signaling pathways [9].

This oncogenic and immunogenic potential, as it turned out, is usually due to the presence of undifferentiated iPSCs. Therefore, before using these cells in therapy, it is important not only to carry out a complete differentiation of all iPSCs, but also to thoroughly clean the material intended for transplantation from pluripotent cells [10, 11]. Many methods of such purification have been developed that significantly reduce the risk of tumor formation [12, 13]. Nevertheless, even the most sophisticated pre-purification is not able to secure the planting of material obtained from iPSCs, since after selective removal of pluripotent cells, they can reappear by converting differentiated cells back into stem cells (which, for example, hypoxia can push) [14, 15]. And because of this, there is again a risk of tumor formation. Usually, such tumors are not detected immediately, but only with prolonged observation [16].

Since the cause of such spontaneous transitions may be the genomic instability of the obtained iPSCs, it is important to reduce the level of DNA damage and the number of genomic rearrangements in them by limiting the replicative stress caused by reprogramming – for example, by increasing the level of checkpoint kinase 1 (CHK1) and adding free nucleosides to the reprogramming medium [17].

In order to protect the body from the risk associated with the possibility of tumor formation during cell therapy using iPSCs or differentiated cells derived from them, an emergency fuse is needed that allows you to get rid of these cells with great reliability if something goes wrong.

Such a fuse could be selective apoptosis of all cells obtained from IPSC, such that the remaining cells of the body would not experience severe intoxication.

For this purpose, an apoptosis activation system was created based on the thymidine kinase (TC) gene of the herpes simplex virus (HSV) and a rather toxic and mutagenic drug ganciclovir, an analog of guanine. When exposed to TC-HSV, ganciclovir is phosphorylated into monophosphate, which is then converted into triphosphate by cellular kinases. The latter inhibits DNA synthesis by competitively suppressing DNA polymerase. The peculiarity of this system is that apoptosis is observed only in dividing cells. In order for apoptosis to affect not all dividing cells, but only pluripotent cells with transcription regulators characteristic only for them, promoters of transcription factor genes Oct-4 [18] or Nanog [19] are used as a promoter for the TC-HSV gene. Tumors formed from such modified pluripotent stem cells can be inhibited by the administration of ganciclovir. However, the disadvantage of such a system is that it allows slowly dividing cells to survive, which is why treatment with a toxic and mutagenic drug has to be stretched for a long time [20], and this reduces the chances of successful treatment.

As an alternative, another apoptosis activation system has been developed. In order to make the use of iPSCs in the clinic safe, it was proposed simultaneously with the reprogramming of the patient's cells to inject the gene of induced caspase 9 (iCasp9) into them on the same cassette as the reprogramming factors. Genetically engineered caspase 9 is activated by a drug (AP1903 or AP20187) that causes dimerization of its molecules. As a result, apoptosis begins in 94-99% of cells containing iCasp9 [21], leading to the death of these cells (Fig. 1).



Figure 1. Model of the system of induced apoptosis. The iCasp9 "cell suicide" gene was constructed from DNA sequences encoding: 1) tacrolimus-binding dimerization domain FKBP12 with mutation F36V, increasing affinity; 2) linker sequence of amino acids (Ser–Gly–Gly–Gly–Ser), which provides flexibility of design; 3) human caspase 9, devoid of physiological dimerization domain (ΔCasp9) [24]. Due to this design, the dimerization of the genetically engineered protein iCasp9, necessary for the induction of enzymatic activity, occurs only with the addition of a small synthetic molecule (for example, AP1903) interacting with the FKBP12 domain. Thus, in order to "turn on" the enzymatic activity of the caspase that triggers the apoptosis cascade, initiation through the preliminary mitochondrial pathway of apoptosis is not needed – it is replaced by a molecule dimerizing the caspase. Such a "fuse" will allow you to get rid of rejuvenated cells after they have fulfilled their therapeutic function or in the case of the formation of a tumor from these cells. Drawing by the author of the article.The technique was originally developed for the rapid removal of therapeutic allogeneic T cells in the case of a patient developing a graft-versus-host reaction during leukemia treatment [22].

It allowed the use of significantly higher concentrations of T cells without fear of consequences: a single injection of a dimerization inducer was enough to destroy cells containing the iCasp9 gene in half an hour.

In addition to the speed of healing, an important advantage of this system is the low toxicity of the dimerizing drug, as well as the fact that it (in the case of AP1903) is not used in any other therapy or in food products. According to preliminary data, the introduction of the iCasp9 gene into iPSCs did not affect their pluripotency in any way: the ability to both proliferate and differentiate into cells of all three germ leaves was preserved.

Since the removal of cells by induced apoptosis was still not 100% - possibly due to the activation of anti–apoptotic proteins like Bcl-2 [23] - further improvement of the IPSC emergency safety system based on suicide genes is necessary. This will bring closer the time when rejuvenated stem cells can be widely used to treat people.

Literature
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20.10.2015
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