Anti-ischemic patch
Cell patches will restore tissue after ischemia
A collaboration of scientists from Lomonosov Moscow State University, the Federal State Medical University of Cardiology of the Ministry of Health of the Russian Federation and Tsing Hua University (Taiwan) has developed a highly effective method of tissue repair after ischemia and successfully tested it on mice. The method is based on the transplantation of layers of genetically modified stem cells. The study showed that the method may be promising for restoring normal blood supply, innervation and regeneration of ischemically damaged muscle tissue. The work was supported by the Russian Science Foundation (RNF) and the Russian Foundation for Basic Research (RFBR), its results were published in the International Journal of Molecular Sciences (Boldyreva et al., Transplantation of Adipose Stromal Cell Sheet Producing Hepatocyte Growth Factor Induces Pleiotropic Effect in Ischemic Skeletal Muscle).
According to the World Health Organization, 17.9 million people died from cardiovascular diseases in 2016 alone, which accounted for 31% of all deaths in the world. In Russia, mortality from cardiovascular diseases is gradually decreasing: if in 2007 1.18 million people died from them, then in 2017 - 860 thousand. However, so far in therapeutic practice neither in Russia nor in the world there are no well-established effective methods for the recovery of patients who have suffered from diseases of the cardiovascular system.
Cell therapy methods may be promising in the recovery of patients with ischemic diseases, such as myocardial infarction, critical lower limb ischemia, ischemic stroke. However, the results of the first generation of cell therapy for these diseases turned out to be very modest. One of the most important reasons for the lack of effectiveness was the death of a significant part of the cells after transplantation when they were administered as a suspension.
Under natural conditions, stem cells in tissues are located in a specific microenvironment – a cellular niche, where their viability is maintained by contacts with other cells and the extracellular matrix. When cells are deprived of these contacts, the mechanism of their death is triggered. To overcome this significant problem in this work, scientists have proposed using several approaches to increase cell survival after transplantation. For this purpose, after cultivation, the cells were not removed from the culture cup with the help of enzymes and were not transferred to a suspension for subsequent injection through a needle (each of these actions leads to the death of part of the cells), but were transplanted in the form of a cell layer in which contacts between cells were preserved, as well as with the extracellular matrix developed by the cells themselves.
Such a patch of cell layers in some way imitated a cell niche – a natural microenvironment. In addition, to increase cell viability and their therapeutic properties, the cells were modified with the safest viral vector based on an adeno–associated virus carrying the hepatocyte growth factor gene – HGF - which has a pleiotropic (multiple) effect on regeneration processes, stimulating the growth of blood vessels and nerves, cell division and suppressing their programmed death – apoptosis. HGF also serves as the most important factor regulating the division and directed movement of mesenchymal stromal cells themselves. Therefore, the hyperproduction of this factor by the autocrine mechanism (that is, by the cells themselves) can maintain the viability of these cells.
"The choice of mesenchymal stromal cells (MSCs), in this case, obtained from adipose tissue, was also important. MSCs by their properties are an ideal tool for regenerative technologies. These cells produce a wide range of biologically active factors and extracellular matrix proteins that stimulate regeneration processes, release extracellular vesicles capable of transferring mRNA and microRNA to other cells, triggering a regenerative program in them. Targeted enhancement of these properties by genetic modification allows us to obtain cells with a high regenerative potential," explained Elena Parfenova, head of the Laboratory of Postgenomic Technologies of the Faculty of Fundamental Medicine of Moscow State University and Director of the Institute of Experimental Cardiology of the NMIC of Cardiology, head of the study.
The work included several stages. First, the scientists took samples of adipose tissue from mice and obtained a culture of mesenchymal stem cells. Then, using a vector system based on viruses, the expression of the gene encoding HGF was increased. Having made sure that the gene modification of stem cells was successful – in 90% of cells, the modification led to the production of a fluorescent protein, the gene of which is embedded in the vector, and in modified cells the production of HGF increased tenfold – the scientists tested the therapeutic potential of the development.
The development has passed the test of neuroprotective properties. To do this, an explant of modified stem cells was placed in the culture of neurons of the dorsal root ganglion and evaluated how axons develop in the latter. In the neurons from the experimental group, the processes grew longer than in the control group, which indicated stimulation of the growth of nerve endings.
Micrograph of axons of neuronal culture of the dorsal radicular ganglion under the influence of conventional MSCs (left) and genetically modified MSCs (right). A drawing from an article in Int. J. Mol. Sci.
Then the scientists moved on to testing the development for effectiveness in tissue repair after ischemia. It is impossible to carry out such tests without the participation of animals, so scientists simulated ischemia of the hind limb in mice. Then the mice were divided into 5 groups: animals from the first and second were treated with a suspension of unmodified and modified MSCs, respectively, the third and fourth with layers of unmodified and modified cells. The fifth group was a control group. Blood flow in the extremities was measured weekly in mice using a laser doppler, and at the end of the experiment, muscle tissue was taken for analysis.
The study showed that in mice from all experimental groups, blood flow was restored better than in animals from the control group. However, the most effective therapy was with the help of layers of genetically modified MSCs: when they were used, the blood flow in the tissues was restored to almost 70% of the original by day 21. In the control group, blood supply was restored by less than 40% by this time. Histological studies have also shown that therapy with modified MSCs layers leads to the restoration of vascularization and innervation in the ischemic limb of mice.
"The results obtained are the basis for further promotion of this cellular technology, first in preclinical studies, in which it is necessary to prove the safety of its use in animals, and then in clinical studies in patients with severe lower limb ischemia, leading to amputations and disability," Elena Parfenova added. "Based on the results of this study, it is planned to create a biomedical cell product for the treatment of these patients."
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