Tissue Engineering and Cell Transplantation for Neurological Diseases

Today we observe the boost in the development of cell therapies and tissue engineering of the brain and spinal cord. This boost seems to be conditioned by the ineffectiveness of the treatment of neurological diseases while the expenses keep growing: the EU reports spending 80 billion euro for the therapy of neurological diseases.    

We would like to illustrate the tissue engineering approach and cell transplantation with the example of the treatment of the spinal cord injury (SCI). Annual expenses of the US for the SCI total 2 billion dollars. Various rehabilitation measures can significantly improve the injury outcome and improve life quality but are unable to remove severe neurological deficit and restore the damaged function.  Surgical methods largely aim at the orthopedic correction of the spine, which is often very efficient in the acute stage, but do not improve the neurological status of the patient 72 hours post injury. The reason for ineffectiveness of the available methods of therapy is clear. Unfortunately, neither rehabilitation, nor surgery removes the key reason of the disease, which is the damage of the structure of the spinal cord and damage of its function. Unfortunately, medicine cannot offer universal methods to restore the spinal cord and its function after the injury.

Use of the cell therapy and tissue engineering is another attempt to cope with the SCI. By now the clinical experience of using cell technologies is not extensive. This is associated with the insufficient theoretical basis, with the difficulties to obtain, to define and to preserve stem cells as well as with the lack of polished methodical and methodological approaches to their use. The mechanism of the cell action, their consequent differentiation, transformation and fate remain unclear. However, only in 2012-2014 over 80 national and private institutes of regenerative medicine have been established in Europe, and each plans to study the issues of the cell therapy and tissue engineering for the brain and spinal damage.

The data of clinical trials demonstrated that it is possible to treat chronic SCI with the transplantation of fetal neural cells (FNCs). The suspension of FNCs is administered intrathecally or intraventricularly or in the course of reconstructive surgeries. However, the use of foreign cell material can be dangerous as it can lead to infections, immunological and transplantation conflict and raises a lot of legal, ethical and religious issues. Embryonic cells now are not used as they can initiate tumors. Being free from such side effect and paramedical aspects, tissue engineering and transplantation of various autologous cells, i.e. the patient’s own cells, hold a great promise for the treatment of the diseases of the Central Nervous System (CNS) including the chronic SCI,  

It is important to note that there is no credible measurement of neurological status for chronic SCI cases. Available measurement scales do not permit detection of the minimal, slowly progressing improvements which sometimes can be observed even in the untreated part of the SCI contingent. Moreover, the measurement scales had not been agreed with the data of neurophysiologic methods that are not scheduled in the diagnostics of neurological disorders in SCI cases. Obviously, steady growth of severe consequences of SCI, high rates of disability and imperfect methods of therapy demand development of the new therapeutic methods.

Thus, tissue engineering of the spinal cord and transplantation of the autologous cells for chronic SCI can be viewed as a new and very promising direction of medicine that requires systemic and multi-vector research.

  1. Cell Transplantation in the Therapy of Brain Injury

Several methods of intraspinal transplantation of the cells have been developed in order to come through or around glial barrier. The first candidates for transplantation were embryonic neural stem cells, Schwann cells and the cells of olfactory sheath (COS). It should be noted that not all strategies of cell transplantation are aimed at the same goals. Non-differentiated nature of the neural stem cells endows them with the potential to mature into neural and glial phenotype. Accordingly, it can be supposed that in the site of injury they can transform either into neurons to transfer the synaptic information through the gap or, into glial cells to provide the neurotrophic support. Similarly, the transplants of fetal neuronal tissues propose opportunity to replace neural or glial cells that were damaged in the course of the primary or secondary damage. The Schwann cells or COSs are considered to provide the neurotrophic support for growth and re-myelinization of the regenerating or remained axons.  The role of neurotrophic factors in the preserving of the cells, their differentiation and elongation makes them important supplement to all strategies of cell transplantation.

Neural stem cells can be obtained from embryonic tissue and consequently are subject to the same ethical limitations or criteria of accessibility, unless they are suddenly found in the mature CNS which is considered to fully consist of post-mitotic cells.  Most of the approaches suppose to obtain the neural stem cells and then to culture them in vitro before transplantation. But recent research study new promising opportunities to stimulate the proliferation, migration and differentiation of the cells in vivo. Pluripotency and therapeutic potential of the cells were demonstrated by John McDonald and the team who transplanted the progenitor cells into the spinal cord of adult rats after contusion. The team observed improved motor functions of the rear limbs conditioned by differentiation of progenitor cells into neurons, oligodendrocytes and astrocytes (J.McDonald, 2005).

Recently, other scientists demonstrated the ability of the embryonic stem cells that were transplanted into spinal cord to differentiate into oligodendrocytes that further are supposed to remyelinize the recipients’ axons. Although, many of the properties of neural and embryomic cells require further research, interest to their basic biology will make them right candidates for future strategies of regeneration.

Long acknowledged favorable effect of peripheral nerves on growth of axons is attributed to the Schwann cells, and relative easiness of their isolation and expansion in culture makes them attractive for regeneration in the chronic SCI cases. Long back in 1966 the research was published that showed regeneration of the corticospinal tract and functional recovery of the  severed spinal cord in a rat with the help of 18 transplants of intercostal nerves stabilized by fibrin glue with fibroblast growth factor. Despite huge interest of the medical community to peripheral nerves and Schwann cells these results were not repeated.

The transplants of Schwann cells showed ability to incorporate into the site of injury, to make the ends of the damaged spinal cord meet, to stimulate growth of axons into the transplant and myelinate entering axons, but weak migration prevents from using them as well as their unwillingness to grow outside the transplant in CNS. Still, these disadvantages can be overcome and it is necessary to continue research of the Schwann cells.

However, a lot of attention is now given to the cells of olfactory sheath (COSs) that can help axons regenerate through the zone of transplant-host interaction.  

The COSs are special glial cells that are similar to Schwann cells and erythrocytes and can be found both in peripheral nervous system and in the CNS along with olfactory axons. The neurons of olfactory epithelium are unique, because they constantly renew and grow axons that are accompanied by COSs from the peripheral nervous system to the mature and often inhibiting CNS.  Their ability to accompany axons in the zone of interaction from peripheral nervous system to CNS presents them as potential solution for the challenges of use of Schwann cells. This new direction in the research culminated in the junction of the completely severed spinal cord of the rat. The researchers demonstrated extended regeneration of corticospinal tract in addition to noradrenergic and serotonergic fibers of distal end of the spinal cord which is associated with the significant functional restoration. The experience of other researchers using COSs in the partial SCI models support the general concept that these cells provide favorable conditions for the growth across the site of injury and, possibly, contribute to myelinization. However, potential contamination with Schwann cells requires further studies of the origin of the observed myelin. Recent research describe isolation and purification of the human olfactory embryonic cells (OECs) and propose that they can remyelinate axons of the CNS of adult rats when transplanted into demyelinized areas.  

The research of the regenerative potential of the OECs caused a lot of discussions just as neural stem cells, but similarly, their basic biology requires further research. As far as the olfactory system allows for isolation of the cells from nasal sheath, it will greatly optimize the use of the technology.

  1. Stem Cells in the Therapy of the Spinal Cord Injury: Fundamental Research and Animal Trials

In the past time several types of cells have been studied and the promising candidates for the CSI therapy has been detected. Embryonic stem cells, autologous glial cells of olfactory sheath, Schwann cells and stromal cells of the bone marrow (SCBM) are the cells types that have therapeutic potential. Adult SCBM, also known as mesenchymal stem cells, locate in the bone marrow and are supposed to provide support to hematopoietic stem cells (HSCs). These cells have progenitor characteristics as they produce certain trophic factors and cytokins. The SCBM and HSCs are described as the cells that can differentiate into several mesenchymal types including muscular, chondral, bone and adipose tissues. They also demonstrate homing-effect which is confirmed by their move to the damaged tissues in the cardiovascular system. It has been proved that transplantation of SCBM and HSCs act beneficially after the traumatic CSI and demyelinating injury.

The HSCs seem especially attractive for clinical practice as they can be isolated from the patients in the hospital and transplanted back to the hosts, providing the autologous model of cell therapy. In 1999 Bjornson showed the ability of the neural stem cells to differentiate into HSCs, including myeloid and lymphoid cells, as well as less mature cells. The cells of the rodents migrate into the brain and during the transplantation to preliminarily radiated recipients differentiate into microglia and astrocytes.

There is also evidence that permits proposal that human and rodent CSBMs can differentiate into the cells that have neuronal markers under the experimental conditions. The cultured stromal cells of bone marrow migrate to the brain and differentiate into astrocytes when transplanted into the lateral ventricle or striatum of mice. There are data that confrm the differentiation of other cell types isolated from the bone mesoderm in the mammalian CNS. In 2000 there was evidence that showed that the cells isolated from bone marrow enter the brain and differentiate into the cells that produce neuronal markers that supports the idea that the cells from mesoderm can accept the characteristics of neuronal cells. Moreover, in 2003 the ex vivo study showed that the cells of bone marrow with the phenotype of HSCs or progenitor cells express molecules that are associated with nervous system. It points to the fact that adult HSCs that have always been considered to have mesodermal origin express neural genes that have ectodermal etiology. Neural transcription factors and antigens in the neural differentiation in the cells of bone marrow can pint to the predisposition to differentiation into neural cells when placed into the brain.

In 2003 Sigurjonsson and the team showed that large part of adult human HSCs that integrated into the spinal cord of chicken embryo differentiates into neurons. Before no unconditioned neuronal differentiation of human HSCs in vivo has been shown. The author comes to the conclusion that the human bone marrow contains population of HSCs that has neurogenetic potential and under the appropriate conditions it can differentiate into neurons. When human HSCs are transplanted into the uninjured spinal cord of a chicken, the neuronal differentiation has not been observed, but the HSCs that entered a small cut of the spinal cord expressed neuronal markers.  Hence, it can be supposed that neuronal differentiation is not conditioned by the embryonic environment but by the specific microenvironment of the regenerating embryonic neural tissue.

Transplantation of HSC fraction of the bone marrow was popularized for the therapy of hematopoietic diseases, such as leukemia. Recent experiments in animals showed that the cells of HSCs fraction of bone marrow differentiate into various non-hematopoietic cells, such as cells of skeletal muscles and hepatocytes, if administered intravenously. If they are administrated directly into the heart, they can differentiate into cardiomyocytes. Intravenous administration of non-fractioned cells of the bone marrow in mice showed that the cells of bone marrow are able to differentiate into astrocytes and neural markers expressing cells.

In 2004 Koshitsuka following Chopps research of 200 demonstrated that transplantation of bone marrow HSCs to the damaged spinal cord of the mice improved functional restoration of their rear limbs. After the transplantation the mice could walk carrying their weight by rear limbs, while the control group could not do that. Moreover, some transplanted HSCs that survived in the site of injury differentiated into glial cells and neural precursors. These data imply that transplantation of the HSCs can be effective therapeutic method and needs further research.

In 2004 it was noted that the CSBMs selectively forward to the damaged tissues of the spinal cord if they are administered in three different ways: lumbar puncture, 2) intravenous injection, 3) intraventricular injection. The animals free from injury did not show the transplanted CSBM around the spinal cord segments. And, on the contrary all samples of the rat models of SCI exhibited the signs of CSBM in the tissue; the CSBM were located either inside or around the damaged parenchyma and were absent in other parts of the spinal cord signifying specific effect of pathotropism. Immunofluorescent tests confirmed that the alkaline positive stain included viable cells with specific morphological features of the cytoplasms and nuclei. The axons around the transplanted cells exhibited growth. These data confirmed the hypothesis that cell transplantation is more beneficial when transplanted into the CSC intraventricularly or intrathecally, than in the case intravenous administrations. Moreover, the amount of the cells in the tissues of the damaged spinal cord increased with the time showing that the more time have passed from the transplantation the more effective the cell transplantation can be.

  1. Stem Cells: Limited Clinical Trials

The discovery of the HSCs to trans-differentiate into the neural lines along with the accessibility of HSCs shifted attention to the use of HSC as promising approach to the cell substitution in the CNS disorders.

A large number of the research was conducted to study the regenerative potential of HSCs in the ischemic damage of the brain by mobilization of autologous HSCs (endogenous approach) or transplantation of HSCs (exogenous approach). There are several sources of HSCs: the cells that were received directly from bone marrow, the cells of umbilical cord blood and the cells from peripheral blood. In 1988 Weissman and the team focused their attention on the set of protein markers of the surface of the mouse blood cells, and these markers were associated with the probability of cell affiliation with long-term HSCs. Four years later the laboratory proposed the comparable set of the markers for HSCs. Weissman proposed other markers as the closest for mouse and human HSCs. There is a well known marker CD 34+ among them. It seems that isolation of HSCs from bone marrow for the therapy will soon become a history. Currently, to transplant HSCs the physicians prefer to isolate the donor cells from peripheral circulating blood. It has been known for a long time that a small number of stem and progenitor cells circulate in blood, but several decades ago the injections of such cytokine as the granulocyte colony-stimulating (G-CSF) factor were used to achieve migration of the cells from bone marrow to blood.  To obtain the cells, G-CSF is injected to the donor several days before the isolation. Then the blood comes through the apheresis system that filters CD34+ and white blood cells. Interestingly, such CNS injuries as acute cerebral ischemia lead to spontaneous three-fold increase of CD34+ in peripheral blood. This change seems insufficient mechanism of self-regeneration and further mobilization of CD34+ with G-CSF seems logical. Besides, G-CSF is known to provide neuroprotective effect after SCI. Recent pre-clinical research marked clinical restoration in the rats with focal cerebral ischemia after subcutaneous injection of G-CSF.

Therefore, the review of literature confirms the benefits of using methods of cell transplantation and tissue engineering in the treatment of chronic SCI patients. However, clinical use of the technology requires research.

The detailed information about cell therapy and tissue engineering for neurological diseases you can get from the monographs of Prof. Andrey Bryukhovetskiy Cell Transplantation and Tissue Engineering in Neurological Diseases, 2003 and  Spinal Cord Injury: Cell Technologies in the Treatment and Rehabilitation, 2010 (in Russian). The books can be purchased in the Contract department of our clinic.

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Andrey S. Bryukhovetskiy



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