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Introduction to Cell Transplantation

Laurance Johnston, Ph.D.

Sponsor: Institute of Spinal Cord Injury, Iceland

 

 

Before discussing specific transplantation procedures, we need to briefly summarize the therapeutic potential of various cells or tissues that are being incorporated into cell-transplantation programs designed to restore some function after SCI. For readers desiring more in-depth information, the following books are recommended: Stem Cell Now (Scott CT. Pi Press; 2006), and  Human Embryonic Stem Cells (Kiessling AA, Anderson SC. Jones and Bartlett Publishers, 2007).

Olfactory Tissue/Cells

Because olfactory tissue is exposed to the air we breathe, it contains cells with considerable turnover potential, including renewable neurons, progenitor stem cells (below), and olfactory ensheathing cells (OECs). Because of the unique nature of this tissue, its regeneration-catalyzing potential is being examined for a variety of neurological disorders, including in addition to SCI, Parkinsonís disease and ALS (amyotrophic lateral sclerosis).

When transplanted into the injured spinal cord, OECs potentially promote axonal regeneration by producing insulating myelin sheaths around both growing and damaged axons, secreting growth factors, and generating structural and matrix macromolecules that lay the tracks for axonal elongation.

Stem Cells

Briefly, stem cells are precursor or progenitor cells that have the potential to transform into a wide variety of tissue. Although often dichotomously categorized as either embryonic or adult, they actually represent a continuum of cell types that can transform into our end-product tissue.

For example, as our central nervous system (CNS) develops, embryonic stem cells generate more specialized tissue-specific neural stem cells. In turn, these tissue-specific stem cells can differentiate into neuron- or glial-restricted precursor cells, the former with the potential to generate neurons and the latter into CNS support cells called oligodendrocytes and astrocytes.

Stem-Cell Differentiation (From Stem Cell Now, CH Scott, 2005)

Omnipotent embryonic stem cells have the greatest potential to differentiate into a wide range of cell types, although it has been difficult to steer them in the desired direction. Adult stem cells are found in most tissues, including, for example, CNS, bone marrow, skin, intestine, liver, muscle, hair follicles, and even teeth. Sometimes, they are robustly expressed, such as the bone-marrowís ongoing production of blood-cell-replenishing stem cells; in other tissue, they are quiescent and need to be coaxed into action.

Although adult stem cells usually differentiate into the specialized cells connected with the originating tissue, when certain cues are provided, they can transform into cells associated with other tissue. For example, under appropriate circumstances, bone-marrow-derived stem cells can differentiate into nerve cells and, indeed, are being used in several SCI-transplantation programs. Furthermore, studies suggest that adult stem cells can reprogram back into a more embryonic state. Finally, although we have emphasized their therapeutic potential, given the wrong cues, stem cells can turn into physiological troublemakers, causing, for example, cancer.

Embryonic Stem Cell Isolation

Basically, after an egg is fertilized, an embryo is formed, which then splits into a two-cell embryo. In Stem Cell Now, author Christopher Scott compares the process to dividing a soap bubble with a knife, creating two smaller bubbles within the confines of the original. Cut again, and it becomes four bubbles or a four-cell embryo. This division goes on, successively creating 8, 16, 32, 64, 128-cell embryo, the total volume changing little.

Embryo Cleavage (From Stem Cell Now, CH Scott, 2005)

Between four and six days, the cells rearrange into two layers: an outer layer which will develop into placental and amniotic tissue and a few dozen cells called the inner-cell mass (ICM) which turns into everything else. Now labeled a blastocyst, the embryo is about 0.1-mm across or the size of the period at the end of this sentence.

Blastocyst & Inner Cell Mass (From Stem Cell Now, CH Scott, 2005)

As the cells continue to develop, they increasingly lose their omnipotent nature. After about two weeks, the ICM cells start to organize into three specific layers that become our various tissues: 1) ectodermal layer (developing into nerve, skin, etc), 2) mesodermal (turning into blood, muscle, bone, etc), and 3) endodermal (differentiating into the gut, liver, pancreas, bladder, etc.).

To obtain ESC, the ICM cells are isolated before they start turning into these layers, and grown in culture. The culturing technology has only recently emerged and requires sophisticated methodology and skill. For example, scientists have had to grow the cells on a layer of animal cells to provide nutrients and the signals needed to keep the cells from further differentiating.

Schwann Cells

Schwann cells are responsible for remyelinating axons in the peripheral nervous system, which, unlike the CNS, has considerable inherent regenerative potential. Over the years there has been much speculation on the potential of these cells to exert similar, regenerative effects when introduced into the injured spinal cord.

Cell Source

Transplantable cells can be obtained from the patient (autologous); genetically different individuals, embryos, or umbilical cords (allogeneic); or different species (xenogeneic). All three types have been transplanted in an effort to restore function after SCI. Because autologous tissue is from the patient, there is no immunological rejection. The undifferentiated nature of embryonic and, to a lesser degree, umbilical cells also minimizes rejection. Overall, cells are not selected based on the theoretical best source or regenerative potential but their isolation ease, such as concentrating blood stem cells.

Site of Transplantation

Donor cells are transplanted back into the patient by a variety of routes, including into the spinal cord or surrounding fluid, intravenously, or intramuscularly. Clearly, itís easier and safer to inject cells into a muscle, blood, or spinal fluid than surgically accessing the spinal cord, albeit perhaps not as effective. Many devil-is-in-the-details questions remain whether the cells transplanted by these divergent routes actually reach the injury site to exert any significant benefit. Although more studies are needed, investigators are beginning to study the fate of transplanted cells in animal models of SCI.

For example, Czech scientists are developing procedures using magnetic resonance imaging (MRI). Basically, with these procedures, very small magnetic iron-oxide particles are attached to the stem cells, making them visible by MRI, and, in turn, allowing them to be followed to some degree after transplantation. The overall research goal is to determine the time course of migration to the injury site and how long the cells persist there.  With such information, we can better understand the optimal time frame for transplantation, the number of cells needed, and the best route of administration. 

In another example, an international team of scientists have used magnetic-imaging procedures to assess the migration of iron-oxide-labeled olfactory ensheathing cells that have been transplanted into the rat spinal cord. The labeled OECs could be observed in the spinal cord for at least two months after transplantation. Although extensive migration of transplanted OECs in both directions was observed in the normal spinal cord, the cells were unable to migrate through the injury-site scar of a transected spinal cord.

The issue was further studied by Japanese scientists in mice experimentally injured at the thoracic T10 level. Mouse-derived neural stem cells, which were labeled with a bioluminescent agent allowing later detection, were transplanted into the injured animals by three different routes: 1) into the spinal-cord injury site, 2) into the intrathecal space surrounding the cord, and 3) intravenous administration. Six weeks later, the location and number of surviving cells were assessed. When the cells were injected directly into the injury site, 10% of them survived, including cells remaining at the injury site. In the case of the intrathecally transplanted cells, although some did, indeed, migrated to the injury site, only a miniscule 0.3 % cumulatively survived after six weeks. Finally, no intravenously transplanted cells were detected at the injury site. However, considerable luminescence showed up in the chest, suggesting pulmonary embolism (a blockage of the lungís pulmonary artery). One third of the mice died immediately after the intravenous transplantation. The investigators concluded that implanting the stem cells directly into the injury site was the most effective and feasible transplantation method.

As a part of related research described later, Drs. Fernando Callera and Claudio de Melo (Brazil) examined the deposition of autologous (i.e., isolated from the patient), magnetically labeled, bone-marrow-derived stem cells transplanted into 10 patients. Age ranged from 21 to 45, and the duration since injury varied from 2 to 13 years. Stem-cell-rich bone marrow was aspirated from the iliac crest of the patientís pelvic bone, and the stem cells isolated and labeled with magnetic nanoparticles. Five hours after aspiration, these magnetically labeled cells were implanted into the patientís spinal canal by lumbar puncture. In five of the ten patients, 20-day post-transplantation MRI assessments (i.e., magnetic resonance imaging) indicated that the labeled stem cells had migrated to the injury site but nowhere else in the central nervous system. In a control group of six individuals with SCI who got an injection of just the magnetic beads without stem cells, no lighted up areas were observed at the injury site. The study demonstrates that at least some of stem cells transplanted by this method truly migrate to the injury site.  

Age & Level of Injury

Other factors that may influence stem-cell therapeutic effectiveness include age and level of injury. As shown by UK researchers, this seems to be the case for bone-marrow stem cells, which have been transplanted in a number of SCI-related programs. In this study, bone marrow was isolated from iliac crest (i.e., an area of the pelvis) of donors with SCI and grown in culture. Donor age ranged from 23-66 years, and all had complete injuries sustained five months to 23 years before the bone-marrow tissue was collected. Although a limited sample size, stem-cell growth in culture was greater when the cells were isolated from younger patients and those with cervical injuries.

Dr. Carlos Lima, who developed procedures for transplanting stem-cell containing olfactory tissue into the injured cord, will not accept patients older than 40 due to the diminution of regenerative potency in this tissue with age.

 

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