Scientific and Ethical Considerations in the Advancement of Stem Cell Research

Rivka Zimm

A SC injury (SCI) is defined as an insult to the spinal cord (SC) resulting in a change, either temporary or permanent, in the cord’s normal motor, sensory or autonomic function. Every year, over a million Americans suffer from SCIs resulting in significant impairment to their ability to work and participate in their activities of daily life.[1] The use of stem cells in the treatment of SCI’s for both functional recovery of cells and regeneration of damaged nerves has seen great advancement in the past several years and continues to advance as the scientific community of the world begins realizing the potential of the stem cell. This paper will review the current advancements and address the ethical issues concerning the use of stem cells in human subjects.

To understand SCI and its treatment, one must first understand the building blocks of the SC itself. Within the SC, there are two broad categories of cells: neurons, which process information and are responsible for transmitting the electrical signals throughout the body, and glia, which support neurons metabolically and mechanically.[2] Each neuron is surrounded by a cell membrane, which is critical for the neuron to be able to send electrical signals. All neurons consist of a cell body, also called a soma, dendrites, and an axon. The cell body contains the nucleus and intracellular organelles. The dendrites are extensions of the cell body that receive chemical signals from the other neurons and pass those signals to the soma of the neuron. The axon transmits information from the soma of the neuron to the dendrites of the next neuron. The connection point of the axon of one neuron and the dendrites of another neuron is called the synapse.[3] Both the dendrites and the axon are extensions of the cell body and are also known as processes.

Within the broad class of neurons, there are three categories of cells: receptors, interneurons, and effectors/motor neurons, that vary based on their functions. Receptors act to receive and encode sensory information. Through this action, receptors begin the process of sensation and perception. Interneurons process information, by sending and receiving signals. Because of this tie to signals, interneurons constitute the bulk of the nervous system. The final kind of neurons are effectors/motor neurons which send signals to the all of the muscles and glands of an organism, thus making them responsible for the behavior of the organism.[4]

The second broad class of neural cells, glial cells, is defined as the glue of the nervous system. These are the accessory cells that are required for neurons to function. Many glial cells come together to fill the space between neurons. The reason that so many of these cells are able to fit between just two neurons is because glia are considerably smaller than neurons; in fact, they outnumber neurons by a factor between 10 and 50. Even so, the miniscule size of glial cells is responsible for the fact that the entire population of glial cells barely accounts for half of the brain’s mass.[5]

There are two main categories of glial cells within the SC and the rest of the central nervous system (CNS): macroglia and microglia. The macroglia of the nervous system, are the larger type of glial cells, and are split into two classes: astrocytes, and oligodendrocytes. Astrocytes are unusual in that they lack organelles. Because of this, astrocytes cannot do the functions of other cells, such as the synthesis of proteins; rather, they provide structural support for their neighboring neurons, and help repair the brain in the case of damage. They also regulate the flow of ions and larger molecules through the synaptic region (the space between two neurons). The cells of the second type of macroglia, oligodendrocytes, have organelles and microtubules. These organelles allow the oligodendrocytes to produce the myelin that makes up the myelin sheaths, which are the insulating coats of axons. Microglia are the macrophages of the CNS and do most of its “housekeeping”.[6] They are the main source of active immune defense for the CNS, and clear out and digest all of the dead cells (mostly neurons) from within the brain and the rest of the CNS.

The macroglia and microglia work mainly in the CNS. There is another type of glial cell specifically found in the peripheral nervous system (PNS), called Schwann cells. Schwann cells act as the oligodendrocytes of the developing PNS. In place of producing myelin, a Schwann cell will wrap itself around the axon of the neuron, and build a myelin sheath. As this happens, the cytoplasm of the SC is pushed forward, leaving only the membrane of the Schwann cell wrapped around the once-naked axon. This process, called myelination, greatly increases the speed with which action potentials are carried along an axon.[7]

In the case of SCI, neurons and glial cells may become damaged, causing loss of functionality within the organism with the injury. This loss is dependent on where along the spinal cord the injury takes place. SCI is categorized as cervical injury, thoracic injury, lumbar injury, and sacral injury. The first, cervical injury, usually results in paralysis or weakness of limbs (arms and legs), and effects all bodily functions below the level of injury, in the neck. Additionally, many with cervical injury find that they have loss of physical sensation, respiratory issues, bowel, bladder, and sexual dysfunction. This area of the SC controls signals to the back of the head, neck and shoulders, arms and hands, and diaphragm.[8] (See Figure 1)

The second type of SCI, thoracic injury, is less common than cervical SCI. This is because the area of injury is found directly beneath the rib cage, which is protective. Thoracic SCI results in paralysis or weakness of legs, and will affect bodily functions below the level of injury. Just as in cervical injury, patients with thoracic injury can experience loss of physical sensation, respiratory issues, and sexual dysfunction. In addition, patients may experience bowel and/or bladder dysfunction. This area of the SC controls signals to some of the muscles of the back and part of the abdomen.[9] (See Figure 1)

The third type of SCI, lumbar SCI, results in the paralysis or weakness of the legs similar to thoracic injury. This area of the SC controls signals to the lower parts of the abdomen and the back, the buttocks, some parts of the external genital organs, and parts of the leg.[10] (See Figure 1)

The fourth and final type of SCI, sacral SCI, results in the paralysis or weakness of hips and legs. Just like thoracic injury, bowel, bladder, and sexual dysfunction are known to occur. This area of the SC controls signals to the thighs and lower parts of the legs, the feet, and genital organs.[11] (See Figure 1)

Figure 2

All types of SCI can be classified as complete or incomplete injury. In the case of complete injury, the patient experiences a loss of functionality throughout all of their axons and/or nerves below the level of injury. The axons and nerves may be still intact, but they are not functioning properly. In contrast, the axons and nerves of an organism with incomplete SCI can still interact with the brain, and convey messages, whether sending them or receiving them. Because of this, sensation and movement below the line of injury is possible for a patient with incomplete SCI.[12]

SCI can be divided into two phases: primary and secondary. The first, the primary phase, takes place at the time of the actual injury, and can be caused by contusions and compressions. When contusion occurs, the vertebral bones shatter, and when compression occurs, there is extreme pressure placed upon the spinal cord. Primary injury occurs most often with lumbar and cervical SCI. It affects both lower and upper motoneurons which play a part in the skeletal system and SC. The primary injury usually results in hyperreflexia (overactive or over responsive reflexes), hypertonia (increased rigidity, tension, and spasticity of the muscles) and muscle atrophy or weakness (atrophy in lower motoneurons, and weakness in upper neurons).[13] Contrary to popular belief, the primary injury phase does not cause the most damage to the nervous system. Rather, the secondary phase of SCI is mostly responsible for long term damage.[14]

The secondary damage phase of SCI results in complex damage at the cellular level. During this phase, massive cell death can occur, due to the inflammatory response of the host immune system. This massive cell death includes both necrosis (the death of most or all of the cells in an organ or tissue due to disease, injury, or failure of the blood supply) and apoptosis (the death of cells that occurs as a normal and controlled part of an organism's growth or development.)[15] Additionally, the hemorrhaging and production of chemokines that occurs during this phase breaks the blood brain barrier (BBB), which usually protects the CNS from a variety of substances that are in circulation throughout the bloodstream. These chemokines include IL 1, which activates and recruits inflammatory cells, thus causing a local inflammatory response.[16]

There are three major phases of the secondary phase of damage of SCI, which follow: the acute phase, the subacute phase, and the chronic phase. The acute phase lasts between two hours and two days, the subacute phase can last for weeks or even months, and the chronic phase may last for months or even years.[17]

Figure 2 (below) summarizes the damage phases, both primary and secondary.[18]

Figure 2

Due to the damage that occurs in both of the phases, (the inflammatory component of SCI, and subsequent demyelination of surviving axons), the treatment of SCI relies largely on inhibiting the inflammatory response of the host, reducing and/or eliminating cell death (necrosis and apoptosis), and enhancing neural regeneration and remyelination.[19] To accomplish these, and promote recovery in the host, many scientists advocate the use of stem cells. To be successful, cell transplantation should accomplish the following: 1) secrete neurotropic molecules (that will nourish nervous tissues and cause cell recovery), 2) act as a scaffold for axon regeneration, and 3) actually replace lost neurons.[20]

There are many different types of cells that are being transplanted into SCI patients in an attempt to achieve the best results regarding SCI treatment. These cells can be broken up into two major categories: embryonic stem cells (ESCs) and adult stem cells (ASCs). The cells of the first category, ESCs, are derived from the inner mass of the cell embryo. They are quite useful, as they are pluripotent, meaning they can differentiate into all cell types and they can replicate indefinitely.[21] They also have anti-inflammatory properties. However, there are many disadvantages to these cells as well. First, there are moral and practical considerations in harvesting the cells, as the harvesting of ESCs requires the death of the embryo that the cells are from. The cells also have karyotypic instability because of repeated freeze thaw cycles (when the cells divide they lose pieces of chromosomes or entire chromosomes). Finally, these cells have been known to be teratogenic in the host, meaning they may cause birth defects in future children of the recipient.[22] However, they are still marginally better than ASCs.[23]

Within the category of ESC there are additional distinctions within the subtypes. These subtypes are induced pluripotent stem cells (iPSCs) and mesenchymal progenitor cells (MPCs). iPSCs can be generated from ASCs and reprogrammed to perform like ESCs. MPCs are embryonic progenitor cells (they can differentiate to form one or more kinds of cells but they cannot divide and reproduce indefinitely) that are isolated from first trimester fetal blood.[24]

The cells of the first type of ESCs, iPSCs, are really ASCs that have been reprogrammed to perform the functions of ESCs. Scientists have discovered that one of the most beneficial parts of iPSCs is the cells that can be derived from them. These include: motoneuron grafts, GAbAergic neurons, and neural supporting cells such as oligodendrocyte progenitors (OPCs).[25] This eliminates the classic ethical considerations with ESCs.

iPSCs appear as if they may become a valuable resource in the future, with the help of more advanced technology. As of yet, the only real sensory and/or motor recoveries that have occurred with the help of iPSCs have been when they were co transplanted with other beneficial cells. These include: adding collagen as a scaffold to add initial support (which actually increased differentiation and motor and/or sensory recovery), the addition of Schwann Cells, which would probably be beneficial on their own, the addition of cells that overexpress NGN-2, and the transplantation of iPSCs, along with sonic hedgehog, and retinoic acid as cofactors.[26] These efforts have actually paid off, as studies have shown that remyelination and axonal regeneration have occurred, functional motor recovery has been obtained, as well as neuronal differentiation.[27]

The motoneuron grafts of iPSCs were extraordinarily useful. They can be used to replace damaged motor neurons, extend axons through muscles to reinnervate them, and recover sensory and motor functions. These cells express active growth factors such as neurotrophins three and four, nerve growth factors, and vascular endothelial derived growth factor.[28] Even the other grafts derived cells were useful. The GAbAergic neurons increased sensory function, and the neural supporting cells caused axon remyelination and motor function improvement.[29]

The simplicity in the method of differentiation of iPSCs is also a concrete advantage. Neurogenins (which are transcription factors involved in neuron differentiation) especially NGN-2, which is essential for the development of the CNS, are added to the blank (reprogrammed) stem cells, creating cells essentially similar to the common ESC cell (the NPC).[30] However, this causes a lack of consistency in the cells that are implanted, in regards to their differentiation and proliferation. Once the cells have become NPCs, they will behave as such, and will no longer fit a scientific mold.[31]

Additionally, the iPSCs have been tumorigenic in the past.[32] They are impractical, as genetic modification and reprogramming is a painstaking process, and they require extensive retrospective classification to ensure that they truly behave like ESCs. Unfortunately, the attempts at genetic modification have not been very effective as of late.[33]

The other type of ESCs, mesenchymal progenitor cells (MPCs), are isolated from fetal blood from the first trimester of pregnancy. This presents a tremendous ethical dilemma. However, they are highly proliferative, and they can undergo repeated freeze thaw cycles without losing their viability, without losing their mesodermal differentiation potential, and without accumulating karyotypic abnormalities. The cells are non-immunogenic, and can even suppress regular immune response, which is good for allogeneic transplantation. Additionally, they are highly pathotropic, as they can secrete a wide range of trophic factors. This promotes neural cell survival, which is especially useful when used for cell rescue and as support cells. MPCs are not only nontumorigenic, but they even have anti-tumor properties, and immunosuppression is not necessary for implantation.[34]

The use of adult stem cells (ASCs) also has advantages and disadvantages. They are morally/ethically, and practically more efficient than ESCs. Additionally, their proliferative and differentiation potential can be raised to compete with ESCs when coadministered with chondroitinase treatment (especially in a subtype of ASCs known as olfactory ensheathing cells, or OECs).[35] However, they are naturally less effective than ESCs, in regards to this proliferative and differentiation potential. They also fall short in regards to post implantation survival, migration and integration within the host/recipient CNS, and in their increased neuropathic pain.[36]

ASCs have three distinct categories, each with two types of stem cells within them. The first category is made up of neural progenitor cells (NPCs). NPCs are like stem cells in that they can differentiate to form one or more kinds of cells. However, they cannot divide and reproduce indefinitely. The second category is made up of cells that originate in bone marrow. These highly important cells include bone marrow mesenchymal/stromal cells (BMSCs), and mesenchymal stem cells (MSCs). The third and final category includes all ASCs that originate outside of the bone marrow. This includes olfactory ensheathing cells (OECs) and Schwann cells (SCs- not to be confused with stem cells or spinal cord).