Neural Stem Cells

In the last decade, interest in the field of stem cell research has intensified. This increased interest has led to the identification and isolation of adult stem cells from a variety of organs including the central nervous system, bone marrow, liver, intestine, retina, skeletal muscle, pancreas, cornea, and skin (Kirschstein and Skirboll, 2001). The potential therapeutic uses of these cells to ease human suffering and provide cures for human disease have been the driving forces for most of the interest in stem cell research.

Although stem cell research is in its infancy, it is hoped that someday stem cells can be used to cure many diseases such as Parkinson’s, amyotrophic lateral sclerosis (Lou Gehrig’s disease), Huntington’s and diabetes mellitus. Stem cells fall into two categories, somatic (adult) and embryonic. All stem cells are undifferentiated, capable of self-renewal and, when given the proper cues, can give rise to fully differentiated mature cell types (Gage, 2000). Even though adult and embryonic stem cells share basic characteristics, there are also very important differences.

One of the differences between adult and embryonic stem cells is their origin. Embryonic stem cells are derived from the inner cell mass of an embryonic blastocyst while adult stem cells are derived from differentiated tissues (Gage, 2000). The different origins of these two types of stem cells is an important social issue. The acquisition of embryonic stem cells results in the loss of a developing embryo, while adult stem cells can be harvested from donors without negatively affecting the person or animal.

Mature mammals have small populations of adult stem cell distributed throughout the body and these cells are thought to play a role in the repair of damaged organs and tissues (Rossi and Cattaneo, 2002). One enticing prospect of adult stem cells is their potential to be used as autographs, which would eliminate the rejection of transplanted cells (Roskams and Tetzlaff, 2005). Another therapeutic potential for adult stem cells is selective in situ activation allowing for repair of damaged tissues and organs without cell transplantation (Roskams and Tetzlaff, 2005).

The other major difference between embryonic and adult cells is their plasticity. Adult stem cells are thought to be much more limited, with respect to what they can differentiate into, when compared to embryonic stem cells. All cells of multicellular organisms are derived from a single cell called the zygote. As this single cell undergoes division the daughter cells slowly become restricted by ‘turning’ genes on and off to generate all of the specialized cells found in multicellular organisms (Clarke et al, 2000).

Even though every cell of the body has the same genetic make up, the functional and morphological characteristics of the cell are dictated by the genes that are expressed. Embryonic stem cells are pluripotent and are capable of differentiating into nearly every cell type of the body. This is due to the fact that these cells are collected from the inner cell mass of the blastocyst and very little cellular specialization has occurred, whereas, adult stem cells have varying degrees of plasticity because they are harvested from tissues (Kirschstein and Skirboll, 2001).

Some types of adult stem cells, mesenchymal stem cells for example, are much more plastic than was originally thought and can differentiate into cell types very different from their tissue of origin (Loveil-Badge, 2001). Neural stem cells It has long been believed that all neurogenesis was confined to the in utero and early postnatal periods of development and no new neurons could be generated in the adult brain. This assumption was based on the fact that neurons present in the adult brain did not have mitotic figures and a lack of neural proliferation following brain injury (Altaian and Das, 1964).

The first evidence that a population of stem cells existed in the central nervous system (CNS) came from Altman and Das in the 1960’s. They demonstrated the presence of dividing cells in the murine hippocampus and olfactory bulb that became neurons. Despite this fact, the presence of a stem cell population giving rise to neurons in the adult mammalian CNS was not widely accepted until the 1990’s. In 1992, Reynolds and Weiss were the first to isolate neural stem cells (NSC) from the adult mammalian CNS.

They found a population of cells isolated from the striatum of the adult mouse brain that could differentiate into neurons and astrocytes after they were exposed to epidermal growth factor. Furthermore, they demonstrated that the differentiated neurons were immunoreactive for substance P and 7-aminobutyric acid indicating that the cells might be functional. Since this initial description of NSCs they have been identified in several regions within the adult CNS including the hippocampus, subventricular zone, olfactory bulb, and spinal cord (Gage, 2000).

The first description that NSC existed in humans occurred in 1998 when Ericksson and colleagues demonstrated that neurogenesis occurred in the adult human hippocampus. The study consisted of five cancer patients that were injected with BrdU and their brains were examined post-mortem. They found a small population of cells that co-labeled for both BrdU and several neuronal markers indicating that the co-labeled cells were created during BrdU exposure. Neural stem cells are an adult type of stem cell because they are derived from differentiated tissue and divide to become other NSC, neuronal progenitor or glial progenitor cells.

It is believed that neuronal progenitor cells are destined to become neurons and glial progenitor cells will give rise to astrocytes and oligodendrocytes (Gage, 2000). It has been demonstrated that NSCs can generate glial and neuronal cells in vitro when they are exposed to certain trophic factors (Zigova et al, 1998), and in vivo after transplantation (Winkler et al, 1998). However, this neural restriction can be over come if NSCs are transplanted into a chick blastocyst where they can contribute to all three germ layers (Seaberg et al, 2003).

This helps elucidate the importance of the external cellular environment in maintaining cellular restriction. Additional work demonstrates that glial progenitor cells that are given the proper cues are capable of becoming more than there progenitor restriction would suggest and can differentiate into neurons (Clarke et al, 2000; Gage, 2000). Oligodendrocyte precursor cells Noble, Raff, and others have shown that the adult optic nerve retains an active population of glial progenitors termed oligodendrocyte precursor cells (OPCs) (Raff et al.

, 1984). OPCs are also called O-2A progenitor cells because initial work demonstrated that they could not only differentiate into oligodendrocytes, but also astrocytes. The astrocyte that was derived from O-2A progenitors was called a type 2 astrocyte to indicate that it came from a separate linage (Franklin and Blakemore, 1995). The demonstration of type 2 astrocytes in vivo has proven to be very difficult and there is some question to their existence (Franklin and Blakemore, 1995).

However, recent research demonstrates that OPCs that are exposed to bone morphogenetic proteins (BMP) promotes type 2 astrocyte differentiation and if followed by basic fibroblast growth factor (bFGF) these cells revert to a state in which they can not only self-renew but are capable of differentiating into oligodendrocytes, astrocytes, and neurons (Kondo and Raff, 2000). Furthermore, if these reverted cells are kept in bFGF for 30 days, approximately 62% (± 5%) of them will differentiate into neurons (Kondo and Raff, 2000).

This is an example of a glial restricted progenitor cell taking on the characteristics of a neural stem cell and these cells have been termed multipotent CNS stem cells or neural stem cell like cells (Kondo & Raff, 2000, 2004a,b). The possibility of neural progenitor contamination from other neurogenic regions is remote because the optic nerve was harvested rostral to the optic chiasm eliminating concerns of any contamination. In work done by Nunes et al (2003) they found that human OPCs have the same potential to convert into a multipotent CNS stem if first exposed to fetal bovine serum, which contains BMPs.

They did not observe the conversion to the type 2 astrocyte, but the BMP in the serum are still believed to be the inducing factor. As stated previously, the conversion of OPCs into a type 2 astrocyte is under question. There is no doubt that these cells express glial fibrillary acidic protein (GFAP), which is considered to be an astrocytic marker, but GFAP expression does not necessarily indicate functional astrocyte differentiation (Garcia et al, 2004). Many cells, in addition to astrocytes, express GFAP and include cells found in the liver, kidney, and gut.

This indicates that GFAP immunoreactivity is not an exclusive astrocyte marker and it also has no bearing on the functional properties of the cells (Garcia et al, 2004). That being said, GFAP expression has been confirmed in other NSC populations and some evidence is beginning to suggest that radial glia act as neural stem cells (Garcia et al, 2004). One of the important genetic steps for conversion of OPCs into multipotent NSC is the induction of Sox2 gene expression (Kondo and Raff, 2004a).

The Sox2 gene belongs to the SOXB1 group of transcription factors which is made up of 3 closely related genes including Soxl, Sox2, and Sox3 (Graham et al, 2003). The SOXB1 genes are widely expressed in vertebrate neural stem cells throughout the developing and adult brain (Graham et al, 2003). In fact, Sox2 is one of the first transcription factors expressed in the developing neural tube (Kondo and Raff, 2004a). The SOXB1 factors that are expressed in NSC have been shown to be important for the maintenance and self-renewal of these cells (Episkopou, 2005).

As NSC differentiate and exit the cell cycle expression of Sox2 decreases. In work done by Graham et al. (2003) they found that NSC that over expressed Sox2 failed to differentiate and cells in which Sox2 expression was blocked exited the cell cycle and differentiated into neurons. The exposure of OPCs to BMP is important for the induction of multipotent neural stem cells. Bone morphogenetic proteins are a subclass of the transforming growth factor /3 superfamily and are known to be important in many developmental processes including differentiation, mophogenesis, lineage commitment, cell survival and apoptosis (Mehler et al.

, 1997). In addition to their role in development, they have been shown to be important in maintaining pluripotency in both mouse embryonic stem cells and NSCs (Nakashima et al, 2001). Nakashima et al, 2001 showed that mouse telencepahlic neural progenitor cells were able to differentiate into astrocytes after exposure to both BMP and leukemia inhibitory factor. After brief exposure to BMP, OPCs are able to convert to a type 2 astrocyte and into a multipotent CNS stem cell (Kondo and Raff, 2000, 2004a,b).

The exact mechanism by which this happens in OPCs is not fully understood but in mouse neural progenitor cells BMP exposure increases the expression of inhibitor of differentiation genes Id 1 and Id 3 (Nakashima et al, 2001). BMP are present in the adult and developing nervous system and yet there is no evidence that OPCs are able to become multipotent CNS stem cells in vivo (Kondo and Raff 2004b). This can partly be explained by Noggin which is a BMP antagonist. Noggin is expressed by oligodendrocytes and type 1 astrocytes and it interferes with the binding of BMP to their receptors (Kondo and Raff 2004b).

Kondo and Raff (2004b) demonstrated that over expression of Noggin by OPCs decreased the number of cells that were able to become multipotent stem cells, and interference of Noggin production by siRNA increased the ability of conversion. This helps demonstrate the importance of environmental cues in maintaining cell restrictions. The effect of exogenous factors on the plasticity of OPCs is important in understanding not only the basic biology of these cells, but also their potential use in therapies.

Oligodendrocyte precursor cells are widely distributed in the CNS and constitute a major cycling population in the parenchyma of the brain and spinal cord (Dawson et al, 2003). In some regions of the adult rat CNS, as many as 70% of the cells that are dividing have been found to be OPCs (Dawson et al, 2003). hi humans, OPCs comprise 3% of the cells in the subcortical white mater (Nunes et al, 2003). The widespread distribution of OPCs and their potential to revert into a cell that can give rise to all three major cell types of the CNS makes them very appealing therapeutic targets (Kondo and Raff, 2004a).

Stem cells as therapeutic candidates Successful repair of any injured tissue involves the removal of the damaged cells followed by replacement of those cells allowing a return to its original structure and function (Rossi and Cattaneo, 2002). The potentials for stem cells to be used as therapeutic agents are varied and include providing replacements for lost cells, activation of endogenous stem cell populations, or transplantation of modified stem cells to release specific trophic factors (Cao et al, 2002).

There are many obstacles to overcome before stem cells can be used effectively as neuronal replacements in the treatment of diseases associated with the CNS. One of them is what is considered a functional neuron? hi order for a stem cell derived neuron to be considered functional it must be stably differentiated, polarized showing a single axon and multiple dendrites, capable of generating an action potential, and not only able to release neurotransmitters but also possess receptors for them (Reh, 2002).

Another problem in the CNS is the integration of the transplanted neurons. Even though you have a functionally differentiated cell the proper integration of replacement neurons is very difficult due to the complex connections that these cells make and the distances over which some of the axons must travel to make proper connections (Rossi and Cattaneo, 2002).

Regardless of the avenue that is chosen for stem cell therapies certain key factors must be met, and these depend on the successful engraftment into recipient tissue which depends on: 1) Survival of the stem cell in the recipient tissue 2) stable phenotypic expression of the differentiated stem cell and 3) proper integration of the cell into recipient tissue (Rossi and Cattaneo, 2002). Stated another way, successful engraftment depends on the intrinsic potential of the stem cell and multiple environmental factors of the recipient tissues.

The role of the local environment in which cells are grafted has been shown to have great influence on the fate of transplanted cells (Gage, 2000). In the case of the CNS, it has been shown that glial cells have a large influence on the recipient environment. A better understanding of how the glia influence the recipient environment and what effects this has on cell receptor expression and differentiation is crucial to further research in the area of stem cell transplantation and manipulation. Works Cited

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