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amaxa eNews #2
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The Appeal of Somatic Stem Cells for Research and Gene Therapy
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Since the first bone marrow transplant approximately 50 years ago, the use of stem cells for regenerative medicine as well as for basic research has caught the attention of the general public. The capability of stem cells for self-renewal and their ability to differentiate into a wide range of cell types has opened up avenues for examining developmental and cell fate determination pathways, as well as the possibility of eventual therapeutic treatments for a wide range of medical conditions.
Much attention has lately been focused on the use of human embryonic stem (ES) cells (and on the associated ethical and moral considerations). However, numerous other types of stem and progenitor cells exist, many with attributes that are likely to make them even more desirable for specific research and therapeutic applications than ES cells.
ES cells have become a favored tool for studying development and cell fate determination, providing an experimental model to investigate stages of development that are inaccessible in vivo, and allowing insight into many of the molecular and cellular mechanisms responsible for the onset and/or progression of human diseases.
The virtually unlimited capability of ES cells for self-renewal and their ability to differentiate into any cell type also seemed to make them ideal candidates for regenerative therapies. But beyond the legal, moral, and ethical considerations of this type of research, the very same qualities which make these cells so attractive, i.e. their proliferative capacity and pluripotency, have also led to concerns about the possibility of tumor development and differentiation of implanted cells into inappropriate cell types.
Indeed, implantation of mouse ES cells into intact or injured rodent brains resulted in only a fraction of the cells expressing neural markers, while some cells expressed markers of other cell types, including muscle and epithelial cells [1,2], and a number of animals developed teratoma-like structures at the implantation sites [1].
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Somatic (adult) stem cells (SSC)
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While SSC may not have the innate versatility of ES cells, it is often these "limitations" which make them particularly desirable for both basic research and ultimately for cell replacement therapies.
The presumptive biological role of SSC is to remain quiescent within tissues until called upon to repair or regenerate damage.
Unlike ES cells which must be actively inhibited from differentiating in culture (by co-culturing with feeder cells and/or appropriate cytokines), SSC remain quiescent unless acted upon by specific proliferative and/or inductive agents and are often already committed to specific lineages. Such qualities are likely to make them more suitable for transplantation purposes, minimizing false differentiation and tumor risk following transplantation, as well as for studying specific differentiation and developmental pathways.
Importantly, SSC can be isolated from adult tissues. This not only avoids the moral issues associated with ES cells, but also allows the possibility of autologous transplants, i.e. extracting cells from the patient, then modifying and re-injecting them into the site of interest.
Many different types of SSC have been isolated from different stages of differentiation, providing researchers with a wide range of choices according to the specific needs of their research.
Epiblast-like stem cells (ELSC)
Pluripotent epiblast-like stem cells (ELSC) [3-4] can be released from connective tissue compartments, skeletal muscle, and dermis. Like ES cells, ELSC can form cells from all three embryonic germ layer lineages. They do not express markers for germ layer lineage stem cells but rather Oct-3/4, which is characteristic of ES cells. However, ELSC differ from ES cells in that they do not differentiate spontaneously, but rather remain quiescent unless activated.
Germ layer lineage cells
Germ layer lineage cells [3] are committed to form cells from a specific embryonic germ layer. They can be released from connective tissue compartments, and express both general and specific lineage markers. They also have extensive capabilities for self-renewal and maintain telomerase activity as long as they remain uncommitted. Once committed to particular tissue or cell type, germ layer lineage cells lose telomerase activity, and they assume the lifespan of the progenitor cells for their species. These cells do not respond to lineage induction agents that act outside their specific germ-layer lineage.
Progenitor cells
Progenitor cells are located near their respective differentiated cell types, express tissue-specific phenotypic markers and are preprogrammed to commit to particular cell types. They do not spontaneously differentiate but remain quiescent unless acted upon by specific proliferative and/or inductive agents. They do not respond to lineage induction agents that have actions outside their specific germ-layer lineage and have finite life spans before replicative senescence and cell death occurs.
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SSC of high interest
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Bone marrow is often used as an easily accessible source of stem cells as it contains both hematopoietic stem cells (HSC) and mesenchymal stem cells (MSC). HSC give rise to all different blood lineages and stroma-derived MSC generate mesenchymal cells of the osteogenic, chondrogenic, adipogenic, myoblastic, and fibroblastic lineages.
A more primitive adherent stem-cell type, multipotent adult progenitor cells (MAPC), is also found in the marrow and can differentiate into MSCs, endothelial, epithelial, and even hematopoietic cells.
Neural stem cells (NSC) are also of particular interest. These multipotent cells have been isolated from the developing and adult central nervous systems of diverse mammalian species. They can be maintained and expanded in an undifferentiated state or differentiated into more restricted precursor cells which in turn generate the main cell classes in the brain: neurons, oligodendrocytes and astrocytes.
NSC and lineage-restricted neural precursors are among the candidates for cell-based therapeutic treatment conditions ranging from spinal cord injuries to Alzheimer's disease, and knowledge gained from studying these cells is already providing insight into a wide range of central nervous system diseases.
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Genetic manipulation of somatic stem cells
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Different strategies for manipulating SSC exist, facilitating examination of cellular and molecular mechanisms through ectopic- or overexpression studies as well as RNA interference-mediated inhibition of gene expression.
Viral vectors
Viral vectors (such as murine oncoretroviruses, adenovirus, herpes simplex virus, lentiviruses and others) are able to transfer genes into various cells with high efficiency, with retroviruses having the particular advantage of facilitating stable gene transfer through integration of the transferred DNA into the cell's chromosomes. However, generating viral vectors is time consuming, and using them is associated with a number of biosafety issues.
Although the newest generations of viral vectors have significantly reduced the risk of generating replication-competent virus, the possibility of producing a pathogenic virus through recombination remains a considerable concern. Retroviruses are a particular concern as they appear to integrate into host DNA in a largely random fashion, which may lead to both disruption of cellular genes and upregulation of proto-oncogenes by viral enhancers.
Additionally, expression of transgenes would be expected to be affected by cis-elements in neighboring host DNA, resulting in inconsistent gene expression between different clones.
Plasmid vectors
Plasmid vectors have a number of advantages over viral systems: they have low immunogenicity, they are less toxic, more cost-efficient, and easier to both scale-up and to control for quality. But they cannot enter cells by themselves and thus need to be combined with an efficient gene-delivery method.
Unfortunately, standard electroporation and biochemical transfection methodologies have not been particularly efficient in transfecting SSC. However, with the development of nucleofection, a technology is now available which can rival the transfection efficiency of viral systems for many cell types, without the associated bio-safety issues.
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Conclusion
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The flexibility of being able to isolate somatic stem cells from multiple lineages and different stages of differentiation, combined with the ability to efficiently transfect these cells promises to greatly increase our understanding of the cellular and molecular mechanisms governing these cells. This will ultimately facilitate the development of advanced cell replacement therapies.
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References
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[1] Björklund et al. (2002) Embryonic stem cells develop into functional dopaminergic neurons after transplantation in a Parkinson rat model. Proc. Natl. Acad. Sci. USA. 99(4):2344-2349.
[2] Deacon et al. (1998) Blastula-stage stem cells can differentiate into dopaminergic and serotonergic neurons after transplantation. Exp Neurol 1998 Jan;149(1):28-41.
[3] Young, H.E., Duplaa, C., Romero-Ramos, M. et al. (2004) Adult Reserve Stem Cells and Their Potential for Tissue Engineering. Cell Biochemistry and Biophysics 40(1): 1-80.
[4] Young,H.E., Duplaa,C., Young,T.M. et al. (2004) Clonogenic analysis reveals reserve stem cells in postnatal mammals: II. Pluripotent epiblast-like stem cells. Anat. Rec., 277A, 178–203.
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Nucleofection of mouse NSC
Primary mouse NSC isolatd from the lateral ventrical wall of an adult mouse were nucleofected using the Mouse NSC Nucleofector Kit, program A-33/A-O33 and a plasmid encoding the enhanced green fluorescent protein eGFP. 48h post nucleofection, the cells were analyzed by light (A) and fluorescence miscroscopy (B). (Photograph courtesy of Dr. L. Wikstrom et al., NeuroNova, Stockholm, Sweden.)
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amaxa's Nucleofection Process, Nucleofector Device and Nucleofector Solutions are covered by PCT Applications PCT/EP01/07348, PCT/DE02/01489, PCT/DE02/01483, and other pending patents, and domestic or foreign applications corresponding thereto.
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