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White Papers |
Fluorescent Stem Cells: Evidence for Transfer of Fluorescent
Probes from Human MSC to Chondrogenic Lineage
Precis:
Vitro Biopharma recently introduced “Tools for Stem Cell and Drug Discovery™”, a series of products to support stem cell research and drug discovery. These products are based on human mesenchymal stem cells (MSC) derived from cord blood and include various cell lines and media optimized to support their growth and self-renewal. We are expanding this line of core products to include culture media to induce differentiation to specific terminally differentiated phenotypes and additional products for use in induced pluripotent stem cell (iPS) research to facilitate development of iPS generation methods that do not require introduction of foreign DNA via transfection. Adult MSC have potential clinical application in the treatment of an expanding list of diseases including cerebral palsy, bone fractures, rheumatoid arthritis and other autoimmune disorders based upon their multipotent differentiation capacity, immunosuppressive properties and other factors as well. An emerging property of these non-hematopoietic stem cells that appears central to therapeutic benefit is homing or migration to specific cellular targets, including damaged cells within specific tissues, cancer stem cells and regions of cellular inflammation. Thus, for example, intravenously injected human MSC presumably migrate to CNS regions through the blood-brain barrier and additional CNS structures to sites of cerebral injury, resulting in recovery of functions lost due to intrauterine stroke, a common pathophysiology leading to cerebral palsy. The tropism of MSC to specific targets is well established even though the underlying mechanisms are under active investigation including the role of cytokine receptors such as cytokine receptor 2 and this research may well lead to other applications of stem cells in the targeted delivery of therapeutic agents. We have recently developed a series of fluorescent labeled human MSC for use as tools to further enhance stem cell tropism research. Our development of these products has led to initial studies of their research applications and we present these findings here that show not only presence of label within stem cells but also provide initial evidence suggesting transfer of fluorescent label to differentiated cells including those of the chondrogenic lineage. Our results are discussed with regard to potential underlying mechanisms and the critical necessity of appropriate stem cell labeling methodology to minimize artefactual results.
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POU5-F1 Expression in Human Mesenchymal Stem Cells
Precis:
Induced pluripotent stem cells (iPS) enable pluripotentiality through the reprogramming of adult cells thus avoiding use of embryonic cells and the associated ethical dilemmas. IPS technology has considerable clinical potential in the generation of personalized, autologous cells with pluripotent differentiation capacity and thus numerous medical applications. Original methods of iPS generation involved transfection with: POU5-F1 (Oct 3-4), Klf4, Sox2 and c-Myc (Wernig, M, et. al, Nature 448:318-24, 2007). However, clinical application of iPS technology will require methods of iPS generation without transfection of target cells with foreign genes to ensure safety. Here, we describe a well-characterized human MSC line expressing POU5-F1, considered a master regulatory element or gatekeeper of pluripotentiality (Pan, GJ, et. al, Cell Res.12: 321-9, 2002), and its application to studies of de-differentiation and differentiation of these modified human MSC.
Our results show that human MSC transfected with an Oct4/POU5F1 expression vector exhibited expanded differentiation capacity as determined by a stem cell potency assay (LumiSTEM™-96) and enhanced capacity for adipogenic differentiation without use of classic differentiation agents. Other groups have also shown similar results in independent investigations. Hans Scholer and colleagues at the Cell and Developmental Biology group at Max Planck Institute for Molecular Biomedicine, showed that adult neural stem cells could be re-programmed to pluripotency by over-expression of Oct-4 only (Kim, JB, et. al, Cell 136: 411-419, 2009). And, Michela Deleidi and colleagues at the Center for Neuoregeneration Research, Harvard Medical School, showed that adult neural stem cells could be induced to form dopamine neurons by expression by Oct-4 induced dedifferentiation. (Keystone Symposium, B-4 Abstract no. 138). Taken together, the work ongoing by these independent groups suggests simplified reprogramming to pluripotency in adult stem cells through induced expression of Oct-4 only and may lead to enhanced methods of iPS generation together with methods which do not require transfection.
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POU5-F1 Expression in Human Mesenchymal Stem Cells
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Copyright by Vitro Diagnostics, Inc. 2009, all rights reserved.