[Show abstract][Hide abstract] ABSTRACT: Adult human mesenchymal stem cells (hMSCs) possess multilineage differentiation potential, and differentiated hMSCs have recently been shown to have the ability to transdifferentiate into other lineages. However, the molecular signature of hMSCs is not well-known, and the mechanisms regulating their self-renewal, differentiation, and transdifferentiation are not completely understood. In this study, we demonstrate that fully differentiated hMSCs could dedifferentiate, a likely critical step for transdifferentiation. By comparing the global gene expression profiles of undifferentiated, differentiated, and dedifferentiation cells in three mesenchymal lineages (osteogenesis, chondrogenesis, and adipogenesis), we identified a number of "stemness" and "differentiation" genes that might be essential to maintain adult stem cell multipotency as well as to drive lineage-specific commitment. These genes include those that encode cell surface molecules, as well as components of signaling pathways. These genes may be valuable for developing methods to isolate, enrich, and purify homogeneous population of hMSCs and/or maintain and propagate hMSCs as well as guide or regulate their differentiation for gene and cell-based therapy. Using small interfering RNA gene inactivation, we demonstrate that five genes (actin filament-associated protein, frizzled 7, dickkopf 3, protein tyrosine phosphatase receptor F, and RAB3B) promote cell survival without altering cell proliferation, as well as exhibiting different effects on the commitment of hMSCs into multiple mesenchymal lineages.
[Show abstract][Hide abstract] ABSTRACT: MicroRNAs (miRNAs) are a group of recently discovered small RNAs produced by the cell using a unique process, involving RNA polymerase II, Microprocessor protein complex, and the RNAase III/Dicer endonuclease complex, and subsequently sequestered in an miRNA ribonucleoprotein complex. The biological functions of miRNAs depend on their ability to silence gene expression, primarily via degradation of the target mRNA and/or translational suppression, mediated by the RNA-induced silencing complex (RISC). First discovered in Caenorhabditis elegans (lin-4), miRNAs have now been identified in a wide array of organisms, including plants, zebrafish, Drosophila, and mammals. The expression of miRNAs in multicellular organisms exhibits spatiotemporal, and tissue- and cell-specificity, suggesting their involvement in tissue morphogenesis and cell differentiation. More than 200 miRNAs have been identified or predicted in mammalian cells. Recent studies have demonstrated the importance of miRNAs in embryonic stem cell differentiation, limb development, adipogenesis, myogenesis, angiogenesis and hematopoiesis, neurogenesis, and epithelial morphogenesis. Overexpression (gain-of-function) and inactivation (loss-of-function) are currently the primary approaches to studying miRNA functions. Another family of small RNAs related to miRNAs is the small interfering RNAs (siRNAs), generated by Dicer from long double-stranded RNAs (dsRNAs), and produced from an induced transgene, a viral intruder, or a rogue genetic element. siRNAs silence genes via either mRNA degradation, using the RISC, or DNA methylation. siRNAs are actively being applied in basic, functional genetic studies, particularly in the generation of gene knockdown animals, as well as in gene knockdown studies of cultured cells. These studies have provided invaluable information on the specific function(s) of individual genes. siRNA technology also presents exciting potential as a therapeutic approach in disease prevention and treatment, as suggested by a recent study targeting apolipoprotein B (ApoB) in primates. Further elucidation of how miRNAs and other small RNAs interact with known and yet-to-be identified gene regulatory pathways in the cell should provide us with a more in-depth understanding of the mechanisms regulating cellular function and differentiation, and facilitate the application of small RNA technology in disease control and treatment.
Birth Defects Research Part C Embryo Today Reviews 06/2006; 78(2):140-9. DOI:10.1002/bdrc.20070 · 2.63 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Much of the knowledge regarding the regulatory pathways for adult stem cell self-renewal and differentiation has been obtained from the results of in vitro cultures. However, it is unclear if adult stem cells are controlled in the same way under physiological conditions. We examined this issue with respect to the migration of stem cells to tissue injury and how switch from a migratory state to one of proliferation wherein they participate in development. Building on our previous identification of multipotent stem cells in trabecular bone, we have examined the in vitro behavior of these cells within the bone milieu. We found that cell proliferation is inhibited within the trabecular bone niche as cells migrate out of the trabecular bone prior to proliferation. Additionally, multiple cell types were detected in adult trabecular bone, including osteoblasts, osteoclasts, endothelial cells, and Stro-1-positive mesenchymal stem cells. Furthermore, we demonstrated that Stro-1-positive cells migrated out of their native bone niche to generate multipotential stem and progenitor cells during in vitro culture. We conclude that self-renewal and differentiation of adult stem cells in connective tissues are tightly controlled and separately orchestrated processes. A regulatory network of extrinsic factors and intrinsic signals acts to stimulate the exit of stem cells from their niche so that they can localize to sites of wound healing, where they participate in development after functional differentiation.
Stem Cells and Development 01/2006; 14(6):712-21. DOI:10.1089/scd.2005.14.712 · 3.73 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Transdifferentiation is a process whereby one cell type committed to and progressing along a specific developmental lineage switches into another cell type of a different lineage through genetic reprogramming. Even though this process has been well studied and established in amphibian systems, it is unclear if mammalian cells possess the same potential. Recent in vivo transplantation studies showed that adult mesenchymal stem cells (MSCs) were able to differentiate into mesoderm-derived cell types as well as cells with neuroectodermal and endodermal characteristics, suggesting that transdifferentiation occurs in mammalian systems. However, there are concerns over these findings because of the possibility of progenitor cell contamination and cell fusion. In this study, we have developed an in vitro differentiation strategy to assess if human MSCs that have differentiated into a given mesenchyme cell lineage can transdifferentiate into other cell types in response to inductive extracellular cues. Our results showed that fully differentiated cells from hMSCs were capable of dedifferentiation and transdifferentiation into cells of another developmental lineage at single cell levels.
The FASEB Journal 07/2004; 18(9):980-2. DOI:10.1096/fj.03-1100fje · 5.04 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Gene delivery is an essential research tool for elucidating gene structure, regulation, and function in biomedical research and is the technological basis for gene therapy. However, the application of nonviral vectors in mammalian cell transfection and gene therapy is limited in that current methods require large amounts of exogenous DNA and/or exhibit high cytotoxicity and low transfection efficiency in primary cells. Here we describe the development of a novel, noninvasive gene delivery protocol using plasmid DNA vectors, based on the principle of electric field-induced molecular vibration. This method enables foreign DNA molecules to penetrate the plasma membrane and enter the cytoplasm of both primary mesenchymal progenitor cells and established cell lines of various species, at high efficiency and with low cell mortality. This procedure requires no special reagents, allows stable expression of transduced DNA, and does not interfere with the normal cellular differentiation activities of human and chick mesenchymal progenitors.