We compared the transcriptomes of marrow-derived mesenchymal stem cells (MSCs) with differentiated adipocytes, osteocytes, and chondrocytes derived from these MSCs. Using global gene-expression profiling arrays to detect RNA transcripts, we have identified markers that are specific for MSCs and their differentiated progeny. Further, we have also identified pathways that MSCs use to differentiate into adipogenic, chondrogenic, and osteogenic lineages. We identified activin-mediated transforming growth factor (TGF)- signaling, platelet-derived growth factor (PDGF) signaling and fibroblast growth factor (FGF) signaling as the key pathways involved in MSC differentiation. The differentiation of MSCs into these lineages is affected when these pathways are perturbed by inhibitors of cell surface receptor function. Since growth and differentiation are tightly linked processes, we also examined the importance of these 3 pathways in MSC growth. These 3 pathways were necessary and sufficient for MSC growth. Inhibiting any of these pathways slowed MSC growth, whereas a combination of TGF-, PDGF, and -FGF was sufficient to grow MSCs in a serum-free medium up to 5 passages. Thus, this study illustrates it is possible to predict signaling pathways active in cellular differentiation and growth using microarray data and experimentally verify these predictions. (Blood. 2008;112:295-307)
We provide evidence that the human DNA ligase III gene encodes a mitochondrial form of this enzyme. First, the DNA ligase III cDNA contains an in-frame ATG located upstream from the putative translation initiation start site. The DNA sequence between these two ATG sites encodes an amphipathic helix similar to previously identified mitochondrial targeting peptides. Second, recombinant green fluorescent protein harboring this sequence at its amino terminus was efficiently targeted to the mitochondria of Cos-1 monkey kidney cells. In contrast, native green fluorescent protein distributed to the cytosol. Third, a series of hemagglutinin-DNA ligase III minigene constructs were introduced into Cos-1 cells, and immunocytochemistry was used to determine subcellular localization of the epitope-tagged DNA ligase III protein. These experiments revealed that inactivation of the upstream ATG resulted in nuclear accumulation of the DNA ligase III protein, whereas inactivation of the downstream ATG abolished nuclear localization and led to accumulation within the mitochondrial compartment. Fourth, mitochondrial protein extracts prepared from human cells overexpressing antisense DNA ligase III mRNA possessed substantially less DNA ligase activity than did mitochondrial extracts prepared from control cells. DNA end-joining activity was also substantially reduced in extracts prepared from antisense mRNA-expressing cells. From these results, we conclude that the human DNA ligase III gene encodes both nuclear and mitochondrial enzymes. DNA ligase plays a central role in DNA replication, recombination, and DNA repair. Thus, identification of a mitochondrial form of this enzyme provides a tool with which to dissect mammalian mitochondrial genome dynamics.For some time it was thought that the mammalian mitochondria lacked the capacity to repair damaged DNA (18). This hypothesis had its origin in the observation that pyrimidine dimers produced within the mitochondrial DNA (mtDNA) of cultured cells were not repaired, in marked contrast to similar lesions produced in the nuclear genomes of these same cells (4,15,21). However, it is now clear that this organelle is not entirely deficient in DNA repair activity. For example, it appears that base excision repair of oxidized DNA occurs in mtDNA of mammalian cells (8,23,34,40). In addition, a number of studies have revealed that mammalian cultured cells can repair mtDNA damage caused by a number of chemical agents, including cisplatin (15), bleomycin (33), streptozotocin (22), and 4-nitroquinoline (36). More recently, it was shown that mammalian mitochondrial extracts possess DNA nonhomologous end-joining and homologous recombination activities (14, 41). Taken together, these findings indicate that mammalian mitochondria likely possess a number of DNA repair pathways. A series of recent observations suggest that mutations to the mitochondrial genome may be associated with a variety of human pathologies (5, 9, 10, 38, 49), thus highlighting the need for a greater understanding of the molecular...
IntroductionHuman multipotent mesenchymal stem cell (MSC) therapies are being tested clinically for a variety of disorders, including Crohn's disease, multiple sclerosis, graft-versus-host disease, type 1 diabetes, bone fractures, and cartilage defects. However, despite the remarkable clinical advancements in this field, most applications still use traditional culture media containing fetal bovine serum. The ill-defined and highly variable nature of traditional culture media remains a challenge, hampering both the basic and clinical human MSC research fields. To date, no reliable serum-free medium for human MSCs has been available.MethodsIn this study, we developed and tested a serum-free growth medium on human bone marrow-derived MSCs through the investigation of multiple parameters including primary cell isolation, multipassage expansion, mesoderm differentiation, cellular phenotype, and gene-expression analysis.ResultsSimilar to that achieved with traditional culture medium, human MSCs expanded in serum-free medium supplemented with recombinant human platelet-derived growth factor-BB (PDGF-BB), basic fibroblast growth factor (bFGF), and transforming growth factor (TGF)-β1 showed extensive propagation with retained phenotypic, differentiation, and colony-forming unit potential. To monitor global gene expression, the transcriptomes of bone marrow-derived MSCs expanded under serum-free and serum-containing conditions were compared, revealing similar expression profiles. In addition, the described serum-free culture medium supported the isolation of human MSCs from primary human marrow aspirate with continual propagation.ConclusionsAlthough the described serum-free MSC culture medium is not free of xenogeneic components, this medium provides a substitute for serum-containing medium for research applications, setting the stage for future clinical applications.
The ability of embryonic stem cells and adult stem cells to differentiate into specific cell types holds immense potential for therapeutic use in cell and gene therapy. Realization of this potential depends on efficient and optimized protocols for genetic manipulation of stem cells. In the study reported here, we demonstrate the use of nucleofection as a method to introduce plasmid DNA into embryonic and adult stem cells with significantly greater efficiency than electroporation or lipid-based transfection methods have. Using enhanced green fluorescent protein (eGFP) as a reporter gene, mouse embryonic stem cells were transfected both transiently and stably at a rate nearly 10-fold higher than conventional methods. The transfected cells retained their stem cell properties, including continued expression of the stem cell markers SSEA1, Oct4, and Rex1; formation of embryoid bodies; differentiation into cardiomyocytes in the presence of appropriate inducers; and, when injected into developing blastocysts, contribution to chimeras. Higher levels of transfection were also obtained with human embryonic carcinoma and human embryonic stem cells. Particularly hard-to-transfect adult stem cells, including bone marrow and multipotent adult progenitor cells, were also transfected efficiently by the method of nucleofection. Based on our results, we conclude that nucleofection is superior to currently available methods for introducing plasmid DNA into a variety of embryonic and adult stem cells. The high levels of transfection achieved by nucleofection will enable its use as a rapid screening tool to evaluate the effect of ectopically expressed transcription factors on tissue-specific differentiation of stem cells. Stem
Many of the currently established human embryonic stem (hES) cell lines have been characterized extensively in terms of their gene expression profiles and genetic stability in culture. Recent studies have indicated that microRNAs (miRNAs), a class of noncoding small RNAs that participate in the regulation of gene expression, may play a key role in stem cell self-renewal and differentiation. Using both microarrays and quantitative PCR, we report here the differences in miRNA expression between undifferentiated hES cells and their corresponding differentiated cells that underwent differentiation in vitro over a period of 2 weeks. Our results confirm the identity of a signature miRNA profile in pluripotent cells, comprising a small subset of differentially expressed miRNAs in hES cells. Examining both mRNA and miRNA profiles under multiple conditions using cross-correlation, we find clusters of miRNAs grouped with specific, biologically interpretable mRNAs. We identify patterns of expression in the progression from hES cells to differentiated cells that suggest a role for selected miRNAs in maintenance of the undifferentiated, pluripotent state. Profiling of the hES cell "miRNA-ome" provides an insight into molecules that control cellular differentiation and maintenance of the pluripotent state, findings that have broad implications in development, homeostasis, and human disease states.
SummaryThe first guidance on Good Cell Culture Practice (GCCP) dates back to 2005. This document expands this to include aspects of quality assurance for in vitro cell culture focusing on the increasingly diverse cell types and culture formats used in research, product development, testing and manufacture of biotechnology products and cell-based medicines. It provides a set of basic principles of best practice that can be used in training new personnel, reviewing and improving local procedures, and helping to assure standard practices and conditions for the comparison of data between laboratories and experimentation performed at different times. This includes recommendations for the documentation and reporting of culture conditions. It is intended as guidance to facilitate the generation of reliable data from cell culture systems, and is not intended to conflict with local or higher level legislation or regulatory requirements. It may not be possible to meet all recommendations in this guidance for practical, legal or other reasons. However, when it is necessary to divert from the principles of GCCP, the risk of decreasing the quality of work and the safety of laboratory staff should be addressed and any conclusions or alternative approaches justified. This workshop report is considered a first step toward a revised GCCP 2.0.
As the number of human embryonic stem cell (hESC) lines increases, so does the need for systematic evaluation of each line's characteristics and potential. Comparisons between lines are complicated by variations in culture conditions, feeders, spontaneous differentiation, and the absence of standardized assays. These difficulties, combined with the inability of most labs to maintain more than a few lines simultaneously, compel the development of reference standards to which hESC lines can be compared. The use of a stable cell line as a reference standard offers many advantages. A line with a relatively unchanging hESC-like gene and protein expression pattern could be a positive control for developing assays. It can be used as a reference for genomics or proteomics studies, especially for normalizing results obtained in separate laboratories. Such a cell line should be widely available without intellectual property restraints, easily cultured without feeders, and resistant to spontaneous changes in phenotype. We propose that the embryonal carcinoma (EC) line 2102Ep meets these requirements. We compared the protein, gene, and microRNA expression of this cell line with those of hESC lines and alternative reference lines such as the EC line NTERA-2 and the karyotypically abnormal hESC line BG01V. The overall expression profiles of all these lines were similar, with exceptions reflecting the germ cell origins of EC. On the basis of global gene and microRNA expression, 2102Ep is somewhat less similar to hESC than the alternatives; however, 2102Ep expresses more hESC-associated microRNAs than NTERA-2 does, and fewer markers of differentiated fates. STEM CELLS 2007;25:437-446
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