The heterogeneity of human mesenchymal stem cell preparations—Evidence from simultaneous analysis of proteomes and transcriptomes

Wagner, Wolfgang; Feldmann, Robert; Seckinger, Anja; Maurer, Martin H.; Wein, Frederik; Blake, Jonathon; Krause, U.; Kalenka, Armin; Bürgers, Heinrich F.; Saffrich, Rainer; Wuchter, Patrick; Kuschinsky, Wolfgang; Ho, Anthony D.

doi:10.1016/j.exphem.2006.01.002

Cited by 175 publications

(124 citation statements)

References 82 publications

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“…20 Adipogenic differentiation characterized by positive staining with oil red O for fat droplets in cells of the adipocyte lineage (Figure 4b) and von Kossa-positive staining for cells of the osteogenic lineage ( Figure 4c) were observed under appropriate conditions for both LV-transduced and non-transduced MSC. Furthermore, eGFP-expressing adipocytes could be generated from LV-transduced MSC as detected by immunofluorescence staining (Figure 4b, lower panel).…”

Section: Puromycin Selection Of Lv-transduced Msc Is Superior To Facsmentioning

confidence: 94%

“…20,21 About 50 ml BM aspirate was obtained from the iliac crest and collected in a syringe containing 10 000 IU heparin to prevent coagulation. Due to the lack of a single definitive marker for MSC, these cells were purified by three criteria: (I) plastic adherence, (II) positive for expression of CD105, CD73, CD90, (III) lack of expression of hematopoietic antigens like CD45, CD34 and markers for monocytes, macrophages and B cells.…”

Section: Methodsmentioning

confidence: 99%

“…We ensured by several control experiments that isolated MSC behave their CD34 À phenotype even after several rounds in culture. 20 In addition, we cultured the MSC in medium for hematological cells (RPMI, 10% fetal calf serum (FCS) or Stemline II medium, thrombospondin (TPO 80 ng ml À1 ), stem cell factor (SCF, 50 ng ml À1 ), FL-3 ligand (50 ng ml À1 )) and did not find differentiation of the MSC to a CD34 þ phenotype. 22 In brief, the mononuclear cell fraction was isolated by Biocoll densitygradient centrifugation (d ¼ 1.077 g cm À3 ; Biochrom, Berlin, Germany).…”

Section: Methodsmentioning

confidence: 99%

“…20,21 After two passages in vitro they represented a mainly homogeneous population of fibroblast-shaped cells as assessed by cell morphology (compare Figures 1c and 2). To determine whether BM-derived MSC could be transduced efficiently with lentiviral vectors, a subconfluent MSC culture was infected with VSV-G-pseudotyped HIV SIN -CMV-eGFP vector particles (Figure 1a).…”

Section: Puromycin Selection Of Lv-transduced Msc Is Superior To Facsmentioning

confidence: 96%

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Improved lentiviral transduction of human mesenchymal stem cells for therapeutic intervention in pancreatic cancer

et al. 2008

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Genetic modification of human bone marrow mesenchymal stem cells (MSC) is highly valuable for their exploitation in basic science and therapeutic applications, for example in cancer. We present here a new, fast and easy-to-use method to enrich a functional population of lentiviral (LV)-transduced MSC expressing enhanced green fluorescent protein (eGFP). We replaced the eGFP gene by a fusion gene of puromycin acetyltransferase and eGFP. Upon LV gene transfer and puromycin selection, we quickly obtained a pure transduced MSC population, in which growth, differentiation capacity and migration preferences were not compromised. Furthermore, we are the first to report the migration velocity of MSC among which 30% were moving and velocity of about 15 mm h À1 was not altered by LV transduction. Manipulated MSC underwent senescence one passage earlier than non-transduced cells, suggesting the use for therapeutic intervention in early passage numbers. Upon tail vein application in nude mice, the majority of LV-transduced MSC could be detected in human orthotopic pancreatic tumor xenografts and to a minor extent in mouse liver, kidney and lung. Together, LV transduction of genes to MSC followed by puromycin selection is a powerful tool for basic research and improves the therapeutic prospects of MSC as vehicles in gene therapy.

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Section: Puromycin Selection Of Lv-transduced Msc Is Superior To Facsmentioning

confidence: 94%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Puromycin Selection Of Lv-transduced Msc Is Superior To Facsmentioning

confidence: 96%

See 2 more Smart Citations

Improved lentiviral transduction of human mesenchymal stem cells for therapeutic intervention in pancreatic cancer

et al. 2008

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“…For example, when MSCs were cultured in the low serum content (2% FBS), the peroxiredoxin family of antioxidant enzymes was more abundantly expressed in this condition. In contrast, genes and proteins in the functional categories of development, morphogenesis, and organogenesis such as actin, tropomyosin, and transgelin are significantly overrepresented in MSCs cultured in a commercially available medium supplemented with 10% FBS [54].…”

Section: Discussionmentioning

confidence: 78%

Differential expression of cell surface proteins in human bone marrow mesenchymal stem cells cultured with or without basic fibroblast growth factor containing medium

Lee

Kim

et al. 2009

Proteomics

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Mesenchymal stem cells (MSCs) are multipotent cells, which have the capability to differentiate into various mesenchymal tissues such as bone, cartilage, fat, tendon, muscle, and marrow stroma. However, they lose the capability of multi-lineage differentiation after several passages. It is known that basic fibroblast growth factor (bFGF) increases growth rate, differentiation potential, and morphological changes of MSCs in vitro. In this report, we have used 2-DE coupled to MS to identify differentially expressed proteins at the cell membrane level in MSCs growing in bFGF containing medium. The cell surface proteins isolated by the biotin-avidin affinity column were separated by 2-DE in triplicate experiments. A total of 15 differentially expressed proteins were identified by quadrupole-time of flight tandem MS. Nine of the proteins were upregulated and six proteins were downregulated in the MSCs cultured with bFGF containing medium. The expression level of three actin-related proteins, F-actin-capping protein subunit alpha-1, actin-related protein 2/3 complex subunit 2, and myosin regulatory light chain 2, was confirmed by Western blot analysis. The results indicate that the expression levels of F-actin-capping protein subunit alpha-1, actin-related protein 2/3 complex subunit 2, and myosin regulatory light chain 2 are important in bFGF-induced morphological change of MSCs.

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Basic Techniques in Human Mesenchymal Stem Cell Cultures: Differentiation into Osteogenic and Adipogenic Lineages, Genetic Perturbations, and Phenotypic Analyses

Bruedigam

Driel

Koedam

et al. 2011

CP Stem Cell Biology

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This unit describes basic techniques in human mesenchymal stem cell (hMSC) cultures. It includes protocols for the differentiation of hMSCs into osteogenic and adipogenic lineages, genetic perturbations, and phenotypic analyses. hMSCs can be differentiated with dexamethasone and β‐glycerophosphate into mineralizing osteoblasts within 2 to 3 weeks, or with dexamethasone, indomethacin, and 3‐isobutyl‐1‐methylxanthine into lipid vesicle–containing adipocytes within 1 to 2 weeks. Phenotypic changes during those highly dynamic differentiation processes can be detected by biochemical and histological assays and gene expression analyses of differentiation markers. In addition, this unit describes an electroporation method that allows the transient genetic perturbation of hMSCs. Curr. Protoc. Stem Cell Biol. 17:1H.3.1‐1H.3.20. © 2011 by John Wiley & Sons, Inc.

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The heterogeneity of human mesenchymal stem cell preparations—Evidence from simultaneous analysis of proteomes and transcriptomes

Cited by 175 publications

References 82 publications

Improved lentiviral transduction of human mesenchymal stem cells for therapeutic intervention in pancreatic cancer

Improved lentiviral transduction of human mesenchymal stem cells for therapeutic intervention in pancreatic cancer

Differential expression of cell surface proteins in human bone marrow mesenchymal stem cells cultured with or without basic fibroblast growth factor containing medium

Basic Techniques in Human Mesenchymal Stem Cell Cultures: Differentiation into Osteogenic and Adipogenic Lineages, Genetic Perturbations, and Phenotypic Analyses

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