Project description:Mesenchymal stromal cells (MSCs) are cultured cells that can give rise to mature mesenchymal cells under appropriate conditions, and secrete a number of biologically relevant molecules that may play an important role in regenerative medicine. Evidence indicates that pericytes (PCs) correspond to mesenchymal stem cells in vivo and can give rise to MSCs when cultured, but a comparison between the gene expression profiles of cultured PCs (cPCs) and MSCs is lacking. We have devised a novel methodology to isolate PCs from human adipose tissue, and compared cPCs to MSCs obtained through traditional methods. Freshly isolated PCs expressed CD34, CD140b, and CD271 on their surface, but not CD146. Both MSCs and cPCs were able to differentiate along mesenchymal pathways in vitro, displayed an essentially identical surface immunophenotype, and exhibited the ability to suppress CD3+ lymphocyte proliferation in vitro. Microarray expression data of cPCs and MSCs formed a single cluster among other cell types. Further analyses showed that the gene expression profiles of cPCs and MSCs are extremely similar, although MSCs differentially expressed endothelial cell-specific transcripts. These results confirm, using the power of transcriptomic analysis, that PCs give rise to MSCs, and suggest that low levels of endothelial cells may persist in MSC cultures established using traditional protocols. Highly purified human adipose-tissue pericytes (AT3G5Cs) were isolated based on the expression of the antigen detected by the 3G5 antibody, lack of expression of CD31, and ability to adhere to tissue-cultured plastic whithin a short time. The in vitro properties of cultured AT3G5Cs were compared to those of adipose tissue mesenchymal stromal cells (ATMSCs) obtained through traditional methods. Gene expression profiles of cultured AT3G5Cs (n = 3 different biological samples) and ATMSCs (n = 3 different biological samples) were compared to each other by statistical analyses. Gene expression profiles of cultured AT3G5Cs and ATMSCs were compared to those of other cell types by clustering analyses.
Project description:Dedifferentiated fat (DFAT) cells, established in vitro from mature adipocytes, exhibit properties of multipotent mesenchymal stem/stromal cells (MSCs), such as the ability to differentiate into multiple mesenchymal lineages. Although DFAT cells exhibit certain properties of proliferative progeny, at present there is only limited knowledge about their characteristics as MSCs because those cells are considered to be potential artifacts of cell culture. To elucidate the identity of DFAT cells, we compared gene expression profiles of human DFAT cells and adipose-derived stem/stromal cells (ADSCs) established using adipose tissue from the same donors. Microarray analysis showed that the global RNA expression profiles of human DFAT cells were very similar to those of ADSCs, a representative MSC, despite being committed adipocyte progenitors. Subcutaneous adipose tissues that were obtained during surgical operation for non-malignant disease were donated by 3 patients after obtaining informed consent. Three sets of DFAT cells and ADSCs, each derived from adipose tissue from the same donor were used for RNA extraction and subsequent microarray analysis.
Project description:Pericytes derived from skin dermis can substantially enhance the short-term tissue-regenerative capacity of human epidermal cells already committed to differentiation; they also display both phenotypic and functional properties of mesenchymal stem cells. In this microarray analysis, we compared the gene expression profile of dermal pericytes to that of the remaining dermal cells of neonatal human foreskin.
Project description:Limbal stromal cells were reported to resemble mesenchymal stem cells (MSCs) with multipotential differentiation cability. However, little is known about their gene expression profiles compared to MSC derived from various sources. In this study, the gene expression profile of limbal stromal cells was compared to bone marrow, adipose stromal cells and foreskin fibroblasts. In addition, we also explored the gene expression changes of ex vivo expanded limbal stromal cells when cultured in two different systems. Expanded limbal stromal cells were divided into two groups; each cultured separately on a matrigel-coated plate in DMEM/F12 medium supplemented with bFGF and LIF and the other on a normal plate in DMEM medium supplemented with 10% fetal bovine serum (FBS). Cryopreserved bone marrow mesenchymal cells, adipose stromal cells and foreskin fibroblasts were cultured-expanded until confluent. Total RNA was extracted from all the samples and subjected to microarray experiments with an Agilent platform by using Human GE 8x60k microarrays. Data analysis was carried out with GeneSpring software. A total of 871 genes were upregulated when the limbal stromal cells were cultured in the matrigel system, whereas 58 genes were consistently differentially expressed in limbal stromal cells compared to other lineages. Besides the long intergenic non-coding RNA and unknown genes, these genes represent gene ontology for cellular components, molecular function and biological process. Samples derived from the same source were closely clustered by Hierachical clustering analysis. The limbal stromal cells have a distinct molecular signature compared to MSCs from other lineages. The culture system affected the gene expression profile of limbal stromal cells tremendously. Derived limbal stromal cells were cultured using two different methods, one with matrigel and the other with FBS. Their gene expression profiles were compared. The gene expression profile of limbal stromal cells that were cultured with FBS also was compared to the gene expression profiles of bone marrow mesenchymal stem cells, adipose stromal cells and foreskin fibroblasts.
Project description:Mesenchymal stromal cells (MSCs) are cultured cells that can give rise to mature mesenchymal cells under appropriate conditions, and secrete a number of biologically relevant molecules that may play an important role in regenerative medicine. Evidence indicates that pericytes (PCs) correspond to mesenchymal stem cells in vivo and can give rise to MSCs when cultured, but a comparison between the gene expression profiles of cultured PCs (cPCs) and MSCs is lacking. We have devised a novel methodology to isolate PCs from human adipose tissue, and compared cPCs to MSCs obtained through traditional methods. Freshly isolated PCs expressed CD34, CD140b, and CD271 on their surface, but not CD146. Both MSCs and cPCs were able to differentiate along mesenchymal pathways in vitro, displayed an essentially identical surface immunophenotype, and exhibited the ability to suppress CD3+ lymphocyte proliferation in vitro. Microarray expression data of cPCs and MSCs formed a single cluster among other cell types. Further analyses showed that the gene expression profiles of cPCs and MSCs are extremely similar, although MSCs differentially expressed endothelial cell-specific transcripts. These results confirm, using the power of transcriptomic analysis, that PCs give rise to MSCs, and suggest that low levels of endothelial cells may persist in MSC cultures established using traditional protocols.
Project description:Dedifferentiated fat (DFAT) cells, established in vitro from mature adipocytes, exhibit certain properties of multipotent mesenchymal stem/stromal cells (MSCs), such as the ability to differentiate into multiple mesenchymal lineages. Although DFAT cells exhibit properties of proliferative progeny, at present there is only limited knowledge about their MSC-specific characteristics because those cells are considered to be potential artifacts of cell culture. To elucidate the identity of DFAT cells, we compared gene expression profiles of human DFAT cells and adipose-derived stem/stromal cells (ADSCs) established using adipose tissue from the same donors. Microarray analysis showed that global mRNA expression profiles of human DFAT cells were very similar to those of ADSCs, a representative MSC, despite being committed adipocyte progenitors.
Project description:The gene expression profile of non-cultured, highly purified human adipose tissue pericytes: transcriptomic evidence that pericytes are stem cells in human adipose tissue
Project description:Pericytes are a key player in vascularization, protecting endothelial cells from external harm and promoting the formation of new vessels when necessary. However, pericytic identity and its relation with other cell types, such as mesenchymal stromal/stem cells, is highly debated. This study compares the role of pericytes and unselected stromal cells in vascularization using multichannel microfluidic chips. In both angiogenesis and vasculogenesis, pericytes promote more vessel formation than stromal cells. Pericytes can wrap around endothelial vessels acting as mural cells, while stromal cells remain separated. Whole-transcriptome sequencing confirms an upregulation of pro-vascularization genes in endothelial cell-pericyte co-cultures, while metabolism increases and inflammation decreases in stromal cell co-cultures. Treatment of stromal-endothelial cell co-cultures with either conditioned media or isolated extracellular vesicles from pericytes replicates the increase in vasculogenesis of the direct co-cultures. Cytokine quantification reveals that IL-6 is significantly increased in pericyte conditions. Blocking it with siltuximab results in a reduction of pericyte vasculogenic potential comparable to stromal cell levels, revealing that pericyte pro-vascularization is mediated by IL-6. This study provides new insights into the relationship between pericytes and endothelial cells and the elusive identity of mesenchymal stromal cells. These findings are relevant for both vascular biology and tissue engineering.
Project description:Despite their key role in immunity our understanding of primary and secondary lymphoid stromal cell heterogeneity and ontogeny remains limited. Here, using genome-wide expression profiling and phenotypic and localization studies, we identify a functionally distinct subset of BP3-PDPN+PDGFRβ+/α+CD34+ stromal adventitial cells in both lymph nodes and thymus that is located within the perivascular niche surrounding PDPN-PDGFRβ+/α-Esam-1+ITGA7+ pericytes. In re-aggregate organ grafts adult CD34+ adventitial cells gave rise to multiple thymic and lymph node mesenchymal subsets including pericytes, FRC-, MRC- and FDC-like cells, the development of which was lymphoid environment dependent. During thymic ontogeny pericytes developed from a transient population of BP3-PDPN+PDGFRβ+/α+CD34-/lo anlage-seeding progenitors that subsequently up-regulated CD34 and we provide evidence suggesting that similar embryonic progenitors give rise to lymph node mesenchymal subsets. These findings extend the current understanding of lymphoid mesenchymal cell heterogeneity and highlight a role of the CD34+ vascular adventitia as a potential ubiquitous source of lymphoid stromal precursors in postnatal tissues. To comprehensively study the differences and similarities between mesenchymal stromal subsets in the thymus and lymph nodes, global gene expression analysis was performed on sorted PDPN-, BP-3-PDPN+ and BP-3+PDPN+ PDGFRb+ lymph node mesenchymal cells (LNMC) as well as PDPN- and BP-3-PDPN+ PDGFRb+ thymic mesenchymal cells (TMC) from 2 w old mice by microarray. Total RNA was prepared from TMC and LNMC (pooled inguinal, brachial and axillary LN) subsets sorted from 3 (TMC) and 10-11 (LNMC) 2 weeks old mice per experiment. Isolated RNA from 3 individual experiments was amplified and prepared for hybridization to the Affymetrix Mouse Gene 1.1 ST Array at a genomics core facility: Center of Excellence for Fluorescent Bioanalytics (KFB, University of Regensburg, Germany)
Project description:Comparative gene expression profiles of human retinal pericytes (HRMPC) and lipoaspirate derived mesenchymal stromal cells (adipose stromal cells, ASC) cultivated either in normal (1g/l) or high (4.5g/l) glucose medium to identify similarities and discrepancies and elucidate high glucose effects considering cell-based therapies in diabetic retinopathy. A hallmark of diabetic retinopathy is pericyte- dropout increased vascular permeability and progressive vascular occlusion. ASC and HRMVPC were cultured in NG or HG for 7 days. Then cells were trypsinized, washed and RNA isolated using Qiagen RNeasy mini Kit (Qiagen, Hilden, Germany). RNA quality was tested by capillary electrophoresis on an Agilent 2100 Bioanalyzer (Agilent) and high quality (RNA integrity values ≥ 9) was confirmed.