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. 2015 Dec 1;24(23):2822-40.
doi: 10.1089/scd.2015.0153. Epub 2015 Aug 19.

Cultured Human Adipose Tissue Pericytes and Mesenchymal Stromal Cells Display a Very Similar Gene Expression Profile

Lindolfo da Silva Meirelles  1   2 , Tathiane Maistro Malta  1 , Virgínia Mara de Deus Wagatsuma  1 , Patrícia Viana Bonini Palma  1 , Amélia Goes Araújo  3 , Kelen Cristina Ribeiro Malmegrim  4 , Fábio Morato de Oliveira  1 , Rodrigo Alexandre Panepucci  3 , Wilson Araújo Silva Jr  1   5 , Simone Kashima Haddad  1 , Dimas Tadeu Covas  1   6
Affiliations

Cultured Human Adipose Tissue Pericytes and Mesenchymal Stromal Cells Display a Very Similar Gene Expression Profile

Lindolfo da Silva Meirelles et al. Stem Cells Dev. .

Abstract

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 (EC)-specific transcripts. These results confirm, using the power of transcriptomic analysis, that PCs give rise to MSCs and suggest that low levels of ECs may persist in MSC cultures established using traditional protocols.

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Figures

<b>FIG. 1.</b>
FIG. 1.
Morphology of 3G5+CD31− cells, 3G5+CD31+ cells, and 3G5−CD31+ cells after 12 h in culture. (A–F) Low-power images demonstrate cells derived from 3G5+CD31− cells (A, D), which were adherent in their majority compared with cells derived from 3G5+CD31+ cells (B, E), or 3G5−CD31+ cells (C, F), which were nonadherent in their majority. (G–L) Higher power images that detail the morphology of the adherent cells derived from the three cell populations examined. (H, K) Adherent cells with a pericytic morphology aggregated with nonadherent cells are visible (white arrows). (H) A cell clump corresponding to a microvessel fragment is indicated by a black arrow. (I) Adherent cells with endothelial morphology are indicated (white arrow). (L) White arrows indicate adherent cells with a cell morphology characteristic of macrophages.
<b>FIG. 2.</b>
FIG. 2.
Morphology and cytogenetics of cAT3G5Cs. (A) On day three after isolation, cAT3G5Cs exhibited various morphologies, with a few or multiple cellular projections. (B) On day 5 after isolation, cAT3G5Cs were already fibrolastoid. This morphology was maintained on day 7 after isolation (C) and after serial passaging. (D) cAT3G5Cs after the first passage. (E) Spectral karyotype of cAT3G5Cs. cAT3G5Cs, cultured adipose tissue-derived 3G5+CD31− cells.
<b>FIG. 3.</b>
FIG. 3.
In vitro functional characterization of cAT3G5Cs and ATMSCs. (A) cAT3G5Cs subjected to adipogenic differentiation, stained with Oil Red O to show lipid-laden vacuoles (visible as dark gray—black in grayscale), and counterstained with Harris hematoxylin. (B) Negative control for (A). (C) Micrograph of cAT3G5Cs subjected to osteogenic differentiation and stained with Alizarin Red S, which highlights calcium in the extracellular matrix in red (visible as dark gray—black in grayscale). (D) Negative control for C. (E) Micrograph of ATMSCs subjected to adipogenic differentiation, stained with Oil Red O, and counterstained with Harris hematoxylin. (F) Negative control for E. (G) ATMSCs subjected to osteogenic differentiation and stained with Alizarin Red S. (H) Negative control for G. (I–N) Macroscopic views of A, E, F, C, G, and H, respectively. Staining intensity is proportional to the intensity of black (I–N). (O) Section of a chondrogenic pellet formed by one cAT3G5C population, stained with Toluidine Blue, which changes color from blue (which would correspond to a dark shade of gray in grayscale) to purple (shown as a light shade of gray) in the presence of sulfated glycosaminoglycans characteristic of cartilage. (P) Myotubes formed in a cAT3G5C culture established from liposuction material without the adherence selection step. (Q) Flow cytometry dot plot showing the presence of a minor population (around 2%) of CD56+ cells (possibly contaminating myogenic cells scraped off muscle during liposuction) in a cAT3G5C culture established without the adherence step. (R) In vitro suppression of CD3+ lymphocyte proliferation by cAT3G5Cs and ATMSCs. ATMSCs, adipose tissue-derived mesenchymal stromal cells.
<b>FIG. 4.</b>
FIG. 4.
Immunophenotype of ATMSCs, cAT3G5Cs, and freshly isolated AT3G5Cs. Solid line histograms depict the expression of the indicated molecules in ATMSCs (A) or cAT3G5Cs (B) from the same donor compared with negative controls (shaded histograms), representative of at least three immunophenotypings, in which ATMSCs and cAT3G5Cs from different donors were compared with each other. (C) Frequencies of cells positive for the indicated surface molecules in ATMSCs and cAT3G5Cs. Bars represent standard deviation. (D) Log(2) means fluorescence intensities (MFIs) of the indicated surface molecules in ATMSCs and cAT3G5Cs. Bars represent standard deviation. (E) Normalized log(2) expression levels of matrix metalloproteinase 3 (MMP3) by ATMSCs, cAT3G5Cs, cAT3G5Cs cultured in MSC medium (cAT3G5Cs DME10), bone marrow MSCs (BMMSCs), dental pulp stem cells (DPSCs), lung fibroblasts (lFBs), human umbilical vein endothelial cells (HUVECs), peripheral blood white blood cells (PBWBCs), and human embryonic stem cells (hESCs). Table cells were colored to reflect MMP3 gene expression intensity shown in the scale below it.
<b>FIG. 5.</b>
FIG. 5.
Immunophenotype of freshly isolated AT3G5Cs. Plastic adherent 3G5+CD31− cells (AT3G5Cs) were isolated from human subcutaneous adipose tissue from two different donors by fluorescence-activated cell sorting, and double-immunostained with antibodies to detect the molecules indicated, or with control antibodies (IgG). (A) Dot plots of AT3G5Cs from donor no. 19. Expression of NG2 is additionally shown as a histogram (solid line) plotted against a control histogram (shaded). (B) Dot plots of AT3G5Cs from donor no. 20. NG2, nerve/glial antigen 2.
<b>FIG. 6.</b>
FIG. 6.
Immunodetection of 3G5+CD31− cells in situ. Cryosections of subcutaneous adipose tissue were stained with an anti-CD31 antibody to identify endothelial cells and with the 3G5 antibody to identify pericytes. (A, B) Micrographs of sections observed using standard fluorescence microscopy; arrows indicate 3G5+ cells. (C–F) Sequence of confocal microscopy micrographs showing nuclei stained with DAPI (C), a 3G5+ cell (D), a microvessel (E), and a composite image (F), with an arrow indicating the 3G5+ cell. DAPI, 4′,6-diamidino-2-phenylindole.
<b>FIG. 7.</b>
FIG. 7.
Hierarchical clustering of cAT3G5Cs, ATMSCs, and other cell types. Microarray data were clustered using Euclidean distance with average linkage. airway FBs, airway fibroblasts; ATMSCs 18 PM, ATMSCs from donor no. 18 cultured in pericyte medium; cAT3G5Cs, adipose tissue-derived 3G5+ cells cultured in pericyte medium; cAT3G5Cs DME10, cAT3G5Cs cultured in ATMSC medium (DMEM + 10% fetal bovine serum); dermal FB iPSCs, dermal fibroblast induced pluripotent stem cells; dermal FBs, dermal fibroblasts; distal lung FBs, distal lung fibroblasts; DMEM, Dulbecco's modified Eagle's medium; ESC, embryonic stem cell (additional letters designate specific cell lines); HMECs, human microvascular endothelial cells; PBWBCs, peripheral blood white blood cells (numbers indicate different samples).
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