Primovert inverted

Characterization of the hDPSC products was examined according to recent studies [21 (link), 22 (link), 27 (link)], as described in the Supplementary Methods. As for cell morphology assay, hDPSC products in a T-75 flask (Corning) were imaged under a Primovert inverted microscope (Carl Zeiss Microscopy). The expression of MSC surface markers including human CD146, CD105, CD73, CD90, CD34, CD45, CD14, and human leukocyte antigen DR (HLA-DR), which were referred from a previous report [28 (link)], in hDPSC products was examined by flow cytometric analysis (FCM). The lineage-specific-gene expression was analyzed in the hDPSC products cultured under the specific inductive conditions into osteoblasts, chondrocytes, and adipocytes by quantitative reverse transcription polymerase chain reaction (qRT-PCR) assay. For secretome assay, hepatocyte growth factor (HGF), interleukin 6 (IL6), monocyte chemotactic protein 1 (MCP1), and sialic acid-binding immunoglobulin-type lectin 9 (SIGLEC9) in the CM of the hDPSC products were analyzed by enzyme-linked immunosorbent assay (ELISA).

The tube formation assays were performed on Matrigel (Corning, Wiesbaden, Germany) using μ-Slide angiogenesis slides (Ibidi, Planegg/Martinsried, Germany) according to the manufacturer’s instructions. Then, BEC or LEC cells were plated at a cell density of 1 × 104/well in a complete endothelial cell medium. One hour later, the cells fully adhered on the Matrigel, and the full medium was replaced with the conditioned media (n = 3). The tube networks formed were photographed after 4 and 16 h using a Zeiss Primo Vert inverted microscope fitted with an AxioCam ERc5s camera (Zeiss, Munich, Germany). The number of branches, loops, and branching points was evaluated using the Image J software (version 1.53, U. S. National Institutes of Health, Bethesda, Maryland, USA). The experiments were performed with LEC and BEC cells of two different passages using supernatants from 3 different donors.