Anti human cd90

To track the injected MSCs in vivo, cell suspensions of human MSCs were labeled with live cell tracker CM-Dil (Cat: C7000; Thermo Fisher Scientific) and incubated in 1 µL of labeling solution per mL of Hank Balanced-Salt Solution (Sciencell, Carlsbad, CA) for 5 minutes at 37°C followed by incubation at 4°C for 15 minutes. After rinsing three times with PBS, cells were plated into a flask and left overnight in the 37°C incubator. This procedure was repeated daily for three consecutive days. A total of 1 × 10⁵ labeled MSCs were subconjuctivally injected into the right eye of rats and 1 × 10⁶ labeled MSCs for rabbits at two sites. An in vivo fluorescent microscope (Axiozoom; Carl Zeiss) was used to visualize the MSCs immediately after injection and weekly up to 12 weeks for rats and up to 2 weeks for rabbits. Enucleated MSC-injected rabbit eyes were cryosectioned, fixed with 4% paraformaldehyde, mounted on a slide with Vectashield mounting medium (Vector Laboratories, Burlingame, California) and imaged using a confocal microscope (LSM 710, Carl Zeiss). Additional tracking of the human MSCs was performed by immunostaining of the rabbit cornea with anti-human CD90 (from Biolegend; cat#328107, FITC antihuman CD90 [Thy1]).

Flow cytometric analysis was carried out by entrusting a biotechnology company
(Jiamai Biotechnology Co., Ltd, Guangzhou, China). HDPSCs were washed and
collected into the flow tube. Then, PBS containing primary antibodies were added
into the sample for incubation for 2 h at room temperature in the dark:
fluorescein isothiocyanate (FITC)-conjugated anti-human Stro-1 (Proteintech,
China, FITC-65184), allophycocyanin (APC)-conjugated anti-human CD45 (Biolegend,
USA, 304011), or anti-human CD90 (Biolegend, USA, 328113) and phycoerythrin
(PE)-conjugated anti-human CD146 (Proteintech, China, PE-65181), anti-human CD29
(Biolegend, USA, 303003), anti-human CD11b (Biolegend, USA, 393111), or
anti-human CD34 (Biolegend, USA, 343505). No primary antibody incubated sample
was used as a negative control. The cell suspensions were washed twice,
resuspended in PBS, and analyzed with a Novocyte D2060R Flow Cytometer (Agilent,
USA). Novoexpress 1.5.6 (Novoexpress Software, USA) was employed for data
analysis and graph plotting.