Fig. 4

Monocytes-released EVs promote survival and differentiation of iPSC. A Schematic presentation of experimental design. On Day -3, monocytes (Monos) were seeded in the inserts in (i) BM; (ii) BM containing 10 µM CHIR and 5 ng/mL noggin (CN), and (iii) in coculture with iPSC in BM supplemented with CHIR and noggin (Co-cult). After 48 h (Day -1), medium from the well was collected, cell debris were removed by centrifugation, and EVs were precipitated overnight (ON). Monocytes were given the same medium (i-iii) once again. On Day 0, exosomes were added to iPSC in BM containing 10 µM CHIR and 5 ng/mL noggin. On Day, 1 EVs were harvested from monocytes and precipitated overnight (ON). On Day 2, exosomes were added to iPSC during medium refreshing with BM containing 10 µM CHIR and 5 ng/mL noggin. On Day 4, CCK8 assay was performed and RNA was extracted from cells for RT-PCR. B Western blotting was performed to confirm the presence of EVs marker proteins in the isolated EVs fraction. Uncropped gels are shown in Additional file 1 Fig. S3. C CCK8 assay was performed on iPSC differentiated in the presence of monocytes-derived EVs on Day 4. (PS)—precipitation solution control. (Mono)—mono-cultured iPSC. D, E Relative expression of late primitive streak marker TBX6 (D) and posterior intermediate mesoderm marker OSR1 (E) by iPSC differentiated in the presence of monocytes-derive EVs was assessed by RT-PCR on Day 4