Fig. 1

Isolation, recondition and characterization of PBMC derived monocytes into RM. a (i) PBMCs were isolated by density gradient centrifugation method, wherein the buffy coat was diluted in RPMI-1640 media (1:3) which was layered over ficoll. The obtained PBMCs were plated overnight. Thereafter, the non-adherent cells were discarded and the adherent monocyte cells were isolated by trypsinization. The monocytes isolated based on adherence showed reduced purity (65.9%) as seen by flow cytometric analysis of CD14+ cells gated in side scatter plot. a (ii) Graphical representation of adherent and sorted CD14⁺ monocyte population showing higher percentage of CD14⁺ cells in sorted culture than adherent culture. Around 54.5 ± 9.39% cells of adherent PBMCs stained positive for CD14 and 79.5 ± 6.24% cells in FACS sorted population were CD14 positive, (n = 10). b Bright field images at × 20 resolution showing kinetics of monocyte recondition and morphology alteration. The small round monocytes at day 1 formed colonies and became large and rounded at day 6 (known as reconditioned monocytes or RM). c Flow cytometric analysis of CD14, a monocyte-specific marker, was downregulated in RM and RNLCs and d CD117 was upregulated in RM as compared to monocytes suggesting molecular and phenotypic changes. e 5-Ethynyl-2′-deoxyuridine (EDU) assay was performed to analyse the proportion of proliferative cells. RM showed significant proliferation capacity as against negligible proliferation in monocytes and sparse proliferation in RNLCs. A graphical representation of the EDU analysis is also shown (n = 5). f Kinetic study through qPCR was performed to analyse the pluripotency and proliferation in RM which indicated that RM had a transient expression marker responsible for pluripotency (CD34, NANOG, c-myc) and proliferation markers (KI67) and the stem cell-like properties peaked at day 6 which declined further (n = 5). *p < 0.05; **p < 0.01; ***p < 0.001