From blood to pluripotency: Fibrocytes as a reprogrammable somatic cell source for bovine iPSCs
Fibrocytes represent a distinct somatic cell type derived from peripheral blood leukocytes, first described as spindle-shaped adherent cells with dual hematopoietic and mesenchymal features.  Although fibrocytes were identified in early descriptive studies across several mammalian systems, most of this work predated modern molecular approaches, and the cells remain incompletely defined at the molecular level and have not previously been derived or characterized in cattle. Seeking somatic cells that could be collected aseptically and reproducibly under field conditions for reprogramming to pluripotency, we recognized fibrocytes as a practical and previously unexplored candidate population. Here, we establish a reproducible method for fibrocyte derivation and expansion from adult bovine blood and define their molecular identity using transcriptomic and network analyses. Principal component and differential expression analyses revealed extensive immune, inflammatory, metabolic, and stress-responsive pathways that distinguished fibrocytes from fibroblasts. Upstream regulator analysis identified a fibrocyte-restricted transcriptional network governed by SPI1, IRF5/IRF7, NFKBIZ, PRDM1, CIITA, and MAFB, supporting a monocyte-derived origin and indicating some retention of hematopoietic lineage memory despite acquisition of mesenchymal features. Optimized fibrocyte medium (FbC; dexamethasone, ascorbate, PDGF-BB, EGF, A83-01, CHIR99021) supported stable proliferation and selectively enhanced cytoskeletal and matrix-constructive programs while attenuating inflammatory tone. When reprogrammed with polycistronic OCT4-SOX2-KLF4-cMYC and SV40 large T antigen, fibrocytes generated induced pluripotent stem cell (iPSC) colonies exhibiting defining molecular and morphological features of pluripotency. These findings establish fibrocytes as a field-adaptable, stably expandable, and reprogrammable somatic cell type with practical applications in induced pluripotent stem cell generation, genetic preservation, and reproductive biotechnology. Overall design: Bovine fibrocytes isolated and cultured in two different mediums -  fibroblast and fibrocyte medium to identify suitable culture cunditions that support decreased inflammatory response and proliferation.