Abstract
Background & Aim Bone-marrow Mesenchymal Stromal Cells (BMSC) are promising therapies for fracture healing. However, undifferentiated BMSC may act only through an inductive paracrine effect without direct bone formation and direct cell-replacement. Here, we developed ALLOB, an off-the-shelf product constituted of human allogeneic bone-forming cells derived from BMSC and performed a side-by-side comparison with their former BMSC by using in vitro and in vivo approaches. Methods, Results & Conclusion At transcriptomic level, ALLOB batches (n=5) have a distinct genomic signature compared to their former BMSC (n=5) (image 1). Among the genes differentially expressed between both cell types (ALLOB vs. BMSC), upregulated genes are related to pathways such as matrix organization, cell and bone differentiation, demonstrating engagement of ALLOB cells towards the osteoblastic lineage. In vivo, a single dose of ALLOB or BMSC (same donor) significantly enhanced bone formation 28 days post-administration over the calvaria of Nude mice compared to control excipient (n=8 mice per group). Histomorphometrical analyses and double immunofluorescence targeting mouse and human type I collagen (Col1) revealed the presence of bone from donor (human) origin only in ALLOB treated mice (image 2). In addition, immunohistochemistry targeting human specific nuclei (hKu80) demonstrated that ALLOB cells engraft, differentiate into osteocytes through a cartilage intermediate state and persist at administration site, thus, demonstrating their ability to supplement target tissue with functional bone forming cells. Interestingly, as the neoformed bone observed in ALLOB treated mice develops through intramembranous and endochondral ossifications which are key processes involved in long bone healing, ALLOB and control excipient (n=8 per group) were injected in a severe long bone fracture model in nude mice. Using X-ray monitoring (every 2 weeks), ALLOB improved fracture healing with a significant reduction of the bone defect size compared to the excipient. Altogether, these results show that ALLOB is distinct from their former BMSC at the molecular and cellular levels and from our knowledge, it's the first time that a cell-based product derived from BMSC is able to produce the desired tissue in vivo without the use of any scaffold, demonstrating its regenerative property. ALLOB is then a good clinical candidate to treat patients suffering of bone disorders that require bone cell replacement.
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