Abstract
Therapeutic potential of human bone marrow stromal/stem cells (hBMSC) must be developed using well defined xenogenic-free conditions. hBMSC were isolated from healthy donors (n = 3) using different isolation and expansion methods. Donor I was isolated and expanded by either bone marrow directly seeded and cells expanded in 10% AB human serum (AB) +5 ng/ml fibroblast growth factor-2 (FGF2) [Direct(AB + FGFlow)] or Ammonium-Chloride-Potassium Lysing Buffer was used before the cells were expanded in 10% AB +5 ng/ml FGF-2 [ACK(AB + FGFlow)] or Lymphoprep density gradient medium was used before the cells were expanded in 10% AB +5 ng/ml FGF2 [Lympho(AB + FGFlow)] or bone marrow directly seeded and cells expanded in 10% pooled platelet lysate plasma (PL) + heparin (2 I/U/mL) [Direct(PL)]. Groups for donors II and III were: Direct(AB + FGFlow) or 10% AB +10 ng/ml FGF2 [Direct(AB + FGFhigh)] or Direct(PL). HBMSCs were assessed for viability, multi-potency, osteogenic, inflammatory response and replicative senescence in vitro after 1 and 3 weeks. Pre-selected culture conditions, Direct(AB + FGFhigh) or Direct(PL), were seeded on biphasic calcium phosphate granules and subcutaneously implanted in NOD/SCID mice. After 1 and 11 weeks, explants were analysed for inflammatory and osteogenic response at gene level and histologically. To identify implanted human cells, in situ hybridisation was performed. hBMSC from all conditions showed in vitro multi-lineage potency. hBMSCs expanded in PL expressed stemness markers in vitro at significantly higher levels. Generally, cells expanded in AB + FGF2 conditions expressed higher osteogenic markers after 1 week both in vitro and in vivo. After 11 weeks in vivo, Direct(AB + FGFhigh) formed mature ectopic bone, compared to immature mineralised tissues formed by Direct(PL) implants. Mouse responses showed a significant upregulation of IL-1α and IL-1β expression in Direct(PL). After 1 week, human cells were observed in both groups and after 11 weeks in Direct(AB + FGFhigh) only. To conclude, results showed a significant effect of the isolation methods and demonstrated a relatively consistent pattern of efficacy from all donors. A tendency of hBMSC expanded in PL to retain a more stem-like phenotype elucidates their delayed differentiation and different inflammatory expressions.
Highlights
Therapeutic potential of human bone marrow stromal/stem cells must be developed using well defined xenogenic-free conditions. hBMSC were isolated from healthy donors (n = 3) using different isolation and expansion methods
Www.nature.com/scientificreports present advantages over other sources of MSC3 and over pluripotent cell types such as induced pluripotent stem cells due to their autologous mode of use, which require less extensive in vitro manipulation or ethical clearance, associated with a lower risk4. hBMSC are rare cells, population ranges from 0.001% to 0.01% of the total number of nucleated cells present in bone marrow[5]
PL has shown superior efficacy when compared to Foetal bovine serum (FBS)-propagated cultures. hBMSC were shown to be more responsive to chondrogenic and adipogenic stimulation when cultured in PL compared to FBS17
Summary
Foetal bovine serum (FBS) is derived from the whole blood of bovine foetuses and it is a rich source of essential growth factors These include platelet derived growth factor (PDGF), transforming growth factor beta 1 (TGFβ-1), fibroblast growth factor 2 (FGF2), vascular endothelial growth factor (VEGF), insulin-like growth factor (IGF), growth hormones and albumin, making it the optimum and most broadly used supplement for expansion of hBMSC9. The concentrations of growth factors in FBS are difficult to control between production batches, and even clinical-grade FBS is reported to show variability between its inherent composite of bioactive factors[9] To address these issues, alternative animal-free strategies are currently being developed for the provision of nutrients and attachment factors for culture and expansion of hBMSC. Concerns of immunological responses and transmission of human infections are valid as for other human-derived media supplements
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
