Abstract
Potency assays are critical for regenerative medicine, addressing the known challenge of functional heterogeneity among human multipotent stromal cells (hMSC). Necessary laboratory cell expansion allows analysis before implantation in the patient. Levels of induction of five signature gene biomarkers, ALPL, COL1A2, DCN, ELN and RUNX2, constituted a previously reported proof-of-principle osteogenic potency assay. We tested assay modification to enhance reproducibility using six consistent bone marrow derived hBM-MSC and explored applicability to three adipose tissue derived hAT-MSC. Using a potent proprietary osteogenic induction factor, the GUSB/YWAHZ reference gene pair provided real time PCR consistency. The novel assay conditions supported the concept that genes encoding extracellular matrix proteins one week after osteogenic induction were informative. Nonetheless, relatively low induction of COL1A2 and ELN encouraged search for additional biomarkers. TGFB2 mRNA induction, important for osteogenic commitment, was readily quantifiable in both hBM-MSC and hAT-MSC. Combined with DCN, TGFB2 mRNA induction data provided discriminatory power for resolving donor-specific heterogeneity. Histomorphometric decorin and TGF-β2 protein expression patterns in eight-week heterotopic bone implants also discriminated the two non-bone-forming hMSC. We highlight progress towards prompt osteogenic potency assays, needed by current clinical trials to accelerate improved intervention with enhanced stem cell therapy for serious bone fractures.
Highlights
Seeking to ameliorate an enormous healthcare burden from bone morbidities through tissue engineering [1], use of autologous culture-expanded osteoprogenitor cells grown on porous bioceramic scaffolds could substantially improve the repair of large defects in long bones [2]
Establishment of hBM-MSC and hAT-MSC Expansion in Culture hBM-MSC and hAT-MSC stored in cryotubes under liquid nitrogen at early passage (n), were carefully thawed from frozen vials and expanded in culture under cGMP-like growth conditions similar to corresponding protocols adopted for clinical trials (Figure 1A)
Heterogeneity among the cells of multipotent differentiation potential derived from connective tissues, generally referred to as “mesenchymal stromal/stem cells” [46], a contested nomenclature
Summary
Seeking to ameliorate an enormous healthcare burden from bone morbidities through tissue engineering [1], use of autologous culture-expanded osteoprogenitor cells grown on porous bioceramic scaffolds could substantially improve the repair of large defects in long bones [2]. Donor-related hMSC heterogeneity [5] can hinder therapeutic efficacy [6] and a consensus view recommends rigorous and thorough supportive protocols [7] This is relevant for a cell-based osteogenic potency assay to confirm appropriate biological activity of the specific therapeutic cells, a required release criterion before final marketing authorization of the patient treatment. Human MSC can be differentiated in vitro by supplementing a growth maintenance medium with osteogenic induction factors [10] that stimulate a chronological series of long-appreciated stepwise cellular changes: a reduced proliferation, extracellular matrix (ECM)
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