A highly standardized and characterized human platelet lysate for efficient and reproducible expansion of human bone marrow mesenchymal stromal cells.

BACKGROUND: Human platelet lysate (hPL) represents a powerful alternative to fetal bovine serum (FBS) for human mesenchymal stromal cell (hMSC) expansion. However, the large variability in hPL sources and production protocols gives rise to discrepancies in product quality, characterization and poor batch-to-batch standardization. METHODS: hPL prepared with more than 200 donors (200+DhPL) or with five donors (5DhPL) were compared in terms of growth factor (GF) contents and biochemical analysis. A multiple protein assay and proteomic analysis were performed to further characterize 200+DhPL batches. We also compared the phenotypic and functional characteristics of bone marrow (BM)-hMSCs grown in 200+DhPL versus FBS+basic fibroblast growth factor (bFGF). RESULTS: By contrast to 5DhPL, industrial 200+DhPL displayed a strong standardization of GF contents and biochemical characteristics. We identified specific plasmatic components and platelet-released factors as the most relevant markers for the evaluation of the standardization of hPL batches. We used a multiplex assay and proteomic analysis of 200+DhPL to establish a proteomic signature and demonstrated the robust standardization of batches. 200+DhPL was shown to improve and standardize BM-hMSC expansion compared with FBS+bFGF. The levels of expression of BM-hMSC membrane markers were found to be much more homogeneous between batches when cells were cultured in 200+DhPL. BM-hMSCs cultured in parallel under both conditions displayed similar adipogenic and osteogenic differentiation potential and immunosuppressive properties. CONCLUSIONS: We report a standardization of hPL and the importance of such standardization for the efficient amplification of more homogeneous and reproducible cell therapy products.

Viral inactivation of human platelet lysate by gamma irradiation preserves its optimal efficiency in the expansion of human bone marrow mesenchymal stromal cells.

BACKGROUND: Human platelet lysate (hPL) represents a powerful medium supplement for human mesenchymal stromal cell (hMSC) expansion. The recently published general chapters of the Pharmacopeia require the addition of a step of viral inactivation during the production process of such raw biological material used for cell-based medicinal products. STUDY DESIGN AND METHODS: The ability of gamma irradiation to inactivate viruses from a panel representative of the virus diversity was evaluated. The impact of gamma irradiation on hPL composition and efficiency as a supplement for hMSC culture was evaluated. RESULTS: An efficient inactivation of all the viruses tested was demonstrated, with the minimum reduction factors obtained being superior to 4.5 log10 for human immunodeficiency virus (HIV) and hepatitis A virus (HAV) and superior to 5 log10 for bovine viral diarrhea virus (BVDV), pseudorabies virus (PRV) and porcine parvovirus (PPV). The gamma irradiation did not affect the content in interesting biochemical factors for cell culture or in growth factors (GF), except to basic fibroblast GF (bFGF) whereas it highly impacted the contents in the factors involved in the coagulation cascade. Finally, gamma irradiated hPL remained as efficient as non-irradiated hPL for the proliferation, clonogenic potential, differentiation potential, and immunosuppressive properties of hMSCs. CONCLUSION: The feasibility of using gamma irradiation to efficiently inactivate viruses in hPL while maintaining its optimal efficacy as a supplement for hMSC expansion was demonstrated. Such an inactivated hPL represents a very attractive raw material for the efficient production of safe cellular therapy products.

Skeletal Muscle Regenerative Potential of Human MuStem Cells following Transplantation into Injured Mice Muscle.

After intra-arterial delivery in the dystrophic dog, allogeneic muscle-derived stem cells, termed MuStem cells, contribute to long-term stabilization of the clinical status and preservation of the muscle regenerative process. However, it remains unknown whether the human counterpart could be identified, considering recent demonstrations of divergent features between species for several somatic stem cells. Here, we report that MuStem cells reside in human skeletal muscle and display a long-term ability to proliferate, allowing generation of a clinically relevant amount of cells. Cultured human MuStem (hMuStem) cells do not express hematopoietic, endothelial, or myo-endothelial cell markers and reproducibly correspond to a population of early myogenic-committed progenitors with a perivascular/mesenchymal phenotypic signature, revealing a blood vessel wall origin. Importantly, they exhibit both myogenesis in vitro and skeletal muscle regeneration after intramuscular delivery into immunodeficient host mice. Together, our findings provide new insights supporting the notion that hMuStem cells could represent an interesting therapeutic candidate for dystrophic patients.

Pathogen reduction through additive-free short-wave UV light irradiation retains the optimal efficacy of human platelet lysate for the expansion of human bone marrow mesenchymal stem cells.

BACKGROUND: We recently developed and characterized a standardized and clinical grade human Platelet Lysate (hPL) that constitutes an advantageous substitute for fetal bovine serum (FBS) for human mesenchymal stem cell (hMSC) expansion required in cell therapy procedures, avoiding xenogenic risks (virological and immunological) and ethical issues. Because of the progressive use of pathogen-reduced (PR) labile blood components, and the requirement of ensuring the viral safety of raw materials for cell therapy products, we evaluated the impact of the novel procedure known as THERAFLEX UV-Platelets for pathogen reduction on hPL quality (growth factors content) and efficacy (as a medium supplement for hMSC expansion). This technology is based on short-wave ultraviolet light (UV-C) that induces non-reversible damages in DNA and RNA of pathogens while preserving protein structures and functions, and has the main advantage of not needing the addition of any photosensitizing additives (that might secondarily interfere with hMSCs). METHODOLOGY / PRINCIPAL FINDINGS: We applied the THERAFLEX UV-Platelets procedure on fresh platelet concentrates (PCs) suspended in platelet additive solution and prepared hPL from these treated PCs. We compared the quality and efficacy of PR-hPL with the corresponding non-PR ones. We found no impact on the content of five cytokines tested (EGF, bFGF, PDGF-AB, VEGF and IGF-1) but a significant decrease in TGF-ß1 (-21%, n = 11, p<0.01). We performed large-scale culture of hMSCs from bone marrow (BM) during three passages and showed that hPL or PR-hPL at 8% triggered comparable BM-hMSC proliferation as FBS at 10% plus bFGF. Moreover, after proliferation of hMSCs in an hPL- or PR-hPL-containing medium, their profile of membrane marker expression, their clonogenic potential and immunosuppressive properties were maintained, in comparison with BM-hMSCs cultured under FBS conditions. The potential to differentiate towards the adipogenic and osteogenic lineages of hMSCs cultured in parallel in the three conditions also remained identical. CONCLUSION / SIGNIFICANCE: We demonstrated the feasibility of using UV-C-treated platelets to subsequently obtain pathogen-reduced hPL, while preserving its optimal quality and efficacy for hMSC expansion in cell therapy applications.