Translation of a standardized manufacturing protocol for mesenchymal stromal cells: A systematic comparison of validation and manufacturing data

Background Many data are available on expansion protocols for mesenchymal stromal cells (MSCs) for both experimental settings and manufacturing for clinical trials. However, there is a lack of information on translation of established protocols for Good Manufacturing Practice (GMP) from validation to manufacturing for clinical application. We present the validation and translation of a standardized pre-clinical protocol for isolation and expansion of MSCs for a clinical trial for reconstitution of alveolar bone. Methods Key parameters of 22 large-scale expansions of MSCs from bone marrow (BM) for validation were compared with 11 expansions manufactured for the clinical trial “Jaw bone reconstruction using a combination of autologous mesenchymal stromal cells and biomaterial prior to dental implant placement (MAXILLO1)” aimed at reconstruction of alveolar bone. Results Despite variations of the starting material, the robust protocol led to stable performance characteristics of expanded MSCs. Manufacturing of the autologous advanced therapy medicinal product MAXILLO-1-MSC was possible, requiring 21 days for each product. Transport of BM aspirates and MSCs within 24 h was guaranteed. MSCs fulfilled quality criteria requested by the national competent authority. In one case, the delivered MSCs developed a mosaic in chromosomal finding, showing no abnormality in differentiation capacity, growth behavior or surface marker expression during long-term culture. The proportion of cells with the mosaic decreased in long-term culture and cells stopped growth after 38.4 population doublings. Conclusions Clinical use of freshly prepared MSCs, manufactured according to a standardized and validated protocol, is feasible for bone regeneration, even if there was a long local distance between manufacturing center and clinical site. Several parameters, such as colony forming units fibroblasts (CFU-F), percentage of CD34+ cells, cell count of mononuclear cells (MNCs) and white blood cells (WBCs), of the BM may serve as a predictive tool for the yield of MSCs and may help to avoid unnecessary costs for MSC manufacturing due to insufficient cell expansion rates.publishedVersio

CD90 is dispensable for white and beige/brown adipocyte differentiation

Brown adipose tissue (BAT) is a thermogenic organ in rodents and humans. In mice, the transplantation of BAT has been successfully used to combat obesity and its comorbidities. While such beneficial properties of BAT are now evident, the developmental and cellular origins of brown, beige, and white adipocytes have remained only poorly understood, especially in humans. We recently discovered that CD90 is highly expressed in stromal cells isolated from human white adipose tissue (WAT) compared to BAT. Here, we studied whether CD90 interferes with brown or white adipogenesis or white adipocyte beiging. We applied flow cytometric sorting of human adipose tissue stromal cells (ASCs), a CRISPR/Cas9 knockout strategy in the human Simpson-Golabi-Behmel syndrome (SGBS) adipocyte model system, as well as a siRNA approach in human approaches supports the hypothesis that CD90 affects brown or white adipogenesis or white adipocyte beiging in humans. Taken together, our findings call the conclusions drawn from previous studies, which claimed a central role of CD90 in adipocyte differentiation, into question

Feasibility and safety of treating non-unions in tibia, femur and humerus with autologous, expanded, bone marrow-derived mesenchymal stromal cells associated with biphasic calcium phosphate biomaterials in a multicentric, non-comparative trial

Background: ORTHO-1 is a European, multicentric, first in human clinical trial to prove safety and feasibility after surgical implantation of commercially available biphasic calcium phosphate bioceramic granules associated during surgery with autologous mesenchymal stromal cells expanded from bone marrow (BM-hMSC) under good manufacturing practices, in patients with long bone pseudarthrosis. Methods: Twenty-eight patients with femur, tibia or humerus diaphyseal or metaphyso-diaphyseal non-unions were recruited and surgically treated in France, Germany, Italy and Spain with 100 or 200 million BM-hMSC/mL associated with 5–10 cc of bioceramic granules. Patients were followed up during one year. The investigational advanced therapy medicinal product (ATMP) was expanded under the same protocol in all four countries, and approved by each National Competent Authority. Findings: With safety as primary end-point, no severe adverse event was reported as related to the BM-hMSC. With feasibility as secondary end-point, the participating production centres manufactured the BM-hMSC as planned. The ATMP combined to the bioceramic was surgically delivered to the non-unions, and 26/28 treated patients were found radiologically healed at one year (3 out of 4 cortices with bone bridging). Interpretation: Safety and feasibility were clinically proven for surgical implantation of expanded autologous BM-hMSC with bioceramic. Funding: EU-FP7-HEALTH-2009, REBORNE Project (GA: 241876).The research leading to these results has received funding from the European Research Council under the European Union's Seventh Framework Programme (FP7/FP7-HEALTH-2009); REBORNE Project (GA: 241876

Translation of a standardized manufacturing protocol for mesenchymal stromal cells: A systematic comparison of validation and manufacturing data

Background Many data are available on expansion protocols for mesenchymal stromal cells (MSCs) for both experimental settings and manufacturing for clinical trials. However, there is a lack of information on translation of established protocols for Good Manufacturing Practice (GMP) from validation to manufacturing for clinical application. We present the validation and translation of a standardized pre-clinical protocol for isolation and expansion of MSCs for a clinical trial for reconstitution of alveolar bone. Methods Key parameters of 22 large-scale expansions of MSCs from bone marrow (BM) for validation were compared with 11 expansions manufactured for the clinical trial “Jaw bone reconstruction using a combination of autologous mesenchymal stromal cells and biomaterial prior to dental implant placement (MAXILLO1)” aimed at reconstruction of alveolar bone. Results Despite variations of the starting material, the robust protocol led to stable performance characteristics of expanded MSCs. Manufacturing of the autologous advanced therapy medicinal product MAXILLO-1-MSC was possible, requiring 21 days for each product. Transport of BM aspirates and MSCs within 24 h was guaranteed. MSCs fulfilled quality criteria requested by the national competent authority. In one case, the delivered MSCs developed a mosaic in chromosomal finding, showing no abnormality in differentiation capacity, growth behavior or surface marker expression during long-term culture. The proportion of cells with the mosaic decreased in long-term culture and cells stopped growth after 38.4 population doublings. Conclusions Clinical use of freshly prepared MSCs, manufactured according to a standardized and validated protocol, is feasible for bone regeneration, even if there was a long local distance between manufacturing center and clinical site. Several parameters, such as colony forming units fibroblasts (CFU-F), percentage of CD34+ cells, cell count of mononuclear cells (MNCs) and white blood cells (WBCs), of the BM may serve as a predictive tool for the yield of MSCs and may help to avoid unnecessary costs for MSC manufacturing due to insufficient cell expansion rates

CD90 Is Dispensable for White and Beige/Brown Adipocyte Differentiation

Brown adipose tissue (BAT) is a thermogenic organ in rodents and humans. In mice, the transplantation of BAT has been successfully used to combat obesity and its comorbidities. While such beneficial properties of BAT are now evident, the developmental and cellular origins of brown, beige, and white adipocytes have remained only poorly understood, especially in humans. We recently discovered that CD90 is highly expressed in stromal cells isolated from human white adipose tissue (WAT) compared to BAT. Here, we studied whether CD90 interferes with brown or white adipogenesis or white adipocyte beiging. We applied flow cytometric sorting of human adipose tissue stromal cells (ASCs), a CRISPR/Cas9 knockout strategy in the human Simpson-Golabi-Behmel syndrome (SGBS) adipocyte model system, as well as a siRNA approach in human approaches supports the hypothesis that CD90 affects brown or white adipogenesis or white adipocyte beiging in humans. Taken together, our findings call the conclusions drawn from previous studies, which claimed a central role of CD90 in adipocyte differentiation, into question