Abstract

We have established human adipose-derived mesenchymal stromal/stem cell line (ASCL) as an expandable cell source to generate megakaryocytes (MKs) releasing platelets for clinical transfusion. The use of ASCL has an advantage in manufacturing platelets, because both establishment of ASCL and its differentiation to MKs and platelets do not require gene transfer, and its endogenous thrombopoietin (TPO) is utilized for their differentiation. Here we report characterization of ASCL and ASCL-derived platelets for clinical application. ASCL retained their proliferation capacity for 6 months. There was no abnormality by karyotype analysis for ASCL, and no gene mutations registered in database for cancer and other diseases were noted by next-generation sequence analysis (n=3). ASCL satisfies the minimal criteria for defining mesenchymal stem cells by The International Society for Cellular Therapy: (1) adherence to plastic in vitro culture, (2) expression (>95% of cells) of CD73, CD90, and CD105 and no expression (<2% of cells) of CD45, CD34, CD14, CD19, and HLA-DR, and (3) capacity to differentiate into osteoblasts, adipocytes, and chondrocytes in vitro. ASCL was cultured in 10 liters of MK lineage induction media using a bioreactor. The frequency of CD41-expressing MK-sized cells increased during this culture with a peak at Day 8 (60-70%). We detected increased gene expression of TPO, p45NF-E2, beta-1 tubulin, VWF, GATA2, PF4, FOG1, and Fli1 during this differentiation. ASCL-derived platelets were obtained with a peak at Day 12 with 30-50 platelets released from a single MK. The expression of intracellular factors, VWF, PF4, and beta-1 tubulin in these platelets (CD42b-positive/anuclear platelet-sized cells) was also observed by immunostaining. We next compared functionality of ASCL-derived platelets and concentrate platelets (CP) from the Japanese Red Cross Society. Upon stimulation with thrombin (0.5 U/mL), surface exposure of PAC-1 in ASCL-derived platelets was higher than that in CP (p<0.05). Similar to PAC-1 surface exposure, the binding of labeled fibrinogen or P-selectin to ASCL-derived platelets was higher than that to CP, though the differences were not statistically significant. A sufficient number of platelets were obtained to perform platelet aggregation study under light transmission aggregometry. Agonist (ADP 20 μM and calcium 10 mM)-induced aggregation was observed in both ASCL-derived platelets and CP. To investigate the kinetics of ASCL-derived platelets and CP, these cells (1 x 107 cells) were infused into irradiated immunodeficient NSG mice (2.0 Gy, 7 days). Blood samples from recipient mouse were analyzed for the presence of human platelets 0 mins, 30 mins, 2 hrs, and 24 hrs after infusion. The kinetics of ASCL-derived platelets was similar to those of CP at the peak with 2 hrs (n=3). We tested thrombus formation under flow condition. Blood samples labeled with anti-CD41 antibody (clone, SZ22) were perfused on a collagen-coated chip. Incorporation of cells expressing human CD41 into the thrombi was observed in both samples from ASCL-derived platelet- and CP-infused mouse. Taken together, manufacturing platelets from ASCL in a large-scale culture and CP showed the similar in vivo kinetics and function, further supporting the clinical application of our approach.

Disclosures

Ono-Uruga:AdipoSeeds Inc.: Equity Ownership. Okamoto:Nippon Shinyaku Co., Ltd.: Research Funding; Eisai Co., Ltd.: Research Funding; Toyama Chemical Co., Ltd.: Research Funding; Astellas Pharma Inc.: Research Funding; Kyowa Hakko Kirin Co., Ltd.: Research Funding; Sumitomo Dainippon Pharma Co., Ltd.: Research Funding; Otsuka Pharmaceutical Co., Ltd.: Honoraria, Research Funding; Chugai Pharmaceutical Co., Ltd.: Research Funding; Teijin Pharma Limited: Research Funding; Shionogi & Co., Ltd.: Research Funding; Bristol-Myers Squibb K.K.: Honoraria, Research Funding; Alexion Pharmaceuticals, Inc.: Research Funding; Asahi Kasei Pharma Corp.: Research Funding; Pfizer Inc.: Honoraria, Research Funding; JCR Pharmaceuticals Co., Ltd.: Research Funding. Ikeda:AdipoSeeds Inc.: Equity Ownership. Matsubara:AdipoSeeds Inc.: Equity Ownership; Kyowa Hakko Kirin Co., Ltd.: Research Funding; Tosoh Corporation: Research Funding; Nissui Pharmaceutical Co., Ltd.: Research Funding; Fujimori Kogyo Co., Ltd.: Research Funding.

Author notes

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Asterisk with author names denotes non-ASH members.

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