A model study for the manufacture and validation of clinical-grade deciduous dental pulp stem cells for chronic liver fibrosis treatment
A model study for the manufacture and validation of clinical-grade deciduous dental pulp stem cells for chronic liver fibrosis treatment
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DOI：
10.1186/s13287-020-01630-w
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发表时间：
2020-03-25
影响因子：
7.5
通讯作者：
Taguchi, Tomoaki
中科院分区：
文献类型：
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作者：
Iwanaka, Tsuyoshi;Yamaza, Takayoshi;Taguchi, Tomoaki
背景人类乳牙牙髓干细胞（hDPSC）具有显著的干细胞潜能，与细胞增殖、间充质多能性和免疫抑制功能相关，并且在多种动物疾病模型中显示出有益的作用。最近的研究表明，hDPSCs在体内表现出抗纤维化和抗炎作用，并在体内肝源性相关的肝再生，这表明hDPSCs可能提供用于治疗肝脏疾病的具有巨大临床需求的有前途的来源。然而，如何制造具有适当质量、安全性和临床前功效保证的离体大规模临床级hDPSC仍不清楚。方法采用集落形成单位成纤维细胞（CFU-F）法从人乳牙牙髓组织中分离hDPSC，在无异种无血清（XF/SF）条件下扩增，并建立主细胞库（MCB）和工作细胞库（WCB）。最终产物是从WCB直接解冻的hDPSC。我们检测了MCB和WCB中最终hDPSC产品和hDPSC产品的安全性和质量检查、干细胞特性和临床前潜力。结果在XF/SF培养条件下，优化了hDPSC的分离和扩增工艺，建立了MCB和WCB。对最终hDPSC产品和MCB和WCB中的hDPSC产品的安全性和质量进行了验证，包括群体倍增能力、染色体稳定性、微生物安全性和干细胞特性，包括形态学、细胞表面标志物表达和多能性。我们还评价了最终hDPSC产品和WCB中hDPSC产品的体内免疫原性和致瘤性，并验证了CCl 4诱导的慢性肝纤维化小鼠模型中肝再生的体内治疗功效。结论该工艺可从微小乳牙牙髓组织中制备出足够数量的临床级hDPSC产品，可提高hDPSC产品在慢性肝纤维化治疗中的临床应用价值。
Background Human deciduous pulp stem cells (hDPSCs) have remarkable stem cell potency associated with cell proliferation, mesenchymal multipotency, and immunosuppressive function and have shown beneficial effects in a variety of animal disease models. Recent studies demonstrated that hDPSCs exhibited in vivo anti-fibrotic and anti-inflammatory action and in vivo hepatogenic-associated liver regeneration, suggesting that hDPSCs may offer a promising source with great clinical demand for treating liver diseases. However, how to manufacture ex vivo large-scale clinical-grade hDPSCs with the appropriate quality, safety, and preclinical efficacy assurances remains unclear. Methods We isolated hDPSCs from human deciduous dental pulp tissues formed by the colony-forming unit-fibroblast (CFU-F) method and expanded them under a xenogeneic-free and serum-free (XF/SF) condition; hDPSC products were subsequently stored by two-step banking including a master cell bank (MCB) and a working cell bank (WCB). The final products were directly thawed hDPSCs from the WCB. We tested the safety and quality check, stem cell properties, and preclinical potentials of final hDPSC products and hDPSC products in the MCB and WCB. Results We optimized manufacturing procedures to isolate and expand hDPSC products under a XF/SF culture condition and established the MCB and the WCB. The final hDPSC products and hDPSC products in the MCB and WCB were validated the safety and quality including population doubling ability, chromosome stability, microorganism safety, and stem cell properties including morphology, cell surface marker expression, and multipotency. We also evaluated the in vivo immunogenicity and tumorigenicity and validated in vivo therapeutic efficacy for liver regeneration in a CCl4-induced chronic liver fibrosis mouse model in the final hDPSC products and hDPSC products in the WCB. Conclusion The manufacture and quality control results indicated that the present procedure could produce sufficient numbers of clinical-grade hDPSC products from a tiny deciduous dental pulp tissue to enhance clinical application of hDPSC products in chronic liver fibrosis.