Surgical Research Unit, CMU-1, University Hospitals of Geneva, Geneva, Switzerland.
Group for Functionalized Biomaterials, Institute of Chemical Sciences and Engineering, Ecole Polytechnique Fédérale de Lausanne, EPFL SB ISIC SCI-SB-SG, Lausanne, Switzerland.
Xenotransplantation. 2021 May;28(3):e12666. doi: 10.1111/xen.12666. Epub 2021 Feb 4.
Neonatal and juvenile porcine islet cell clusters (ICC) present an unlimited source for islet xenotransplantation to treat type 1 diabetes patients. We isolated ICC from pancreata of 14 days old juvenile piglets and characterized their maturation by immunofluorescence and insulin secretion assays. Multipotent mesenchymal stromal cells derived from exocrine tissue of same pancreata (pMSC) were characterized for their differentiation potential and ability to sustain ICC insulin secretion in vitro and in vivo. Isolation of ICC resulted in 142 ± 50 × 10 IEQ per pancreas. Immunofluorescence staining revealed increasing presence of insulin-positive beta cells between day 9 and 21 in culture and insulin content per 500IEC of ICC increased progressively over time from 1178.4 ± 450 µg/L to 4479.7 ± 1954.2 µg/L from day 7 to 14, P < .001. Highest glucose-induced insulin secretion by ICC was obtained at day 7 of culture and reached a fold increase of 2.9 ± 0.4 compared to basal. Expansion of adherent cells from the pig exocrine tissue resulted in a homogenous CD90 , CD34 , and CD45 fibroblast-like cell population and differentiation into adipocytes and chondrocytes demonstrated their multipotency. Insulin release from ICC was increased in the presence of pMSC and dependent on cell-cell contact (glucose-induced fold increase: ICC alone: 1.6 ± 0.2; ICC + pMSC + contact: 3.2 ± 0.5, P = .0057; ICC + pMSC no-contact: 1.9 ± 0.3; theophylline stimulation: alone: 5.4 ± 0.7; pMSC + contact: 8.4 ± 0.9, P = .013; pMSC no-contact: 5.2 ± 0.7). After transplantation of encapsulated ICC using Ca -alginate (alg) microcapsules into streptozotocin-induced diabetic and immunocompetent mice, transient normalization of glycemia was obtained up to day 7 post-transplant, whereas ICC co-encapsulated with pMSC did not improve glycemia and showed increased pericapsular fibrosis. We conclude that pMSC derived from juvenile porcine exocrine pancreas improves insulin secretion of ICC by direct cell-cell contact. For transplantation purposes, the use of pMSC to support beta-cell function will depend on the development of new anti-fibrotic polymers and/or on genetically modified pigs with lower immunogenicity.
新生和幼年猪胰岛细胞簇 (ICC) 为胰岛异种移植治疗 1 型糖尿病患者提供了无限的来源。我们从 14 天大的幼年仔猪的胰腺中分离出 ICC，并通过免疫荧光和胰岛素分泌测定来鉴定其成熟度。从同一胰腺的外分泌组织中分离出多能间充质基质细胞 (pMSC)，并对其分化潜能和在体外和体内维持 ICC 胰岛素分泌的能力进行鉴定。ICC 的分离得到了每个胰腺 142±50×10 IEQ。免疫荧光染色显示，在培养的第 9 天至 21 天之间，胰岛素阳性β细胞的存在逐渐增加，并且 ICC 中每 500IEC 的胰岛素含量从第 7 天到第 14 天逐渐增加，从 1178.4±450µg/L 增加到 4479.7±1954.2µg/L，P<0.001。ICC 获得的最高葡萄糖诱导的胰岛素分泌是在培养的第 7 天获得的，与基础相比达到了 2.9±0.4 的折叠增加。从猪外分泌组织中扩增的贴壁细胞产生了同质的 CD90、CD34 和 CD45 成纤维样细胞群，分化为脂肪细胞和成软骨细胞证明了其多能性。在 pMSC 的存在下，ICC 的胰岛素释放增加，并且依赖于细胞-细胞接触（葡萄糖诱导的折叠增加：ICC 单独：1.6±0.2；ICC+pMSC+接触：3.2±0.5，P=0.0057；ICC+pMSC 无接触：1.9±0.3；茶碱刺激：单独：5.4±0.7；pMSC+接触：8.4±0.9，P=0.013；pMSC 无接触：5.2±0.7）。使用 Ca-藻酸盐 (alg) 微胶囊将封装的 ICC 移植到链脲佐菌素诱导的糖尿病和免疫功能正常的小鼠中后，移植后第 7 天血糖得到短暂正常化，而与 pMSC 共封装的 ICC 则不能改善血糖，并且囊周纤维化增加。我们得出的结论是，来自幼年猪外分泌胰腺的 pMSC 通过直接的细胞-细胞接触改善了 ICC 的胰岛素分泌。对于移植目的，使用 pMSC 来支持β细胞功能将取决于新的抗纤维化聚合物的开发和/或具有较低免疫原性的基因修饰猪。
