Sharma Anshu, Rani Rajni
Molecular Immunogenetics Group, National Institute of Immunology, New Delhi, 110067, India.
Systems Biology Group, CSIR-Institute of Genomics and Integrative Biology, New Delhi, 110025, India.
Stem Cell Res Ther. 2017 Jul 12;8(1):167. doi: 10.1186/s13287-017-0615-1.
Type 1 diabetes (T1D) is a multifactorial autoimmune disorder where pancreatic beta cells are lost before the clinical manifestations of the disease. Administration of mesenchymal stem cells (MSCs) or MSCs differentiated into insulin-producing cells (IPCs) have yielded limited success when used therapeutically. We have evaluated the immunoprophylactic potentials of precursors to insulin-producing cells (pIPCs) and IPCs in nonobese diabetic (NOD) mice to ask a basic question: do we need to differentiate MSCs into IPCs or will pIPCs suffice to attenuate autoimmune responses in T1D?
Bone marrow-derived MSCs from Balb/c mice were characterized following the International Society for Cellular Therapy (ISCT) guidelines. MSCs cultured in high-glucose media for 11 to 13 passages were characterized for the expression of pancreatic lineage genes using real-time polymerase chain reaction. Expression of the PDX1 gene in pIPCs was assessed using Western blot and fluorescence-activated cell sorting (FACS). Triple-positive MSCs were differentiated into IPCs using a three-step protocol after sorting them for cell surface markers, i.e. CD29, CD44, and SCA-1. Nonobese diabetic mice were administered pIPCs, IPCs, or phosphate-buffered saline (PBS) into the tail vein at weeks 9 or 10 and followed-up for 29-30 weeks for fasting blood glucose levels. Two consecutive blood sugar levels of more than 250 mg/dl were considered diabetic.
MSCs grown in high-glucose media for 11 to 13 passages expressed genes of the pancreatic lineage such as PDX1, beta2, neurogenin, PAX4, Insulin, and glucagon. Furthermore, Western blot and FACS analysis for PDX-1, a transcription factor necessary for beta cell maturation, confirmed that these cells were precursors of insulin-producing cells (pIPCs). NOD mice administered with pIPCs were better protected from developing diabetes with a protective efficacy of 78.4% (p < 0.009); however, administration of IPCs gave protective efficacy of 55% at the end of 28-30 weeks.
Precursors to insulin-producing cells seem to have better potential to arrest autoimmune response in type 1 diabetes when administered before the onset of the disease in NOD mice. When translated to humans, autologous mesenchymal stem cells grown in high-glucose media for 10 to 13 passages may have beneficial effects in individuals at high risk of developing type 1 diabetes.
1型糖尿病（T1D）是一种多因素自身免疫性疾病，在疾病临床表现出现之前胰腺β细胞就已丧失。间充质干细胞（MSCs）或分化为胰岛素产生细胞（IPCs）的MSCs在治疗应用时取得的成功有限。我们评估了胰岛素产生细胞前体（pIPCs）和IPCs在非肥胖糖尿病（NOD）小鼠中的免疫预防潜力，以探讨一个基本问题：我们是否需要将MSCs分化为IPCs，还是pIPCs就足以减弱T1D中的自身免疫反应？
按照国际细胞治疗协会（ISCT）指南对来自Balb/c小鼠的骨髓源性MSCs进行鉴定。使用实时聚合酶链反应对在高糖培养基中培养11至13代的MSCs进行胰腺谱系基因表达的鉴定。使用蛋白质免疫印迹法和荧光激活细胞分选（FACS）评估pIPCs中PDX1基因的表达。对细胞表面标志物即CD29、CD44和SCA-1进行分选后，采用三步方案将三阳性MSCs分化为IPCs。在第9周或第10周给非肥胖糖尿病小鼠尾静脉注射pIPCs、IPCs或磷酸盐缓冲盐水（PBS），并随访29 - 30周以检测空腹血糖水平。连续两次血糖水平超过250mg/dl被视为糖尿病。
在高糖培养基中培养11至13代的MSCs表达胰腺谱系基因，如PDX1、β2、神经生成素、PAX4、胰岛素和胰高血糖素。此外，对β细胞成熟所必需的转录因子PDX - 1进行的蛋白质免疫印迹法和FACS分析证实这些细胞是胰岛素产生细胞前体（pIPCs）。接受pIPCs治疗的NOD小鼠对糖尿病的发展具有更好的保护作用，保护效力为78.4%（p < 0.009）；然而，在28 - 30周结束时，给予IPCs的保护效力为55%。
在NOD小鼠疾病发作前给予胰岛素产生细胞前体似乎在阻止1型糖尿病自身免疫反应方面具有更好的潜力。如果应用于人类，在高糖培养基中培养10至13代的自体间充质干细胞可能对有发展为1型糖尿病高风险的个体具有有益作用。
