In vitro and in vivo effects of insulin-producing cells generated by xeno-antigen free 3D culture with RCP piece
In vitro and in vivo effects of insulin-producing cells generated by xeno-antigen free 3D culture with RCP piece
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DOI：
10.1038/s41598-019-47257-7
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发表时间：
2019-07-24
影响因子：
4.6
通讯作者：
Shimada, Mitsuo
中科院分区：
文献类型：
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作者：
Ikemoto, Tetsuya;Feng, Rui;Shimada, Mitsuo
为了建立广泛的1型糖尿病细胞治疗，我们的目的是开发一种有效的方案，从脂肪来源的干细胞（ADSCs）产生胰岛素产生细胞（IPC）。我们使用人重组肽（RCP）花瓣状μ片和无异种抗原试剂建立了3D培养物。简言之，我们采用我们的两步方案在96孔培养皿中分化ADSC，并在含有0.1mg/mL RCP μ片段的无异种抗原试剂中培养细胞7天（步骤1），然后加入组蛋白脱乙酰酶抑制剂14天（步骤2）。在第21天，用抗胰岛素抗体双硫腙对生成的IPC进行强染色，并通过电子显微镜检测到类似胰岛素分泌颗粒的显微结构。3D培养细胞的葡萄糖刺激指数（最大值为4.9）和MAFA mRNA表达均显著高于常规培养细胞（P < 0.01和P < 0.05）。将96个IPC移植于肾包膜下或肠系膜内后14天左右，链脲佐菌素诱导的糖尿病裸鼠的高血糖状态转变为正常血糖状态。组织学评价显示，胰岛素和C肽阳性结构存在于120天。我们建立的用于产生IPC的无异种抗原和RCP花瓣状μ片3D培养方法可能适合于临床应用，因为在体外和体内证明了有效性。
To establish widespread cell therapy for type 1 diabetes mellitus, we aimed to develop an effective protocol for generating insulin-producing cells (IPCs) from adipose-derived stem cells (ADSCs). We established a 3D culture using a human recombinant peptide (RCP) petaloid mu-piece with xeno-antigen free reagents. Briefly, we employed our two-step protocol to differentiate ADSCs in 96-well dishes and cultured cells in xeno-antigen free reagents with 0.1 mg/mL RCP mu-piece for 7 days (step 1), followed by addition of histone deacetylase inhibitor for 14 days (step 2). Generated IPCs were strongly stained with dithizone, anti-insulin antibody at day 21, and microstructures resembling insulin secretory granules were detected by electron microscopy. Glucose stimulation index (maximum value, 4.9) and MAFA mRNA expression were significantly higher in 3D cultured cells compared with conventionally cultured cells (P < 0.01 and P < 0.05, respectively). The hyperglycaemic state of streptozotocin-induced diabetic nude mice converted to normoglycaemic state around 14 days after transplantation of 96 IPCs under kidney capsule or intra-mesentery. Histological evaluation revealed that insulin and C-peptide positive structures existed at day 120. Our established xeno-antigen free and RCP petaloid mu-piece 3D culture method for generating IPCs may be suitable for clinical application, due to the proven effectiveness in vitro and in vivo.