Levin Gabriel, Zuber Samuel M, Squillaro Anthony I, Sogayar Mari Cleide, Grikscheit Tracy C, Carreira Ana Claudia O
Cell and Molecular Therapy Center (NUCEL), School of Medicine, University of São Paulo, São Paulo, Brazil.
Interunits Graduate Program in Biotechnology, Institute of Biomedical Sciences, University of São Paulo, São Paulo, Brazil.
Front Bioeng Biotechnol. 2020 Jun 5;8:476. doi: 10.3389/fbioe.2020.00476. eCollection 2020.
Cell therapy and tissue engineering has recently emerged as a new option for short bowel syndrome (SBS) treatment, generating tissue engineered small intestine (TESI) from organoid units (OU) and biodegradable scaffolds. The recombinant human R-Spondin 1 (rhRSPO1) protein may be a key player in this process due to its mitogenic activity in intestinal stem cells. Aiming at optimizing the TESI formation process and advancing this technology closer to the clinic, we evaluated the effects of rhRSPO1 protein on OU culture and TESI formation. Intestinal OU were isolated from C57BL/6 mice and cultured in Matrigel in the presence or absence of recombinant human rhRSPO1. Throughout the culture, OU growth and survival rates were evaluated, and cells were harvested on day 3. OU were seeded onto biodegradable scaffolds, in the presence or absence of 5 μg of rhRSPO1 and implanted into the omentum of NOD/SCID mice in order to generate TESI. The explants were harvested after 30 days, weighed, fixed in formalin and embedded in paraffin for histological analysis and immunofluorescence for different cell markers. After 3 days, rhRSPO1-treated OU attained a larger size, when compared to the control group, becoming 5.7 times larger on day 6. Increased survival was observed from the second day in culture, with a 2-fold increase in OU survival between days 3 and 6. A 4.8-fold increase of non-phosphorylated β-catenin and increased relative expression of mRNA in the rhRSPO1-treated group confirms activation of the canonical Wnt pathway and suggests maintenance of the OU stem cell niche and associated stemness. After 30 days of maturation, rhRSPO1-treated TESI presented a larger mass than constructs treated with saline, developing a more mature intestinal epithelium with well-formed villi and crypts. In addition, the efficiency of OU-loaded rhRSPO1-treated scaffolds significantly increased, forming TESI in 100% of the samples ( = 8), of which 40% presented maximum degree of development, as compared to 66.6% in the control group ( = 9). rhRSPO1 treatment improves the culture of mouse intestinal OU, increasing its size and survival , and TESI formation , increasing its mass, degree of development and engraftment.
细胞疗法和组织工程最近已成为治疗短肠综合征（SBS）的一种新选择，即利用类器官单元（OU）和可生物降解支架生成组织工程小肠（TESI）。重组人R-spondin 1（rhRSPO1）蛋白可能在此过程中起关键作用，因为它对肠道干细胞具有促有丝分裂活性。为了优化TESI的形成过程并使该技术更接近临床应用，我们评估了rhRSPO1蛋白对OU培养和TESI形成的影响。从小鼠C57BL/6中分离出肠道OU，并在有或无重组人rhRSPO1的情况下于基质胶中培养。在整个培养过程中，评估OU的生长和存活率，并在第3天收获细胞。将OU接种到有或无5μg rhRSPO1的可生物降解支架上，并植入NOD/SCID小鼠的大网膜中以生成TESI。30天后收获外植体，称重，用福尔马林固定并包埋在石蜡中，用于组织学分析和不同细胞标志物的免疫荧光检测。3天后，与对照组相比，rhRSPO1处理的OU体积更大，在第6天时增大了5.7倍。从培养第二天开始观察到存活率增加，在第3天至第6天之间OU存活率增加了2倍。rhRSPO1处理组中未磷酸化的β-连环蛋白增加了4.8倍，且mRNA相对表达增加，这证实了经典Wnt途径的激活，并表明OU干细胞生态位及其干性得以维持。成熟30天后，rhRSPO1处理的TESI比用盐水处理的构建体质量更大，并发育出具有良好形成的绒毛和隐窝的更成熟的肠上皮。此外，负载OU的rhRSPO1处理支架的效率显著提高，在100%的样本（n = 8）中形成了TESI，其中40%呈现出最大发育程度；相比之下，对照组（n = 9）为66.6%。rhRSPO1处理改善了小鼠肠道OU的培养，增加了其大小和存活率以及TESI的形成，增加了其质量、发育程度和植入率。
