Department of Clinical Dentistry, Centre for Translational Oral Research (TOR), University of Bergen, 5009, Bergen, Norway.
Department of Clinical Medicine, University of Bergen, Bergen, Norway.
Stem Cell Res Ther. 2023 Aug 9;14(1):199. doi: 10.1186/s13287-023-03403-7.
Induced pluripotent stem cells (iPS) can be generated from various somatic cells and can subsequently be differentiated to multiple cell types of the body. This makes them highly promising for cellular therapy in regenerative medicine. However, to facilitate their clinical use and to ensure safety, iPS culturing protocols must be compliant with good manufacturing practice guidelines and devoid of xenogenic products. Therefore, we aimed to compare the efficiency of using humanized culture conditions, specifically human platelet lysate to fetal bovine serum, for iPS generation from different sources, and to evaluate their stemness.
iPS were generated via a platelet lysate or fetal bovine serum-based culturing protocol from matched dermal, buccal and gingival human fibroblasts, isolated from healthy donors (n = 2) after informed consent, via episomal plasmid transfection. Pluripotency, genotype and phenotype of iPS, generated by both protocols, were then assessed by various methods.
More attempts were generally required to successfully reprogram xeno-free fibroblasts to iPS, as compared to xenogenic cultured fibroblasts. Furthermore, oral fibroblasts generally required more attempts for successful iPS generation as opposed to dermal fibroblasts. Morphologically, all iPS generated from fibroblasts formed tight colonies surrounded by a reflective "whitish" outer rim, typical for iPS. They also expressed pluripotency markers at both gene (SOX2, OCT4, NANOG) and protein level (SOX2, OCT4). Upon stimulation, all iPS showed ability to differentiate into the three primary germ layers via expression of lineage-specific markers for mesoderm (MESP1, OSR1, HOPX), endoderm (GATA4) and ectoderm (PAX6, RAX). Genome analysis revealed several amplifications and deletions within the chromosomes of each iPS type.
The xeno-free protocol had a lower reprogramming efficiency compared to the standard xenogenic protocol. The oral fibroblasts generally proved to be more difficult to reprogram than dermal fibroblasts. Xeno-free dermal, buccal and gingival fibroblasts can successfully generate iPS with a comparable genotype/phenotype to their xenogenic counterparts.
诱导多能干细胞（iPS）可从各种体细胞中产生，随后可分化为身体的多种细胞类型。这使它们在再生医学中的细胞治疗中具有很高的应用前景。然而，为了促进其临床应用并确保安全性，iPS 培养方案必须符合良好生产规范指南，且不含有异源产品。因此，我们旨在比较使用人源化培养条件（特别是人血小板裂解物替代胎牛血清）从不同来源生成 iPS 的效率，并评估其干细胞特性。
通过基于血小板裂解物或胎牛血清的培养方案，从经知情同意后分离自健康供体（n=2）的匹配的皮肤、口腔和牙龈人成纤维细胞中生成 iPS，通过外源性质粒转染。然后通过各种方法评估两种方案生成的 iPS 的多能性、基因型和表型。
与使用异源培养的成纤维细胞相比，成功将无动物源成纤维细胞重编程为 iPS 通常需要更多的尝试。此外，与皮肤成纤维细胞相比，口腔成纤维细胞通常需要更多的尝试才能成功生成 iPS。形态上，所有由成纤维细胞生成的 iPS 均形成紧密的集落，周围环绕着反射性的“白色”外边缘，这是 iPS 的典型特征。它们还在基因（SOX2、OCT4、NANOG）和蛋白质水平（SOX2、OCT4）上表达多能性标志物。在刺激下，所有 iPS 均通过表达中胚层（MESP1、OSR1、HOPX）、内胚层（GATA4）和外胚层（PAX6、RAX）的谱系特异性标志物显示出分化为三个原胚层的能力。基因组分析显示每种 iPS 类型的染色体都存在多个扩增和缺失。
无动物源方案的重编程效率低于标准异源方案。口腔成纤维细胞通常比皮肤成纤维细胞更难以重编程。无动物源的皮肤、口腔和牙龈成纤维细胞可以成功生成与异源成纤维细胞具有相似基因型/表型的 iPS。
