Phermthai Tatsanee, Thongbopit Sasiprapa, Pokathikorn Puttachart, Wichitwiengrat Suparat, Julavijitphong Suphakde, Tirawanchai Nednapis
Stem Cell Research and Development Unit, Department of Obstetrics & Gynecology, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok, Thailand.
Stem Cell Research and Development Unit, Department of Obstetrics & Gynecology, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok, Thailand.
Cytotherapy. 2017 Aug;19(8):990-1001. doi: 10.1016/j.jcyt.2017.04.004. Epub 2017 May 26.
Human amniotic mesenchymal stromal cells (hAMSCs) are a potent and attractive stem cell source for use in regenerative medicine. However, the safe uses of therapeutic-grade MSCs are equally as important as the efficiency of MSCs. To provide efficient, clinic-compliant (safe for therapeutic use) MSCs, hAMSC lines that completely eliminate the use of animal products and have been characterized for carcinogenicity and biosafety are required.
Here, we have efficiently generated 10 hAMSC lines under human umbilical cord blood serum (hUCS)-supplemented medium (xeno-free culture) and fetal bovine serum (FBS)-supplemented medium (standard culture) and investigated carcinogenicity and immunosuppressive properties in the resultant hAMSC lines. All hAMSC lines were examined for efficiency (growth kinetics, cryopreservation, telomere length, phenotypic characterization, differentiation potential), carcinogenicity (proto-oncogene and tumor suppressor gene and epigenomic stability) and safety (immunosuppressive properties).
Stem cell characteristics between the xeno-free hAMSC lines and the cell lines generated using the standard culture system showed no differences. Xeno-free hAMSC lines displayed normal growth proliferation potential, morphological, karyotypic, phenotypic differentiation properties and telomere lengths. Additionally, they retained normal immunosuppressive effects. As a marker of carcinogenicity and biosafety, proto-oncogenes expression levels showed no differences in xeno-free hAMSCs, and we detected no SNP mutations on hotspot codons of the P53 tumor suppressor gene and stable epigenomic imprinting in xeno-free hAMSC lines.
Xeno-free hAMSC lines retain essential stem cell characteristics, with a high degree of certainty for meeting biosafety and carcinogenicity standards for a xeno-free system supplemented with allogenic hUCS. The cell lines are suitable and valuable for therapeutic purposes.
人羊膜间充质基质细胞（hAMSCs）是再生医学中一种强大且有吸引力的干细胞来源。然而，治疗级间充质干细胞的安全使用与间充质干细胞的效率同样重要。为了提供高效、符合临床要求（治疗使用安全）的间充质干细胞，需要完全消除动物产品使用且已进行致癌性和生物安全性表征的hAMSC系。
在此，我们在添加人脐带血血清（hUCS）的培养基（无动物源培养）和添加胎牛血清（FBS）的培养基（标准培养）中高效生成了10个hAMSC系，并研究了所得hAMSC系的致癌性和免疫抑制特性。对所有hAMSC系进行了效率（生长动力学、冷冻保存、端粒长度、表型特征、分化潜能）、致癌性（原癌基因和肿瘤抑制基因以及表观基因组稳定性）和安全性（免疫抑制特性）检测。
无动物源hAMSC系与使用标准培养系统生成的细胞系之间的干细胞特征无差异。无动物源hAMSC系表现出正常的生长增殖潜能、形态、核型、表型分化特性和端粒长度。此外，它们保留了正常的免疫抑制作用。作为致癌性和生物安全性的标志物，原癌基因表达水平在无动物源hAMSCs中无差异，并且我们在无动物源hAMSC系中未检测到P53肿瘤抑制基因热点密码子上的单核苷酸多态性突变以及稳定表观基因组印记。
无动物源hAMSC系保留了基本的干细胞特征，高度确定符合添加同种异体hUCS的无动物源系统的生物安全性和致癌性标准。这些细胞系适用于治疗目的且具有价值。
