Alves-Paiva Raquel M, do Nascimento Sabrina, De Oliveira Denise, Coa Larissa, Alvarez Kelen, Hamerschlak Nelson, Okamoto Oswaldo Keith, Marti Luciana C, Kondo Andrea T, Kutner Jose Mauro, Bortolini Maria Augusta Tezelli, Castro Rodrigo, de Godoy Juliana A Preto
Department of Hemotherapy and Cellular Therapy, Hospital Israelita Albert Einstein, São Paulo, Brazil.
Experimental Research Laboratory, Hospital Israelita Albert Einstein, São Paulo, Brazil.
Front Cell Dev Biol. 2022 Apr 4;10:858996. doi: 10.3389/fcell.2022.858996. eCollection 2022.
Mesenchymal stem cells (MSCs) are multipotent cells found in various tissues and are easily cultivated. For use in clinical protocols, MSCs must be expanded to obtain an adequate number of cells, but a senescence state may be instituted after some passages, reducing their replicative potential. In this study, we report a case where MSC derived from an elderly donor acquired a senescence state after three passages. The bone marrow was aspirated from a female patient submitted to a cell therapy for the incontinency urinary protocol; MSCs were cultivated with DMEM low glucose, supplemented with 10% autologous serum (AS) plus 1% L-glutamine and 1% antibiotic/antimycotic. Senescence analysis was performed by β-galactosidase staining after 24 and 48 h. Controls were established using BM-MSC from healthy donors and used for senescence and gene expression assays. Gene expression was performed using RT-PCR for pluripotency genes, such as , , , and MSC telomere length was measured by the Southern blotting technique, and MSCs were also analyzed for their capacity to differentiate into adipocytes, chondrocytes, and osteocytes. The patient's MSC expansion using AS displayed an early senescence state. In order to understand the role of AS in senescence, MSCs were then submitted to two different culture conditions: 1) with AS or 2) with FBS supplementation. Senescence state was assessed after 24 h, and no statistical differences were observed between the two conditions. However, patients' cells cultured with AS displayed a higher number of senescence cells than FBS medium after 48 h ( = 0.0018). Gene expression was performed in both conditions; increased expression of was observed in the patient's cells in comparison to healthy controls ( = 0.0016); reduced gene expression was observed for ( = 0.0016) and ( = 0.0014) genes. Telomere length of the patient's cells was shorter than that of a healthy donor and that of a patient of similar age. Osteocyte differentiation seemed to be more diffuse than that of the healthy donor and that of the patient of similar age. MSCs could enter a senescence state during expansion in early passages and can impact MSC quality for clinical applications, reducing their efficacy when administered.
间充质干细胞（MSCs）是存在于各种组织中的多能细胞，且易于培养。在临床方案中使用时，必须扩增MSCs以获得足够数量的细胞，但经过若干传代后可能会进入衰老状态，从而降低其增殖潜力。在本研究中，我们报告了一例源自老年供体的MSCs在传代三次后进入衰老状态的病例。从一名接受尿失禁细胞治疗方案的女性患者身上抽取骨髓；将MSCs用低糖DMEM培养，并补充10%自体血清（AS）、1% L-谷氨酰胺和1%抗生素/抗真菌剂。在24小时和48小时后通过β-半乳糖苷酶染色进行衰老分析。使用来自健康供体的骨髓间充质干细胞（BM-MSC）建立对照，并用于衰老和基因表达测定。使用逆转录聚合酶链反应（RT-PCR）检测多能性基因（如 、 、 、 ）的表达。通过Southern印迹技术测量MSCs的端粒长度，并分析MSCs分化为脂肪细胞、软骨细胞和骨细胞的能力。使用AS对患者的MSCs进行扩增显示出早期衰老状态。为了了解AS在衰老中的作用，随后将MSCs置于两种不同的培养条件下：1）添加AS或2）添加胎牛血清（FBS）。在24小时后评估衰老状态，两种条件之间未观察到统计学差异。然而，48小时后，与FBS培养基相比，用AS培养的患者细胞显示出更多的衰老细胞（ = 0.0018）。在两种条件下均进行了基因表达检测；与健康对照相比，患者细胞中 基因的表达增加（ = 0.0016）； （ = 0.0016）和 （ = 0.0014）基因的表达降低。患者细胞的端粒长度短于健康供体以及年龄相仿患者的端粒长度。与健康供体和年龄相仿患者相比，骨细胞分化似乎更为分散。MSCs在早期传代扩增过程中可能进入衰老状态，这会影响用于临床应用的MSCs质量，降低其给药时的疗效。
