Gao Peng, Han Peilin, Jiang Dapeng, Yang Shulong, Cui Qingbo, Li Zhaozhu
Department of Pediatric Surgery, The 2nd Affiliated Hospital of Harbin Medical University, Harbin, China.
Department of Pediatric Surgery, Harbin Children's Hospital, Harbin, China.
Cytotechnology. 2017 Oct;69(5):751-763. doi: 10.1007/s10616-017-0084-5. Epub 2017 Apr 13.
To study the effects of the donor age on the application potential of human urine-derived stem cells (hUSCs) in bone tissue engineering, by comparing proliferation, senescence and osteogenic differentiation of hUSCs originated from volunteers with different ages. The urine samples were collected from 19 healthy volunteers (6 cases from children group aged from 5 to 14, 5 cases from middle-aged group aged from 30 to 40, and 8 cases from the elder group aged from 65 to 75), and hUSCs were isolated and cultured. The cell morphology was observed by microscope and the cell surface markers were identified by flow cytometry. Their abilities to undergo osteogenic, adipogenic and chondrogenic differentiation were determined in vitro, and cell proliferation analyses were performed using Cell Counting Kit-8 (CCK8) Assay. The senescence of hUSCs among three groups was assessed by senescence-associated β galactosidase staining. After osteogenic differentiation, the alkaline phosphatase (ALP) activity of hUSCs was measured and expression of osteogenic-related runt-related transcription factor 2 (RUNX2) and osteocalcin (OCN) was determined by quantitative real-time polymerase chain reaction (qRT-PCR) and western blot. The hUSCs isolated from urine samples were adherent cells displayed "rice gain"-like and "spindle-shaped" morphology, expressing surface markers of mesenchymal stem cells (MSCs) (CD73, CD90, CD105) and the peripheral cell marker (CD146), but not hematopoietic stem cell markers (CD34, CD45) or the embryonic stem cell marker (OCT3/4). The obtained hUSCs could be induced into osteogenic, adipogenic or chondrogenic differentiation. The hUSCs from the children group showed higher proliferation and lower tendency to senescence than those from the middle-aged and elder groups. After osteogenic induction, the ALP activity and RUNX2 and OCN expression of hUSCs from the children group were higher than those from the elder group. While no significant differences were observed when comparing the middle-aged group with the children group or the elder group. Donor age could influence the potency of hUSCs on proliferation, senescence and capacity of osteogenic differentiation. hUSCs from children group have shown higher proliferation, lower tendency to senescence, and stronger osteogenic capacity, which means to be more suitable for basic research and have better clinical application. Furthermore, hUSCs from all groups suggest the application potential in bone tissue engineering as seed cells.
为研究供体年龄对人尿液来源干细胞（hUSCs）在骨组织工程中应用潜力的影响，通过比较不同年龄志愿者来源的hUSCs的增殖、衰老和成骨分化情况。收集了19名健康志愿者的尿液样本（5至14岁儿童组6例，30至40岁中年组5例，65至75岁老年组8例），并分离培养hUSCs。通过显微镜观察细胞形态，用流式细胞术鉴定细胞表面标志物。体外测定其成骨、成脂和成软骨分化能力，使用细胞计数试剂盒-8（CCK8）法进行细胞增殖分析。通过衰老相关β半乳糖苷酶染色评估三组hUSCs的衰老情况。成骨分化后，测定hUSCs的碱性磷酸酶（ALP）活性，通过定量实时聚合酶链反应（qRT-PCR）和蛋白质免疫印迹法检测成骨相关的 runt相关转录因子2（RUNX2）和骨钙素（OCN）的表达。从尿液样本中分离出的hUSCs为贴壁细胞，呈“米粒样”和“纺锤形”形态，表达间充质干细胞（MSCs）的表面标志物（CD73、CD90、CD105）和外周细胞标志物（CD146），但不表达造血干细胞标志物（CD34、CD45）或胚胎干细胞标志物（OCT3/4）。所获得的hUSCs可被诱导成骨、成脂或成软骨分化。儿童组的hUSCs比中年组和老年组的hUSCs具有更高的增殖能力和更低的衰老倾向。成骨诱导后，儿童组hUSCs的ALP活性以及RUNX2和OCN表达高于老年组。而中年组与儿童组或老年组比较时未观察到显著差异。供体年龄可影响hUSCs的增殖、衰老和成骨分化能力。儿童组的hUSCs表现出更高的增殖能力、更低的衰老倾向和更强的成骨能力，这意味着更适合基础研究且具有更好的临床应用前景。此外，所有组的hUSCs均显示出作为种子细胞在骨组织工程中的应用潜力。
