Introduction

Adipose-derived stromal/stem cells (ASCs) have attracted attention as a promising material for regenerative medicine. Previously, we reported an age-related decrease in the adipogenic potential of ASCs from human subjects and found that the individual difference in this potential increased with age, although the mechanisms remain unclear. Recently, other groups demonstrated that a secreted antagonist of bone morphogenetic protein (BMP) signaling, Gremlin 2 (GREM2), inhibits the differentiation of bone marrow-derived mesenchymal stem cells (BMSCs) into osteoblasts and the adipogenesis of 3T3-L1 cell. Here, we examined the effects of GREM2 on the differentiation of ASCs into adipocytes.

Methods

To examine changes in GREM2 expression levels with age, immunohistochemistry was performed on subcutaneous adipose tissues from subjects 12–97 years of age. Next, GREM2 gene expression levels in ASCs collected from subjects 5–90 years of age were examined by RT-PCR, and the change with age and correlation between the expression level and the adipogenic potential of ASCs were analyzed. In addition, to assess whether GREM2 affects adipogenesis, ASCs (purchased from a vendor) were cultured to induce adipogenesis with recombinant GREM2 protein, and siRNA-induced GREM2 knockdown experiment was also performed using aged ASCs.

Results

In adipose tissues, GREM2 expression was observed in cells, including ASCs, but not in mature adipocytes, and the expression level per cell increased with age. GREM2 expression levels in ASCs cultured in vitro also increased with age, and the individual differences in the level increased with age. Of note, partial correlation analysis controlled for age revealed that the adipogenic potential of ASCs and the GREM2 gene expression level were negatively correlated. Furthermore, based on a GREM2 addition experiment, GREM2 has inhibitory effects on the adipogenesis of ASCs through activation of Wnt/β-catenin signaling. On the other hand, GREM2 knockdown in aged ASCs promoted adipogenesis.

Conclusions

The GREM2 expression level was confirmed to play a role in the age-related decrease in adipogenic potential observed in ASCs isolated from adipose tissues as well as in the enhancement of the individual difference, which increased with age. GREM2 in adipose tissues increased with age, which suggested that GREM2 functions as an inhibitory factor of adipogenesis in ASCs.

中文翻译：

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人类脂肪来源的基质/干细胞中随着年龄的增长，gremlin 2 的增加及其对脂肪生成的抑制作用。

介绍

脂肪来源的基质/干细胞（ASCs）作为一种有前途的再生医学材料引起了人们的关注。以前，我们报道了人类受试者 ASC 的脂肪形成潜力与年龄相关的下降，并发现这种潜力的个体差异随着年龄的增长而增加，尽管机制仍不清楚。最近，其他研究小组证明，一种分泌的骨形态发生蛋白 (BMP) 信号拮抗剂 Gremlin 2 (GREM2) 可抑制骨髓间充质干细胞 (BMSCs) 分化为成骨细胞和 3T3-L1 细胞的脂肪生成。在这里，我们检查了 GREM2 对 ASC 分化为脂肪细胞的影响。

方法

为了检查 GREM2 表达水平随年龄的变化，对来自 12-97 岁受试者的皮下脂肪组织进行免疫组织化学。接下来，通过 RT-PCR 检查从 5-90 岁的受试者收集的 ASC 中 GREM2 基因的表达水平，并分析了表达水平与 ASC 的脂肪形成潜力之间的年龄变化和相关性。此外，为了评估 GREM2 是否影响脂肪生成，培养 ASC（从供应商处购买）以用重组 GREM2 蛋白诱导脂肪生成，并使用老化的 ASC 进行 siRNA 诱导的 GREM2 敲低实验。

结果

在脂肪组织中，在包括 ASC 在内的细胞中观察到 GREM2 表达，但在成熟脂肪细胞中没有观察到，并且每个细胞的表达水平随着年龄的增长而增加。体外培养的 ASCs 中 GREM2 的表达水平也随着年龄的增长而增加，并且水平的个体差异随着年龄的增长而增加。值得注意的是，控制年龄的偏相关分析显示，ASCs 的脂肪形成潜力与 GREM2 基因表达水平呈负相关。此外，基于 GREM2 添加实验，GREM2 通过激活 Wnt/β-catenin 信号传导对 ASCs 的脂肪生成具有抑制作用。另一方面，老年 ASC 中的 GREM2 敲低促进了脂肪生成。

结论

证实 GREM2 表达水平在从脂肪组织分离的 ASC 中观察到的与年龄相关的脂肪生成潜力降低以及个体差异的增强中起作用，个体差异随着年龄的增长而增加。脂肪组织中的 GREM2 随年龄增长而增加，这表明 GREM2 是 ASC 中脂肪生成的抑制因子。