Department of Orthopaedic Surgery, University of Texas Health Science Center at Houston, Houston, TX, 77054, USA.
Institute of Molecular Medicine, University of Texas Health Science Center at Houston, Houston, TX, 77054, USA.
Stem Cell Res Ther. 2018 Nov 21;9(1):316. doi: 10.1186/s13287-018-1066-z.
Human muscle-derived stem cells (hMDSCs) have been shown to regenerate bone efficiently when they were transduced with Lenti-viral bone morphogenetic protein 2 (LBMP2). However, whether the age of hMDSCs and the animal host affect the bone regeneration capacity of hMDSCs and mechanism are unknown which prompted the current study.
We isolated three gender-matched young and old populations of skeletal muscle stem cells, and tested the influence of cells' age on in vitro osteogenic differentiation using pellet culture before and after Lenti-BMP2/green fluorescent protein (GFP) transduction. We further investigated effects of the age of hMDSCs and animal host on hMDSC-mediated bone regeneration in a critical-size calvarial bone defect model in vivo. Micro-computer tomography (CT), histology, and immunohistochemistry were used to evaluate osteogenic differentiation and mineralization in vitro and bone regeneration in vivo. Western blot, quantitative polymerase chain reaction (PCR), and oxidative stress assay were performed to detect the effects of age of hMDSCs on cell survival and osteogenic-related genes. Serum insulin-like growth factor 1 (IGF1) and receptor activator of nuclear factor-kappa B ligand (RANKL) were measured with an enzyme-linked immunosorbent assay (ELISA).
We found LBMP2/GFP transduction significantly enhanced osteogenic differentiation of hMDSCs in vitro, regardless of donor age. We also found old were as efficient as young LBMP2/GFP-transduced hMDSCs for regenerating functional bone in young and old mice. These findings correlated with lower phosphorylated p38MAPK expression and similar expression levels of cell survival genes and osteogenic-related genes in old hMDSCs relative to young hMDSCs. Old cells exhibited equivalent resistance to oxidative stress. However, both young and old donor cells regenerated less bone in old than young hosts. Impaired bone regeneration in older hosts was associated with high bone remodeling due to higher serum levels of RANKL and lower level of IGF-1.
hMDSC-mediated bone regeneration was not impaired by donor age when hMDSCs were transduced with LBMP2/GFP, but the age of the host adversely affected hMDSC-mediated bone regeneration. Regardless of donor and host age, hMDSCs formed functional bone, suggesting a promising cell resource for bone regeneration.
当人类肌肉源性干细胞（hMDSCs）被慢病毒骨形态发生蛋白 2（LBMP2）转导时，已显示其能够有效地再生骨骼。然而，hMDSCs 的年龄和动物宿主是否会影响 hMDSCs 的骨再生能力以及其机制尚不清楚，这促使我们开展了当前的研究。
我们分离了三组性别匹配的年轻和老年骨骼肌干细胞，并在慢病毒 BMP2/绿色荧光蛋白（GFP）转导前后使用微球培养法测试了细胞年龄对体外成骨分化的影响。我们进一步在体内临界尺寸颅骨骨缺损模型中研究了 hMDSCs 的年龄和动物宿主对 hMDSC 介导的骨再生的影响。使用微计算机断层扫描（CT）、组织学和免疫组织化学方法来评估体外成骨分化和矿化以及体内骨再生。Western blot、定量聚合酶链反应（PCR）和氧化应激测定法用于检测 hMDSCs 年龄对细胞存活和成骨相关基因的影响。使用酶联免疫吸附测定（ELISA）测量血清胰岛素样生长因子 1（IGF1）和核因子-kappa B 受体激活剂配体（RANKL）。
我们发现 LBMP2/GFP 转导显著增强了 hMDSCs 的体外成骨分化，而与供体年龄无关。我们还发现，LBMP2/GFP 转导的老年 hMDSCs 与年轻 hMDSCs 一样能够有效地在年轻和老年小鼠中再生功能性骨骼。这些发现与老年 hMDSCs 中磷酸化 p38MAPK 表达较低以及细胞存活基因和成骨相关基因表达水平相似相关。老年细胞表现出对氧化应激的同等抗性。然而，年轻和老年供体细胞在老年宿主中再生的骨骼较少。在老年宿主中，由于 RANKL 血清水平较高和 IGF-1 水平较低，骨重塑增加，导致骨再生受损。
当 hMDSCs 被 LBMP2/GFP 转导时，hMDSC 介导的骨再生不受供体年龄的影响，但宿主年龄的增加会对 hMDSC 介导的骨再生产生不利影响。无论供体和宿主年龄如何，hMDSCs 都形成了功能性骨骼，这表明 hMDSCs 是一种有前途的骨再生细胞资源。
