Stem cell injections are an attractive therapeutic tool. It has been demonstrated that injected stem cells promote tissue repair and regeneration via paracrine mechanisms. However, the effects of injected stem cells continue for far longer than they are present. We hypothesized that the effects of injected stem cells are prolonged due a sequential paracrine relay mechanism. Conditioned media was collected from mesenchymal stem cells (MSCs) after 24hr. This media was then added to RAW264.7. Media was collected from the macrophages after 24hrs and was then added to endothelial cells (ECs). This conditioned macrophage media, but not control media, promoted wound healing and induced EC differentiation. Similar results were observed with primary macrophages. To identify the active paracrine factors released by macrophages in response to stimulation by MSC conditioned media we used an antibody array; identifying increased expression of the angiogenesis-related proteins stromal cell-derived factor 1 (SDF1) and plasminogen activator inhibitor-1 (PAI-1). Knockdown of either protein inhibited the ability of conditioned media derived from MSC paracrine factor stimulated macrophages to induce EC differentiation both in vitro and in vivo. Conditioned media derived from P7 mouse macrophages induced EC differentiation. Moreover, SDF1 and PAI-1 levels were >120 higher in P7 macrophages compared to adult macrophages; suggesting that MSC paracrine factors promote adult macrophages to adopt a juvenile phenotype. These results indicate that MSC paracrine factors induce macrophages to secrete SDF1 and PAI-1; in-turn inducing endothelial cells to differentiate. Identification of a sequential paracrine mechanism opens new therapeutic avenues for stem cell therapy.

中文翻译：

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顺序旁分泌机制对于干细胞疗法的治疗益处是必需的

干细胞注射是一种有吸引力的治疗工具。已经证明注射的干细胞通过旁分泌机制促进组织修复和再生。但是，注射的干细胞的作用持续的时间比它们存在的时间长得多。我们假设由于顺序的旁分泌中继机制，注射的干细胞的作用得以延长。24小时后从间充质干细胞（MSC）收集条件培养基。然后将该媒体添加到RAW264.7。24小时后从巨噬细胞中收集培养基，然后将其加入到内皮细胞（EC）中。这种条件性的巨噬细胞培养基而非对照培养基促进了伤口愈合并诱导了EC分化。用原代巨噬细胞观察到相似的结果。为了鉴定巨噬细胞响应MSC条件培养基刺激而释放的活性旁分泌因子，我们使用了抗体阵列；鉴定血管生成相关蛋白基质细胞衍生因子1（SDF1）和纤溶酶原激活物抑制剂1（PAI-1）的表达。两种蛋白质的组合均抑制了来自MSC旁分泌因子刺激的巨噬细胞的条件培养基在体外和体内诱导EC分化的能力。源自P7小鼠巨噬细胞的条件培养基诱导EC分化。此外，与成人巨噬细胞相比，P7巨噬细胞的SDF1和PAI-1水平高出120倍；提示MSC旁分泌因子促进成年巨噬细胞采用少年表型。这些结果表明，MSC旁分泌因子诱导巨噬细胞分泌SDF1和PAI-1。依次诱导内皮细胞分化。顺序旁分泌机制的鉴定为干细胞治疗开辟了新的治疗途径。