Low Glucose Culture Environment Can Enhance the Wound Healing Capability of Diabetic Adipose-derived Stem Cells

Application of autologous adipose-derived stem cells (ASC) for diabetic chronic wounds has become an emerging treatment option. However, ASCs from diabetic individuals showed impaired cell function and suboptimal wound healing effects. We proposed that adopting a low glucose level in the culture medium for diabetic ASCs may restore their pro-healing capabilities. ASCs from diabetic humans and mice were retrieved and cultured in high-glucose (HG, 4.5 g/L) or low-glucose (LG, 1.0 g/L) conditions. Cell characteristics and functions were investigated in vitro. Moreover, we applied diabetic murine ASCs cultured in HG or LG condition to a wound healing model in diabetic mice to compare their healing capabilities in vivo. Human ASCs exhibited decreased cell proliferation and migration with enhanced senescence when cultured in HG condition in vitro. Similar findings were noted in ASCs derived from diabetic mice. The inferior cellular functions could be partially recovered when they were cultured in LG condition. In the animal study, wounds healed faster when treated with HG or LG-cultured diabetic ASCs relative to the control group. Moreover, higher collagen density, more angiogenesis and cellular retention of applied ASCs were found in wound tissues treated with diabetic ASCs cultured in LG condition. In line with the literature, our study showed that a diabetic milieu exerts an adverse effect on ASCs. Adopting LG culture condition is a simple and effective approach to enhance the wound healing capabilities of diabetic ASCs, which is valuable for the clinical application of autologous ASCs from diabetic patients. Human adipose-derived stem cells (hASCs) were extracted from two non-diabetic and two diabetic female donors for the scRNA-seq processing.

自体脂肪来源干细胞（adipose-derived stem cells, ASC）应用于糖尿病慢性创面已成为一种新兴的治疗选择。然而，来自糖尿病个体的ASC功能受损，创面修复效果欠佳。我们提出，为糖尿病来源的ASC采用低糖培养基培养，可恢复其促创面修复能力。

我们获取了人类及小鼠来源的糖尿病ASC，并分别在高糖（high-glucose, HG，4.5 g/L）与低糖（low-glucose, LG，1.0 g/L）培养条件下进行培养。体外实验中，我们对细胞的特征与功能进行了检测；此外，我们将经高糖或低糖培养的糖尿病小鼠ASC植入糖尿病小鼠创面修复模型，以体内比较二者的修复能力。

体外实验显示，在高糖条件下培养的人类ASC增殖、迁移能力下降，细胞衰老程度加剧。糖尿病小鼠来源的ASC也观察到了相似的结果。而在低糖条件下培养时，这些受损的细胞功能可得到部分恢复。

动物实验结果表明，与对照组相比，植入经高糖或低糖培养的糖尿病ASC的创面愈合速度更快。此外，植入低糖培养的糖尿病ASC的创面组织中，胶原密度更高、血管生成更为显著，且移植的ASC细胞存留率也更高。

与已有文献结论一致，本研究证实糖尿病微环境对ASC具有不利影响。采用低糖培养条件是一种简单有效的手段，可提升糖尿病来源ASC的创面修复能力，这对于糖尿病患者自体ASC的临床应用具有重要价值。

我们从2名非糖尿病女性供者与2名糖尿病女性供者体内提取了人类脂肪来源干细胞（human adipose-derived stem cells, hASCs），用于单细胞RNA测序（single-cell RNA sequencing, scRNA-seq）实验处理。