Pluripotent reprogramming arises from a unique subset of pre-disposed human dermal fibroblasts

Induced cellular reprogramming to the pluripotent state offers a novel stem cell source for autologous transplantation. While recent studies have explored the role of factors required for induced pluripotent stem cell (iPSC) induction, the cellular and molecular basis of reprogramming from human fibroblasts remains elusive. Here, we have identified a subset of human dermal-derived fibroblasts that shares hallmark molecular and epigenetic features with pluripotent cells. Functional studies demonstrate that these cells contribute to the majority of human iPSCs generated from dermal fibroblasts and are dependent on heterogeneous fibroblast microenvironment for reprogramming competency. Molecular characterization indicated these predisposed fibroblasts were unique to other dermal derived stem cells and possessed features of proliferative selfrenewal. Our study reveals human fibroblasts are not equivalently capable of cellular reprogramming, and suggests that reprogramming factors overcome commitment steps that allow predetermined dermal fibroblasts to establish stable pluripotent state. Samples were obtained from human dermal fibroblasts (hFibs) transduced with a EOS lentiviral vector containing trimerized Oct4 enhancer elements that allow expression of GFP. Upon EOS transduction, we observed a small frequency of adult breast-derived dermal fibroblasts expressing GFP, fibroblasts were sorted based on GFP expression on FACS Aria (BD Pharmagin). 2 samples of GFP-positive and 2 samples of GFP-negative cells from transduced dermal fibroblasts and 1 sample of skin-derived precursors were obtained for this study.

诱导多能性细胞重编程为自体移植提供了新型干细胞来源。尽管近期研究已探讨了诱导多能干细胞（induced pluripotent stem cell, iPSC）诱导所需因子的作用，但人类成纤维细胞重编程的细胞与分子基础仍未阐明。本研究鉴定出一类人类真皮来源成纤维细胞亚群，其具有多能细胞标志性的分子与表观遗传特征。功能实验证实，该亚群构成了真皮成纤维细胞来源的人类iPSC的绝大多数，且其重编程能力依赖于异质性成纤维细胞微环境。分子特征分析表明，这类具有重编程倾向的成纤维细胞与其他真皮来源干细胞存在显著差异，并具备增殖自我更新的特性。本研究揭示，人类成纤维细胞并非均等地具备细胞重编程能力，并提示重编程因子可克服既定的细胞分化步骤，使预先设定的真皮成纤维细胞得以建立稳定的多能状态。本研究的样本来自转导了携带三聚体Oct4增强子元件、可驱动绿色荧光蛋白（GFP）表达的EOS慢病毒载体的人类真皮成纤维细胞（human dermal fibroblasts, hFibs）。经EOS载体转导后，我们在成人乳腺来源的真皮成纤维细胞中观察到少量表达GFP的细胞；随后通过BD Pharmagin的FACS Aria流式细胞仪基于GFP表达水平对成纤维细胞进行分选。本研究共获取了转导后成纤维细胞的GFP阳性样本2份、GFP阴性样本2份，以及1份皮肤来源前体细胞样本。