Nucleic Acid and Non-Nucleic Acid-Based Reprogramming of Adult Limbal Progenitors to Pluripotency

Reprogramming somatic cells to a pluripotent state by nucleic acid based (NAB) approaches, involving the ectopic expression of transcription factors, has emerged as a standard method. We recently demonstrated that limbal progenitors that regenerate cornea are reprogrammable to pluripotency by a non-NAB approach through simple manipulation of microenvironment thus extending the possible therapeutic use of these readily accessible cells beyond the proven treatment of corneal diseases and injury. Therefore, to determine the validity and robustness of non-cell autonomous reprogramming of limbal progenitors for a wider clinical use, here, we have compared their reprogramming by non-NAB and NAB approaches. We observed that both approaches led to (1) the emergence of colonies displaying pluripotency markers, accompanied by a temporal reciprocal changes in limbal-specific and pluripotency gene expression, and (2) epigenetic alterations of Oct4 and Nanog, associated with the de-novo activation of their expression. While the efficiency of reprogramming and passaging of re-programmed cells were significantly better with the NAB approach, the non-NAB approach, in contrast, led to a regulated reprogramming of gene expression, and a significant decrease in the expression of Hormad1, a gene associated with immunogenic responses. The reprogramming efficiency by non-NAB approach was influenced by exosomes present in conditioned medium. Cells reprogrammed by both approaches were capable of differentiating along the three germ lineages and generating chimeras. The analysis suggests that both approaches are effective in reprogramming limbal progenitors but the non-NAB approach may be more suitable for potential clinical applications by averting the risk of insertional mutagenesis and immune responses associated with the NAB approach.借助基于核酸（nucleic acid based, NAB）的重编程策略——该策略通过异位表达转录因子将体细胞重编程至多能状态——现已成为一项成熟的标准技术。我们近期的研究证实，可通过非NAB策略，借助对微环境的简单操控，将具有角膜再生能力的角膜缘祖细胞重编程至多能状态，这一发现将这类易于获取的细胞的潜在治疗用途拓展至了角膜疾病与损伤的常规临床治疗之外。因此，为验证非细胞自主性重编程角膜缘祖细胞策略的有效性与稳定性，以拓展其更广泛的临床应用场景，本研究对比了非NAB与NAB两种重编程策略的效果。研究结果显示，两种策略均可实现以下效果：(1) 形成表达多能性标志物的细胞克隆，同时伴随角膜缘特异性基因与多能性基因表达的时序性互变；(2) 引发Oct4与Nanog的表观遗传修饰改变，进而实现这两个基因的从头激活表达。尽管NAB策略在重编程效率与重编程细胞的传代培养表现上显著更优，但非NAB策略可实现基因表达的精准调控，且能显著下调与免疫原性应答相关的Hormad1基因的表达水平。非NAB策略的重编程效率受条件培养基中外泌体的调控影响。经两种策略重编程得到的细胞，均可分化为三胚层谱系细胞，并可用于构建嵌合体动物。综合分析表明，两种策略均可有效实现角膜缘祖细胞的重编程，但非NAB策略可规避NAB策略所伴随的插入诱变风险与免疫原性问题，因此更适用于潜在的临床应用场景。