In the current issue of Acta Physiologica , Fan et al report that light activation enhances and prolongs the regenerative properties of adipose‐derived stromal/stem cells in a murine hindlimb ischaemia model through mechanisms involving secretion of angiogenic and vasculogenic factors.1 To understand why this finding merits a highlight, it helps to take a flashlight into the tunnels of time and get a historical perspective.

In 2001, human ASC took centre stage in the spotlight of regenerative medicine with a landmark publication from the University of Pittsburgh and the University of California‐Los Angeles.2 In this seminal study, Patricia Zuk, Adam Katz and their colleagues presented compelling evidence documenting the robust ability of ASC to differentiate along the adipocyte, chondrocyte, myocyte and osteoblast lineage pathways.2 This supported the authors’ novel hypothesis that adipose tissue, harvested from healthy donors during elective liposuction surgery, was a rich reservoir for cells with regenerative properties. While Alexander Friedenstein had first described bone‐marrow‐derived mesenchymal stromal/stem cells (MSC) with similar or identical properties in the 1960s, their frequency within a bone marrow aspirate was one to three orders of magnitude less abundant.

In 2004, three laboratories in rapid succession shed light on the endothelial cell functionality of ASC. First, Planat‐Bernard et al showed that human stromal vascular fraction (SVF) cells, the precursors of culture expanded ASC, formed vasculogenic structures when cultured in three‐dimensional Matrigel scaffolds in vitro.3 Furthermore, when injected intramuscularly in vivo, the SVF cells enhanced circulatory recovery in a nude mouse model of hindlimb ischemia.3 Next, Rehman et al reported that human ASC secreted angiogenic cytokines including hepatocyte growth factor (HGF), vascular endothelial growth factor (VEGF) and transforming growth factor β (TGFβ) in vitro and postulated that these accounted for the pro‐angiogenic effects of ASC when injected intramuscularly into a hindlimb ischemic mice.4 Shortly thereafter, Miranville et al independently confirmed that human SVF cells differentiated into endothelial cells in vitro and improved vasculogenic recovery when injected intramuscularly into a murine hindlimb ischemic model.5 By 2006, Zvonic et al began using mass spectrometry to define the heterogeneous content of the ASC secretome, detecting adiponectin, angiotensinogen, stromal‐derived growth factor and serpins, among other proteins.6

Flashing forward to 2008, Tomchuck, Betancourt and colleagues showed that it was possible to educate bone marrow MSC by manipulating their exposure to toll‐like receptor (TLR) ligands.7 In the presence of endotoxin or lipopolysaccharide (LPS), the agonist for TLR‐4, MSC secreted pro‐inflammatory cytokines while in the presence of single chain DNA, an agonist for TLR‐3, MSC secreted immunosuppressive or anti‐inflammatory cytokines. This team went on to distinguish these two phenotypes as MSC1 (pro‐inflammatory) and MSC2 (immunosuppressive), similar to the distinction between type 1 (M1) and type 2 (M2) macrophages.7 Betancourt and her colleagues have since founded their biotechnology company, Commence Bio, based on this intellectual property and have begun exploring MSC1 therapy for cancer targets and MSC2 immunosuppressive therapy for inflammatory bowel disease and related autoimmune disorders.

By combining these individual glimmers from the past, Fan et al have now extended and advanced the stromal/stem cell field by demonstrating a non‐invasive physical means to educate and manipulate the regenerative functionality of ASC.1 After exposure to photoactivation for 30 minutes, murine ASC displayed increased proliferative potential in vitro compared to untreated controls (figure 1).1 When a murine hindlimb ischaemia model was injected intravenously with phosphate‐buffered saline alone, blood flow did not return to normal. While injection of untreated and photoactivated ASC significantly improved blood flow within 2 weeks, this increase persisted up to 38 days only with the photoactivated ASC (figure 3). Analyses of the skeletal muscle at this final timepoint by qRT‐PCR indicated a significant increase in mRNA encoding angiopoietin 2, platelet‐derived growth factor‐BB, and VEGFA and VEGFC (figure 4). Subsequent unbiased mass spectrometry analysis of the conditioned medium of human ASC detected >80 proteins associated with angiogenesis as well as heat shock protein 90 (HSP90) isoforms which were selectively induced following light exposure (table 1). Based on their findings, Fan et al conclude that photoactivation can serve as a low‐cost physical manipulation of ASC to improve their efficacy as a cell therapy for peripheral artery disease, a frequent complication in type 2 diabetic and atherosclerotic patients.

While the use of ASC and MSC in regenerative medicine is still in its infancy, adult cell therapy has gained some momentum. There is a growing industry to isolate, expand and package human stromal/stem cells in accordance with the Good Manufacturing Practice (GMP) standards necessary to address regulatory authority concerns regarding patient safety and product efficacy. Since the ASC and MSC release both cytokines and exosomes that contribute paracrine regenerative effects, there is a need to further explore and understand the secretome content with respect to tissue repair mechanisms. The secretome can potentially be harvested, cryopreserved and lyophilized as a stable, off the shelf product that can be delivered at point of care to patients as a substitute or supplement to live cell therapies. The ability to standardize and quantify the process of cell manipulation and media conditioning using physical as opposed to biological or chemical means has practical applications. Thus, the ability to insure a proliferative and secretory advantage to ASC by photoactivation has commercial and clinical translational appeal. Theoretically, such a treatment regimen could be applied to any cell therapy, including autologous SVF cells isolated and processed using a closed‐system device at the point of care by a surgeon practicing medicine within an operating room. Before such an approach can be put into practice, further studies will be necessary. The time course and dosage of light exposure to the ASC and SVF cells will need to be validated in vitro using cells prepared from multiple donors, not just one lot isolated from a single individual as reported in the current manuscript. Likewise, the ability of photoactivation to persist following cryopreservation of the cell product will likewise merit investigation as this will have value in generating a stable, off the shelf product. Fortunately, there is precedent to suggest that this may be a likely outcome. Studies by have demonstrated that ASC exposed to a physical temperature of 43°C prior to cryopreservation display induced levels of HSP and enhanced viability post‐thaw relative to untreated controls. Thus, the concept of photoactivation as described by Fan et al in this issue of Acta Physiologica holds potential clinical translation as a quantifiable and low budget method for educating and modulating the functionality of ASC and other cell types for a wide range of regenerative medical therapies.

在当前的《生理学报》中，Fan等人报道光激活通过涉及血管生成和血管生成因子分泌的机制增强和延长了小鼠后肢缺血模型中脂肪来源的基质/干细胞的再生特性。1要了解为什么这一发现值得强调，它有助于将手电筒带入时间的隧道中并获得历史的眼光。

2001年，匹兹堡大学和加利福尼亚大学洛杉矶分校发表了具有里程碑意义的出版物，人类ASC在再生医学的关注中占据了中心位置。2在这项开创性研究中，帕特里夏·祖克（Patricia Zuk），亚当·卡茨（Adam Katz）及其同事提供了令人信服的证据，证明了ASC沿脂肪细胞，软骨细胞，肌细胞和成骨细胞谱系分化的强大能力。2这支持了作者的新假说，即在选择性吸脂手术期间从健康供体中收获的脂肪组织是具有再生特性的细胞的丰富储库。亚历山大·弗里登斯坦（Alexander Friedenstein）在1960年代首次描述了具有相似或相同特性的骨髓间充质基质/干细胞（MSC），但它们在骨髓穿刺物中的频率却少了1至3个数量级。

2004年，三个连续快速的实验室揭示了ASC的内皮细胞功能。首先，Planat-Bernard等人表明，当在三维Matrigel支架中进行体外培养时，人间质血管部分（SVF）细胞（培养物的前体）会扩展ASC，形成血管生成结构。3此外，在体内肌肉注射时，SVF细胞增强了后肢缺血裸鼠模型的循环恢复。3接下来，Rehman等人报道了人ASC在体外分泌了包括肝细胞生长因子（HGF），血管内皮生长因子（VEGF）和转化生长因子β（TGFβ）在内的血管生成细胞因子，并推测这些原因可解释ASC的促血管生成作用肌肉注射后肢缺血小鼠。4此后不久，Miranville等人独立地证实人SVF细胞在体外分化为内皮细胞，并在肌肉内注射到鼠后肢缺血模型中时改善了血管生成的恢复。5到2006年，Zvonic等人开始使用质谱来定义ASC分泌组的异质含量，检测脂联素，血管紧张素原，基质衍生生长因子和丝氨酸蛋白酶抑制剂，以及其他蛋白质。6

快闪到2008年，Tomchuck，Betancourt和他的同事们表明，可以通过操纵它们接触toll样受体（TLR）配体来教育骨髓MSC。7在存在内毒素或脂多糖（LPS）的情况下，MSC会分泌促炎细胞因子，而在存在单链DNA时会存在TLR-3的激动剂，MSC会分泌免疫抑制或抗炎细胞因子。该小组继续区分这两种表型，如MSC1（促炎性）和MSC2（免疫抑制性），类似于1型（M1）和2型（M2）巨噬细胞之间的区别。7 Betancourt和她的同事自此以来基于此知识产权建立了他们的生物技术公司Commence Bio，并已开始探索针对癌症靶标的MSC1治疗和针对炎症性肠病和相关自身免疫性疾病的MSC2免疫抑制治疗。

通过结合过去的这些个体微光，Fan等人现在通过展示一种非侵入性的物理方法来教育和操纵ASC的再生功能，从而扩展并推进了基质/干细胞领域。1暴露于光激活状态30分钟后，与未经处理的对照相比，鼠ASC在体外显示出增加的增殖潜能（图1）。1个当仅用磷酸盐缓冲盐水静脉内注射鼠后肢缺血模型时，血流量未恢复正常。尽管注射未经治疗和光活化的ASC可以显着改善2周内的血流，但是这种增加仅在使用光活化的ASC的情况下可持续长达38天（图3）。通过qRT-PCR在此最后时间点分析骨骼肌，结果表明编码血管生成素2，血小板源性生长因子-BB，VEGFA和VEGFC的mRNA显着增加（图4）。随后对人ASC的条件培养基进行的无偏质谱分析检测到> 80种与血管生成相关的蛋白质，以及在光照后选择性诱导的热休克蛋白90（HSP90）同工型（表1）。根据他们的发现，

虽然ASC和MSC在再生医学中的使用仍处于起步阶段，但成人细胞治疗已获得一定动力。根据解决对患者安全和产品功效的监管要求的必要的良好生产规范（GMP）标准，分离，扩增和包装人基质/干细胞的行业正在不断发展。由于ASC和MSC均释放有助于旁分泌再生作用的细胞因子和外泌体，因此有必要就组织修复机制进一步探索和了解分泌蛋白的含量。分泌蛋白组可以潜在地作为稳定的现成产品收获，冷冻保存和冻干，可以在护理时作为活细胞疗法的替代品或补充品运送给患者。使用物理方法而不是生物学方法或化学方法来标准化和量化细胞操作和培养基调节过程的能力具有实际应用。因此，通过光活化确保对ASC的增殖和分泌优势的能力具有商业和临床翻译吸引力。从理论上讲，这种治疗方案可以应用于任何细胞疗法，包括在手术室由外科医生在手术室中进行医学护理时使用封闭系统设备分离和处理的自体SVF细胞。在将这种方法付诸实践之前，有必要进行进一步的研究。需要使用多个供体制备的细胞在体外验证ASC和SVF细胞的光照时间和剂量。不仅限于当前手稿中的与单个人孤立的很多。同样，在细胞产物冷冻保存后光活化持续的能力同样值得研究，因为这对于产生稳定的现成产品具有价值。幸运的是，有先例表明这可能是可能的结果。研究表明，相对于未处理的对照，ASC在冷冻保存之前暴露于43°C的物理温度下显示出诱导的HSP水平，并且融化后的活力增强。因此，Fan等人在本期杂志中描述了光激活的概念 冷冻保存细胞产物后光活化的持续能力将同样值得研究，因为这对于产生稳定的现成产品具有价值。幸运的是，有先例表明这可能是可能的结果。研究表明，相对于未经处理的对照，ASC在冷冻保存之前暴露于43°C的物理温度下显示出诱导的HSP水平，并且融化后的活力增强。因此，Fan等人在本期杂志中描述了光激活的概念 冷冻保存细胞产物后光活化的持续能力将同样值得研究，因为这对于产生稳定的现成产品具有价值。幸运的是，有先例表明这可能是可能的结果。研究表明，相对于未经处理的对照，ASC在冷冻保存之前暴露于43°C的物理温度下显示出诱导的HSP水平，并且融化后的活力增强。因此，Fan等人在本期杂志中描述了光激活的概念 研究表明，相对于未经处理的对照，ASC在冷冻保存之前暴露于43°C的物理温度下显示出诱导的HSP水平，并且融化后的活力增强。因此，Fan等人在本期杂志中描述了光激活的概念 研究表明，相对于未经处理的对照，ASC在冷冻保存之前暴露于43°C的物理温度下显示出诱导的HSP水平，并且融化后的活力增强。因此，Fan等人在本期杂志中描述了光激活的概念生理学杂志（ACTA Physiologica）具有潜在的临床翻译功能，可作为一种可量化的低成本方法，用于教育和调节ASC和其他细胞类型的功能，以用于各种再生医学治疗。