Enabling a robust scalable manufacturing process for therapeutic exosomes through oncogenic immortalization of human ESC-derived MSCs.
Enabling a robust scalable manufacturing process for therapeutic exosomes through oncogenic immortalization of human ESC-derived MSCs.
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DOI：
10.1186/1479-5876-9-47
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发表时间：
2011-04-25
影响因子：
7.4
通讯作者：
Lim SK
中科院分区：
文献类型：
--
作者：
Chen TS;Arslan F;Yin Y;Tan SS;Lai RC;Choo AB;Padmanabhan J;Lee CN;de Kleijn DP;Lim SK
人胚胎干细胞来源的间充质干细胞(hESC-MSCs)的外切体或分泌的双脂小泡(hESC-MSCs)在动物模型中被证明可以减轻心肌缺血/再灌注损伤。然而，由于hESC-MSCs不是无限可扩展的，大规模生产这些外体需要通过hESCs衍生补充hESC-MSC，并产生测试和验证每个新批次的经常性成本。因此，我们的目的是研究MYC永生化hESC-MSC是否会在不影响产生治疗有效的外切体的情况下绕过这一限制。用携带MYC基因的慢病毒感染hESC-MSCs。对转化细胞进行MYC基因整合、转录本和蛋白质水平、表面标志、细胞周期、端粒酶活性、核型、全基因组基因表达和分化潜能的分析。用高效液相色谱分离得到外切体，在小鼠心肌缺血/再灌注损伤模型上进行检测，并用Evans‘s蓝染色和TTC染色进一步评估心肌梗死范围。MYC转化的MSCs与亲本的hESC-MSCs基本相似，主要区别在于塑性黏附减少，生长更快，不衰老，MYC蛋白表达增加，体外成脂能力丧失，从技术上讲，转化后的细胞成为非MSCs。出乎意料的是，在心肌缺血/再灌注损伤的小鼠模型中，来自MYC转化的MSCs的外切体能够减少相对梗塞范围，这表明产生治疗性外切体的能力被保留了下来。我们的结果表明，MYC转化是一种实用的策略，可以确保无限供应细胞，以生产毫克范围内的外切体，无论是作为治疗剂还是输送载体。此外，通过MYC转化提高了增殖率，减少了细胞生产时间，从而降低了生产成本。
Exosomes or secreted bi-lipid vesicles from human ESC-derived mesenchymal stem cells (hESC-MSCs) have been shown to reduce myocardial ischemia/reperfusion injury in animal models. However, as hESC-MSCs are not infinitely expansible, large scale production of these exosomes would require replenishment of hESC-MSC through derivation from hESCs and incur recurring costs for testing and validation of each new batch. Our aim was therefore to investigate if MYC immortalization of hESC-MSC would circumvent this constraint without compromising the production of therapeutically efficacious exosomes. The hESC-MSCs were transfected by lentivirus carrying a MYC gene. The transformed cells were analyzed for MYC transgene integration, transcript and protein levels, and surface markers, rate of cell cycling, telomerase activity, karyotype, genome-wide gene expression and differentiation potential. The exosomes were isolated by HPLC fractionation and tested in a mouse model of myocardial ischemia/reperfusion injury, and infarct sizes were further assessed by using Evans' blue dye injection and TTC staining. MYC-transformed MSCs largely resembled the parental hESC-MSCs with major differences being reduced plastic adherence, faster growth, failure to senesce, increased MYC protein expression, and loss of in vitro adipogenic potential that technically rendered the transformed cells as non-MSCs. Unexpectedly, exosomes from MYC-transformed MSCs were able to reduce relative infarct size in a mouse model of myocardial ischemia/reperfusion injury indicating that the capacity for producing therapeutic exosomes was preserved. Our results demonstrated that MYC transformation is a practical strategy in ensuring an infinite supply of cells for the production of exosomes in the milligram range as either therapeutic agents or delivery vehicles. In addition, the increased proliferative rate by MYC transformation reduces the time for cell production and thereby reduces production costs.
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影响因子：
3.7
作者：
Nagai, Atsushi;Kim, Woo K.;Lee, Hong J.;Jeong, Han S.;Kim, Kwang S.;Hong, Seok H.;Park, In H.;Kim, Seung U.
通讯作者：
Kim, Seung U.
影响因子：
5
作者：
Lai, Ruenn Chai;Arslan, Fatih;Lim, Sai Kiang
通讯作者：
Lim, Sai Kiang
影响因子：
4
作者：
Caplan, Arnold I.;Dennis, James E.
通讯作者：
Dennis, James E.
影响因子：
37.8
作者：
PFEFFER, MA;BRAUNWALD, E
通讯作者：
BRAUNWALD, E
影响因子：
4.8
作者：
Gnecchi, Massimiliano;He, Huamei;Dzau, Victor J.
通讯作者：
Dzau, Victor J.