| Direct Reprogramming of Mouse Embryonic Fibroblasts to Conventional Type 1 Dendritic Cells by Enforced Expression of Transcription Factors
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Author:
Date:
2020-05-20
[Abstract] Ectopic expression of transcription factor combinations has been recently demonstrated to reprogram differentiated somatic cells towards the dendritic cell (DC) lineage without reversion to a multipotent state. DCs have the ability to induce potent and long-lasting adaptive immune responses. In particular, conventional type 1 DCs (cDC1s) excel on antigen cross-presentation, a critical step for inducing CD8+ T cell cytotoxic responses. The rarity of naturally occurring cDC1s and lack of in vitro methodologies for the generation of pure cDC1 populations strongly hinders the study of cDC1 lineage specification and function. Here, we describe a protocol for the generation of induced DCs (iDCs) by lentiviral-mediated expression of the transcription factors PU.1, IRF8 and ...
[摘要] [摘要] 转录因子组合的异位表达最近被证明可以将分化的体细胞重编程为树突状细胞（DC）谱系，而不会回复到多能状态。DC具有诱导有效和持久的适应性免疫应答的能力。在特定的常规类型1的DC（cDC1s）练成抗原交叉呈递，用于诱导CD8的关键步骤+ T细胞的细胞毒性应答。天然存在的cDC1的稀有性和缺乏用于生成纯cDC1群体的体外方法论，严重阻碍了对cDC1谱系规格和功能的研究。在这里，我们描述了用于生成感应DC（iDC）的协议 慢病毒介导的转录因子PU.1，IRF8和BATF3在小鼠胚胎成纤维细胞中的表达。iDC 在9天内获得DC形态，cDC1表型和转录特征。使用此协议生成的iDC 具有对炎症刺激，吞噬死细胞，将抗原加工并交叉呈递给CD8 + T细胞的功能。DC重新编程提供了一个简单易处理的系统，可以生成大量的cDC1类细胞用于高内涵筛选，从而开辟了新途径，可以更好地了解cDC1的规格和功能。将来，在成纤维细胞中忠实诱导cDC1命运可能会导致产生患者特定的疫苗接种DC。
[背景技术树突状细胞（DC）是专业的抗原呈递细胞，专门用于识别，加工和呈递T细胞抗原，在诱导适应性免疫应答和免疫记忆中起关键作用（Me rad 等，2013）。DC可以分为4个主要子集：浆细胞样DC（pDC ），大量1型干扰素的产生者，循环单核细胞衍生的单核细胞衍生DC 和常规DC（cDC ...
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| Isolation and Primary Culture of Adult Human Adipose-derived Stromal/Stem Cells
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Author:
Date:
2017-03-05
[Abstract] Adipose-derived stromal/stem cells (ASCs) are multipotent cells that can be isolated from adipose tissue. Studies have shown that cells have the capacity to self-renew and differentiate into adipocyte, chondrocyte, myocyte, and osteoblast lineages. Thus, significant interest regarding their use for regenerative purposes to restore aging or damaged tissue has grown in recent decades. These cells have also been shown to immunomodulate the microenvironment and secrete abundant growth factors, which minimize inflammation and aid repair and regeneration. ASCs can be readily isolated from the stromal vascular fraction (SVF) of lipoaspirates. Given their ease of accessibility, bountiful source, and potential application in regenerative medicine and tissue engineering, there is growing interest ...
[摘要] 脂肪来源的基质/干细胞（ASCs）是可以从脂肪组织分离的多能细胞。研究表明，细胞具有自我更新和分化成脂肪细胞，软骨细胞，肌细胞和成骨细胞谱系的能力。因此，近几十年来，对再生用途恢复老化或损伤组织的兴趣越来越大。这些细胞也被证明可以免疫微环境并分泌丰富的生长因子，从而使炎症最小化并辅助修复和再生。 ASCs可以容易地从脂质体的基质血管分数（SVF）中分离出来。鉴于其易于获取，丰富的来源和在再生医学和组织工程中的潜在应用，对于ASC的表征和利用越来越感兴趣。该方案描述了从成人人类脂肪组织中分离的ASC以及用于培养维持的方法，包括扩增和低温保存。
背景 脂肪来源的基质/干细胞（ASCs）表现出干细胞领域的巨大潜力。根据造血干细胞移植的治疗奇迹，ASCs代表干细胞的未来，因为它们更容易获得源 - 脂肪组织。 ASCs自我更新和分化成各种组织谱系（包括脂肪细胞，软骨细胞，肌细胞和成骨细胞谱系）的能力允许恢复损伤的组织。另外，推测ASCs有可能在体外复制组织。器官将允许更容易获得新颖药物的评估，从而显着降低药物生产成本。然而，隔离，维护和冷冻保存过程中的不一致，禁止集体分析世界各地不同实验室的结果。用于分离和培养ASC的标准方案对于确保一致的数据分析是必要的。
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| Efficient Isolation of Influenza Specific CTLs
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Author:
Date:
2016-06-20
[Abstract] Human antigen-specific CD8+ T-cell clones are valuable tools for dissecting CD8+ T-cell responses against antigens derived from infectious agents, cancer and self antigens. Here we describe a protocol for isolating human antigen-specific CD8+ T cells. This protocol uses surface capture of IFNγ to identify antigen responsive cells that are then cloned by single-cell sorting. Here we use CD8+ T-cell responses to influenza matrix protein (MP) as an example, but this approach can be applied to any antigen specificity.
[摘要] 人抗原特异性CD8 + T细胞克隆是用于解剖来自感染剂，癌症和自身抗原的抗原的CD8 + T细胞应答的有价值的工具。 在这里我们描述了用于分离人抗原特异性CD8 + T细胞的方案。 该协议使用IFNγ的表面捕获来鉴定抗原反应性细胞，然后通过单细胞分选克隆。 这里我们使用CD8 + T细胞对流感基质蛋白（MP）的反应作为例子，但这种方法可以应用于任何抗原特异性。
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