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诱导性多能干细胞 (iPSC)

多能干细胞 (PSC) 具有分化能力,可作为各种细胞组织开发、药物发现和毒理学、疾病进展和再生医学研究的起点。

就细胞疗法来说,源自“通用”供体的诱导性多能干细胞 (iPSC) 库有望提供成本更低、获取更快捷、控制更严格的同种异体疗法。

Bio-Techne 的使命是提供创新性解决方案,使细胞和基因疗法能够触及更多的患者。

stem cell research
关于多能干细胞和细胞疗法

再生医学和免疫细胞疗法为治疗退行性疾病、致病性基因缺陷、组织损伤和癌症提供了革新性的思路。为了进行这些细胞疗法,会从患者(自体疗法)或免疫相容的供体(同种异体疗法)中回收体细胞,然后使用小分子将它们重编程至未分化状态。所得的 iPSC 能够分化为内胚层、中胚层和外胚层的多种细胞系。

有效的细胞疗法产品需要长期具有活力与功能、能够归巢到正确的靶组织,以及能够规避宿主的免疫排斥。基于活细胞的细胞和基因疗法被归类为前沿治疗药物 (ATMP),由于其固有的变异性,需要进行广泛的质量控制检验。

Listen as Tenielle Ludwig, Director of the WiCell Stem Cell Bank, defines correct use of the terms stemness and pluripotency. These terms are often used interchangeably by researchers and within scientific publications. Dr. Ludwig discusses how consistent use of this nomenclature can positively impact the field of pluripotent stem cell research.

Somatic Cell Isolation and Selection

iPSC Isolation icon

终末分化的体细胞可以轻松地从各种组织(例如,皮肤成纤维细胞或外周 T 细胞)中回收,并用于生成 iPSC。

Somatic Cell Reprogramming to iPSC

iPSC Reprogram icon

iPSC Reprogramming

There are four general groups to categorize an iPSC reprogramming strategy:

  • Integrative Reprogramming techniques require the reprogramming factors to be inserted permanently into the host cell genome. This strategy can be further divided into viral and non-viral methods. The pioneering iPSC experiments by Takahashi & Yamanaka (2006) were conducted by integrating four transgenes using Retroviral Vectors.
  • Viral reprogramming  cells to iPSCs, using lentiviral or retroviral-based transduction methods are the most efficient but have distinct drawbacks for clinical and translational applications
  • Non-viral transposons technology such as TcBuster™ is a next-generation solution that avoids some of the pitfalls of virus transductions.
  • Non-integrative Reprogramming techniques are the preferred methodology for clinical and translational iPSC generation. They require no genomic integration, and therefore have significantly reduced chance of introducing harmful mutations. Small molecules are widely used in non-integrative reprogramming.
Diagram of iPSC Reprogramming Groups

After reprogramming cells to iPSC, confirm their phenotype by the detection of appropriate stemness markers. See Cell Characterization below on this page.  Cell Banking Reagents Including Cryopreservation Media, CEPT Cocktail Kit (Catalog # 7991), and ROCK Inhibitors.

graphic of somatic cell reprogramming to iPSC

采用多色流式细胞术验证干性标志物表达。使用人/小鼠多能干细胞多色流式细胞术试剂盒中的试剂对人类细胞进行染色,同时分析 SSEA-1SSEA-4Oct-3/4SOX2。SSEA-4 和 Oct-3/4 大量表达,但 SSEA-1 未表达,这表明这些细胞处于未分化状态。

iPSC Expansion and Culture

images of stemness marker expression on iPSC
graphs of stemness marker expression on iPSC

ExCellerate™ iPSC 扩增培养基中培养的人 iPSC,在长期培养中维持了干性标志物的表达。这些细胞表达未分化干细胞标志物 Oct-3/4(红色)和 TRA-1-60(红色)以及纤维状肌动蛋白(F-Actin,绿色)和 DAPI(蓝色)(A)。经人/小鼠多能干细胞多色流式细胞术试剂盒评估,iPSC 系表达高水平的 Oct-3/4、SSEA-4SOX2,不表达 SSEA-1 (B-C)。传代超过 45 代后,4 个细胞系中未分化干细胞标志物表达 >97%。图中显示了平均值 ± 标准差。

Gene Engineering for iPSC

iPSC Gene Engineering icon

对细胞进行改造设计,以使其不受到宿主的免疫排斥、改善组织归巢和植入,以及引入新的功能,如嵌合抗原受体 (CAR)。

image of TcBuster-transposed gene in iPSC by DNAscope

使用 DNAscope™ 分析直接可视化 TcBuster 转位 iPSC。检测基于用 DNAscope 探针靶向 TcBuster 载体骨架。野生型 iPSC (A),TcBuster 转导 iPSC 的混合细胞群 (B),从混合细胞群中分离的选定克隆 (C)。

Pluripotent Stem Cell Differentiation

iPSC Differentiation icon

使用严格符合批次间一致性要求的试剂,简化扩展细胞分化过程中的批次桥接。

神经元分化

immunocytochemistry of neuronal differentiation from iPSC

免疫细胞化学分析展现了从使用 StemXVivo 神经祖细胞分化试剂盒得到的 iPSC 分化而来的神经元细胞。用神经元特异性 β-III 微管蛋白 (TUJ1) 表达来指示神经元 (A)、用 Pax6 来指示神经祖细胞 (B),以及用 Oct-3/4 来指示未分化 iPSC。在第 10 天和第 32 天,对在 VitronectinExCellerate iPSC 扩增培养基中生长的神经元分化的图像进行量化。

心肌细胞分化

immunocytochemistry of cardiomyocytes differentiated from iPSC

免疫细胞化学分析展现了从使用 StemXVivo 心肌细胞分化试剂盒分化的 iPSC 的情况。将细胞维持在 ExCellerate iPSC 扩增培养基中并分化为心肌细胞,用人肌钙蛋白 T(心脏)表达 (cTNT1) 来加以指示。

肝细胞分化

immunocytochemistry of hepatocytes differentiated from iPSC

免疫细胞化学分析展现了从使用 StemXVivo 肝细胞分化试剂盒分化的 iPSC 的情况。将细胞维持在 ExCellerate iPSC 扩增培养基中并分化为肝细胞,用白蛋白HHNF-4α 表达来加以指示。

无血清和无动物源细胞培养

 

在进行再生医学和细胞疗法项目的转化研究时,提高细胞培养的一致性。采用这些培养基将

  • 减少培养基成分差异
  • 简化监管指南合规
  • 简化原材料变更的比较测试

Cell Characterization

Verification of pluripotency by mmunocytochemistry/ immunofluorescence. (Left) Confocal immunofluorescence analysis of Mouse Anti-Human Nanog Antibody (1E6C4) (Catalog # NBP1-47427) (green). Actin filaments have been labeled with DY-554 phalloidin (red). Nanog staining was confined to the nucleus. (Right) ADLF1 induced pluripotent stem cell line stained with Mouse Anti-Human TRA-1-60 (TRA-1-60) (Catalog # NB100-730) and Anti-Mouse IgG Secondary Antibody (red) and counterstained with DAPI (blue). TRA-1-60 staining was confined to the cell surface.

iPSC verification image showing cell markers
Single-Cell Western data showing neuron differentiation from iPSC

iPSC 神经元分化的 Single-Cell Western 分析。每个点代表单个细胞。用 GMP SB 431542GMP 重组人 Noggin 处理 iPSC,然后用 GMP 重组人 FGFGMP N-2 MAX 培养基补充剂 (100X) 和抗坏血酸进行终末分化。对细胞中的 Pax6神经元特异性 β-III 微管蛋白 (Tuj) 进行了分析。在 iPSC 中,检测不到 Pax6,且 46% 细胞表达了 Tuj,而 85% 的神经元为 Tuj+ Pax6+。请参阅我们的应用说明了解更多详情。

iPSC Applications

Disease research often relies on the use of animal models or two-dimensional (2D) in vitro culture systems. Though extremely useful, animal models are limited in their ability to recapitulate complex diseases and accurately model human cellular responses to new drugs and therapies. Traditional in vitro culture systems rely on examining cellular responses in a contrived 2D environment, with cells grown either in a monolayer plastic dish or in suspension surrounded by culture media. Advancements in cell culture techniques to include organoid and 3D cultures that more closely recapitulate in vivo tissue microenvironment, exponentially expand the applications for iPSCs.

Illustration highlighting the application of iPSCs

Diverse applications of iPSCs. Somatic cells are harvested from patient and reprogrammed into iPSCs. The resulting patient specific iPSCs can then be used in disease modeling and drug screening to generate disease and patient-specific therapies. Additionally, patient-specific iPSCs can be modified to repair genetic mutations. These repaired iPSCs can then be transplanted into the patient to restore tissue functionality.

Resources

Custom Solutions

We’re committed to providing optimized solutions to optimize your iPSC workflow. Our custom services team will work with you to deliver reagents and immunoassays that fit your process. Importantly, we have experience developing certified animal-free (AF) reagents as custom projects in cases where AF grade is not otherwise available. We’re experts in the requirements for regulatory compliance as well as custom formulation, vialing, and packaging.

Translational Programs for Cell Therapy

When it’s time to advance your cell therapy product to clinical manufacturing, partner with us for reliable, quality, and/or custom services. We will work with you to provide reproducible production of reagents and assays at clinical scale, with complete documentation. We offer GMP reagents as well as 21 CFR Part 11-compliant analytical instruments for automation and high throughput. We can help you streamline the manufacture of your cell therapies.

Background Information

Luminex 是 Luminex Corporation 的注册商标。