Induced Pluripotent Stem Cells (iPSC)

Cyagen possesses advanced somatic cell reprogramming technology, capable of reprogramming somatic cell samples collected from blood to produce non-integrating human iPSCs.

iPSC Reprogramming Service Process

iPSC Reprogramming Service

iPSC Detection and Functional Identification

⮚ Cell Morphology

⮚ Karyotyping

⮚ STR Profiling

⮚ Cell-Specific Markers

⮚ Flow Cytometry Analysis

⮚ In Vitro Tri-lineage Differentiation

⮚ Teratoma Formation Assay

iPSC Reprogramming Service Advantages

• Somatic cells extracted from blood are used to produce high-quality iPSCs with a success rate of up to 99%.
• Utilization of stable episomal vectors in non-integrative reprogramming ensures non-integrated human IPS cells while preserving downstream experiments.
• Standardized operational procedures compatible with various culture systems enable large-scale production of high-purity cells.
• We provide complete customization options to meet researchers' specific needs.

When combined with gene editing techniques, iPSCs can be used to explore the mechanisms of disease, and develop effective new drugs and cell therapies. Cyagen’s iPSC disease model research platform has mature gene editing technologies and stem cell culture systems; it has overcome many difficulties related to iPSC cultivation, genetic modification, and monoclonalization needed for modern translational and regenerative medicine research. Our proprietary process has improved upon all nearly all aspects of custom iPSC modeling: upgraded delivery vector provides up to 50% HDR efficiency, over 50% transfection efficiency, transfection viability more than 80%, and RNP delivery achieving up to 90% KO efficiency.

Our process is empowered by powerful AI algorithms, including the the RNA splicing model tool developed with support from the Rare Disease Data Center (RDDC), which can assist in screening WB-negative clones. Additionally, the Smart-CRISPR™ cell gene editing system enables rapid design of gene knockout and other strategies, yielding an editing efficiency as high as 90%.

iPSC Gene Editing Technology Process

iPSC Gene Editing Services

iPSC Gene Editing Service Case Study: EGFP Knock-in AAVS1 Cell Line Model

Using gene editing technology, the EGFP gene was knocked into the AAVS1 locus in induced pluripotent stem cells (iPSCs). As shown in the diagram, sgRNA and donor were transfected into iPSCs via the RNP method, and the EGFP sequence was inserted into the AAVS1 locus through the HDR pathway.

Figure 1. Design of EGFP Knock-in Strategy

PCR and sequencing identification was used to confirm that a homozygous iPSC cell line with EGFP knocked into the AAVS1 locus was obtained, with 100% EGFP expression observed under fluorescence microscopy. Chromosomal karyotyping analysis conducted using G-banding after cell culture showed a normal chromosome count of 46 with no obvious structural abnormalities. Immunofluorescence staining for the three pluripotent markers NANOG, OCT4, and SOX2 detected positive signals, indicating the pluripotency (stemness) of the knocked-in cells.

Figure 2. Gel Electrophoresis Identification Results of iPSC-EGFP Knock-in

Note: The primer design strategy aimed to amplify partial sequences upstream and downstream of the entire EGFP and its genomic location. The band without inserted EGFP was 762bp, while the band with successfully inserted EGFP was observed at 3194bp. The results indicate that in four monoclonal lines, bands appeared at 3194bp, indicating successful insertion of EGFP and homozygous clones.

Figure 3. Sequencing Results of iPSC-EGFP Knock-in

Figure 4. Fluorescence Image of iPSC-EGFP Knock-in Homozygous Clone (EGFP Expressed in 100% of iPSC Homozygous Clones)

Figure 5. Karyotype Analysis of iPSC-EGFP Knock-in (Chromosomal Karyotyping Analysis conducted using G-banding after cell culture, showing a normal chromosome count of 46 with no obvious structural abnormalities)

Figure 6. iPSC-EGFP Immunofluorescence - Pluripotency Detection: Immunofluorescence staining conducted for the pluripotent markers NANOG, OCT4, and SOX2 detected positive signals, indicating the pluripotency of the knocked-in cells.

Advantages of Our iPSC Gene Editing Services

• Advanced Gene Editing Technology Platform: With over tens of thousands of successful gene editing projects ranging from in vivo animal experiments to in vitro cell experiments, we offer comprehensive services including cellular gene expression regulation, cell function validation, development of mouse disease models, and phenotype analysis.
• Optimized and Upgraded iPSC Gene Editing System: Our newly upgraded delivery vectors achieve HDR efficiency of up to 50%, transfection efficiency of >50%, and transfection viability of >80%.
• Empowered by Powerful AI Algorithms: Supported by the Rare Disease Data Center (RDDC), our RNA splicing model tools assist in screening WB-negative clones. Our Smart-CRISPR™ Cell Gene Editing System enables easy implementation of various strategies such as gene knockout and gene knock-in, with editing efficiency reaching up to 90%.
• Professional Project Management: We provide proposals within 24 hours, offer regular updates on project progress, and have a team of doctoral experts providing technical support and detailed delivery reports. We also offer delivery of different clones (homozygous, heterozygous, and controls) according to project requirements.

Directed differentiation refers to guiding iPSCs through specific experimental conditions and cell culture systems to transform them into specific types of somatic cells, such as neurons, cardiomyocytes, hepatocytes, etc. Cyagen can provide a variety of disease model development and drug screening capabilities, offering high-quality iPSC model services to researchers.

iPSC Directed Differentiation Services

iPSC Directed Differentiation Service Case Study: iPSC Model Differentiation Capacity

• Neural Progenitor Cell (NPC) Differentiation - Neural Disease and Drug Models

  

• Motor Neuron (MN) Differentiation - Neurological Diseases such as ALS and Drug Models

  

• Hematopoietic Stem Cell (CD34+) Differentiation - Can be used for differentiation into hematopoietic lineage cells such as NK cells, T cells, etc.