Factors for iPSC Generation

Generation of induced pluripotent stem cells (iPSCs) via the ectopic expression of reprogramming factors is a simple, advanced, yet often inefficient, slow, stochastic technology due to the overexpression of multiple genes. Scientists of Creative Biolabs have used almost all available approaches for the delivery of reprogramming factors. Creative Biolabs has developed streamed-line protocols for efficient iPSC generation with viral vectors, DNA (plasmid), RNA and recombinant proteins. Each of these services will be provided with a comprehensive report suitable for publications.

Introduction to Generation of iPSCs

In 2006, Yamanaka's team first produced iPSCs from mouse embryonic fibroblasts (MEFs) by introducing four factors Oct4, Sox2, KLF4, and c-myc. iPSCs have similar pluripotency and self-renewal capacity to embryonic stem cells (ESCs). When establishing a method for efficiently generating and differentiating iPSCs, regenerative medicine with transplantation of iPSCs-derived cells, tissues, or organs will approach reality. One advantage of using autografts from patient-derived iPSCs is that the risk of immune rejection is very low. iPSCs can be generated from various types of cells with transduction of defined transcription factors.

Generation of iPS Cells from Hematopoietic Cells

Due to the high reprogramming efficiency, hematopoietic cells can be obtained in a minimally invasive way and will be a good donor source for establishing iPSCs. Various methods for establishing iPSCs from hematopoietic cells have been reported.

• Generation of iPS cells from B lymphocyte
• Generation of iPS cells from hematopoietic stem/progenitor cells obtained from peripheral blood or umbilical cord blood
• Generation of iPS cells from peripheral T lymphocytes and myeloid cells

Fig.1 A schematic illustration of iPS cell research and the applications. (Mochiduki, 2012)

Generation of iPS Cells from Hematological Malignancy

iPSCs can be generated not only from normal cells but also from several types of tumor cells. Disease-specific iPSCs, especially from hematological malignancies, are useful because primary samples of hematological malignancies are usually difficult to be expanded. After the establishment of iPSCs with malignant cell genome abnormalities, iPSCs with genetic abnormalities can be distinguished and continuously obtained. They will be used in studies that require large numbers of living cells, proteomes, epigenome and transcriptome profiling, leukemia stem cell analysis or drug screening assays. iPSCs from hematological malignancies have been established from myeloproliferative neoplasms including chronic myelogenous leukemia (CML) and JAK2-V617F mutation-positive polycythemia vera (PV). iPSC technology has great potential to promote oncology research based on patient samples.

Services at Creative Biolabs

Generally steps in a reprogramming experiment include tissue selection, proceeding through iPSC generation, possible transgene excision to produce iPSC cells that are ready for use in a translational setting. Creative Biolabs is dedicated to providing several viable and cost-effective methods for pluripotent stem cells (iPSC) reprogramming. We employ advanced iPSC reprogramming factor delivery strategy by virus, iPSC reprogramming factor delivery by episomal vectors, as well as other iPSC reprogramming methods (mRNA, protein) to help you obtaining the desired iPSC.

iPSC Factor Reprogramming

Conventional iPSC factor reprogramming is based on integrating vectors with the problems of cell death, residual expression, and re-activation of reprogramming factors, immunogenicity, uncontrolled silencing of transgenes, and insertional mutagenesis. Methods of factor reprogramming fall into two broad categories: chemical and transgene reprogramming. It has been reported that many small molecules promote reprogramming when used with the classical reprogramming factors.

Transgene Reprogramming

There are genomic integration methods and integration-free methods that can be used for the induction of reprogramming factors. The genomic integration methods can induce iPS cells easily and efficiently. Therefore, they are useful for basic research. The induction methods are also divided into four categories based on the types of vector being used, i.e. a virus, DNA (plasmid), RNA or protein.

• Factor Delivery by Viral Vectors
• Retrovirus vector. The reprogramming factors transfected using a retrovirus system integrate into the host genome and show strong and stable expression.
• Lentivirus vector. The reprogramming factors induced by lentivirus systems integrate into the host genome and strongly and stably express the transgenes.
• Adenovirus vector. The reprogramming factors by DNA adenovirus is used to avoid integration of transgenes into the reprogrammed genomes.
• Sendai virus vector. The Sendai virus is a minus strand RNA virus. The reprogramming factors induced by Sendai virus systems are replicated in the cytoplasm and stably expressed in infected somatic cells.
• Factor Delivery by DNA (plasmid) Vectors
• piggyBac transposon vector. The piggyBac transposon vector system can achieve a non-viral integration of reprogramming factors into the host genome and leads to the stable expression of reprogramming factors.
• Episomal plasmid vector. Epstein Barr virus (EBV)-based self-replicating episomal vectors need only one transfection for successful reprogramming due to their ability to replicate and partition in mammalian cells.
• Factor Delivery by RNA Vectors
• Direct delivery of synthetic mRNAs. Direct delivery of synthetic mRNAs encoding reprogramming factors has been reported to generate iPS cells with high efficiency.
• Mature double-stranded miRNAs. This reprogramming method is free from viruses and genomic integration. In addition, this method is technically easier than the method using synthetic mRNAs.
• Factor Delivery by Protein

Chemical Reprogramming

Small molecules that regulate specific targets involved in signaling, metabolic, transcriptional, and epigenetic mechanisms have become valuable tools for detecting basic stem cell biology and manipulating stem cell fate, state, or function in vitro and in vivo. Rational design and/or screening of useful compounds have been used for enhancing cell-based therapy and/or promoting the development of therapeutic drugs targeting endogenous stem and progenitor cells to treat degenerative diseases, cancer, and injuries.

Small molecules have many unique advantages over genetic manipulations:

• More convenient to use
• Rapid and reversible
• Effects confined to different cell or tissue compartments
• Effects can be fine-tuned by varying their concentrations and combination

Fig.2 Chemical approaches to stem cell biology and therapeutics. (Li, 2013)

With 100% success rate, Creative Biolabs is guaranteed to provide highly efficient generation services of different cells into iPSC with our advanced technology. contact us today to discuss your iPSC generation project with a technical specialist.

Features of Our Services

• Customized Reprogramming - We offer highly customized reprogramming services depending on your preferred donor cell type and reprogramming method. We work with a diverse range of cell types and samples, including blood, tissue, or specified cell samples.
• High Quality Standards – Our stem cell reprogramming methods are in accordance with scientific standards. We adhere to strict quality control measures ensuring the generation of highly pure iPSCs free from genomic alterations.
• Comprehensive Packages - Our full-service package includes cellular characterization and functional testing of generated iPSCs. We also offer downstream developmental lineage differentiation and screening services for in-depth analysis of your iPSC lines.
• Expertise & Experience - Our team has a robust background in stem cell research and biotechnology, and extensive experience in generating, characterizing, and manipulating iPSCs. We provide excellent customer service, offering consultation on the best iPSC generation strategy that fits the specific objectives and requirements of your project.

FAQs

• Q: How long does the iPSC Generation process typically take?
  A: The duration of the iPSC Generation process varies depending on the starting material and the method used for reprogramming. However, the entire process from cell collection to iPSC colony picking typically takes around 4 to 6 weeks. The entire process, including quality control tests, can take several months.
• Q: What tests are done to confirm successful reprogramming of the cells?
  A: To confirm successful reprogramming, multiple tests are carried out. These include gene expression analysis for pluripotency markers, assessment of proper cell morphology, absence of reprogramming factors, and the ability to differentiate into all three germ layers, among other tests.
• Q: What are the commonly used reprogramming factors for iPSC generation?
  A: The most commonly used reprogramming factors are Oct4, Sox2, KLF4, and c-Myc, known as the Yamanaka factors. However, different combinations of factors can be used for different cell types, and additional factors can be added to improve efficiency or quality.
• Q: What kind of support does your service provide during the iPSC generation process?
  A: Our service provides full support during the iPSC generation process. This includes consultation on design, regular updates throughout the process, troubleshooting if issues arise, and detailed reports on the results. We also offer follow-up services, including iPSC expansion and differentiation.

Scientific Resources

iPSC Reprogramming

Learn More

References

1. Mochiduki, Y.; Okita, K. Methods for iPS cell generation for basic research and clinical applications. Biotechnol J. 2012, 7(6): 789-97.
2. Li, W.; et al. Chemical approaches to stem cell biology and therapeutics. Cell Stem Cell. 2013, 13(3): 270-83.

For Research Use Only. Not For Clinical Use.