Custom iPS Cell Reprogramming by mRNA

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Custom iPS Cell Reprogramming by mRNA

Non-integrative mRNA reprogramming has proven to be the most efficient and clinically-relevant method for human induced pluripotent stem cells (iPSCs) reprogramming. Armed with advanced platform and expertise, Creative Biolabs provides the most rapid integration-free method for iPSC reprogramming. Our pipeline is built to meet the customer’s project and assay-specific requirements for cellular characteristics and functionality. The scalability of our iPSCs ranges from one million to one billion cells and can be delivered on a predictable schedule.

Application of iPSC

The derivation of iPSCs sparked widespread enthusiasm for the development of new models of human disease, enhanced platforms for drug discovery and more widespread use of autologous cell-based therapy. The emergence of reprogramming technology enabled the use of disease-specific iPSCs in established directed-differentiation protocols. Improvements in reprogramming methodologies have increased the efficiency of iPSC derivation and the quality of iPSC lines in use. Noncorrected cells differentiated from patient-derived iPSCs have been used in drug screening. Currently, a handful of candidate drugs identified in iPSC-based systems are under study in human trials.

iPSCs in disease modeling and drug discovery. Fig.1 iPSCs in disease modeling and drug discovery. (Rowe, 2019)
  • iPSC models of host-pathogen interactions
  • It has been shown that cells terminally differentiated from human iPSCs are susceptible to infection with human pathogens, opening a new frontier for probing host-pathogen interactions. Human iPSC-based systems overcome the limitation of species specificity of infectious pathogenicity and inflammatory responses.

  • Drug screen
  • iPSC-based systems have recently provided platforms to screen for novel antimicrobials.

  • Organoids
  • Upon the development of patient-specific iPSCs, related techniques were applied to disease modeling to derive an affected cell lineage.

  • Human-animal chimaeras
  • iPSC-based chimeric disease modeling by xenotransplantation has emerged as a reliable tool to fuel these advances by faithfully modeling human diseases such as glioblastoma, stroke, osteosarcoma, Parkinson disease.

iPSC Reprogramming by mRNA

A ready source of iPSCs is critical to the effective study of differentiation pathways and the investigation of the therapeutic potential of iPSCs. Since the discovery that human iPSCs could be generated by inducing expression of specific reprogramming factors, many different reprogramming technologies have emerged to generate iPSCs. The evolution of reprogramming technologies has culminated in the development of synthetic mRNA-mediated reprogramming. In that way, engineered synthetic self-replicating mRNA is used to mimic cellular RNA to generate human iPSCs. The single RNA strand contains several reprogramming factors and enables extremely efficient reprogramming using a single transfection step without any viral intermediates or host genome integration.

Methods Contents Advantages and Disadvantages
First-generation technologies Retroviral and lentiviral systems
  • Allowed for highly efficient reprogramming events but lacked the necessary control over host genome integrations.
  • Cure-excisable lentiviral systems offered a solution to genome integration but required lengthy sub-cloning procedures and screening to ensure excision of the reprogramming factors.
Second-generation technologies Non-integrating episomal DNA plasmids
  • Transgene-free but lacked the high reprogramming efficiencies of earlier retroviral and lentiviral techniques.
Third-generation technologies Negative sense, non-integrating RNA viruses (Sendai virus)
  • Produce high reprogramming efficiencies and were easy to use, but residual Sendai virus was difficult to clear from cells, resulting in the requirement for multiple rounds of clonal expansion and analysis.
Next-generation technologies Synthetic self-replicating RNA engineered to mimic cellular RNA
  • Without any viral intermediates or host genome integration.
  • Footprint-free
  • Integration-free
  • Efficient and rapid
  • The RNA can easily be selectively eliminated.

What Can We Do for You?

For iPSCs creation, expression of reprogramming factors as mRNAs is available zero-footprint technology. The single RNA strand, containing the OCT-4, KLF-4, SOX-2 and GLIS1, enables extremely efficient reprogramming using a single transfection step without any viral intermediates or host genome integration. Once iPSCs are generated, the RNA can easily be selectively eliminated by removing the interferon-gamma inhibitor, B18R from the cell culture medium.

Schematic workflow of iPSC reprogramming by mRNA. Fig.2 Schematic workflow of iPSC reprogramming by mRNA.

Technical Edge

  • Patient-specific solutions
  • Ability to source tissue and generate iPSCs from a specific patient clinical profile.

  • RNA reprogramming services
  • Integration-free, non-viral, safe and efficient.

  • Differentiation services
  • Readily available lineages offered at scale, coupled with flexible, efficient and iterative custom solutions.

  • Consultative science
  • Industry-leading scientific experts will help to develop a cost-effective and milestone driven project to meet your research needs.

  • Rigorous Quality Control Protocols
  • Protocols include but are not limited to assessing cell count, viability, genomic stability, and expression of lineage markers at the transcriptome and protein levels.

  • Concierge customer services and technical support
  • Start-to-finish assistance and support throughout the entire process are provided by a dedicated technical team.

Our advanced generation technology can be used to transform samples from skin, blood and urine. The resultant iPSCs possess low transgene persistence, increased chromosomal stability and reduced clonal variation. As RNA reprogramming leads to decreased copy number variation, it also reduces the risk of oncogenesis and abnormal karyology. Creative Biolabs is dedicated to providing first-class iPSC reprogramming services for our customers. Our services will contribute greatly to the success of your iPSC programs. Please contact us for more information and a detailed quote.

Reference

  1. Rowe, R. G.; Daley, G. Q. Induced pluripotent stem cells in disease modeling and drug discovery. Nature Reviews Genetics. 2019, 20(7): 377-388.
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