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.

Reprogramming Steps

Typically, during human disease modeling and cell-based therapies, the capacity to reprogram somatic cells to induce iPSCs has provided great value. Nowadays, iPSC derivatives have already reached the stage of clinical testing in human patients. Using different techniques to produce induced iPSCs needs that scientists choose the suitable reprogramming method for generating iPSCs, thus the resulting iPSCs enable to be transitioned towards clinical applications effectively. 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.

Fig 1. Schematic workflow of iPSC Reprogramming.

Reprogramming by Virus

• Retro-and lentivirus: Retro- and lentiviral delivery vectors have been used to generate iPSCs almost every lab, because this viral transduction is easily implemented in most biomedical research labs. Using this delivery system, the reprogramming efficiency was 0.01–0.02% in human cells with iPSC colonies occurring between 25 and 30 days post-infection. As retroviruses only infect dividing cells, lentiviral delivery system was more popular so that both dividing and non-dividing cells could be infected in the hopes of improving reprogramming efficiency.
• Lentiviral plus cre-lox mediated transgene excision: To date, the first production of transgene free iPSCs were performed with lentiviral vectors containing loxP sites in the 5′and 3′LTR of the viral vectors. The existence of loxP sites gave a substrate to remove most of the transgene sequences by Cre-mediated recombination.
• Adenovirus: Adenovirus is a non-integrating virus that does not infect replicating cells. The first known research reporting the production of iPSCs with an adenoviral vector was reprogrammed mouse tail tip fibroblasts to iPSCs. However, the reprogramming efficiency was in the range of 0.001–0.0001%, because the expression window of reprogramming factors is too narrow to induce the expression of endogenous factors required to transition somatic cells.

Reprogramming by Episomal Vectors

Episomal Vectors for iPSC reprogramming are vectors developed to offer the preeminent system for performing transgene-free and virus-free induced iPSCs in a feeder-free environment. Until now, a great number of different somatic cell types have been successfully reprogrammed by using episomal iPSC reprogramming vectors. This advanced system possesses several advantages:

• Absolute security during your iPSC project development
• Wide range of application for a great number of somatic cell types
• Optimized for feeder-free reprogramming

Reprogramming by Protein

It has proven that direct expression of reprogramming factors as proteins also enable to generate footprint-free iPSCs. Therefore, this approach may be another good choice for the generation of iPSCs appropriate for studies in translational medicine.

Fig 2. Schematic diagram of the generation of human iPSCs from somatic cells of a healthy or diseased individual.

Reprogramming by mRNA

For iPSCs creation, expression of reprogramming factors as mRNAs is also an available zero-footprint technology. However, the strong immunogenic response elicited in cells by the introduction of synthetic mRNA became a bid problem to reprogramming with mRNA. Scientists have solved this problem through modified the RNA bases by substituting 5-methylcytidine for cytidine and pseudouridine for uridine, as well as added the interferon inhibitor B18R into a cell culture media. These modification extremely reduced cell death which derived from the strong antiviral response that is formed when cells come into contact with mRNA containing standard ribonuclease bases in a common cell culture medium.

Reprogramming by Minicircle Vectors

Minicircle vectors are circularized vectors and their plasmid backbone has been released leaving only the eukaryotic promoter and cDNA(s) that are to be expressed. A minicircle vector composed of Lin28, GFP, Nanog, Sox2, and Oct4, and enables to be used to reprogram human adipose stem cells. Usually, the reprogram steps contain three transfections of the minicircle vector: an initial electroporation, followed by sorting of GFP+ cells, then two lipid-based transfections. With professional scientists devoted themselves in iPSC reprogramming, Creative Biolabs is dedicated to providing the 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.

References

1. Rao, M. S., (2012). “Assessing iPSC reprogramming methods for their suitability in translational medicine.” Journal of cellular biochemistry, 113(10), 3061-3068.
2. Dash, B. C., (2015). “Induced pluripotent stem cell-derived vascular smooth muscle cells: methods and application.” The Biochemical journal, 465(2), 185.