Generation and Transfection of iPSCs

What are induced pluripotent stem cells?

Pluripotent stem cells (PSCs) such as embryonic stem cells (ESC) or induced pluripotent stem cells (iPSC) can give rise to any fetal or adult cell type and have the ability to self-renew indefinitely.

In contrast to human embryonic stem cells (ESCs) which develop during embryogenesis from the inner cell mass of the human blastocyst, induced pluripotent stem cells (iPSCs) are pluripotent cells artificially derived from an already differentiated cell type by a process called reprogramming. Reprogramming is achieved by introducing stem cell-specific transcription factors into a pre-mature or mature somatic cell type.
The resulting iPSCs phenotypically and functionally resemble embryonic stem cells (ESCs), e.g. they can indefinitely self-renew in culture and have the potential to differentiate into cell types from all three germ layers, i.e. any cell type.

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The power of iPSCs

Due to their ESC-like capabilities, iPSCs are a powerful tool for many developmental or disease-related applications. As they can be “made” from a person’s own skin or blood, reprogramming can provide a source of patient-specific, genetically identical cells that would not be rejected by the immune system.

Potential applications comprise:

Basic research: iPSCs can help scientists to further identify mechanisms of development or disease pathogenesis.
Human disease models: Cells derived from human iPSCs could serve as novel human disease models for performing in vitro drug screening and toxicity studies. Patient-specific iPSC-derived cells may help to evaluate drug effects on individual genomic differences.
Regenerative medicine: Moreover, iPSCs also hold great potential for being a new tool in regenerative medicine, e.g. for the treatment of degenerative diseases, including diabetes, Parkinson’s and a number of cardiovascular diseases. They could serve as a renewable source of material for producing therapeutic cells for transplantation. Because they can be derived from any individual, iPSCs may be useful for generating autologous therapeutic cells for transplantation for replacing damaged or diseased cells.

The raise of iPSCs makes the usage of pluripotent stem cells independent from the availability of embryonic cells and thus independent of ethic controversy and patient-/tissue-matching issues.

iPSC Generation

For human cells, iPSC generation by cellular reprogramming was first described in 2007 by two independent research groups:

Yu J et al. (2007) Induced pluripotent stem cell lines derived from human somatic cells. Science 318 (5858): 1917–1920
Takahashi K et al. (2007) Induction of pluripotent stem cells from adult human fibroblasts by defined factors. Cell 131 (5): 861–872

In 2012, Sir John Gurdon and Shinya Yamanaka were honored with the Nobel Prize for “the discovery that mature cells can be reprogrammed to become pluripotent”.

Induced PSCs can be generated in the lab via introducing stem cell specific transcription factors into primary somatic cells by either viral transduction or non-viral transfection. Typical reprogramming factors used in different combinations are Oct4, Sox2, Klf4, c-Myc, Nanog and Lin28. Expression of these factors in various adult cell types including fibroblasts, peripheral blood mononuclear cells (PBMCs), fibroblasts or CD34+ hematopoietic progenitor was shown to be able to revert those cells back into a pluripotent stem cell state.

Nucleofector^® Technology for iPSC generation

For generating iPSCs from a somatic cell, one needs to introduce the relevant transcription factors into the cell to be reprogrammed. Initially, these reprogramming factors were delivered into the somatic cell by retroviral transduction, thereby diminishing their applicability to the clinic because of the permanent integration into the genome. Potential inactivation of tumor suppressor genes by random integration and long-term presence of reprogramming factors may give rise to tumor growth.

Thus, researchers have explored alternatives by using non-integrating RNA viruses or non-viral delivery of episomal vectors or even mRNA. Lonza’s Nucleofector® Technology has been demonstrated to be a convenient, efficient, and cost-effective non-viral alternative for iPSC generation and is currently being used by leading scientists around the world.

Benefits of the Nucleofector® Technology for iPSC generation

Simple, single-step procedure to introduce episomal vectors or RNA
Integration-free, footprint-free reprogramming
Successfully tested for generation of iPSCs from various cell types
Proven for efficient iPSC transfection, e.g. for genome editing using ZFN, TALEN, and CRISPR systems

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Guidelines for iPSC generation from various starting cell types

Nucleofector® Technology has been efficiently used in reprogramming the most common starting cell types, i.e. human peripheral blood mononuclear cells (PBMCs), dermal fibroblasts or CD34+ hematopoietic progenitors, as well as more rare starting cell types like adipose-derived stem cells or keratinocytes. Download our Guidelines for iPSC Generation from Various Starting Cell Types that provides information on which Nucleofector® Kits to use for the transfection of reprogramming vectors into the most commonly used cell types. In addition, for setting up your protocol, our primary cell types with corresponding optimized culture media may serve as a positive control in accordance with Lonza’s Terms and Conditions.

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Transfection of iPSCs

Reference Guide

For studying differentiation pathways in human induced pluripotent stem cells (iPSCs) or modifying such pathways, it is important to find a transfection method that enables efficient transfer of the substrate of interest while maintaining pluripotency. Lonza’s non-viral Nucleofector® Technology has proven to be well suited for both iPSC generation (see above) as well as iPSC transfection. Refer to our Technical Reference Guide Transfection of Human Induced Pluripotent Stem Cells using Nucleofector® Technology to start your iPSC transfection experiments.

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Convenient and efficient non-viral iPSC generation

Webinar

This webinar introduces the Nucleofector® Technology as an efficient, convenient and cost-effective, non-viral alternative for iPSC generation.

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Selected publications

Tanudjojo et al. (2021) Phenotypic manifestation of α-synuclein strains derived from Parkinson's disease and multiple system atrophy in human dopaminergic neurons Nat Commun, 12(1):3817

Byrne et al., (2015) Multi-kilobase homozygous targeted gene replacement in human induced pluripotent stem cells Nucleic Acids Res 43(3): e21

Byrne and Church, (2015) CRISPR-mediated Gene Targeting of Human Induced Pluripotent Stem Cells Curr Prot Stem Cell Biol, 35(Suppl 35):5A.8.1-5A.8.22

Chavez et al., (2015) Highly efficient Cas9-mediated transcriptional programming Nat Methods, 12(4):326-8

Grobarczyk et al., (2015) Generation of Isogenic Human iPS Cell Line Precisely Corrected by Genome Editing Using the CRISPR/Cas9 System Stem Cell Rev Rep, 11(5):774-87

Yang et al., (2013) Optimization of scarless human stem cell genome editing Nucleic Acids Res, 41(19):9049-61

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Important note: The user bears the sole responsibility for determining the existence of any third party rights, as well as obtaining any necessary licenses, including using CRISPR/Cas9.