iPSC generation - Human iPSC colonies expressing pluripotency markers

iPSC Generation 

For iPSC generation, various adult cell types including peripheral blood mononuclear cells (PBMCs), fibroblasts or CD34+ hematopoietic progenitor cells can be reprogrammed to a pluripotent state by introduction of defined transcription factors.  The resulting induced pluripotent stem cells (iPSCs) appear to be phenotypically and functionally equivalent to human embryonic stem cells (hESCs). The ability of hESCs and iPSCs to self-renew in culture and their potential to differentiate into cell types from all three germ layers makes them a powerful research tool.  


Potential iPSC applications:

  • Identifying the mechanisms of human development
  • Generating new models to study human disease pathogenesis
  • Production of specific cell types for in vitro drug screening and toxicity studies  

Moreover, iPSCs also have great potential as a renewable source of material for producing therapeutic cells for transplantation in regenerative medicine. Because they can be derived from any individual, iPSCs may be useful for generating autologous therapeutic cells for transplantation.  


Nucleofector™ Technology for iPSC Generation

For human cells, this cellular reprogramming process was first described in 2007 by two independent research groups (Yu J et al. (2007) Science 318 (5858): 1917–1920; Takahashi K et al. (2007) Cell 131 (5): 861–872). These reprogramming factors were initially transferred into the somatic cells by retroviral transduction, thereby diminishing their applicability to the clinic. 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 include:

  • Simple, single-step procedure to introduce DNA/RNA, e.g. episomal vectors
  • Successfully tested for generation of iPSCs from various cell types
  • Also proven for efficient iPSC transfection, e.g. for genome editing using ZFN, TALEN, and CRISPR systems

 

Guidelines for iPSC Generation from Various Starting Cell Types

The most common starting cell types for iPSC generation are human peripheral blood mononuclear cells (PBMCs), dermal fibroblasts or CD34+ hematopoietic progenitors.  In addition to these three main cell types the Nucleofector™ Technology has also been used to reprogram various other cell types. Lonza does not have a specific reprogramming protocol for any of these cell types, but there are various references listed below which provide some guidelines. The lists below also contain information on Lonza products that may support this application.

a) Human PBMCs

Positive control cells:   Human Peripheral Blood Mononuclear Cells
Cell-specific media:   LGM-3™ Lymphocyte Growth Medium 
Nucleofector™ Kit:   Nucleofector™ Kits for Human T Cells (also suited for PBMCs)
iPSC Culture:   L7™ hPSC Matrix, L7™ hPSC Passaging Solution
References or protocols:  
  • Reprogramming protocol - Available soon!
  • Tran HT et al. (2014) – A Case Study (white paper)

   

b) Human fibroblasts

Positive control cells:    Human Dermal Fibroblasts 
Cell-specific media:    FGM™ Medium 
Nucleofector™ Kit:    Nucleofector™ Kits for Human Fibroblasts  
iPSC Culture:    L7™ hPSC Matrix, L7™ hPSC Passaging Solution
References or protocols:   

 

c) Human CD34+ hematopoietic progenitors

Positive control cells:   

Human Bone Marrow CD34+ Cells

Human Cord Blood CD34+ Cells

Cell-specific media:    HPGM™ Hematopoietic Growth Medium
Nucleofector™ Kit:    Nucleofector™ Kits for Human CD34 
iPSC Culture:    L7™ hPSC Matrix, L7™ hPSC Passaging Solution
References or protocols:   

 

d) Other cell types

Positive control cells:   

Human Keratinocytes

Human Bone Marrow Mononuclear Cells

Human Neural Progenitor Cells 

Human Adipose-derived Stem Cells

Cell-specific media:    See respective cell type or reference 
Nucleofector™ Kit:    See Nucleofector™ Kits for Primary Cells 
iPSC Culture   L7™ hPSC Matrix, L7™ hPSC Passaging Solution
References or protocols:   

 

Lonza provides a range of products to aid iPSC generation, from the most commonly used primary cell types for reprogramming to the Nucleofector™ Technology for efficient, non-viral delivery of reprogramming factors.