Since the discovery of reprogramming factors in 2006 and the boom of CRISPR gene editing strategies, induced pluripotent stem cells (iPSC) have emerged as new cellular models. The development of 3D cell culture technologies has also contributed to the generation of induced Pluripotent Stem Cell (iPSC) derived cells, with unique applications from patient-specific drug responses testing, to regenerative medicine. I would like to introduce in this post a selection of reagents in this domain, a combination of both routine and innovative quality reagents, that I consider as bringing something extra to your stem cell research projects.
We can’t talk about iPSC without discussing cell line engineering strategies. My aim here as a non-specialist is to demystify the word CRISPR for the non-experts by presenting a simple strategy.
iPS Cell Line Engineering for Stem Cell Research
Using a CAS9 + gene specific sgRNA expressing vector (All-in-One) in association with an optimized transfection reagent like the Polyjet DNA will already give you very high transfection efficiency, a good start for a successful CRISPR project :=). The association with a donor clone for a homologous DNA repair knock in or knock out, is a simple method that overcomes the complexity of monoclonal selection by using antibiotic selection instead. The right hand side image illustrates this strategy. For more details, take a look at the guide to the best CRISPR-Cas9 strategies written by my colleague Dimitri.
If we go one step back in the iPSC generation process, we will face the need for reprogramming tools. Molecules tend to be quite popular to avoid the use of animal derived products, especially when the end application is therapeutic purposes. I am actually quite fond of this interactive webpage as a pathway based user guide for molecule selection. Molecules are listed in the blue boxes, sorted by pathway, with a link towards the product page for easy selection and purchasing.
Weekend-free iPSC Culture
Once reprogrammed, the challenge is to maintain pluripotent Embryonic Stem cells (ES)/iPS stem cells into an undifferentiated state. Although feeder free stem cell culture is quite trendy, feeder cells are still very much in use to provide essential nutrients that support healthy cell culture while preventing cell differentiation. I would advise on our own MitoC treated MEF cells produced in our laboratories based in Europe for optimal viability and yield.
Culturing iPSCs is more tricky compared with regular mammalian cell culture. Having a defined media providing consistent results over time is essential to be successful, especially when performing gene editing on these cells. Ajinomoto, experts in amino-acids, have developed such medium, the StemFit-Basic02, which also allows a week-end free culture, always appreciated :=). This medium is specially designed to support efficient single cell expansion, a crucial step in the cloning stage of CRISPR strategies.
iPSC Biomarker Monitoring
Talking about challenges to maintain the undifferentiated state, biomarker monitoring is a standard means for evaluating the changes. The availability of reliable antibodies is essential. I have selected for you two suppliers of well characterised antibodies, Rockland and Reprocell, for stem markers like Oct4; Sox2; Nanog; SSE4…).
Distinguish Pluripotent Stem Cells from Differentiated Cells
While the above applications are antibody based, the distinction between human iPS cells and/or human ES cells from differentiated cells can also be monitored via live cell imaging. The Kp-1 chemical probe allows such differentiation in live cell imaging and flow cytometry application. It’s based on a simple principle: KP-1 has cell permeability and localizes in the mitochondria in the human pluripotent stem cells regardless of mitochondrial membrane potential. After the differentiation of the cells, KP-1 is eliminated from the cells by the ABC transporters which do not work in the pluripotent stem cells, and the cells become non-fluorescent.
Multiplex biomarker identification can also be done with profiling tools like antibody arrays to screen a larger number of proteins in a single experiment. RayBio® G-Series Human Stem Cell Array 1 Kit allows semi-quantitative measurement of 15 human stem cell biomarkers (RayBiotech). It is suitable for all liquid sample types (10-100 µl per array), with a large dynamic range and sensitivity. Just contact me if you wish to get more details on this technology. We can even do it in our own lab for you.
As I do not want this post to become a long listing of reagents (as innovative and exciting as they are!), I will stop here for the day, although I cannot conclude without talking about 3D cell culture which is extremely popular in Stem cell research, notably to promote full differentiation in iPSC derived cells. Among many popular applications and devices, I have selected the Alvetex technology, a highly porous polystyrene scaffold designed for 3D cell culture, which won the Technology Innovator Award in September 2016. Its high porosity and unique 200μm thickness reproduces the closest in vivo like environment for cells to grow in. What better than a video to convince you of its uniqueness versus other technologies like hanging drop, gels or scaffolds?!
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