In this interview, Yvonne Mica, Business Development Consultant – Stem Cells, Thermo Fisher Scientific (MA, USA), discusses the challenges in developing cell culture systems for the rapidly evolving field of iPSCs.
In this interview, Yvonne Mica, Business Development Consultant – Stem Cells, Thermo Fisher Scientific (MA, USA), explains how induced pluripotent stem cell (iPSC) research has evolved and the challenges of historical cell culture systems keeping pace with these advances.
Yvonne Mica completed her doctoral studies in the laboratory of Lorenz Studer at Memorial Sloan Kettering Cancer Center (NY, USA) where she established a novel system for modeling pigmentation disorders using patient iPSC-derived melanocytes. She joined Life Technologies (now a part of Thermo Fisher Scientific) as a Technical Sales Specialist in 2013 to provide guidance and support to scientists in academia and pharma interested in stem cell research. Since 2015, Yvonne has been active as a global Business Development Consultant supporting the stem cell portfolio and applications. She manages strategic alliances with key opinion leaders in the field and emerging markets and works closely with global cross-functional teams including research and development (R&D), product management, marketing and business units to provide market feedback and identify opportunities for the development of products and technologies that will accelerate stem cell research from the bench to the clinic.
How has stem cell research evolved since you first started working in the lab?
I started my graduate studies back in 2007 in Lorenz Studer’s lab at Memorial Sloan Kettering Cancer Center (NY, USA). This was right after the publication of Shinya Yamanaka’s seminal paper first describing the derivation of mouse iPSCs and the same year that the generation of human iPSCs was first reported. Needless to say, reprogramming technology was entirely new and still technically very challenging. In many ways, deriving new iPSC lines felt like more of an art than a science and it was a process really only being done by a few labs with extensive prior expertise working with PSCs. In contrast, today reprogramming as a workflow has been fairly democratized. Reprogramming technologies are highly efficient and user friendly so that many more labs have incorporated iPSCs into their projects.
Beyond reprogramming itself, the things that scientists actually do with stem cells has evolved significantly. Differentiation protocols to generate a lab’s favorite cell type were initially lengthy and costly endeavors, often relying on co-cultures or embryoid body formation. Recent protocols tend to be much more defined, robust, and efficient in terms of yield.
Gene editing now also often goes hand in hand with PSC research. The ability to introduce and correct genetic changes in PSC lines and produce isogenic controls has truly changed how groups are developing PSC disease models. This has led to an improved understanding of disease mechanism across a spectrum of disorders and is opening new doors for drug screening paradigms.
A great deal of this progress has been driven by a desire to use PSC systems not just for disease modeling, taking advantage of patient specific iPSCs, but also with an eye towards clinical research and potential cell replacement therapies.
Overall, the last 10 years has seen an explosion in how PSCs are being adopted to address novel experimental questions, a fantastic evolution in the precision with which we can differentiate and edit PSCs, and a need for more controlled and defined systems for maintaining and manipulating PSCs.
In terms of next generation applications (gene editing and single cell passaging, for example) that require significant and harsh manipulations, what challenges are there with traditional cell culture systems?
Many of the most common culture systems for maintaining human PSCs preceded the paradigm shifts we’ve seen in this research area over the last 10 years. Feeder-dependent culture using Gibco KnockOut Serum Replacement is still routinely used in many labs because it is a robust system that works across PSC lines. However, the presence of xeno- and poorly defined- components can lead to variability and makes this system less well suited for any kind of translational research. Similarly, some of the historical feeder-free culture systems like StemCell Technologies’ mTeSR-1 still contain BSA and are optimized for use with Matrigel, an animal derived substrate.
The field clearly recognized the need for a more defined culture system which is why there was so much excitement when Jamie Thomson’s group (University of Wisconsin; WI, USA) reported on the development of the fully defined Essential 8 formulation that works with the defined, recombinant substrate vitronectin. This defined culture system was a huge step forward in terms of increasing reproducibility and reducing cost. With the movement of PSC research towards clinical applications, these features were well received. However, the field has since come to realize that leaner media like Essential 8 may in turn not be best suited for some of the more challenging applications that are being routinely implemented, such as single cell passaging and gene editing. In short, the way in which we use stem cells, the applications and assays that scientists are pursuing, has progressed rapidly, but our PSC media systems have not kept pace.
In your role as a strategic alliance manager with Thermo Fisher Scientific, you meet with stem cell researchers from around the world. What are some of the challenges you encounter most often?
Meeting with stem cell researchers from around the world is certainly one of my favorite aspects of my role. When you meet with so many different groups from such a diverse background in terms of expertise, research interests, and goals you quickly realize that many of the challenges that labs are struggling with are nearly universal. All labs absolutely require a high degree of reproducibility in their experiments. This applies both to their cells and assays as well as the reagents and technologies that they are using. Routine maintenance and complex workflows need to work routinely across multiple cell lines and for multiple users within and between labs.
With more de novo labs adopting PSCs into their projects, ease of use is also often a challenge. Labs want technologies and culture systems that don’t require a high learning curve or level of expertise.
We’ve already touched on the need for robust gene editing, using TALEN and CRISPR technologies. While there have been huge strides in the actual technology for editing, there has still been an acute need for media that would support the routine use of these editing technologies in PSCs. This has been a real pain point for many labs.
Finally, I would say that with the field progressing so rapidly and new PSC protocols, reagents, and technologies being developed constantly there has also been a sense that these developments need to fit into a lab’s existing workflow. Most groups already have established their own best practices for passaging and differentiation etc, and new technologies need to fit into these workflows seamlessly and allow for an easy transition. No one wants to have to reevaluate and reoptimize all their SOPs every time a new technology is adopted in the lab.
How did Thermo Fisher Scientific approach these challenges with the development of the new Gibco StemFlex Medium?
Our R&D had a clear goal to develop a medium that would be lean enough to minimize variability in routine expansion protocols and limit components that interfere with some applications like reprogramming while also being rich enough to enable superior performance in challenging applications like gene editing and single cell passaging. We also wanted a culture system that would allow for flexibility and not rely on specific passaging methods or special substrates. A lot of emphasis was placed on truly refining the optimal concentrations of each component, through multiple iterations of the prototype formulations. This resulted in the development of our new StemFlex medium — a feeder-free PSC medium that delivers enhanced flexibility and superior performance in today’s stem cell research.
What kind of validation was used to develop StemFlex?
When developing a new media, such as StemFlex, we require absolute confidence that the system will be robust and work not just in the hands of our R&D scientists but across all users and with the many different PSC lines being used in those labs. As a result, we place a lot of emphasis and effort on validating new reagents vigorously.
Once we settled on our top candidate formulations for StemFlex, we reached out in the final step of our development process to groups at Harvard Stem Cell Institute (MA, USA) and Centre for Commercialization of Regenerative Medicine (CCRM; ON, Canada) for extensive evaluation and feedback. This feedback allowed us to identify the final formulation that would truly enable the most robust performance across workflows. Prior to launch we also worked closely with several groups at Harvard, CCRM, and the University of Melbourne (Australia) to test the final StemFlex reagent in their own hands as well.
As a result of these close collaborations, even though StemFlex was only launched on January 15th, we can already say that StemFlex has been tested with not just several dozen PSC lines, but also been evaluated by multiple highly experienced PSC groups.
How is StemFlex medium different than other culture systems available?
The StemFlex formulation has been developed to support not just routine culture of PSCs but to truly excel in challenging applications such as gene editing and single cell passaging which require harsh manipulation of cells. The formulation contains an optimized level of BSA and other components that makes it more robust than Gibco Essential 8 media but leaner than mTeSR. Unlike other culture systems, it also provides unprecedented flexibility in compatibility with different passaging reagents and substrates and can even be used for single cell passaging.
What has been resonating about StemFlex with researchers you speak to?
The data demonstrating the performance of StemFlex medium in gene editing and single cell passaging applications really speaks for itself and has been generating a lot of excitement among researchers who have been struggling with these bottlenecks. For scientists doing CRISPR gene editing in PSCs, the recovery after singularization and electroporation with the Cas9 protein/gRNA complex as well as the subsequent clonal expansion of edited lines had presented two significant pain points. StemFlex shows exceptional recovery at both of these steps and doesn’t require the use of specialized substrates or the addition of a rock inhibitor.
Researchers are also very receptive to the weekend free feed option. The principal investigators I’ve spoken to tend to be more excited about the cost savings associated with the reduced media consumption while the grad students and post docs are of course more motivated by the idea of getting their weekends back!
What are some of the common questions researchers have before evaluating StemFlex medium?
Researchers want to know that their cells will still be pluripotent and behave as expected after long term culture in StemFlex medium. I’m always happy to show them the long term stability and differentiation data that our excellent R&D team has generated.
I also get questions about how easy it will be to transition from current culture systems to StemFlex medium. The good news is that we have data showing that the transition from feeder-dependent culture as well as historical feeder-free systems like mTeSR is quite painless and can be successful after just two passages.
Finally, we get a lot of inquiries as to whether aliquots of StemFlex medium can be frozen. The answer is yes, aliquots of both the supplement and the fully reconstituted, complete StemFlex Medium can be made and frozen for future use.
- Yvonne Mica is employed by Thermo Fisher Scientific