Induced pluripotent stem (iPS) cells, or adult cells that are genetically reprogrammed to a pluripotent state, are powerful tools because scientists can differentiate them to become entirely different cell types. iPS cells are already showing great promise in areas such as drug screening, tissue regeneration, and disease modeling. Here’s a look at how scientists are using today’s iPSC advances with an eye toward applications that may fulfill the promise of these amazing cells.
Advances in the media used to culture, differentiate, and cryopreserve iPS cells is helping to drive many iPS fields forward. Though it sounds simple, media that keeps iPS cells healthy and minimizes stress can make a big difference. One example is Thermo Fisher Scientific’s StemFlex™ Medium, which was developed to overcome common challenges often experienced with current media systems: daily feeding, single-cell cloning and genome editing (tool delivery and clonal isolation of cells with desired edits) are tedious and often difficult with iPS cells.
Though it sounds simple, media that keeps iPS cells healthy and minimizes stress can make a big difference.
Maintenance of pluripotency of iPSCs cultured in StemFlex Medium after electroporation and recovery. Cultures transfected with Cas9–gRNA complexes targeting the HPRT gene were assessed by qualitative immunocytochemistry of OCT4 and TRA-1-60 expression. Image courtesy of Thermo Fisher.
“Stem cells don’t like to be alone, but StemFlex Medium promotes superior survival of single cells,” says Erik Willems, staff scientist in the Cell Biology R&D group at Thermo Fisher Scientific. “Genome editing with iPS cells is also challenging because it’s stressful to the cells, but StemFlex Medium mitigates that stress,” says Willems.
Aside from the biology, StemFlex also mitigates stress for researchers by allowing a flexible cell feeding schedule—researchers can skip two consecutive days of feeding their iPS cell cultures—so that they don’t necessarily have to come in to work on the weekend. StemFlex medium supports full flexibility for common types of matrix and passaging reagents used.
Immunofluorescence analysis of H1 hES cells cultured in NutriStem® hPSC Medium. The cells stain positive for the expression of typical pluripotency markers; SSEA-4 (red), OCT4 (green), and DAPI nuclear staining (blue). Image courtesy of Biological Industries.
With the potential of life-saving applications of iPS cells, Biological Industries offers clinical-grade media that are serum- and xeno-free for iPS cell culture. Their NutriStem hPSC Medium and CryoStem Freezing Medium are compatible with downstream clinical applications, and can be used for growth, expansion, and cryopreservation. These media are made under cGMP guidelines, meeting clinical regulatory standards, says Daniel Haus, application development scientist at Biological Industries.
NutriStem hPSC Medium is especially good for culturing single human iPS cells, and can be customized to allow researchers more control over their cells’ culture environment. CryoStem Freezing Medium gives excellent cell survival from thaw, says Haus: “Human iPS cells are notoriously difficult to freeze and thaw, and this is a small step that can have a big impact on downstream production of iPS cells for therapeutic use.”
Differentiation and disease modeling
The use of iPS cells in clinical applications requires the avoidance of genetic vectors or transgenes that could pose unknown risks in human. Thus Cellular Dynamics International (CDI), a FUJIFILM company, focuses on using episomal reprogramming of iPS cells instead of vectors or transgenes. CDI’s iCell® products include iPS cells that have been differentiated into 13 different cell types, as well as 2 progenitor cell types. For specific research needs, they also offer custom reprogramming and differentiation.
In a collaboration with Roche, CDI used iPS cells to create an in vitro model for diabetic cardiomyopathy. “By applying a high concentration of glucose, cortisol and a peptide (endothelin-1) to our iCell Cardiomyocytes, we induced structural and functional disarrays that are similar to those found in iPS cell-derived cardiomyocytes from diabetic patients,” says Natsuyo Aoyama, an application scientist at CDI. This in vitro model is used as an assay system to screen compounds for studying and treating diabetic cardiomyopathy.
Naive stem cells and other disease applications
Not all stem cells are created equal, and in fact, some are more naive than others. Minerva Biotechnologies developed human “naive state” iPS cells using a growth factor (NME7) they discovered that keeps iPS cells in a naive state, as opposed to the more common “primed” state. While other methods exist for culturing naive iPS cells (such as adding cocktails of biochemical inhibitors to standard FGF media), Cynthia Bamdad, CEO of Minerva Technologies, says that their naive cells differentiate better and have stable karyotypes.
A collaborator, Min-Joon Han, director of the Human iPSC Core at the St. Jude Children’s Research Hospital, verified criteria of the naive state (such as a lack of spontaneous differentiation), and found that Minerva’s naive cells have much higher mitochondrial function than primed cells. He prefers using Minerva’s naive cells over other types because “you only need to add one growth factor, and cells are easy to maintain and have normal karyotyping.” Han also appreciates that the cells are “easy to differentiate, and give more reproducible data.”
“Naive cells are like a clean slate, with no methylation marks, no acetylation marks,” adds Bamdad. “They are free of cell fate decisions.” Bamdad believes this is why their naive cells differentiate more easily in response to differentiating factors—with a higher yield of differentiated cells, and higher expression of the markers for the desired cell type. Another benefit of naive iPS cells is that they have a much higher cloning efficiency than primed state cells, which is especially advantageous when developing iPS-based therapies. Minerva Biotechnologies’ AlphaSTEM® Culture System offers tools for using their naive iPS cells.
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Minerva Biotechnologies is using its iPS technology in two application areas: drug discovery and stem cell therapies such as for the treatment of Parkinson’s disease (PD). They are currently developing neurons from patient-derived iPS cells that can be transplanted into PD patients in future trials.
Scientists at Minerva also found that they can reprogram cancer cells (as if they were stem cells) in a different approach to treating solid tumor cancers. In their CAR T cancer immunotherapy program, patient immune cells are isolated from a blood sample, engineered to kill cancer cells while leaving normal cells alone, then injected back into the patient. “There is so much excitement about cancer immunotherapy because of the recent success that companies have had treating people with blood cancers,” says Bamdad. “Yet blood cancers only make up about 7% of cancers—the other 93% are solid tumor cancers.” Bamdad expects that Minerva’s cancer immunotherapy against solid tumor cancers will be in clinical trials next year.
Even with the start clinical applications, iPS cells remain an imperfect tool. According to Aoyama, a big challenge is genetic heterogeneity in iPS cell lines, which “interferes with understanding how true genetic variants contribute to specific traits.” Aoyama believes that this could be overcome by developing industry standards that include “a systemic generation of iPS cell lines with high-quality cell culture practice.” In the meantime, the exciting journey of iPS cells into clinical research has already seen an auspicious beginning.
Image: iCell(R) Cardiomyocytes are the gold standard iPSC-derived human cell model for interrogating cardiac biology. Image courtesy of Cellular Dynamics International - A FUJIFILM Company.