Researchers grow and use pluripotent stem cells (PSCs) for a variety of purposes, from understanding the basic biology of the pluripotent stem cell to using them as stepping stones to generate more differentiated cells, tissues and organs.
Because stem cells can be derived from different populations—male or female or different ethnicities, for example, or from a donor with a genetic abnormality—and be genetically edited, PSCs hold enormous potential for both therapeutics and drug discovery.
This was never more evident than last week in San Francisco as Biocompare attended the International Society for Stem Cell Research (ISSCR) annual meeting. This is the world’s largest professional organization of stem cell scientists, bringing together the global scientific community to share and discuss the latest innovations in stem cell research. According to current ISSCR president Sean J. Morrison, director of the Children’s Medical Center Research Institute at University of Texas Southwestern and investigator at Howard Hughes Medical Institute, “The advances in cellular immunotherapy and using engineered T cells (CAR T-cells) are exciting and groundbreaking research that will lead to better gene and cell therapies for patients.” During a scientific panel discussion at the meeting, Michel Sadelain, director of the Center for Cell Engineering and Gene Transfer (and Stephen and Barbara Friedman Chair) at Memorial Sloan Kettering Cancer Center shared exciting research using “living drugs” —engineered stem cells as a potential and effective immunotherapy to fight certain types of cancer.
What was once a highly specialized field has become more democratized as a variety of protocols, reagents and other tools enable researchers to de-differentiate cells to a pluripotent state (making induced pluripotent stem cells (iPSCs) virtually indistinguishable from embryonic stem cells), to grow these cells nearly in perpetuity and to differentiate them nearly at will. Here we look at the ways researchers maintain human PSCs.
Lay it down
PSCs don’t like to be alone in an unfamiliar place—to thrive, they need some semblance of home.
Many researchers use a layer of mitotically inactivated mouse embryonic fibroblast (MEF) feeder cells to support improved growth of PSC colonies [1]. Yet MEFs display and secrete an undefined mixture of xenogeneic proteins and other substances, leading some researchers to seek alternative matrices. Among these are a host of human-derived embryonic and adult cells. While these latter are not xenogeneic, they too will introduce undefined factors into the culture—which may or may not pose a problem for many basic research applications. This well-trodden path is often the easiest entrée into PSC culture.
Perhaps the most commonly used substrate for pluripotent stem cells is Matrigel, a solubilized basement membrane extract derived from mouse sarcoma (produced and marketed by Corning but provided by other vendors under alternative trade names). Yet like MEFs, Matrigel is a xeno product; it’s not completely defined and is batch-to-batch inconsistent, says Luis Villa-Diaz, assistant professor of biological sciences at Oakland University. “It works well, but it has some variability.”
Some researchers have taken to recombinant proteins as their matrix: “Laminin and fibronectin seem to be the most popular for human stem cell research now, and vitronectin has also been pretty popular,” notes Lia Kent, technical training manager and scientific support at Biological Industries. Specific isoforms (such as laminin 521) or fragments are often used, as are combinations of these.
Synthetic substrates are being used, as well. For example, Corning offers culture flasks, multiwell plates and microcarriers that have been coated with Synthemax, as well as concentrated Synthemax II-SC Substrate, “an easy-to-use, self-coating material for creating a unique synthetic surface that mimics the natural cell environment,” according to the company’s website.
Other research groups are exploring different, far less expensive, avenues. A group led by Jeong Beom Kim at Ulsan National Institute of Science and Technology (UNIST) in South Korea, for example, has found that diffusion-assisted synthesis-grown nanocrystalline graphene (DAS-NG) can maintain PSCs in long-term culture [2]. Paul Krebsbach’s group at the University of Michigan has reported that its zwitterionic hydrogel, poly2-(methacryloyloxy)ethyl dimethyl-(3-sulfopropyl)ammonium hydroxide (PMEDSAH) can sustain long-term PSC culture, as well.
Alert the media
The problem with PMEDSAH—a completely defined synthetic polymer—is that “it works better with media that is not completely defined,” says Villa-Diaz, who as part of Krebsbach’s group helped create it. “So we use human-cell-conditioned media” from human fibroblast or mesenchymal stem cell cultures.
Often, stem cell cultures make use of serum that contains a wide variety of factors, some of which are unknown. Others use a more (but not completely) defined serum replacement—generally containing serum albumin and other animal-derived products—that can be made in-house or purchased commercially from several vendors. Kim’s graphene work, for example, made use of GIBCO™ KnockOut™ Serum (provided by Thermo Fisher Scientific).
Serum has “properties to maintain a population in renewal but at the same time has properties to induce differentiation, so it becomes a balance to maintain,” Villa-Diaz explains. He equates trying to elucidate mechanical and molecular pathways under such conditions to fishing in murky waters, not knowing where to throw the hook. “If you have xeno-free, chemically defined conditions, so you know all the components in your media and your substrate, now you are fishing in clear water. Whatever treatment you do, you know the variables that can be affected.”
There has been a big push in the field to accomplish just that, largely led by James Thomson at the University of Wisconsin. His lab reported a formulation, termed TeSR, to support the derivation and growth of PSCs in the complete absence of xeno proteins. Variations of the formulation have been commercialized. Yet “the inclusion of human serum albumin and human sourced matrix proteins makes those conditions prohibitively expensive, impractical for routine use, and not truly completely defined,” his group wrote in a paper describing Essential 8 (E8) medium [3].
Thomson’s group used a “daunting” combinatorial approach to create E8, whittling TeSR’s 18 components down to the eight which, when added to basal DMEM/F12 medium, can support PSC growth just as well as TeSR. “A major improvement is the lack of a serum albumin component, as variations in either animal or human sourced albumin batches have previously plagued human ES and iPS cell culture with inconsistencies,” they wrote.
Complete media based on the E8 formulation can be purchased in ready-to-use bottles or in kits of basal media-plus-supplements from vendors such as Cell Applications. One of the principal ingredients, fibroblast growth factor (FGF), has a short half-life, notes Cell Applications’ vice president of sales and marketing Daniel Schroen: Once the formulation is thawed and diluted, “the clock starts ticking.” Kent recommends freezing aliquots and slowly thawing just what’s needed in the refrigerator: “The water bath is probably the worst thing to do for any PSC media.”
Corning Inc. subsidiary Mediatech, Inc., and Biological Industries, Ltd., recently announced a jointly branded xeno-free human pluripotent stem cell medium under the name NutriStem®. This ready-to-use product provides cell therapy and gives manufacturing researchers a solution for raising their PSCs.
Colonies or bust
Unlike those from mouse, human PSCs that do not have contact with other cells will soon begin to undergo anoikis, a form of programmed cell death. This is a problem not only when passaging and subcloning single cells but also when freezing, thawing and sorting cells. Thus cells are typically passaged as clumps or colonies, or treated with an anti-apoptosis agent such as a Rho-associated protein kinase (ROCK) inhibitor.
But ROCK inhibitors are “powerful drugs which have some side effects on the culture,” says Kevin Chen, staff scientist in the NIH Stem Cell Unit, National Institute of Neurological Disorders and Stroke, and an adjunct associate professor at Georgetown University, noting that the long-term effects have not been evaluated. Chen and others have been devising different ways around the need for ROCK inhibitors, including combinations of substrate that mimic contact with neighboring cells. Chen is not a fan of culturing cells as colonies, favoring instead a non-colony type monolayer (NCM), which he finds remains more homogenous, is easier to genetically manipulate and produces higher cell yields.
There is also considerable interest in scaling up PSCs as suspension cultures. To this end, STEMCELL Technologies will soon be releasing mTeSR3D, which has been adapted and optimized for fed-batch protocol. The media “allows one to produce that magic number of 109 cells in just two weeks, starting from a single six-well plate,” explains senior product marketing manager Simon Hilcove.
A continually evolving list of tools is available for PSC culture, ranging from gentle and xeno-free dissociation and cryopreservation reagents to assay kits for pluripotency markers. Also available is a burgeoning set of protocols from academic, biotech, pharma and clinical labs eager to advance the field.
References
[1] Chen, KG, et al., “Human Pluripotent Stem Cell Culture: Considerations for Maintenance, Expansion, and Therapeutics,” Cell Stem Cell, 14(1):13-26, January 2, 2014. [PMID: 24388173]
[2] Lee, H, et al., “Establishment of feeder-free culture system for human induced pluripotent stem cell on DAS nanocrystalline graphene,” Sci Rep, 6:20708, February 5, 2016. [PMID: 26846167]
[3] Chen, G, et al., “Chemically defined conditions for human iPS cell derivation and culture,” Nat Methods, 8(5):424-429, May 2011. [PMID: 21478862]
Image: Shutterstock Images