The Evolution of Human Pluripotent Stem Cell Culture Systems
Human pluripotent stem (hPS) cells can proliferate for years or more as self-replicating, unspecialized cells, yielding an exponentially expanding number of stem cells in culture — each with the ability to differentiate into any cell or tissue in the body. These attributes make hPS cells a unique tool in the field of regenerative medicine, offering potential to heal damaged tissue and treat cell-based diseases, such as heart disease, diabetes, and spinal cord injuries. Pluripotent stem cells are also commonly used in drug discovery, toxicity screening, and studying the underlying factors of disease.
The derivation of human embryonic stem (hES) cells and the generation of induced pluripotent stem (hiPS) cells were breakthrough scientific advancements. Since the first publication of hES cells in 1998, the field has continuously improved aspects of the culture system for a more consistent and clinically-relevant environment.
The ideal hPS cell culture environment would be made of all known components, with little or no variables involved. We strive for a culture system that is completely defined, meaning that the chemical composition and exact quantity is known for every element in the environment. One step further in reducing variables is to use xeno-free or animal component-free reagents. Xeno-free reagents are derived from human origin, while animal component-free reagents are more defined in nature, and encompass synthetic or recombinant materials.
The first hES cells were cultured using techniques borrowed from mouse ES cells. The original hES cell medium contained fetal bovine serum (FBS), but it was quickly evident that the rich, undefined components in the serum easily triggered the sensitive cells to spontaneously differentiate. Instead, the basic medium recipe now includes a serum replacement, which, although still undefined, is a fraction of the whole serum solution, and contains less protein and variation that can cause unwanted differentiation. In addition to the general components in cell culture media (sugars, serum replacement, glutamine and other amino acids, cytokines, buffers, etc.), hPS cells also require basic fibroblast growth factor (bFGF) to help maintain pluripotency.
The first hES cells were co-cultured with inactivated mouse embryonic fibroblast (MEF) feeder cells. The fibroblasts in the feeder-dependent system support the stem cells by modifying the microenvironment, but the exact way this benefits the hPS cells is still largely unknown. There are certain drawbacks to the feeder-dependent cultures system, most notably the introduction of animal-derived cells to the human stem cell environment. There can also be incredible variability in the quality of the MEF feeder cells and their ability to support healthy hPS cells. The use of human fibroblast feeder cells can minimize the dependency on animal products in culture, but human feeder cells still remain highly variable and contribute to an undefined system.
The first major step in optimizing the hPS cell culture environment was to culture the stem cells independently from the feeder cells. This is done by culturing the hPS cells on a mouse-derived attachment substrate (Matrigel), while culturing the feeder cells in a separate flask. A feeder-independent system is based on the use of conditioned medium, where the hPS cell medium is first “conditioned” in the feeder cells, then collected and used to feed the hPS cells in a separate dish. Although physically separating the hPS cells the fibroblasts has some advantages, the main disadvantage of using conditioned medium is that it remains dependent on the undefined and highly variable feeder cells.
Feeder-free culture of hPS cells has great advantages, and offers a much more streamlined system. Without feeder cells, the culture is entirely dependent on the quality of the medium for growth and proliferation of healthy stem cells. The first medium designed for feeder-free culture of hPS cells was mTeSR1. The combination of mTeSR1 medium and Matrigel® substrate creates a very rich environment rich to support hPS cells. However, mTeSR1® medium contains animal-derived components and relies on very high amounts of bFGF to support undifferentiated hPS cells.
In 2008, a more streamlined medium for feeder-free cell culture was developed, called NutriStem® hPSC Medium. NutriStem hPSC Medium is completely defined, xeno-free, and contains very low levels of growth factors and other proteins, including bFGF. The low protein composition avoids potential bias, inhibition, or other effects on subsequent differentiation of the cells. For regenerative medicine applications, NutriStem hPSC Medium has proven to be a clinically-relevant stem cell medium, produced under cGMP guidelines, with a specific Drug Master File (DMF) registered with the FDA.
Feeder-free culture has now become the basis for most human stem cell labs. Eliminating feeder cells provides simple, clean cultures and introduces fewer variables. Many hPS cell lines are still cultured on Matrigel, a reconstituted mixture of extracellular matrix proteins extracted from a mouse EHS tumor, but there are now other substrate options in use as well. Purified attachment proteins, such as recombinant human vitronectin and recombinant human laminin, are now common in many labs, making it possible to culture hPS cells in a xeno-free and chemically defined environment.
There are clear advantages of a defined, xeno-free, and feeder-free culture system for all stem cell cultures, since consistency in the formulations prevents lot-to-lot variability. Eliminating all non-human components reduces health risks for downstream applications, and a completely xeno-free environment reduces potential immunological reactions from stem cells or their derivatives.
Ever since the first hES cell lines were generated, the stem cell field has produced incredible new discoveries at an extremely fast pace. Researchers are continually working to improve the general ease-of-use and scale-up potential of these unique and sensitive cells. Further optimization will provide an increased yield of high quality, healthy cells, while maintaining a pure and clinically-relevant culture environment.
Want more information? Learn more about the basic stem cell definitions, properties of human pluripotent stem cells, morphology and growth characteristics of these cells in culture, and more details about how different culture systems have evolved over time in the comprehensive webinar “Human Pluripotent Stem Cells (hPSC) — An Overview of Morphology and Culture Methods.”