Human pluripotent stem cells are capable of becoming any tissue in the body, making them promising for numerous medical applications, all of which require large numbers of cells of high quality. Now scientists report a simple, efficient, scalable 3D system for producing human pluripotent stem cells and their progeny in a manner compatible with standard industry practices. These findings are detailed in the Proceedings of the National Academy of Sciences.
Pluripotent stem cells include both embryonic stem cells and induced pluripotent stem cells — mature cells reprogrammed into becoming pluripotent. Investigators are exploring their potential use in cell replacement therapies, tissue and organ engineering, and high-throughput pharmacology and toxicology screening.
The applications researchers would like to use pluripotent stem cells for typically require huge numbers of cells. For instance, to treat a patient with Parkinson’s disease, myocardial infarction or type I diabetes, physicians would probably require at least 100,000 dopaminergic neurons, 1 billion heart muscle cells or 1 billion insulin-producing beta cells to survive in the body, respectively. Even more cells are needed before transplantation, as many do not survive after they get transplanted into the body — for instance, only about 6 percent of transplanted dopaminergic neurons or 1 percent of injected heart muscle cells survive in lab rodents several months after transplantation. Large numbers of cells are also needed for tissue engineering — for instance, 10 billion liver cells or heart muscle cells would be needed for an artificial human liver or heart, respectively — and roughly 10 billion cells are needed to screen a million potential drugs once.
However, expanding pluripotent stem cells — that is, increasing their numbers — and differentiating them into the more specialized cells that make up the body remains a challenge. Current systems for culturing these cells are typically 2D, growing them on flat dishes, and these cells require biological signals from their surroundings and each other that may best be generated in a 3D environment.
Scientists had previously devised a number of 3D culturing systems for these cells, but these often had a number of problems, explain bioengineers Yuguo Lei and David Schaffer at the University of California, Berkeley. For instance, the matrix the cells were embedded in often possessed components from human or animal tissue, which can be limited in quantity and pose the risk of infecting or inflaming cells with pathogens or antigens. These systems could also lead cells to clump up in abnormal ways that kill them or make them differentiate in undesirable ways. In addition, the shear forces these systems often subject cells to by shaking in order to simulate natural movements inside the body could damage cells.
Now Lei and Schaffer have developed a 3D system for culturing pluripotent stem cells that can overcome these challenges. The cells are expanded and differentiated in a gel that is liquid at low temperatures but becomes an elastic solid when warmed. The cells are mixed evenly throughout the liquid at cool temperatures, grown in a solid environment at body temperature, and harvested by reliquifying the gel at low temperatures. This provides a 3D environment for growth that is porous enough for nutrients to diffuse inside. At the same time, it prevents the cells from clumping and isolates them from damaging shear forces. In addition, the system is free of any materials taken from humans or animals that might contaminate the cells, instead only using pure proteins.
All in all, the researchers found they could expand pluripotent stem cell numbers equivalent to a factor of 10^72 over 60 passages — that is, episodes of culturing — while still maintaining pluripotency over 280 days. Moreover, the system enabled controlled differentiation of these cells into multiple lineages — for instance, roughly 80 million dopaminergic progenitors per milliliter of gel.
Although the system works quite well at differentiating human pluripotent stem cells into neurons, “we still need to do more work to make it better for differentiating into other specialized cell types,” Schaffer says. These other cell types may include pancreas cells and cardiac cells, “to enable better therapies in those tissues.”
Schaffer adds that they reached the combinations of compounds used in their system through an empirical trial-and-error approach. They would now “like to understand the mechanistic reasons why these conditions worked so well,” he says.