Embryoid bodies (EB) are the three-dimensional aggregates formed in suspension by pluripotent stem cells (PSC), including embryonic stem cells (ESC) and induced pluripotent stem cells (iPSC). EB differentiation is a common platform to generate specific cell lineages from PSCs. However, most EB formation protocols contain undefined components, such as Fetal Bovine Serum (FBS), Knock-Out Serum Replacement (KOSR) or albumin product. These animal-sourced components significantly limit the application of EB formation to generate potentially clinically relevant cell products. At the same time, the undefined composition of the above components can lead to inconsistent outcomes in experiments due to batch differences in their production. This inconsistency also severely affects researchers’ ability to further improve procedure and its final products. Meanwhile, unlike their mouse counterparts, human PSCs usually cannot survive in suspension unless in aggregates or under ROCK inhibitor treatment. As such, it is essential that EB formation and further differentiation can be conducted in chemically defined, animal product-free conditions. This will allow better consistency as well as an easier route to translate into clinically relevant production. We previously developed a fully chemically defined medium Essential 8 (or E8) for the maintenance and expansion of human pluripotent stem cells in the clinical grade environment. In this protocol, we describe a set of optimized procedures to produce EBs from human PSCs in E8 or E8-based media.
Assessing pluripotency in human cells is inherently an intractable problem. In animal systems, pluripotency can be verified through direct means: pluripotent stem cells can be introduced into an developing embryo and thus the cellular developmental potential of any given in vitro preparation can be directly determined by observing the amount of chimaerism or viability of organisms partially or fully derived from in vitro stem cells.
This protocol was developed in the Salk STEM Cell Core to enable the formation of uniform and large embryoid bodies (EBs) from pluripotent human stem cells. It assumes the cells have previously been cultured on Matrigel and typically requires 1–2 wells of a fairly confluent 6-well plate as starting material.
Human embryonic stem cells and human induced-pluripotent stem cells are uniquely defined by their pluripotent differentiation potential and endless self-renewing ability. This capability to become any somatic cell type within the human body has garnered significant attention and interest in the fields of cell biology and regenerative medicine.