Clinical transplantation requires large quantities of functional target cells and most of the existing strategies are difficult to implement at a large scale or have a low differentiation efficiency, therefore posing barriers to further research. Compared to flat culture systems, EB-derived differentiation culture is kept in a relatively fixed position, which offers this method an obvious advantage in quantity and differentiation efficiency[14-16]. A variety of cell lineages have been generated from hEBs such as brain, cornea, heart, liver, and blood (Table 1). In our study, we used a suspension EB-based system to generate iPSC-derived melanocytes and achieved a significantly higher differentiation efficiency compared to that in flat culture systems and these induced melanocytes showed long-term in vivo functionality after transplantation[17]. In short, differentiation from EB to specific cell lineages is an efficient method that is likely to yield large populations of functional cells.

Recently, due to the features of their three germ layer structure[18], hEBs have become useful models for developing human ESC- and iPSC-derived organoids by stimulation with a cocktail of biological agents (Table 1). These EB-derived organoids not only contain some cell subtypes but also show distinct stratification which is similar to the in vivo structure of the tissues or organs developing[19,20]. For example, Jo et al[21] observed an identical organization structure in 3D cultured human midbrain-like organoids (hMLOs) compared with human postmortem midbrain tissue under the electron microscope. Furthermore, these EB-derived organoids are functional. Qian et al[22]found that EB-derived midbrain organoids not only expressed a wider range of characteristic markers common to normal midbrain tissue compared with direct differentiation from iPSCs, but also demonstrated firing action potentials in response to current injection which can be used to establish a disease model of microcephaly[22]. These EB-derived organoids could be used to understand unique features of specific human organs and to gain insights into different disorders.

There is a remarkable difference in differentiation capability in distinct iPSC lineages, and it is urgently necessary to predict the differentiation potential in an early stage of the long differentiation period to avoid unnecessary resource losses. Because EBs can potentially develop into three germ layer tissues for multi-directional differentiation[23,24], it can be hypothesized that they may be suitable candidates for predicting iPSC differentiation potential. Kim et al[25]classified hESCs-derived EBs into several types including cystic, bright cavity and dark cavity according to their morphology. By detecting the expression of specific markers, they showed that most of the cells in cystic EBs are endoderm-lineage populations and both bright and dark cavity EBs own cells from all of the three germ layers. Besides the morphology, EB size can also be used determine the differentiation direction of iPSC. For example, the larger EBs (450 μm diameter) often contained cells of the cardiovascular system, while endothelial cells were usually generated in smaller EBs (150 μm diameter) and such differentiation potentials are mainly caused by the noncanonical WNT pathway[26]. Moreover, gene expression profiles of hPSCs and EBs recovered by high-throughput RNA sequencing can also be used for differentiation prediction, and the results is parallel to the germ layer development in vivo[27].

The novel methods described above have some limitations. Simple morphology or size-dependent prediction system is somewhat subjective, and it is also closely related to the viability of the cells inside EB[28,29], whereas high-throughput RNA sequencing is expensive and complicated. In our study, we developed a simple and practical system and identified a set of parameters combining EB formation, maintenance, and germ layer-specific gene expression in EB stage to predict the differentiation tendency of the various iPSC lines. The validity of this evaluation system was finally confirmed by differentiating these iPSC lines into melanocytes[24]. Recently, we summarized the current methods related to iPSC differentiation prediction and we also postulated that the use of EBs could be more efficient when combined with other modified detection methods such molecular probes[34].Thus, due to their special morphology and functionality, EBs have played an important role in the differentiation prediction related studies.

The remarkable functionality and application prospects of EBs are a result of their unique 3D microstructure and cell communication. Frequent passage in the adherent culture system limits the cell–cell communication in both cell number and time. The suspension culture system for EBs provides extensive material exchange and increases both cell–cell interactions and cell–matrix communication in the aggregate, which is conducive to the transmission and action of cell signals[35,36]. In general, intercellular communication is increased both in space and time at the 3D level. Additionally, differentiation of EBs is an asynchronous process and 3D culture produces distinct layers of spheroids (EBs) as a result of concentration gradients in the culture medium[37]. The differentiation of the outer endoderm of EB caused by growth factors in the medium will further induce endoderm differentiation by producing signals[38]. This unbalanced phenomenon also explains the heterogeneity throughout the differentiation process in EBs. Moreover, more intricate gene profiles could be expressed in a 3D culture system, reflecting original expression in vivo more accurately[16]. Jo et al[21] found that gene profiles detected by RNA sequencing analysis are analogously expressed between hMLOs and the human prenatal midbrain. However, there is a significant difference between the 2D dopamine neurons and normal tissue. The integrity of gene expression is a considerable advantage of the 3D culture system, providing a better foundation for future structural and functional studies. Meanwhile, for the hiPSC-based differentiation, the cadherin-β-catenin-Wnt pathway was found to be involved in the development of EBs by intercellular adhesion[39]. In addition, the activation or inhibition of relevant signaling pathways can yield a definitive lineage differentiation[22,40,41]. Therefore, controlling the dose and duration of cytokines regulates the differentiation of iPSCs by activating various pathways.

Compared with a flat culture, microgravity has been found to be a subsidiary factor that can regulate cell proliferation and survival in a 3D culture system[29-32]. Therefore, bioreactors and random positioning machines have been used to simulate states of microgravity and weightless condition for EB-derived differentiation[42,43].

In conclusion, EBs have shown great application potential in the generation of various functional cells and organoids at a large scale and also in the prediction of iPSC differentiation capability. These EB-derived cells and organoids highly resemble the in vivo status in both gene expression and function display as a result of the structure and microenvironment in the EB.

To generate high quality EBs, the initial step is the pluripotency maintenance in iPSCs where the addition of various cytokines and inhibitors is essential. Two studies[15,44]demonstrated that basic fibroblast growth factor (bFGF) induced hES cell derived fibroblast-like cells produce insulin-like growth factor II and transforming growth factor β family of factors and indirectly establish a balanced stem cell niche of hESCs, contributing to maintain the undifferentiated state of hESCs. In addition, Y-27632 was confirmed to benefit the expression of POU class 5 homeobox 1 (OCT4) in EB formation[45-48]. Compared with the conventional methods, single cell culture system can reduce the complexity caused by feeder cells. However, it is more difficult to maintain undifferentiated status of iPSC when single-cell passage is used. Tsutsui et al[45]developed a unique combination of inhibition of Rho-associated kinase, glycogen synthase kinase and mitogen-activated protein kinase with bFGF for hES cells maintenance and they found these cells to present good pluripotency and genetic integrity in long-term maintenance (> 20 passages).

In general, EBs can be spontaneously generated with hiPSC clusters[8,46] or single cells[41,49] in 3D culture systems using EB-specific medium. When conventional methods such as static suspension culture and the hanging drop method[50,51]are chosen, EB-derived differentiation seems unstable, which is in part associated with the heterogeneity of EBs[52,53]. Therefore, it is necessary to control the generation of uniform EBs for stable and reproducible differentiation. Recently, different types of micro-pattern plates have been developed and widely applied for the generation of EBs with uniform size and morphology using single hiPSCs (Table 1) and on this basis, a variety of culture systems such as the micro-space system[50,51,54], spinner flasks[22], and the National Aeronautics and Space Administration developed rotary cell culture system (NASA rotary system)[55] have also been designed for scale-up.

Besides morphological uniformity, the efficiency and direction of differentiation are also affected by EB size[56-58] and which size is more appropriate depends on the specific cell lineages. It has been demonstrated that a larger amount of cardiomyocytes was found in hESC-derived differentiation using EBs with100 μm diameter when compared to larger ones (300 μm)[59]. However, we found that 300-400 μm might be a more suitable diameter for melanocytes differentiation compared to those bigger or smaller. Since the size and quality of EB during culture maintenance are affected by the number of inoculated cells, micro-pattern plates can be used to control EB size by adjusting cellular density. However, inoculated cell density also depends on the EB culture plate type and culture duration before differentiation. In a study of iPSC differentiation into cardiomyocytes, input of 1.5 × 10⁴ to 4.0 × 10⁴ cells per EB was determined to be the proper density to form homogenous and synchronized EBs. By contrast, we found that 5.0 × 10² to 1.0 × 10³ cells were enough for each EB generation using an Elplasia^TM micro-well plate and this density tended to yield stable and intact EBs (Figure 1) which could be maintained over a relatively long term for differentiation. Overly high cell density (2.0 × 10³ cells/EB) will lead to breakage or fusion in a short time (Figure 1). In short, EBs within a certain size range are more conducive to differentiation which can be modified by cell density adjustment. However, size usually varies across different differentiation assays and needs to be optimized in accordance to the specific conditions.

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Figure 1 Static suspension culture and 24-well Elplasia^TM micro-well culture with human induced pluripotent stem cells. A-C:Induced pluripotent stem cell (iPSC)-derived embryoid bodies (EBs) formed under a suspension system and cultured with mTeSR™, respectively, on day 1 (A), day 3 (B), and day 7 (C); D-L: iPSC-derived EBs formed with single hiPSCs in micro-space cell culture plates at different cell densities: D-F: 2.5 × 10⁵ cells/mL; G–I: 5.0 × 10⁵ cells/mL; and J–L: 1.0 × 10⁶ cells/mL, respectively, on day 1 (D, G, and J), day 3 (E, H, and K), and day 7 (F, I, and L). Scale bars: A, D, G, and J 100 μm; resident graphs, 250 μm.