Engineering Strategies for the Formation of Embryoid Bodies from Human Pluripotent Stem Cells

Abstract

Human pluripotent stem cells (hPSCs) are powerful tools for regenerative therapy and studying human developmental biology, attributing to their ability to differentiate into many functional cell types in the body. The main challenge in realizing hPSC potential is to guide their differentiation in a well-controlled manner. One way to control the cell differentiation process is to recapitulate during in vitro culture the key events in embryogenesis to obtain the three developmental germ layers from which all cell types arise. To achieve this goal, many techniques have been tested to obtain a cellular cluster, an embryoid body (EB), from both mouse and hPSCs. Generation of EBs that are homogeneous in size and shape would allow directed hPSC differentiation into desired cell types in a more synchronous manner and define the roles of cell-cell interaction and spatial organization in lineage specification in a setting similar to in vivo embryonic development. However, previous success in uniform EB formation from mouse PSCs cannot be extrapolated to hPSCs possibly due to the destabilization of adherens junctions on cell surfaces during the dissociation into single cells, making hPSCs extremely vulnerable to cell death. Recently, new advances have emerged to form uniform human embryoid bodies (hEBs) from dissociated single cells of hPSCs. In this review, the existing methods for hEB production from hPSCs and the results on the downstream differentiation of the hEBs are described with emphases on the efficiency, homogeneity, scalability, and reproducibility of the hEB formation process and the yield in terminal differentiation. New trends in hEB production and directed differentiation are discussed.

A Systemized Approach to Investigate Ca2+ Synchronization in Clusters of Human Induced Pluripotent Stem-Cell Derived Cardiomyocytes

“…Furthermore, the decreased number of dissociated IPS-CM was not due to reduced post-EB cell proliferation since the extent of cell division following the disaggregation of all EBs was extremely low and we recorded just one cell division event during the entire study (Supplementary Movie 8 ). Thus, in our view, the most likely explanation for the decreased numbers of IPS-CM following disaggregation of later EBs (>2 weeks) is that changes in the nature of intercellular coupling via modulation of gap junction (connexin), tight-junction (ZO-1 and 2) and adherens junction proteins with EB maturation (Oyamada et al, 1996 ; Komura et al, 2008 ; Phua et al, 2014 ; Pettinato et al, 2015 ) limits the efficiency of IPS-CM dissociation from older EBs. The notion of enhanced cell-to-cell coupling in these older EBs is supported by the comparative functional “adult-like” maturity of the resulting IPS-CM clusters which typically lack spontaneous oscillatory behavior (Figure 8A ) yet respond to elevated [Ca 2+ ] ext (Figures 8D,E ).…”

Sleeping beauty transposon‐mediated poly(A)‐trapping and insertion mutagenesis in mouse embryonic stem cells

“…To explore the differentiation potential of syngap1 +/− cells, normal mES cells and syngap1 +/− cells were collected for formation of embryoid bodies (EBs) according to the suspension method (Pettinato et al, ). After suspension culture for 48 hours, EBs were well formed (Fig.…”

Three-Dimensional Organoid System Transplantation Technologies in Future Treatment of Central Nervous System Diseases

“…After 4-day suspension culture, ESCs or PSCs aggregate and form embryoid/embryoid-like bodies. The procedure in vitro culture recapitulates the key events of embryogenesis in vivo to obtain the three developmental germ layers from which all cell types arise [ 4 , 17 , 50 , 72 ]. The cell pellets are entrapped in a droplet of matrigel or collagen to coculture for and differentiate to develop a specific organ in a specifying differentiation strategy.…”

“…Different from other organoid induction approaches, this approach resorts to acquire specific organoid with full layer structure. Researchers adopt this approach to investigate natural organ development procedures or mechanisms involving diseases [ 4 , 17 , 50 , 72 ]. Unlike EB-like aggregation in SFEBq procedure, this approach prolongs the culturing time of EB-like population.…”