ROCK Inhibitor Is Not Required for Embryoid Body Formation from Singularized Human Embryonic Stem Cells

We report a technology to form human embryoid bodies (hEBs) from singularized human embryonic stem cells (hESCs) without the use of the p160 rho-associated coiled-coil kinase inhibitor (ROCKi) or centrifugation (spin). hEB formation was tested under four conditions: +ROCKi/+spin, +ROCKi/-spin, -ROCKi/+spin, and -ROCKi/-spin. Cell suspensions of BG01V/hOG and H9 hESC lines were pipetted into non-adherent hydrogel substrates containing defined microwell arrays. hEBs of consistent size and spherical geometry can be formed in each of the four conditions, including the -ROCKi/-spin condition. The hEBs formed under the -ROCKi/-spin condition differentiated to develop the three embryonic germ layers and tissues derived from each of the germ layers. This simplified hEB production technique offers homogeneity in hEB size and shape to support synchronous differentiation, elimination of the ROCKi xeno-factor and rate-limiting centrifugation treatment, and low-cost scalability, which will directly support automated, large-scale production of hEBs and hESC-derived cells needed for clinical, research, or therapeutic applications

BG01V/hOG cells formed hEBs in the microwells at a cell seeding density of 15,000 hESCs per microwell.

<p>(<b>a</b>) Condensation of the hESC suspension within the microwells was progressive and evident after 6 hrs of incubation. (<b>b</b>) hEBs were compact and spherical and be able to be extracted intact from the microwells after 24 hrs of incubation. (<b>c</b>) The freshly extracted hEBs formed under four different conditions (ROCKi: ROCK inhibitor; Spin: centrifugation). (<b>d</b>) Internal structural organization among the cells within the freshly extracted hEBs (formed under -ROCK/-spin condition) was demonstrated by the presences of cell-cell junctions, i.e., adherence junctions (AJ), gap junctions (GJ), desmosomes (D), and tight junctions (TJ), in TEM images. All treatments were performed with aliquots from the same cell suspension. Microwell diameter 820 µm. Scale bars 500 µm.</p

H9 hESCs formed hEBs in the microwells at a cell seeding density of 25,000 hESCs per microwell.

<p>(<b>a</b>) Freshly extracted hEBs formed under four different conditions in microwells after 24 hrs of incubation. (<b>b</b>) A closer look at hEBs formed under -ROCK/-spin condition after 24 hrs of incubation before extraction indicated the presences of a compact core (arrows) and a loosely-aggregated corona. (<b>c</b>) Live-dead staining of the formed hEBs under -ROCK/-spin condition after 24 hrs of incubation before transfer to suspension culture indicated high viability (>90%) of hESCs both at the core and the corona (SYTO 10 green-fluorescent nucleic acid stain for all cells; DEAD Red (ethidium homodimer-2) nucleic acid stain for dead cells). (<b>d</b>) Confocal microscopic images of live/dead staining of freshly extracted hEBs (formed under -ROCK/-spin condition) indicated high viability (>85%) of the hESCs. Note that cells at the corona of the hEBs were sloughed off during the transfer process. Scale bars 500 µm.</p

H9 hEBs (formed under -ROCK/-spin condition) differentiated into pancreatic lineage at 18 days when treated with the pancreatic differentiation protocol.

<p>(<b>a</b>) RT-PCR analysis demonstrated expression of key pancreatic lineage-specific genes. (<b>b</b>) Over 90% of the cells were insulin-positive, as evidenced by positive staining for dithizone (DTZ) in dark red. (<b>c</b>) Immunostaining of the cells revealed co-localization (in yellow) of insulin and C-peptide. Cell nuclei were stained in blue with DAPI.</p

Cross-sectional areas (µm²) of the hEBs formed after 24 hrs of incubation.

<p>Cross-sectional areas (µm²) of the hEBs formed after 24 hrs of incubation.</p

BG01V/hOG hEBs (formed under -ROCK/-spin condition) differentiated into tissue lineages specific to each of the three embryonic germ layers.

<p>(<b>a</b>) RT-PCR analysis demonstrated gene expression profiles that are specific to representative lineages associated with each germ layer, i.e., neural (ectoderm), pancreatic (endoderm), and cardiac (mesoderm) lineages. (<b>b</b>) At 25 days when treated with the cardiac differentiation protocol, western blot confirmed expressions of cardiac α-actin and cardiac troponin-C proteins that are representative of cardiac lineage.</p

hEBs (formed under -ROCK/-spin condition) expressed proteins characteristic of all the three developmental germ layers.

<p>At 20 days in suspension culture, the hEBs were positive for Alpha Fetoprotein (AFP, endoderm-specific), SOX1 (ectoderm-specific), and brachiury (mesoderm-specific) for both the (<b>a</b>) BG01V/hOG and (<b>b</b>) H9 cell lines. Cell nuclei were stained in blue with DAPI.</p