Formation of well-defined embryoid bodies from dissociated human induced pluripotent stem cells using microfabricated cell-repellent microwell arrays

Abstract

A simple, scalable, and reproducible technology that allows direct formation of large numbers of homogeneous and synchronized embryoid bodies (EBs) of defined sizes from dissociated human induced pluripotent stem cells (hiPSCs) was developed. Non-cell-adhesive hydrogels were used to create round-bottom microwells to host dissociated hiPSCs. No Rho-associated kinase inhibitor (ROCK-i), or centrifugation was needed and the side effects of ROCK-i can be avoided. The key requirement for the successful EB formation in addition to the non-cell-adhesive round-bottom microwells is the input cell density per microwell. Too few or too many cells loaded into the microwells will compromise the EB formation process. In parallel, we have tested our microwell-based system for homogeneous hEB formation from dissociated human embryonic stem cells (hESCs). Successful production of homogeneous hEBs from dissociated hESCs in the absence of ROCK-i and centrifugation was achieved within an optimal range of input cell density per microwell. Both the hiPSC- and hESC-derived hEBs expressed key proteins characteristic of all the three developmental germ layers, confirming their EB identity. This novel EB production technology may represent a versatile platform for the production of homogeneous EBs from dissociated human pluripotent stem cells (hPSCs).

Figure 1. Time lapse of hEB formation from hiPSCs in the hydrogel microwells at a seeding density of 35,000 hiPSCs/microwell under the no-ROCKi-treatment condition.

Condensation of the cell suspension was evident after 6 hr incubation in the hydrogel microwells and continued until hEB extraction after 24 hr incubation. Gross morphology of hEBs that were freshly extracted vs. 24 hrs after extraction.

Figure 2. Examination of the effect of input cell density per microwell on hEB formation from dissociated hiPSCs (A) or BG01V/hOG hESCs (B) under the no-ROCK-i-treatment condition.

(A1) The probability of EB formation as a function of input hiPSC cell density per microwell; (A2) Gross morphology of hEBs formed from hiPSCs in the hydrogel microwells at different input cell density per microwell; (B1) The probability of EB formation as a function of input hESC cell density per microwell; (B2) Gross morphology of hEBs formed from hESCs in the hydrogel microwells at different input cell density per microwell. Scale bar = 500 µm.

Figure 3. Transmission electron microscopy examination of the internal structural organization of the hEBs formed from hiPSCs (A) or BG01V/hOG hESCs (B) in the hydrogel microwells under the no-ROCK-i-treatment condition at different time points after extraction (A1, B1) freshly extracted; (A2, B2) 1 day; and (A3, B3) 7 days after extraction. (AJ, adherence junction; GJ, gap junction; D, desmosome; TJ, tight junction).

Figure 4. Time course of the sizes (cross-sectional areas) of hiPSC hEBs after extraction from the hydrogel microwells.

–ROCKi: hEBs formed under the no-ROCKi condition; +ROCKi: hEBs formed in the presence of ROCKi. For both conditions, hiPSCs seeding density was 35000 cells per microwell. n = 25.

Figure 5. Expression of molecular markers for the three developmental germ layers by the hiPSC EBs (A) or the BG01V/hOG hESC EBs (B) both at gene and protein levels after 20 days of spontaneous differentiation in suspension culture.

–ROCKi: hEBs formed under the no-ROCKi condition; +ROCKi: hEBs formed in the presence of ROCKi. (A1, B1) RT-PCR analysis for gene expression (full-length gel and blot are included in the supplementary information); (A2, B2) Triple immunofluorescence staining showing protein co-expression on a single EB cluster for each cell line under the –ROCKi condition. AFP: alpha feta protein (endoderm-specific), SOX1 (ectoderm-specific), and BRACHYURY (mesoderm-specific). Scale bar = 50 µm.

Figure 6. Differentiation of hiPSC hEBs into insulin-secreting cells.

(A) When treated with the pancreatic differentiation protocol for 18 days, hEBs differentiated into islet-like clusters that appeared uniform in sizes and spherical; Scale bar = 1 mm; (B) Over 85% of the cells differentiated from hEBs (formed under no-ROCK-i condition) were pancreatic β-cells, as evidenced by the positive staining for Dithizone (DTZ) in dark crimson red; Scale bar = 200 µm; (C) RT-PCR examination of the expressions of pancreatic lineage-specific genes by the cells after pancreatic differentiation; (D) Western blot analysis of PDX-1, another marker for pancreatic differentiation, in the cells; (E, F, G) Immunostaining of the cells revealed co-localization of insulin (in green) and C-peptide (in red), insulin (in green) and Glut-2 (in red), as well as C-Peptide (in red) and PDX-1 (in green); (H) Co-staining for Glucagon (in red) and Insulin (in green) revealed the absence of glucagon in insulin-secreting cells. Cell nuclei were stained in blue with DAPI. Scale bar = 50 µm; (I) ELISA for insulin secretion assay indicated glucose-responsive insulin secretion from the differentiated cells in a physiologic manner.