Rapid Induction of Cerebral Organoids From Human Induced Pluripotent Stem Cells Using a Chemically Defined Hydrogel and Defined Cell Culture Medium

Abstract

Tissue organoids are a promising technology that may accelerate development of the societal and NIH mandate for precision medicine. Here we describe a robust and simple method for generating cerebral organoids (cOrgs) from human pluripotent stem cells by using a chemically defined hydrogel material and chemically defined culture medium. By using no additional neural induction components, cOrgs appeared on the hydrogel surface within 10-14 days, and under static culture conditions, they attained sizes up to 3 mm in greatest dimension by day 28. Histologically, the organoids showed neural rosette and neural tube-like structures and evidence of early corticogenesis. Immunostaining and quantitative reverse-transcription polymerase chain reaction demonstrated protein and gene expression representative of forebrain, midbrain, and hindbrain development. Physiologic studies showed responses to glutamate and depolarization in many cells, consistent with neural behavior. The method of cerebral organoid generation described here facilitates access to this technology, enables scalable applications, and provides a potential pathway to translational applications where defined components are desirable.

Significance: Tissue organoids are a promising technology with many potential applications, such as pharmaceutical screens and development of in vitro disease models, particularly for human polygenic conditions where animal models are insufficient. This work describes a robust and simple method for generating cerebral organoids from human induced pluripotent stem cells by using a chemically defined hydrogel material and chemically defined culture medium. This method, by virtue of its simplicity and use of defined materials, greatly facilitates access to cerebral organoid technology, enables scalable applications, and provides a potential pathway to translational applications where defined components are desirable.

Keywords: Adrenoleukodystrophy; Brain; In vitro techniques; Organoids; Stem cells.

Figure 1.

Gross appearance, histology, and immunofluorescence of cerebral organoids (cOrgs) derived from human induced pluripotent stem cells (hiPSCs) embedded in Cell-Mate3D matrix and cultured in E8 medium. (A): Gross photograph of day 28 cerebral organoid (arrows) attached to matrix. Scale bar = 1 mm. (B): Neural rosettes (arrows) in cOrg derived from WT3 induced pluripotent stem cell line at day 14 (D14). Hematoxylin and eosin stain. Scale bar = 50 µm. (C): Neural tube-like structure in D14 cOrg derived from ALD1 hiPSC line. Hematoxylin and eosin stain. Scale bar = 50 µm. (D): D14 cOrg derived from ALD1 hiPSC line and immunohistochemically stained for β-3 tubulin. Scale bar = 1,236 µm. (E): Immunofluorescent stain showing colocalization of Sox1 (red) and nestin (green) in a cell of a neural tube-like structure and surrounding area in a cOrg from WT1 hiPSC line at D14. Scale bar = 60 µm. (F): Immunofluorescent stain showing Sox2 (green) and nestin (red) colocalization in neural rosettes in a cOrg from ALD2 hiPSC line at D14. Scale bar = 18 µm. (G–I): Immunofluorescent staining of neurocortical-like regions in D28 cOrgs for Tbr1 (ALD3) (G), doublecortin (ALD1) (H), and reelin (WT3) (I). Scale bars = 30 µm (G) and 15 µm (H, I).

Figure 2.

Representative immunohistochemical staining panels for neural markers on day 14 cerebral organoids (cOrgs) derived from control (WT1 and WT3) or cerebral childhood adrenoleukodystrophy patient (ALD2) human induced pluripotent stem cells (hiPSCs). The majority of cells within all cOrgs derived from either control or adrenoleukodystrophy (ALD)-patient hiPSCs stained for β-3 tubulin, Sox2, and nestin. More localized expression of Nurr1 and Pax6 was seen in most cOrgs. Tyrosine hydroxylase positive cells, which often exhibited neuronal-like processes, were found in loose groupings in many of the cOrgs. Scale bars = 50 µm. Specific immunohistochemical stains (brown stain) with hematoxylin (blue) nuclear stain. Abbreviation: Nurr1, nuclear receptor related 1.

Figure 3.

Quantitative reverse-transcription polymerase chain reaction analysis of gene expression for forebrain, hindbrain, and corticogenesis markers in undifferentiated human induced pluripotent stem cells, day 14 cerebral organoids (cOrgs), and day 28 cOrgs. Values indicated are mean ± SD. Data are given for gene expression for each individual cell line at D0 (undifferentiated induced pluripotent stem cell), D14, and D28, and the “overall” analysis included pooled data from all four cell lines (WT1, WT3, ALD1, and ALD3) at each time point. Abbreviations: DCX, doublecortin; EGR2, early growth response 2; FOXG1, Forkhead box G1; ISL2, islet 2; SIX3, sine oculis homeobox 3.

Figure 4.

Physiologic response of cells within a cerebral organoid derived from control human induced pluripotent stem cell line WT1 to glutamate and potassium (K⁺). Intracellular calcium concentration increased upon bath application of glutamate (100 µM) and elevated potassium concentration (50 K; 50 mM). Upper: False color images showing Fluo-3 fluorescence intensity before (1), at the peak (2), and after recovery (3) from the response to glutamate. Image at far right is an IR transmitted light image of the same field. Scale bar = 10 µm. Lower: Plot of fluorescence intensity over time showing Fluo-3 fluorescence in the region indicated by the circle on the images above. Glutamate and elevated potassium were bath applied during the times indicated by the black bars. Abbreviation: Glut, glutamate.