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. 2020 Jun 25;15(1):256–273. doi: 10.1016/j.stemcr.2020.06.001

Figure 1.

Figure 1

Generation and Characterization of HSs from AD Patient and Control Human iPSCs

(A) Schematic model of the differentiation paradigm to generate HSs from iPSCs. LDN, LDN-193189, SB, SB-431542, XAV, XAV-939, CHIR, and CHIR-99021.

(B) Immunostaining for medial pallium marker LEF1 in APP variant, PS1 variant, and gender-matched control EBs aged 30 DIV. Scale bar, 150 μm.

(C) Immunostaining for brain cell subtype markers GFAP, O4 and MAP2, hippocampal markers ZBTB20 and PROX1, and neuronal markers TBR1, calretinin, and calbindin in APP variant, PS1 variant, and gender-matched control cultures at DIV 56. Scale bar, 200 μm.

(D–F) Quantification of GFAP-positive, O4-positive, and MAP2-positive cells (D), ZBTB20-positive and PROX1-positive cells (E), and PAX6-positive, TBR1-positive, calretinin-positive, and calbindin-positive cells (F) expressed relative to the total number of DAPI-labeled cells in APP variant, PS1 variant, and gender-matched control cultures at DIV 56. Results are presented as mean ± SEM. n = 1–4 independent differentiations per clone for N = 3 iPSC clones per genotype. ∗p < 0.05; N/D means not detected. Statistical analysis by two-tailed t test.

(G) Immunostaining for human nuclei marker and hippocampal markers ZBTB20 and PROX1 in control cells 5 weeks after transplantation into mouse hippocampus. Host mouse ZBTB20 and PROX1-positive cells are not colocalized with human nuclei marker. Scale bars, 100 μm.

(H) Immunostaining for human nuclei marker, human cell surface marker NCAM, and neuronal markers doublecortin and calretinin in control cells 5 weeks after transplantation into mouse hippocampus. Host mouse doublecortin and calretinin-positive cells are not colocalized with human markers. Scale bar, 100 μm.

(I) Bright-field images showing representative examples of APP variant, PS1 variant, and gender-matched control HSs at DIV 100. Scale bar, 200 μm.

(J) Confocal images showing representative examples of immunostaining for brain cell subtype markers GFAP, O4 and MAP2, hippocampal markers ZBTB20, PROX1, and neuronal markers TBR1, calretinin, and calbindin in APP variant, PS1 variant, and gender-matched control HSs at DIV 100. Scale bar, 50 μm.

(K) Immunostaining for neuronal marker MAP2, hippocampal granule neuron marker PROX1 and pre-synaptic marker synaptophysin in control HSs at DIV 100 and 3D surface reconstruction of confocal z stacks showing PROX1+/synaptophysin+/MAP2+ granule neuron in control HSs. Scale bars, 100 μm (HS confocal images) and 10 μm (3D reconstruction).