Human organoid model of pontocerebellar hypoplasia 2a recapitulates brain region-specific size differences

Abstract

Pontocerebellar hypoplasia type 2a (PCH2a) is an ultra-rare, autosomal recessive pediatric disorder with limited treatment options. Its anatomical hallmark is hypoplasia of the cerebellum and pons accompanied by progressive microcephaly. A homozygous founder variant in TSEN54, which encodes a tRNA splicing endonuclease (TSEN) complex subunit, is causal. The pathological mechanism of PCH2a remains unknown due to the lack of a model system. Therefore, we developed human models of PCH2a using regionalized neural organoids. We generated induced pluripotent stem cell (iPSC) lines from three males with genetically confirmed PCH2a and subsequently differentiated cerebellar and neocortical organoids. Mirroring clinical neuroimaging findings, PCH2a cerebellar organoids were reduced in size compared to controls starting early in differentiation. Neocortical PCH2a organoids demonstrated milder growth deficits. Although PCH2a cerebellar organoids did not upregulate apoptosis, their stem cell zones showed altered proliferation kinetics, with increased proliferation at day 30 and reduced proliferation at day 50 compared to controls. In summary, we generated a human model of PCH2a, providing the foundation for deciphering brain region-specific disease mechanisms. Our first analyses suggest a neurodevelopmental aspect of PCH2a.

Keywords: Apoptosis; Cerebellum; Differentiation; Organoid; PCH2a; Rare disease.

Fig. 1.

Generation of PCH2a iPSCs. (A) Experimental scheme of PCH2a induced pluripotent stem cell (iPSC) generation (scheme was created with BioRender.com). (B) Magnetic resonance imaging (MRI) scans of the brain of a normally developing infant (6 months of age) and (C) an infant with PCH2a (6 months of age, donor of iPSC line PCH02). T2-weighted coronal images (B,C, left) show cerebellar hemispheres severely reduced in size (indicated by arrows) in the PCH2a child compared to the control individual. T1-weighted sagittal images (B,C, right) illustrate the severe pontine and cerebellar hypoplasia in the PCH2a child (indicated by arrows). The facial regions of the MRI scans are covered in order to protect the privacy of both individuals. (D) Sanger sequencing results of PCH2a iPSCs verifies the TSEN54 c.919G>T point mutation, whereas control lines show the c.919G genotype (homozygous). (E) Alkaline phosphatase (ALP) staining of undifferentiated iPSCs. (F,G) Immunocytochemical staining of undifferentiated iPSCs for the pluripotency markers OCT4 (F) and TRA-1-81 (G) and DAPI (nuclei) demonstrates the pluripotency of generated cell lines. (H-J) Immunocytochemical staining of iPSCs spontaneously differentiated into the three germ layers illustrates the differentiation potential of the generated iPSCs. Cells were stained for Tuj1 (tubulin βIII marker, ectoderm) (H), smooth muscle actin (SMA, mesoderm) (I) and forkhead box A2 (FOXA2, endoderm) (J), as well as DAPI (nuclei). Representative images show iPSCs from the cell line PCH01.

Fig. 2.

PCH2a-derived and control iPSCs do not differ in expression of pluripotency, proliferation and apoptosis markers. (A,C) Immunocytochemical staining of PCH2a and control iPSCs for the pluripotency marker OCT4 (A) and the proliferation marker Ki67 (C) confirmed their expression in all iPSC lines [representative images are of iPSCs from the cell lines CO22, passage (P) 19, and PCH01, P17 (A), and CO57, P20, and PCH03, P20 (B)]. (B,D) Quantification of OCT4⁺ cells (B) and Ki67⁺ cells (D) normalized to DAPI-based cell count showed no significant difference in the number of OCT4⁺ or Ki67⁺ cells between PCH2a and control iPSCs (assessment of three passages per cell line). (E) Immunocytochemical staining of PCH2a and control iPSCs for the apoptosis marker cCas3 showed low expression levels in all iPSC lines (representative images are of iPSCs from CO60, P18, and PCH02, P18). (F) Quantification of cCas3⁺ cells, normalized to DAPI-based cell count showed no significant difference in number of cCas3⁺ cells between PCH2a and control iPSCs (assessment of three passages per cell line). (G) Visualization of cell proliferation by click-chemistry detection of EdU in PCH2a and control iPSCs after 1 and 4 h of incubation with EdU (representative images are of iPSCs from CO60, P18, and PCH02, P18). (H) Quantification of EdU-positive cells, normalized to DAPI-based cell count after 1 and 4 h of EdU incubation showed no significant difference in the number of EdU-positive cells between PCH2a and control iPSCs (assessment of three passages per cell line). ns, P>0.05 [two-tailed unpaired t-test with Welch's correction assuming unequal standard deviations (SDs)].

Fig. 3.

PCH2a organoids are significantly smaller than control organoids. (A) Representative brightfield images of cerebellar and neocortical organoids in culture at day (D) 30 and D90 of differentiation illustrate the differences in size. (B,C) Growth curves of PCH2a and control cerebellar (B) and neocortical (C) organoids, differentiated from three different cell lines per condition (derived from three different individuals), show the area of the organoids in the images pictured in A during the culture period of 90 days. Cerebellar organoids (B) differed significantly in size from D10 of differentiation, with discrepancies increasing over time. Neocortical organoids (C) showed significant differences from D30 of differentiation. n>8 organoids per cell line, timepoint and differentiation. Note that PCH01 neocortical differentiation is absent due to a contamination. Points represent the mean, error bars represent s.e.m. ****P<0.05 (two-tailed unpaired t-test with Welch's correction assuming unequal SDs). (D) The ratio between mean sizes of control and PCH2a organoids calculated from data presented in B,C. The ratios increased over time and were higher within cerebellar differentiation.

Fig. 4.

PCH2a and control cerebellar organoids show differentiation into the cerebellar lineage. Immunohistochemistry of control and PCH2a cerebellar organoid sections at D30 and D90 showed differentiation into the cerebellar lineage. (A,B) Expression of the early neuronal marker Tuj1 (green), the neural precursor marker SOX2 (cyan) and the glutamatergic precursor marker BARHL1 (magenta) in D30 control (A) and PCH2a (B) cerebellar organoids (representative images show organoids derived from CO22, P19, and PCH02, P17). (C,D) The GABA-ergic precursor marker KIRREL2 (magenta) is expressed in D30 cerebellar control (C) and PCH2a (D) organoids together with Tuj1 (green) and SOX2 (cyan) (representative images are of iPSCs from CO22, P19, and PCH01, P19). (E,F) Control (E) and PCH2a (I) cerebellar organoids show calbindin (CALB, cyan) in postmitotic Purkinje cells and neuronal marker MAP2 (magenta) expression (representative images are of iPSCs from CO57, P18, and PCH03, P19).

Fig. 5.

Expression of the apoptotic marker cCas3 is not altered in PCH2a organoids. (A,B) Confocal microscopy images of immunohistochemistry on cerebellar (A) and neocortical (B) organoid sections at D30 and D50 of differentiation show expression of the neural precursor marker SOX2 and the apoptotic marker cCas3 in rosette-like structures of organoids. Representative images of cerebellar organoids (A) are from CO22, P19, and PCH03, P19 (D30/D50), and those for neocortical organoids (B) are from CO57, P18, and PCH02, P17 (D30/50). (C,D) Quantification of the cCas3-positive area over DAPI signal showed no significant difference in cCas3 expression between PCH2a and control in cerebellar (C) and neocortical (D) organoids at D30 and D50 of differentiation. (E,F) Quantification of cCas3-positive area within the SOX2⁺ area showed no significant difference in cCas3 expression between PCH2a and control in cerebellar (E) and neocortical (F) organoids at D30 and D50 of differentiation. ns, not significant, P>0.05 (two-tailed unpaired t-test with Welch's correction assuming unequal SDs).

Fig. 6.

PCH2a cerebellar organoids show earlier establishment of dense SOX2⁺ structures, whereas neocortical organoids demonstrate no difference in SOX2⁺ structures. (A,B) Epifluorescence images of immunohistochemistry on cerebellar (A) and neocortical (B) organoids at D30 and D50 of differentiation show the expression of SOX2 (NPCs) in control (left) and PCH2a (right) organoids. Representative images of cerebellar organoids (A) are from CO57, P18, and PCH02, P17 (D30); and CO57, P18, and PCH03, P19 (D50); those for neocortical organoids (B) are from CO57, P18, and PCH02 P17 (D30); and CO22, P19, and PCH02, P17 (D50). (C,D) Quantitative analysis of the area covered by SOX2⁺ structures normalized to the area of the organoid. (C) PCH2a cerebellar organoids showed a significantly higher proportion of dense SOX2⁺ structures at D30, whereas control organoids showed these structures at D50. (D) Neocortical organoids did not show significant differences at D30 and D50. (E,F) Confocal images of immunohistochemistry against Ki67 (magenta) and Tuj1 (green) in cerebellar (E) and neocortical (F) organoid sections at D50 illustrate the expression of Ki67 in PCH2a (right) and control (left) organoids within SOX2⁺ structures. Representative images for cerebellar organoids (E) are from CO57, P18, and PCH03, P19 (D30); and CO57, P18, and PCH03, P19 (D50); and those for neocortical organoids (F) are from CO60, P19, and PCH02, P17 (D30); and CO60, P19, and PCH02, P17 (D50). The PCH2a cerebellar organoid at D30 is also used to illustrate quantification in Fig. S5A-C. (G,H) Quantitative analysis of percentage of Ki67⁺ area normalized to SOX2⁺ area in cerebellar and neocortical organoids at D30 and D50 of differentiation. (G) PCH2a cerebellar organoids showed a significantly higher proportion of Ki67⁺ area at D30. This difference was reversed at D50 of differentiation. (H) Neocortical organoids did not demonstrate significant differences at D30 and D50 of differentiation in the Ki67⁺/SOX2⁺ area. ns, not significant, P>0.05; *P<0.05; ***P<0.001 (two-tailed unpaired t-test with Welch's correction assuming unequal SDs).