Axonal Extensions along Corticospinal Tracts from Transplanted Human Cerebral Organoids

Abstract

The reconstruction of lost neural circuits by cell replacement is a possible treatment for neurological deficits after cerebral cortex injury. Cerebral organoids can be a novel source for cell transplantation, but because the cellular composition of the organoids changes along the time course of the development, it remains unclear which developmental stage of the organoids is most suitable for reconstructing the corticospinal tract. Here, we transplanted human embryonic stem cell-derived cerebral organoids at 6 or 10 weeks after differentiation (6w- or 10w-organoids) into mouse cerebral cortices. 6w-organoids extended more axons along the corticospinal tract but caused graft overgrowth with a higher percentage of proliferative cells. Axonal extensions from 10w-organoids were smaller in number but were enhanced when the organoids were grafted 1 week after brain injury. Finally, 10w-organoids extended axons in cynomolgus monkey brains. These results contribute to the development of a cell-replacement therapy for brain injury and stroke.

Keywords: axonal extension; cell transplantation; cerebral cortex; cerebral organoid; corticospinal tract; developmental stage; graft overgrowth; human pluripotent stem cell; nonhuman primate; subcerebral projection neuron.

Graphical abstract

Figure 1

Characterization of Cerebral Organoids at Different Stages Based on Cellular Components (A) Schematic of the conditions used to induce cerebral organoids from hESCs. (B) Bright-field images of 6w- and 10w-organoids induced from hESCs. Arrowheads: organoids shown in (C). Scale bars, 1 mm. (C) Immunohistochemistry for FOXG1, MAP2, PAX6, CTIP2, KI67, and SATB2 in 6w- and 10w-organoids. In 10w-organoids, the layer of CTIP2⁺ cells was thicker, and SATB2⁺ cells appeared. Right three columns: higher-magnification images of the corresponding boxed areas. Scale bars, 500 μm (left four columns) and 50 μm (right three columns). (D) Thickness of the total epithelium, the layer of PAX6⁺ cells, and the layer of CTIP2⁺ cells in 6w- and 10w-organoids. Average thickness in each aggregate was used for the analysis. n = 10 for 6w-organoids, and n = 13 for 10w-organoids (n, number of aggregates). ^∗∗∗∗p < 0.0001; ns, not significant, Mann-Whitney test. Results are presented as the mean ± standard deviation. (E) Percentages of PAX6⁺ cells, KI67⁺ cells, CTIP2⁺ cells, and SATB2⁺ cells in 6w- and 10w-organoids. The whole area of each aggregate was analyzed. n = 10 for 6w-organoids, and n = 13 for 10w-organoids (n, number of aggregates). ^∗∗∗∗p < 0.0001, Mann-Whitney test. Results are presented as the mean ± standard deviation. See also Figure S1.

Figure 2

Graft Overgrowth after Transplantation was Obvious in 6w-Organoids (A) Bright-field images of 6w- and 10w-organoids before and after cutting into pieces. Scale bars, 1 mm. (B) The number of cells contained in 1-mm pieces of 6w- and 10w-organoids. n = 6 for both organoids (n, number of aggregates). ns, not significant; Mann-Whitney test. Results are presented as the mean ± standard deviation. (C) Schematic of the procedure for the transplantation of cerebral organoids into the bilateral frontal and parietal cortices of 7-day-old mice (one piece into each cavity). (D) Immunohistochemistry for hNCAM in sagittal and coronal sections of mouse brains at 12 wpt. Upper left, lateral 1.08 mm; lower left, lateral 1.44 mm; right, Bregma +1.26 mm. Scale bars, 1 mm. (E) The total volume of all survived grafts in each mouse at 12 wpt. n = 8 for each organoid (n, number of mice). ^∗∗∗p < 0.001, Mann-Whitney test. Results are presented as the mean ± standard deviation. (F) The total volume of all survived grafts in the frontal or parietal cortices of each mouse at 12 wpt. n = 8 for each organoid (n, number of mice). ns, not significant; Wilcoxon test. Results are presented as the mean ± standard deviation. See also Figure S5.

Figure 3

Engrafted Cerebral Organoids Obtained Vascularization from the Host (A) Immunohistochemistry for hNCAM and CD31 in engrafted 6w- and 10w-organoids. Cx, cerebral cortex. The right three columns show higher-magnification images of the boxed areas. Scale bars, 500 μm (leftmost column) and 100 μm (other columns). (B) The percentage of the CD31⁺ area to the hNCAM⁺ area in engrafted 6w- and 10w-organoids. n = 8 for each organoid (n, number of mice). ns, not significant; Mann-Whitney test. Results are presented as the mean ± standard deviation.

Figure 4

Cerebral Projection Neurons Were Observed in Engrafted Cerebral Organoids, but 6w-Organoids Contained a Higher Percentage of Proliferative Cells (A) Immunohistochemistry for hNuclei, PAX6, and KI67 in 6w- and 10w-organoids engrafted in the cerebral cortex (Cx). Right column is a magnification of the white squares. Scale bars, 500 μm (left and middle columns) and 20 μm (right column). (B) Percentages of PAX6⁺ cells and KI67⁺ cells in hNuclei⁺ cells in the engrafted 6w- and 10w-organoids. n = 8 for each organoid (n, number of mice). ^∗∗∗p < 0.001, Mann-Whitney test. Results are presented as the mean ± standard deviation. (C) Immunohistochemistry for CTIP2, SATB2, and PAX6 in the engrafted 6w- and 10w-organoids. Right column is a magnification of the white squares. Scale bars, 500 μm (left column) and 20 μm (right column). (D) Percentages of CTIP2⁺ cells and SATB2⁺ cells in hNuclei⁺ cells in the engrafted 6w- and 10w-organoids. n = 8 for each organoid (n, number of mice). ^∗∗∗p < 0.001, Mann-Whitney test. Results are presented as the mean ± standard deviation. See also Figures S2 and S5.

Figure 5

6w-Organoids Extended More Axons along the Host CST (A) Immunohistochemistry for human hNCAM after the transplantation of 6w- and 10w-organoids into the frontal cortex (sagittal section) shows graft-derived axonal extensions to the cerebral cortex (Cx), corpus callosum (CC), and striatum (Str). Scale bars, 200 μm. (B) Immunohistochemistry for hNCAM after the transplantation of 6w- and 10w-organoids into the parietal cortex (coronal section) shows graft-derived axonal extensions to the Cx and CC. Scale bars, 200 μm. (C) Schematic showing sagittal sections of mouse brains, including the (1) Str, (2) internal capsule (IC) and cerebral peduncle (CP), and (3) contralateral spinal cord (SC), and IHC for hNCAM showing graft-derived axonal extensions along the host CST after the transplantation of 6w- and 10w-organoids. Scale bars, 100 μm (left three columns) and 20 μm (rightmost column). (D) Number of hNCAM⁺ axons in the Str, IC, CP, and SC of the host after the transplantation of 6w- and 10w-organoids. 6w-organoids provided significantly more axons along the host CST. Average numbers in both hemispheres in each mouse were used for the analyses. n = 8 for each organoid (n, number of mice). ^∗p < 0.05, ^∗∗p < 0.01, Mann-Whitney test. Results are presented as the mean ± standard deviation. See also Figures S3–S5.

Figure 6

Axonal Extensions from 10w-Organoids were Enhanced by Delayed Transplantation (A) Schematic of the procedure for the transplantation of 10w-organoids into the frontal cortex of 6-week-old mice with or without 1-week (1w) delay after lesioning. n = 6 for both groups (n, number of mice). (B) Immunohistochemistry for hNCAM in coronal sections of mouse brains at 12 wpt with or without 1w delay. Bregma +0.18 mm. Scale bars, 1 mm. (C) The graft volume at 12 wpt. All mice with graft survival were analyzed (n = 4 for no-delay group, and n = 6 for 1w-delay group). ^∗∗p < 0.01, Mann-Whitney test. Results are presented as the mean ± standard deviation. (D) Immunohistochemistry for hNCAM and CD31 in engrafted tissues. The right three columns show higher-magnification images of the boxed areas. Scale bars, 500 μm (leftmost column) and 100 μm (other columns). (E) Percentage of the CD31⁺ area to the hNCAM⁺ area in the engrafted tissues. n = 4 for no-delay group, and n = 6 for 1w-delay group (n, number of mice). ns, not significant; Mann-Whitney test. Results are presented as the mean ± standard deviation. (F) Immunohistochemistry for hNuclei, PAX6, and KI67 in the engrafted tissues at 12 wpt with or without 1w delay. Scale bars, 500 μm. (G) Percentages of PAX6⁺ cells and KI67⁺ cells in hNuclei⁺ cells in the engrafted tissues at 12 wpt with or without 1w delay. n = 4 for no-delay group, and n = 6 for 1w-delay group (n, number of mice). ns, not significant; Mann-Whitney test. Results are presented as the mean ± standard deviation. (H) Immunohistochemistry for CTIP2, SATB2, and PAX6 in the engrafted tissues at 12 wpt with or without 1w delay. Scale bars, 500 μm. (I) Percentages of CTIP2⁺ cells and SATB2⁺ cells in hNuclei⁺ cells at 12 wpt with or without 1w delay. n = 4 for no-delay group, and n = 6 for 1w-delay group (n, number of mice). ns, not significant; Mann-Whitney test. Results are presented as the mean ± standard deviation. (J) Immunohistochemistry for hNCAM in sagittal sections of mouse brain shows graft-derived axonal extensions in the striatum (Str), internal capsule (IC), and cerebral peduncle (CP) of the host at 12 wpt with or without 1w delay. Scale bars, 100 μm. (K) Number of hNCAM⁺ fibers in the Str, IC, and CP of the host at 12 wpt with or without 1w delay. n = 4 for no-delay group, and n = 6 for 1w-delay group (n, number of mice). ^∗∗p < 0.01, Mann-Whitney test. Results are presented as the mean ± standard deviation. See also Figure S6.

Figure 7

Transplantation of 10w-Organoids into Monkey Cerebral Cortex (A) Axial (left) and coronal (middle) T2-weighted images (T2WI) and coronal T1-weighted image (T1WI) immediately after the transplantation. Arrows mark the site of the transplantation. Scale bars, 5 mm. (B) Coronal T2WI and T1WI at 12 wpt, and immunohistochemistry for STEM121 in coronal sections of monkey brain. Scale bars, 5 mm. (C) Immunohistochemistry for STEM121, TUJ1, PAX6, CTIP2, and SATB2 in the engrafted tissue in monkey cerebral cortex. Scale bars, 1 mm (except inset) and 20 μm (inset). (D) Percentages of PAX6⁺ cells, KI67⁺ cells, CTIP2⁺ cells, and SATB2⁺ cells in intragraft cells at 12 wpt. n = 2 (n, number of monkeys). (E) Immunohistochemistry for TUJ1 and CD31 in the engrafted tissue and the host cerebral cortex. The right three columns show higher-magnification images of the boxed areas. Scale bars, 1 mm (leftmost column) and 100 μm (other columns). (F) The percentage of the CD31⁺ area to total graft area. n = 2 (n, number of monkeys). (G) Immunohistochemistry for STEM121 shows graft-derived axonal extensions to the cerebral cortex and in the subcortical tissues toward the corpus callosum and striatum. Scale bars, 5 mm (low magnification images) and 100 μm (high magnification images). See also Figure S7.