Vascularization and Engraftment of Transplanted Human Cerebral Organoids in Mouse Cortex - PubMed (original) (raw)
Vascularization and Engraftment of Transplanted Human Cerebral Organoids in Mouse Cortex
Nicolas Daviaud et al. eNeuro. 2018.
Abstract
Neural stem cells (NSCs) hold great promise for neural repair in cases of CNS injury and neurodegeneration; however, conventional cell-based transplant methods face the challenges of poor survival and inadequate neuronal differentiation. Here, we report an alternative, tissue-based transplantation strategy whereby cerebral organoids derived from human pluripotent stem cells (PSCs) were grafted into lesioned mouse cortex. Cerebral organoid transplants exhibited enhanced survival and robust vascularization from host brain as compared to transplants of dissociated neural progenitor cells (NPCs). Engrafted cerebral organoids harbored a large NSC pool and displayed multilineage neurodifferentiation at two and four weeks after grafting. Cerebral organoids therefore represent a promising alternative source to NSCs or fetal tissues for transplantation, as they contain a large set of neuroprogenitors and differentiated neurons in a structured organization. Engrafted cerebral organoids may also offer a unique experimental paradigm for modeling human neurodevelopment and CNS diseases in the context of vascularized cortical tissue.
Keywords: CNS injury; cerebral organoid; neural stem cell transplant; vascularization of intracerebral graft.
Figures
Graphical abstract
Figure 1.
Schematic depiction of intracranial transplantation of human cerebral organoids. A, hES cells were differentiated into NPC or cerebral organoids. Stereotactic surgery was performed to transplant one single cerebral organoid in the lesioned frontoparietal cortex in P8–P10 mice. For NPC transplantation, dissociated NPC were implanted into identical cortical region by stereotactic injection. B, Experimental timeline. P8–P10 immunocompetent mice were used as recipients for transplantation of dissociated human NPC or human cerebral organoids. Histologic analyses of the grafts were performed two or four weeks after grafting. C, Schematic diagrams (top) and intraoperative photographs (bottom) of cerebral organoid transplantation. Briefly, from left to right, once scalp is reflected, a small craniotomy window was opened with the bone flap hinged at the anterior base, and a small piece of the frontoparietal cortex (∼1 mm3) was removed. A single cerebral organoid was then implanted into the prelesioned mouse cortex, followed by return of the bone flap to close the craniotomy window, sealed with fibrin glue, followed by scalp closure.
Figure 2.
Characterization of GFP-labeled NPC and cerebral organoids derived from hESC. A, Diagram of lentiviral vector expressing EGFP (enhanced GFP) driven by EF-1a promoter to label hES cells. B, Phase-contrast and fluorescent images of GFP-labeled hES cells (left) and expression of pluripotency markers Oct4 (green) and Nanog (red; right). C, Timeline of differentiation of hES cells into NPC. D, Phase-contrast and fluorescent images of human NPC derived from GFP-labeled hES cells. E, Representative immunofluorescence images of NPC stained for proliferation marker Ki67 and the indicated neural markers. F, Timeline of derivation of cerebral organoids from hESC. G, left panels, Phase-contrast and fluorescence images of GFP-labeled EB or Matrigel-embedded cerebral organoids. Right panel, Immunofluorescent images of sectioned cerebral organoids stained for GFP. H, Phase-contrast image of cerebral organoid at day 42 of culture and immunofluorescent images of sectioned day 42 cerebral organoid stained for the indicated markers. V: ventricle-like structures. Note the proliferative zone (Ki67+) and Sox2+ neuroprogenitors at the VZ/SVZ, and DCX+ neuroblasts in the outer layer.
Figure 3.
Engraftment and survival of NPC and cerebral organoid transplants. A, Representative immunofluorescence images of NPC transplant (left) and cerebral organoid transplant (right) at the indicated time points post-grafting. Enlarged images of the boxed area are shown at the bottom. D: dorsal, V: ventral, M: medial, L: lateral. B, Quantification showing the relative size of GFP-positive area of NPC and organoid transplants at two and four weeks after grafting. A significant decrease of the graft size of the NPC transplants was detected at four weeks as compared to two weeks after grafting. C, Representative immunofluorescence images for hMito (human mitochondria) and AC3 in NPC transplants (left) or in cerebral organoid transplants (right) at the indicated time points after transplantation. D, Quantification showing the number of AC3-positive apoptotic cells per unit area of hMito-positive grafts at the indicated time points. E, Representative immunofluorescence images showing a high number of apoptotic cells (AC3+) in stage-matched cerebral organoids in culture at six and eight weeks. F, Representative immunofluorescence images of NPC transplant (left) and cerebral organoid grafts (right) 3 or 5 d after transplantation. At these early time points, grafts had not yet been firmly integrated into host brains. Dashed white lines delineate the graft areas; *p < 0.05; n.s., non-statistically significant. Two-way ANOVA followed by a Tukey post hoc test; n = 3 mice for each time point and two images from each mouse.
Figure 4.
Host immune response following NPC and organoid transplants. A, Representative immunofluorescence images of Iba1 in hMito-labeled grafts, at two and four weeks after transplantation. Note the hypertrophied Iba1+ microglia inside the NPC graft, as well as in host brain tissue adjacent to the NPC graft (white arrowhead). B, Representative immunofluorescence images of CD45 in GFP-labeled grafts at indicated time points. Dashed white lines delineate the graft areas. Enlarged images of the boxed area are shown on the right. D: dorsal, V: ventral, M: medial, L: lateral.
Figure 5.
Vascularization of engrafted cerebral organoids. A, Representative immunofluorescence images demonstrate penetration of host CD31-positive blood vessels into hMito+ NPC grafts (left panels) or cerebral organoid grafts (right panels) at the indicated time points post-transplantation. Notice that CD31-positive endothelial cells inside the grafts are hMito-negative (white arrowheads). White arrows: host vasculature. Dashed white lines delineate the graft areas. Enlarged images of the boxed area are shown on the right. D: dorsal, V: ventral, M: medial, L: lateral. B, Quantifications of the number (left) and the average length (right) of CD31-positive blood vessels in hMito-labeled grafts demonstrate a higher number of vasculatures in the engrafted cerebral organoids compared to NPC transplants at four weeks after transplantation, but no significant difference in the average vascular length; *p < 0.05; n.s., non-statistically significant; n = 3 mice for each cohort, and at least two images analyzed from each mouse. Two-way ANOVA followed by a Tukey post hoc test.
Figure 6.
Cell proliferation and NSC pool in cerebral organoid transplants. A, Representative immunofluorescence images of NPC (left panels) and cerebral organoid transplants (right panels) stained for GFP and proliferation marker Ki67. D: dorsal, V: ventral, M: medial, L: lateral. B, Quantification (top) showing a higher density of Ki67+ cells per unit GFP+ area in cerebral organoid than in NPC transplants at both two and four weeks after transplantation. Bottom quantification: percentage of Ki67+/DAPI+ cells within the GFP+ cerebral organoid and NPC grafts. C, Representative immunofluorescence images of NPC (left panels) and cerebral organoid transplants (right panels) showing abundant engrafted cells (GFP+) expressing stem cell marker Sox2 (red). White arrows: Sox2+ OPC in host cortical tissue. D, Quantification (top) showing no significant difference of the density of Sox2+ cells per unit GFP+ area between NPC and cerebral organoid transplants at either time points. Bottom quantification: percentage of Sox2+/DAPI+ cells within the organoid and NPC grafts showing no significant difference between the two types of transplants at either timepoint. Dashed white lines delineate the graft areas. Enlarged images of the boxed area are shown on the right; *p < 0.05. **p < 0.01; n.s., non-statistically significant. Two-way ANOVA followed by a Tukey post hoc test; n = 3 mice for each time point and at least two images from each mouse.
Figure 7.
Stage-matched in vitro cerebral organoid characterization. A, Representative immunofluorescence images of cultured cerebral organoids at six or eight weeks of maturation show layered organization of cortical-like tissue with proliferating cells (Ki67+) and NPC (SOX2+) mainly localized in the VZ/SVZ and neurons (CTIP2+) localized in the outer layer. B, Representative immunofluorescence images of cerebral organoids after six or eight weeks of in vitro maturation. Left, A low number of astrocytes (GFAP+) was present in the organoids after eight weeks of maturation (white arrow), while no Olig2+ cells were detected. Right, Abundant neuroblasts (DCX+) were found at both time points while low level of expression of NF-H was detected at eight weeks of maturation.
Figure 8.
Neurodifferentiation of the engrafted C-organoids. A, Representative immunofluorescent images of DCX-positive neuroblasts in NPC (left panels) and cerebral organoid transplants (right panels) at the indicated time points. D: dorsal, V: ventral, M: medial, L: lateral. B, Quantification of DCX immunointensity per unit area of GFP+ grafts. A significant stronger staining intensity of DCX was measured in organoid compared to NPC transplants after four weeks. C, Representative immunofluorescence images of GFAP+ cells in hMito-labeled NPC (left) and organoids grafts (right) at the indicated time points. White arrows denote colocalization of hMito and GFAP markers in transplants. D, Quantification of GFAP immunointensity per unit area of hMito+ grafts. A significant stronger staining intensity of GFAP was measured in organoid transplants after four weeks. E, Representative immunofluorescence images of Olig2+ cells in the engrafted cerebral organoids at the indicated time points. White arrows: Olig2+ cells in host cortical tissue. F, Quantification shows no significant difference in the percentage of Olig2+/DAPI+ cells in the organoid grafts between two and four weeks after grafting. Dashed white lines delineate the graft areas. Enlarged images of the boxed area are shown on the right; *p < 0.05. **p < 0.01. ***p < 0.001; n.s., non-statistically significant. Two-way ANOVA followed by a Tukey post hoc test and Student’s t test; n = 3 mice for each time point and two images from each mouse.
Figure 9.
Differentiation of cerebral organoid transplants. A, Representative immunofluorescence images showing cells expressing intermediate progenitor marker TBR2 in the engrafted cerebral organoids at two and four weeks after transplantation. Notice no TBR2+ cells were observed in host cortical tissue and a slight decline of the number of TBR2+ cells in the hMito+ organoid grafts from two to four weeks after transplantation. B, Representative immunofluorescence images showing cells expressing deep layer neuronal marker CTIP2 in the engrafted cerebral organoids at two and four weeks after transplantation. Notice CTIP2+ cells in neighboring host cortex (white arrows), while in the hMito+ organoid grafts, there was an increase of CTIP2+ cells from two to four weeks after transplantation. C, Representative immunofluorescence images show expression of NF-H in the transplanted organoids after two and four weeks. White arrowheads denote colocalization of NH-H and hMito in organoid transplants. Dashed white lines delineate the graft areas. D: dorsal, V: ventral, M: medial, L: lateral.
References
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