Human-derived neural progenitors functionally replace astrocytes in adult mice

Abstract

Astrocytes are integral components of the homeostatic neural network as well as active participants in pathogenesis of and recovery from nearly all neurological conditions. Evolutionarily, compared with lower vertebrates and nonhuman primates, humans have an increased astrocyte-to-neuron ratio; however, a lack of effective models has hindered the study of the complex roles of human astrocytes in intact adult animals. Here, we demonstrated that after transplantation into the cervical spinal cords of adult mice with severe combined immunodeficiency (SCID), human pluripotent stem cell-derived (PSC-derived) neural progenitors migrate a long distance and differentiate to astrocytes that nearly replace their mouse counterparts over a 9-month period. The human PSC-derived astrocytes formed networks through their processes, encircled endogenous neurons, and extended end feet that wrapped around blood vessels without altering locomotion behaviors, suggesting structural, and potentially functional, integration into the adult mouse spinal cord. Furthermore, in SCID mice transplanted with neural progenitors derived from induced PSCs from patients with ALS, astrocytes were generated and distributed to a similar degree as that seen in mice transplanted with healthy progenitors; however, these mice exhibited motor deficit, highlighting functional integration of the human-derived astrocytes. Together, these results indicate that this chimeric animal model has potential for further investigating the roles of human astrocytes in disease pathogenesis and repair.

Figure 6. Transplantation of astrocytes from patients with ALS results in mouse movement deficits.

(A) Mice receiving ALS astrocyte transplant show decreased grip strength (mean ± SEM) at 9 months after transplantation, as compared with those receiving WT astrocyte transplant (n = 6, *P < 0.05, 1-way ANOVA followed by Tukey’s multiple comparison tests). (B–E) Treadscan analysis reveals (B) decreased stride length, (C) average print area, (D) decreased maximum longitudinal deviation, and (E) increased stance time in mice receiving ALS astrocyte transplant, as compared with those receiving WT astrocyte transplant (n = 6, *P < 0.05, 1-way ANOVA followed by Tukey’s multiple comparison tests). Data represent mean ± SEM.

Figure 5. Integration of astrocytes from patients with ALS and its effect on mouse MNs.

(A) Longitudinal distribution of hGFAP-expressing astrocytes (red) as well as endogenous astrocytes (green) in the spinal cord. Scale bar: 500 μm. (B) Human astrocytes (from iPSCs) in the gray matter and white matter exhibit typical astrocyte morphologies. Scale bar: 50 μm. (C) Mouse MNs decreased in number in the ALS astrocyte–transplanted group compared with those in the WT astrocyte group. ChAT⁺ cells in the boxed regions of the ChAT/hGFAP images are magnified to their right, and the boxed ChAT images are further magnified to their right; arrows indicates a MN with lower ChAT intensity. Scale bar: 50 μm. (D) Quantification of mouse MNs (mean ± SEM) on the transplanted versus contralateral side (n = 6, *P < 0.05, 1-way ANOVA followed by Tukey’s multiple comparison tests).

Figure 4. Human astrocytes structurally integrate into the host tissue.

(A) In the gray matter, the hGFAP⁺ astrocytes (from iPSCs) presented with a star-shaped morphology, surrounding neurons, including ChAT⁺ MNs (inset). (B) In the white matter, the human astrocytes (from iPSCs) extended long processes that line up with the neurofilament-positive (NF⁺) axons. (C) The distribution of GLT1 is similar between the transplanted side and the untransplanted side, with a higher density in the gray matter than in the white matter. (D) In the gray matter, CX43 was predominantly in the hGFAP⁺ astrocyte processes, especially in the area surrounding neuronal cell bodies. (E) In the white matter, CX43 immunoreactivity was present in the cytoplasm. (F) Human astrocytes (GFP⁺/GFAP⁺, from ESCs) projected to the blood vessel, with their end feet closely surrounding the blood vessel. (G) The AQP4 signals were largely concentrated on the astrocytic end feet along the vessels. Scale bar: 50 μm (A–C); 10 μm (D–G).

Figure 3. Mouse astrocytes undergo cell death in the adult spinal cord.

(A) The mouse cells (hNu^–) and human cells (hNu⁺, from iPSCs) were positive for caspase-3 at 3, 5, and 9 months after transplantation. Arrows indicate mouse cells positive for caspase-3; arrowheads indicate human cells positive for caspase-3. Scale bar: 50 μm. HO, Hoechst 33258. (B) Mouse astrocytes (GFAP⁺/GFP^–, arrows) and human astrocytes (GFAP⁺/GFP⁺, arrowheads, from ESCs) were positive for caspase-3 at 3, 5, and 9 months after transplantation. Scale bar: 10 μm. (C) Few mouse cells in untransplanted side were positive for caspase-3 at 3, 5 and 9 months. Scale bar: 50 μm. (D) Quantification of caspase-3–positive populations (mean ± SEM) (numbers above bars) in human (hNu⁺) and mouse (hNu^–) cells in transplanted side and contralateral side at 3, 5, and 9 months (n = 3).

Figure 2. Human neural progenitors differentiate into astrocytes and replace endogenous astrocytes.

(A and B) hGFAP-expressing astrocytes began to appear at 3 months and became the predominant population by 9 months. (A) The GFP overlapped with (B) hGFAP at 5 and 9 months. (C) The longitudinal distribution of hGFAP-expressing astrocytes (red) as well as endogenous astrocytes (green) in the spinal cord. Scale bar: 500 μm. (D) Human astrocytes (from iPSCs) in the gray matter and white matter exhibit typical astrocyte morphologies. Scale bar: 50 μm. (E) Quantification (mean ± SEM) of all astrocytes (GFAP⁺) in the transplanted and nontransplanted sides (n = 3, P > 0.05, t test), and the average population of mouse astrocytes (GFAP⁺/hGFAP^–) and human astrocytes (GFAP⁺/hGFAP⁺) in the grafted spinal cord at 9 months.

Figure 1. Human cells survive and migrate in the adult spinal cord.

(A) Distribution of hNu⁺ and GFP⁺ cells (from ESCs) in the spinal cord over time (0.5 to 9 months). (B) The dividing human cells (from iPSCs) were revealed by Ki67⁺/hNu⁺ over time. Scale bar: 50 μm. (C) Human cells (marked by GFP) in the spinal cord migrated longitudinally to both rostral (A) and caudal (P) areas, and the distance (mean ± SEM) that hNu⁺ cells spread in the spinal cord was measured (n = 6), with “O” indicating the injection location. (D) Quantification of Ki67⁺/hNu⁺ cells (mean ± SEM) in the spinal cord over time. Ki67 expression in the mouse spinal cord was used as an endogenous control (n = 6). (E) Longitudinal view of the distribution of hNu⁺ cells along the spinal cord. Scale bar: 500 μm.