Forebrain engraftment by human glial progenitor cells enhances synaptic plasticity and learning in adult mice.

Human astrocytes replace host glia in mice engrafted with human glial progenitors(A) Schematic outlining the procedure for magnetic cell sort-based isolation (MACS) of human glial progenitors, tagging with EGFP, and xenografting at P1. The chimeric mice brains were analyzed in 0.5-20 months old chimeric mice. (B) Representative dot map showing the distribution of human nuclear antigen (hNuclei)+ cells in 3 coronal sections from a 10 months-old human chimeric mice. (C) The complex fine structure of human astrocytes in chimeric brain replicates the classical star-shaped appearance of human astrocytes labeled with hGFAP in situ. Most cells in the field are EGFP+/hNuclei+/hGFAP+ (hGFAP, red). (D) At 5 months, EGFP+ cells typically infiltrated corpus callosum and cortical layers V and VI. All EGFP+ cells labeled with an antibody directed against human nuclear antigen (hNuclei) and most of the human cells were also labeled with an antibody directed against human GFAP (hGFAP, red). (E) At 11 months, many areas of cortex were infiltrated by evenly distributed EGFP+ /hNuclei+ cells. (F) The hippocampus was also populated with EGFP+/hNuclei+ cells in a 14 months old animal, with the highest density in the dentate. (G) Human EGFP+/hNuclei+/GFAP+ cells (green arrow) were significantly larger than host murine astrocytes (red arrow). The anti-GFAP antibody cross-reacted with both human and mouse GFAP (red). Inset shows same field in lower magnification. (H) Histogram compares the diameter of mouse cortical astrocytes to human cortical astrocytes in situ (freshly resected surgical samples) and xenografted human astrocytes in cortex of chimeric mouse brain. The maximal diameter of mouse and human astrocytes (in situ and in chimeric mice) was determined in sections stained with an anti-GFAP antibody that labels both human and mouse GFAP. (n=50-65; **, p<0.01, Bonferroni t-test. (B-F): EGFP (green); hNuclei (white and white arrow); DAPI (blue).Scale: 50 μm (C); 100 μm (D-F); and 10 μm (G). Data graphed as means ± SEM.See also: Supplementary Figure S1.

Human astrocytes retain hominid-specific morphology in chimeric miceHuman protoplasmic astrocytes matured in a cell-autonomous fashion in the chimeric mouse brain environment, retaining the long GFAP+, mitochondrial-enriched processes of native human astroglia. (A) An EGFP+/hGFAP+ astrocyte makes contact with the vasculature in a one month-old chimeric mice. (B) Long, unbranched EGFP+ and hGFAP+ astrocytic processes terminated (dashed circle) on the vasculature (laminin; white) 16 days after implantation. (C) The tortuous shape of EGFP+/hGFAP+ processes in chimeric brains replicate the appearance of GFAP+ processes of interlaminar astroglia in intact human tissue. (D) An example of EGFP+/GFAP+ process that spans > 600 μm and penetrates the domains of at least 14 host murine astrocytes (white arrow), (GFAP, red). (E) Long EGFP+ processes contain a large number of mitochondria (white) in an 11 month-old chimeric mouse. (F) An EGFP+/hGFAP+ human astrocyte express C×43 (white) gap junction plaques (left panel). An EGFP+ cell (green arrow) loaded with a small gap junction permeable tracer, Alexa 594 (MW 760) in a cortical slice (P15). Alexa 594 (red) diffused into multiple neighboring EGFP- cells (red arrow). (G) Co-existence of hGFAP+ (red)/hNuclei+ (white) cells (red arrow) and hNG2+(green)/hNuclei+ cells (green arrow) in the dentate of a 12 months old chimeric mouse. (A-F): EGFP (green); (A-C): hGFAP (red).Scale bars: 10 μm (A, C); 20 μm (B, E,G); 50 μm (D) and (F) right panel; 5 μm (F) left panel.See also: Supplementary Figure S2.

Functional properties indicate high-density host engraftment by both human glial progenitors and astrocytes(A) Large and symmetric EGFP+ cell (green) in an acute cortical slice prepared from a mouse engrafted with human glial progenitors 4 months earlier. Inset: lower magnification of the same field. The EGFP+ cell was loaded filled with Rhod2 (red) by a path pipette. Rhod2 diffused into several neighboring EGFP- cells (white arrows, top panel). Cell identity was verified by immunolabeling against GFAP (red, below panel). Neighboring cells were GFAP+ and their shape characteristic of mouse astrocytes indicating that the human EGFP+/GFAP+ astrocytes was couple by functional gap junctions to host GFAP+ astrocytes. (B) I-V curves from host mouse astrocytes n=17; smaller, less complex EGFP+ cells, presumably glial progenitor cells, n=14; and large and symmetric EGFP+ cells, presumably astrocytes, n=37. (C, D) Comparison of the input resistance and gap junction coupled cells detected as number of neighboring cells labeled with Alexa 594. Mouse and large EGFP+ cells (presumed human astrocytes) have a low input resistance, and are extensively coupled by gap junctions. In contrast, small EGFP+ cells - presumed human GPCs - exhibited high input resistance and were not gap junction coupled. n=14-37, *; p < 0.05, Steel-Dwass test. (E) Photolysis of caged Ca2+ in an EGFP+ astrocytic process. white “X” shows initiated point; white arrowhead shows Ca2+ propagation. (F) Top panel, line scan position across the length of a mouse astrocyte filled with NP-EGTA and rhod2. Below, line scan image of an intra-astrocytic Ca2+ wave initiated by photolysis of the cell body. White dashed line indicates the velocity of the intracellular Ca2+ wave. (G) Line scan image of a human astrocyte in a chimeric mouse (H) Comparison of velocities of intracellular Ca2+ waves in host murine and engrafted human EGFP+ astrocytes, and in human astrocytes in freshly resected surgical tissue. n=8-35, *; p < 0.05, Steel-Dwass test.Scale: 30 μm (A); 100 μm (A) insert; 20 μm (B); 10 μm (E). Data graphed as means ± SEM.

Strengthening of excitatory transmission and synaptic plasticity in murine brain by engrafting of human glial cells(A) Comparison of field EPSPs (fEPSPs) in humanized chimeric mice and their unengrafted littermate and mouse GPC allografted controls. The slopes of fEPSP were significantly increased in human chimeric mice. (n=3-40; F=3.15, by two-way ANOVA with Bonferroni post hoc t test; *p<0.05) (B) Induction of LTP by 2-trains of high frequency stimulation (each train consisted of 100 pulses at 100 Hz, 30 s between bursts) in human chimeric mice, but not in unengrafted littermates and allografted mice. (n=7 mice each group); *, p < 0.05, t test compared between before and 60 min after the stimulation for each group (C) Relative decreased percentage of fEPSP by addition of NMDA receptor antagonist APV (50μM) in each group (n=15-27). (D) The adenosine A1 receptor antagonist, DPCPX failed to increase the fEPSP slope in unengrafted rag2 controls (100 nM DPCPX, n=8, p>0.05, Bonferroni test). (E) The adenosine A1 receptor antagonist, DPCPX did not decrease the threshold for induction of LTP in unengrafted controls; the fEPSP slope returned to 101.9 ± 3.6% by 60 min after HFS, similar to the rate of extinction in untreated slices (n = 8, t test).Data graphed as means ± SEM.See also: Supplementary Figure S3.

Astrocytic TNFα contributes to LTP facilitation in chimeric mice, which is attenuated by thalidomide(A) Hippocampal slices prepared from littermate control rag1 mice exhibit a potentiation of fEPSP in response to TNFα (n=6, 12-16 months, *p<0.05; **: p<0.01; Bonferroni post hoc t test). Inset: fEPSP slopes plotted as a function of fiber volley amplitude. (B) Hippocampal slices exposed to TNFα (600 nM; 2-4 hrs.) exhibited an increase in the intensity of GluR1 subunit of AMPA receptors immunolabeling, but not of the NR1 subunit of NMDA receptors (n=5, 9-11months, **p<0.01, t-test). (C) Human chimeric mice exhibit higher intensity of immunolabeling against TNFα and GluR1, but not of NR1 (n=7, 7-20 months, *p<0.05, **p<0.01, t-test). (D) Thalidomide also decreased the immunolabeling of TNFα and GluR1, but not of NR1 in chimeric mice (n=6, 12-16 months, *; p<0.05, **; p<0.01, t-test). (E) The facilitation of LTP in chimeric mice was impaired by thalidomide (n=6, 12.6 ± 0.3 vs. 12.5 ± 0.5 months-old respectively, means ± SEM; p<0.05, t test). (F) Thalidomide did not change the contribution of NMDA receptor activation to fEPSP. Recordings of fEPSPs were obtained before and after addition of the NMDA receptor antagonist APV (50μM), and the difference calculated (n=4). (G) Phosphorylation of the Ser831 site of GluR1 was increased in chimeric mice compared with unengrafted littermate controls. Thalidomide attenuated the increase in phosphorylation of the Ser831 site of GluR1, but had no effect in unengrafted littermate controls, white arrows shows hNuclei+ cells. (n=6, 9-16 months, *p<0.05, t-test).Scale bar: 100 μm (B,C,D,G). All data graphed as means ± SEM.See also: Supplementary Figure S4.

Humanized chimeric mice learn faster than controls(A) Auditory fear conditioning assessed in a cohort of human chimeric, mouse chimeric, and unengrafted control rag1 mice. Chimeric mice exhibit prolonged freezing behavior in test chamber 2, during exposure to the tonal conditioned stimulus when compared to unengrafted mice and allografted mice (n = 5-20; *, p<0.05; **, p<0.01; two-way repeated measures ANOVA with Bonferroni test; means ± SEM). This difference persisted throughout all 4 days. (B) Contextual fear conditioning in human glial-chimeric mice and littermate controls. Freezing behavior was quantified for chimeric and unengrafted littermate controls during the two minutes of acclimatization period (n = 6; *, p<0.05; **, p<0.01; two-way repeated measures ANOVA with Bonferroni test). In addition the mean discrimination ratio for each day was obtained from freezing scores in the training chamber and the alternative chamber (freezing in training chamber/ total freezing time). Chimeric mice demonstrated significantly higher abilities to discriminate the chambers (n = 8-13; *, p<0.05; **, p<0.01; two-way repeated measures ANOVA with Bonferroni test). (C) Barnes maze testing in chimeric and unengrafted littermate controls. Chimeric mice demonstrated a significant learning advantage, as reflected in a shorter latency and fewer errors in solving the maze (n = 6; *, p<0.05; **, p<0.01; two-way repeated measures ANOVA with Bonferroni test). (D) Object-Location Memory Task (OLT) in chimeric mice and their unengrafted littermate controls demonstrated a learning advantage in chimeric mice via enhanced recognition of the novel displaced object. Thalidomide eliminated the learning advantage of chimeric mice suggesting the learning enhancement was TNF-α mediated (n = 7; **, p < 0.01; one-way ANOVA with Bonferroni test).All data plotted as means ± SEM.See also: Supplementary Figure S5.