Astrocytes Transplanted during Early Postnatal Development Integrate, Mature, and Survive Long Term in Mouse Cortex

Transplanted immature cortical astrocytes integrate into the developing cortex, display complex morphologies, and survive for greater than one year in vivo

To investigate how cortical astrocytes integrate, mature, and survive in the developing and mature brain, we developed a transplantation approach allowing us to transplant astrocytes from genetically modified donor mice to recipient mice (Fig. 1A). We prepared immature cortical astrocytes from postnatal day 4 to 8 (P4–P8) mice containing a tamoxifen-inducible CreERT2 allele driven by the astrocyte-specific aldehyde dehydrogenase family 1, member L1 (Aldh1l1) promoter (Srinivasan et al., 2016) and a floxed-stop tandem-dimer Tomato allele (Madisen et al., 2010; Ai9, now referred to as Tomato; Fig. 1B). Before transplantation, astrocytes were cultured, passaged, and treated with tamoxifen to induce Tomato expression (Fig. 1C) and analyzed by immunolabeling and confocal microscopy (Fig. 1D,E). Since Aldh1l1-CreERT2 drives Cre-mediated recombination specifically in astrocytes (Srinivasan et al., 2016), we expected that the majority of Tomato-positive (Tom+) cells would express the astrocytic marker Sox9 following tamoxifen induction. Consistent with this, 97.6 ± 1.5% of Tomato+ cells were positive for the astrocytic protein Sox9 (Fig. 1E), indicating a high degree of specificity of Cre recombination in astrocytes. Importantly, however, 83.7 + 0.7% of Sox9+ astrocytes expressed Tomato, indicating that Cre-mediated recombination in astrocytes was high, but not complete. Given the lineage relationships between astrocytes and oligodendrocyte precursors (OPCs) (Kirdajova et al., 2021), we also labeled for the proteoglycan NG2 to test for the presence of Sox9+/NG2+ intermediate astrocytes (Zhu et al., 2012; Zuo et al., 2018), and Sox9−/NG2+ OPCs. We found that a fraction of Tom+/Sox9+ cells were also NG2+ (11.16 ± 3.23%); however, only 1 ± 0.5% Tom+/Sox9− cells expressed NG2. The remaining fraction of Tom+ cells (1.5 ± 1%) were SOX9− and NG2− and presumably precursors that have not differentiated into Sox9-positive astrocytes. These findings indicated that the majority of cells in our cultures were astrocytes and expressed Tomato following Cre-mediated recombination.

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Figure 1.

Cortical astrocytes transplanted into the immature cortex display protoplasmic astrocyte morphology starting within a few days after transplantation. A, Overview of the experimental design for transplantation of cortical astrocytes into the cerebral cortex of mouse pups. Tam = 4-hydroxytamoxifen. Figure created with BioRender. B, Schematic showing the genetic background of the mice used as transplant donors. Tamoxifen = 4-hydroxytamoxifen. C, Timeline of culture preparation. D, Cultured astrocytes expressing Tomato following tamoxifen administration. Tom = Tomato. E, Percentage of Tomato+ cells in vitro expressing Sox9 (astrocytes), Sox9 and NG2 (intermediate astrocytes), NG2 (OPCs), or neither marker (“Other”). F, Timeline of astrocyte transplantation. G, Transplanted astrocytes at different days post-transplantation (DPT). Transplanted cells are labeled with Tomato (TOM) and astrocytes are detected by SOX9 immunolabeling (green). Arrows indicate astrocyte endfeet. 35 DPT = 35–47 DPT, 110 DPT = 110–130 DPT. Scale bar: 20 µm (D) and 50 and 10 µm (G, top panels, and G, middle and bottom panels, respectively).

Following in vitro analysis, astrocytes were then harvested and transplanted into the cortex of P3–P7 mice, a time point when astrocytes are actively proliferating and developing in cortex (Ge et al., 2012; Clavreul et al., 2019). The brains of recipient mice were subsequently recovered at different time points after transplantation and analyzed for their expression of known astrocytic proteins (Fig. 1F). Analysis of recipient brains at 7 d post-transplantation (DPT) revealed the presence of Tom+/Sox9+ astrocytes in the cortex (Fig. 1G). Transplanted astrocytes were found in multiple cortical layers and showed elaborated processes typical of developing astrocytes in the brain (Bushong et al., 2004). In addition, we encountered many Tom+/Sox9+ cells adjacent to the pial surface, likely representing transplanted astrocytes that escaped the injection site and attached to the pial surface (Figs. 1G, 2A). These represented 59.8 ± 4.41% (7 DPT) and 61.05 ± 3.39% [368–570 (368+) DPT] of transplanted astrocytes, excluding the astrocytes trapped at the injection site. Quantifying the distribution of parenchymal Sox9+ astrocytes in brains collected at 35–47 DPT, we found that most transplanted astrocytes (77.76%) were localized in layer 1. A smaller fraction of transplanted astrocytes was observed in deeper layers (∼22%): layers 2–3 (10.04%), layer 4 (4.42%), and layers 5–6 (7.78%; Fig. 2B). At 35–47 DPT and 110–130 DPT, transplanted astrocytes were retained in the cortex, developed morphologic features characteristic of mature protoplasmic astrocytes, including formation of large, complex arbors (Fig. 1G) and specialized endfeet surrounding blood vessels (Fig. 1G, arrows), and found to have similar lateral migration distances as those found at 7 DPT (Fig. 2C,D) indicating that transplanted astrocytes mostly reach their final position in the cortex by 7 DPT. At 368 DPT, Tomato+ astrocytes were present and continued to maintain their complex morphologies and association with cerebrovasculature (Fig. 1G).

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Figure 2.

Distribution of transplanted Tomato+ astrocytes in cortex. A, Image montage representing the injection track (arrow) in a transplanted cortex at 35–47 DPT. B, Distribution of transplanted astrocytes across cortical layers at 35–47 DPT. Circles represent mean astrocyte number values per mouse for each layer region. L1 = layer 1, L2/3 = layers 2 and 3, L4 = layer 4, L5/6 = layers 5 and 6. C, The lateral migration distance (LMD; yellow dashed line) was defined as the maximum distance between transplanted astrocytes measured along the medio-lateral axis in sections without an injection track. In sections with injection track, the lateral migration distance was measured on the direction perpendicular to the injection track (data not shown). D, Lateral migration distance of transplanted astrocytes at different times post-transplantation. 35 DPT = 35–47 DPT, 110 DPT = 110–130 DPT. Gray circles represent mean LMD values per mouse. The mean LMD for each experimental group (magenta lines) are 0.816 ± 0.075 mm at 7 DPT, 0.676 ± 0.101 mm at 35–47 DPT, and 0.689 ± 0.087 mm at 110–130 DPT. No significant differences were detected between LMD values across post-transplantation times (p = 0.562, one-way ANOVA). Scale bar: 50 µm (A) and 100 µm (C). Data are mean ± SEM.

The majority of parenchymal Tomato+ cells encountered showed morphologic properties typical of protoplasmic astrocytes (Fig. 3A,B). However, on occasion, we detected cells that did not have astrocytic morphology, but instead resembled other types of glia including OPCs (Fig. 3C,D). To account for these cells, we quantified the percentage of Tomato+ cells that were either Sox9+ (astrocytes), NG2+ (OPCs), or Sox9−/NG2− (neither astrocytes or OPCs), across all times points (7, 35–47, 110–130, and 368 DPT). Cells found along the pial surface were excluded from this analysis as it was difficult to resolve individual cells. At all time points, Sox9+ astrocytes with highly ramified morphologies were the most abundant transplanted cell type detected (90.5 ± 4.9% at 7DPT; 90.8 ± 8.4% at 35–47 DPT; 79.5 ± 11.9% at 110–130 DPT; 77.4 ± 9.8% at 368 DPT) with OPCs comprising between 5.6% and 16.1% of transplanted cells (5.6 ± 3.2% at 7 DPT; 8.5 ± 7.7% at 35–47 DPT, 16.1 ± 9% at 110–130 DPT; 8.7 ± 3.8% at 368+ DPT; Fig. 3E). A small group of cells were neither Sox9+ nor NG2+ and often displayed a small round cell body and few thin processes, potentially representing more mature oligodendrocytes (Fig. 3D). These data showed that the vast majority of parenchymal Tomato+ transplanted cells were protoplasmic astrocytes.

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Figure 3.

Characterization of transplanted cells. A–D, While most transplanted Tomato+ cells display a protoplasmic astrocytic morphology (B), some display less ramified, longer processes typical of oligodendrocyte precursor cells (C, OPCs; see also arrows in A) or immature oligodendrocytes (D). E, Cell-specific markers were used to identify the percentage of transplanted cells as Sox9+ astrocytes (B), NG2+ OPCs (C), or cells not expressing either Sox9 or NG2 referred to as “other” cells (D). F, Cultured cells expressing Sox9 and the glial precursor marker Olig2. Tom = Tomato. G, Percentage of TOM+ cultured cells positive for Sox9 only, Sox9 and Olig2, or Olig2 only. Cells not expressing either Sox9 or Olig2 were referred as “other” cells. H, Sox9 expression in transplanted cells at 1 DPT and 3 DPT. I, Left panels, Olig2 expression in transplanted cells at 1 DPT and 3 DPT. Right panel, Percentage of Olig2+ cells among transplanted astrocytes at 1 DPT and 3 DPT. Circles and bars represent mean values per mouse and mean values per group, respectively. The percentage of Olig2+/Sox9+ cells among transplanted astrocytes was significantly lower at 3 DPT compared with 1 DPT (1 DPT, 22.73 ± 1.37; 3 DPT 5.6 ± 0.7; *p = 0.0004 Student’s t test). J, Most transplanted astrocytes have a simple, fusiform shape at 1 DPT (arrowhead), and take on a ramified stellate morphology at 3 DPT (arrow). K, Quantification of cell proliferation (Ki67+ nuclei) among transplanted astrocytes at different times after transplantation. Circles represent mean values per mouse. The mean values per experimental group (bars) are 7.21 ± 2.38% at 1 DPT, 2.67 ± 0.91% at 3 DPT, 2.11 ± 1.26% at 7 DPT and 0 at 35–47 DPT. Significant differences were not detected between the mean values per experimental group across times post-transplantation (p = 0.09, one-way ANOVA). L, Number of transplanted astrocytes per section at 7 DPT and 368–570 DPT (1 YPT). Circles and bars represent mean values per mouse and mean values per group, respectively. Scale bar: 20 µm (A, H–J), 10 µm (B–D), and 50 µm (F). Data are mean ± SEM.

Given our previous result showing a large proportion of immature astrocytes and a small proportion of OPCs in pretransplantation cultures (Fig. 1D,E), we further investigated the fate of transplanted cells following their transplantation. We first examined in vitro the degree of co-expression of Sox9 with Olig2, a marker of glial progenitors (Marshall et al., 2005; Clavreul et al., 2019) in pretransplantation cultures. We found that the majority of Tom+ cells (78.77 ± 2.21%) were positive for Sox9 and Olig2 (Fig. 3F,G), while only a small proportion of cells were Olig2+ and Sox9− (6.91 ± 0.86%), indicating that most pretransplantation cells in culture are Olig2+ immature astrocytes. Interestingly, analysis in vivo found that only 22.73 ± 1.37% of transplanted astrocytes were Sox9+/Olig2+ at 1 DPT and that this proportion decreased to 5.6 ± 0.7% by 3 DPT (Fig. 3H,I), suggesting progressive astrocytic maturation during the first few days in vivo following transplantation. This maturation was consistent with the finding that transplanted cells at 1 DPT showed a fusiform, elongated morphology typical of migrating cells. However, by three DPT, most of transplanted cells showed a complex and ramified morphology, typical of more mature astrocytes (Fig. 3J). We also immunolabeled for the cell proliferation marker Ki67 to estimate the fraction of proliferating Tomato+ astrocytes that may give rise to daughter cells through cell division events. This showed that Ki67+ proliferating astrocytes accounted for <10% of transplanted astrocytes at all time points examined (1, 3, 7, and 35–47 DPT; Fig. 3K). We also compared the number of Tomato+ astrocytes per section between brains collected at 7 DPT and 368+ DPT. Importantly, we found no significant difference in the density of transplanted cells/section between these two time points, indicating that there is no significant loss of transplanted astrocytes by 368 DPT (Fig. 3L).

Transplanted astrocytes develop unique morphologic territories and have normal levels of GFAP

Recent studies have shown that astrocytes can display scavenging activity toward damaged cells, opening the possibility that astrocytes phagocytize cellular material from adjacent astrocytes (Wakida et al., 2018). In light of these findings, it was important to determine whether some Tomato+ cells in recipient brains had acquired Tomato labeling by engulfing fragments of injected cells. At the same time, it was important to establish whether transplanted astrocytes could form mutually exclusive territories or “tile” the brain neuropil like astrocytes in normal brain (Ogata and Kosaka, 2002; Bushong et al., 2004). To answer these questions, we transplanted Tomato+ astrocytes into Aldh1l1-eGFP mice which express GFP specifically in astrocytes (Fig. 4A–C). To quantify the overlap between the territories of Tomato+ and GFP+ astrocytes, we acquired images containing both transplanted and nontransplanted astrocytes and calculated the degree of colocalization using Manders’ coefficients (M1 and M2). M1 and M2 represent the fraction of GFP signal colocalized with Tomato and the fraction of Tomato signal colocalized with GFP, respectively. The mean values of these coefficients across all mice (0.037 ± 0.009 for M1 and 0.065 ± 0.014 for M2) indicated that the overlap between transplanted and resident astrocytes constituted <10% of the total area covered by Tomato+ and GFP+ astrocytes (Fig. 4D). This small amount of overlap is in line with that described for adjacent astrocytes in the brain of adult mice (Ogata and Kosaka, 2002). To further validate these results, we calculated the Manders’ coefficients values for astrocytic signals that are expected to display a high degree of overlap. For this, we chose Tomato and the astrocyte marker glutamine synthetase (GS), because both proteins are expressed in the astrocytic cytoplasm. In these measurements, M1 represents the fraction of GS signal colocalized with Tomato, while M2 represents the fraction of Tomato signal colocalized with GS. The mean values obtained for the two coefficients (0.316 ± 0.019 for M1 and 0.956 ± 0.013 for M2; Fig. 4E) were larger than those describing the overlap between Tomato+ transplanted and GFP+ nontransplanted astrocytes. A value lower than 1 for M1 is in line with the fact that in each field used for the coefficient calculation, only transplanted astrocytes expressed both Tomato and GS, and they occupied only a fraction (50% or less) of the field. The M2 value was very close to 1 indicating that Tomato and GS signals overlap almost completely in Tomato+ cells. These results showed that transplanted astrocytes acquired cortical space and formed complementary territories with GFP+ astrocytes. Furthermore, these data confirmed that Tomato+ cells were truly transplanted astrocytes, ruling out the possibility that nontransplanted astrocytes uptake Tomato, which would complicate interpretation of the findings (Coyne et al., 2006).

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Figure 4.

Transplanted astrocytes develop unique morphologic territories and have normal levels of GFAP. A, In the cortex of transplanted Aldh1L1-eGFP mice, the GFP+ and Tomato+ astrocytes show complementary organization. Tom = Tomato. B, C, Intensity values for GFP and Tomato signals were measured along a line drawn across adjacent transplanted and resident astrocytes. GFP and Tomato intensity profiles showed little overlap between the two signals (C). D, E, Manders’ coefficients representing colocalization between GFP and Tomato (D) and colocalization between glutamine synthetase (GS) and Tomato (E) calculated for three mice (Ms1–Ms3) at 35–47 DPT. Gray circles are Manders’ coefficient values calculated for single image fields, whereas lines represent mean coefficient values computed for each transplanted mouse (magenta) and across all mice in the same experimental group (black). In D, M1 and M2 represent the fraction of GFP signal colocalized with Tomato (GFP&TOM/GFP) and the fraction of Tomato signal colocalized with GFP (GFP&TOM/TOM), respectively. In E, M1 and M2 represent the fraction of glutamine synthetase (GS) signal colocalized with Tomato (GS&TOM/GS) and the fraction of Tomato signal colocalized with GS (GS&TOM/TOM), respectively. F, GFAP expression levels were compared between transplanted (magenta dashed line) and nontransplanted astrocytes (green dashed line) by measuring GFAP intensity in territories occupied by the two kinds of astrocytes. Astrocytic processes that were potentially identified as endfeet (asterisk) were excluded from the quantification. G, Transplanted/nontransplanted ratio values for GFAP expression calculated for single image fields (gray circles) in four mice (Ms1–Ms4) at 110–130 DPT. Magenta lines/circles represent mean values per mouse. The mean value across all mice (black line) was not significantly different from 1 (1.007 ± 0.018; p = 1, Wilcoxon rank-sum test). H, GFAP immunolabeling at 35–47 DPT in the superficial layer of the cortex (mouse transplanted at P3–P7). Similar to nontransplanted astrocytes, transplanted astrocytes displayed high GFAP levels close to the pial membrane (arrow) and lower GFAP levels deeper in cortex (arrowhead). I, GFAP immunolabeling at 35–47 DPT at the injection site (mouse transplanted at P3–P7). Transplanted astrocytes express high levels of GFAP at the injection site (arrow). Scale bar: 20 µm (A, H, I) and 10 µm (B, F). Data are mean ± SEM.

To better understand whether transplanted astrocytes replace astrocytes from recipient mice or simply interlace among these cells, we compared the density of astrocytes for cortical regions which received astrocyte transplantation versus the contralateral cortex as a control. Interestingly, we found that astrocytic density was significantly higher in the transplanted cortex compared with the contralateral cortex (ratio of astrocyte density in transplanted/contralateral cortex = 1.66 ± 0.14, p = 0.021, Wilcoxon rank-sum test; data not shown), indicating that astrocytes were more densely packed in the transplanted cortex. Thus, while transplanted astrocytes do establish cortical territories that “tile” with adjacent astrocytes (transplanted or nontransplanted), they do so by adding to the complement of astrocytes in the recipient cortex.

In response to challenges to the CNS, astrocytes undergo several morphologic, molecular, and functional changes and adopt a reactive phenotype (Escartin et al., 2021). One hallmark of astrocyte reactivity is the up-regulation of glial fibrillary acidic protein (GFAP). In normal conditions, this intermediate filament component is expressed sporadically in subsets of cortical astrocytes, including those located at the glial limitans in the outermost layer of the cortex, and those contacting brain vasculature at their endfeet (Lewis and Cowan, 1985; Kacem et al., 1998). In the presence of brain injury or disease, reactive astrocytes increase GFAP expression, particularly at the injury site or at the core of inflammation/cellular degeneration areas.

We were interested in determining whether transplanted astrocytes displayed aspects of a reactive phenotype. We found that transplanted astrocytes showed generally low levels of GFAP. To quantitatively assess this, we compared GFAP intensity between transplanted and nontransplanted astrocytes at 110–130 DPT. We identified regions of interest occupied by transplanted and nontransplanted astrocytes in confocal microscopy images (Fig. 4F) and calculated the transplanted/nontransplanted ratio for GFAP immunofluorescence. Since basal levels of GFAP in the cortex vary depending on the distance of astrocytes from the pia membrane (Fig. 4H), we selected a region of interest for a nontransplanted astrocyte that closely associated with that of the transplanted astrocyte. We also ensured that the two regions of interest were at a similar distance from the site where transplanted cells had been injected, as this region may be populated by clusters of reactive astrocytes displaying very high levels of GFAP (Fig. 4I). The mean transplanted/nontransplanted ratio for GFAP across all mice was not significantly different from 1 (1.007 ± 0.018; Fig. 4G), indicating that transplanted astrocytes displayed similar levels of GFAP to nontransplanted astrocytes. This low level of GFAP expression in transplanted astrocytes, combined with their typical protoplasmic morphology and with absence of morphologic hypertrophy (Fig. 1G), suggested that transplanted astrocytes were not constitutively in a reactive state.

Transplanted astrocytes express similar levels of glutamate transporter EAAT2/GLT1 but show elevated levels of EAAT1/GLAST that resolve after one year in vivo

Having observed that transplanted astrocytes develop morphologic properties that match nontransplanted astrocytes in the cortex of recipient mice, we next investigated the molecular features of transplanted astrocytes. Glutamate transporters, also named excitatory amino acid transporters (EAATs), are expressed by astrocytes and neurons in the brain. EAAT2/GLT1 and EAAT1/GLAST are abundant transporters observed in astrocytes and play an important role in removing extracellular glutamate (Sattler and Rothstein, 2006; Rose et al., 2018). To determine whether transplanted astrocytes expressed levels of EAAT2/GLT1 and EAAT1/GLAST similar to nontransplanted astrocytes, we immunolabeled for each glutamate transporter and acquired images containing both Tomato+ transplanted astrocytes and neighboring nontransplanted astrocytes (Fig. 5A,B). For each image field we calculated the ratio between the EAAT2/GLT1 or EAAT1/GLAST intensity measured in the transplanted versus adjacent nontransplanted astrocyte (Fig. 5C–E). For EAAT2/GLT1, the mean transplanted/nontransplanted ratio was not significantly different from 1 at either 35–47 DPT (0.979 ± 0.037; Fig. 5C) or 110–130 DPT (1 ± 0.020; Fig. 5C), indicating that this glutamate transporter was expressed in comparable amounts by transplanted and nontransplanted astrocytes as early as 35–47 DPT. For EAAT1/GLAST, the mean transplanted/nontransplanted ratio across all mice in the same experimental group was significantly higher than 1 at 35–47 DPT (1.073 ± 0.011; Fig. 5D) and at 110–130 DPT (1.044 ± 0.017; Fig. 5D). To understand whether the increase was sustained long-term, we analyzed the transplanted/nontransplanted ratio for EAAT1/GLAST at 368–570 DPT. However, by this time, the transplanted/nontransplanted ratio normalized and was not different from 1 (1.011 ± 0.013; Fig. 5E). This indicated that transplanted astrocytes expressed higher levels of EAAT1/GLAST compared with nontransplanted astrocytes during the few months following transplantation. However, this upregulation resolved at later time points. EAAT2/GLT1 expression levels, in contrast, remained similar between transplanted and nontransplanted astrocytes at all time points analyzed. Overall, these results showed that transplanted astrocytes acquired and retained levels of EAAT expression similar to nontransplanted astrocytes.

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Figure 5.

Expression of glutamate transporters EAAT2/GLT1 and EAAT1/GLAST in transplanted and nontransplanted astrocytes. A, B, Representative ROIs used to measure EAAT2/GLT1 (A) and EAAT1/GLAST (B) intensity in territories occupied by transplanted (magenta dashed line) and nontransplanted (green dashed line) astrocytes. Tom = Tomato. C–E, Transplanted/nontransplanted ratio values for GLT1 and GLAST at different times after transplantation. Ratio values for both glutamate transporters were calculated at 35–47 DPT and at 110–130 DPT (C, D), and an additional time point for GLAST was added at 368–570 (368+) DPT (E). Gray circles and magenta lines/circles represent ratio values per image field, and mean ratio values per mouse (Ms), respectively. The mean ratio values for each experimental group (black lines) are 0.979 ± 0.037 for GLT1 at 35–47 DPT (p = 0.656, Wilcoxon rank-sum test), 1 ± 0.020 for GLT1 at 110–130 DPT (p = 0.22, Wilcoxon rank-sum test), 1.073 ± 0.011 for GLAST at 35–47 DPT (*p = 0.007, Wilcoxon rank-sum test), 1.044 ± 0.017 for GLAST at 110–130 DPT (*p = 0.003, Wilcoxon rank-sum test), and 1.011 ± 0.013 for GLAST at 368–570 DPT (p = 0.68, Wilcoxon rank-sum test). Scale bar: 10 µm (A, B). Data are mean ± SEM.

Transplanted astrocytes acquire layer-specific expression of GFAP and Kir4.1

Cortical astrocytes display heterogenous gene expression patterns associated with cortical layering (Lanjakornsiripan et al., 2018; Batiuk et al., 2020; Bayraktar et al., 2020). We were interested to know whether astrocytes acquire layer-specific molecular features following their transplantation. We first assessed astrocyte heterogeneity in our cultures, by immunolabeling them for GFAP and for the potassium channel Kir4.1(Fig. 6A), an astrocytic marker that displays complementary expression pattern to GFAP in vivo in mouse cortex. In the mouse cortex, GFAP is predominantly expressed in layer 1, whereas Kir4.1 is more abundantly expressed in lower layers. We found that GFAP and Kir4.1 signal intensity for Tomato+ cells in our cultures showed some variable expression across culture batches (Fig. 6B,C), but did not show inversely correlated expression patterns (Fig. 6D). We then quantified GFAP and Kir4.1 expression levels in transplanted and nontransplanted astrocytes at 35–47 DPT (Fig. 6E–H) and observed that transplanted astrocytes, like nontransplanted astrocytes, express higher levels of GFAP in layer 1 of cortex compared with astrocytes positioned in layers 2–3 (Fig. 6E,F). Transplanted astrocytes also displayed expression of Kir4.1 similar to nontransplanted astrocytes, with higher expression in layers 2–3 versus layer 1 (Fig. 6G,H). Thus, despite the lack of inverse expression of GFAP and Kir4.1 in culture, transplanted astrocytes acquire differential expression of GFAP and Kir4.1 in vivo.

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Figure 6.

Transplanted astrocytes acquire layer-specific expression of GFAP and Kir4.1. A, Immunolabeling for GFAP and Kir4.1 in cultured astrocytes. Tom = Tomato. B, C, Distribution of GFAP (B) and Kir4.1 (C) intensity levels in four batches of cultured astrocytes. D, Plot of Kir4.1 intensity versus GFAP intensity for cultured astrocytes collected from four different batches. Colored lines represent the best-fit lines for each cell batch. E–H, Analysis of the distribution of GFAP and Kir4.1 in vivo. E, G, Necab1 and Sox9 immunolabeling were used to identify cortical layers (dashed yellow lines and captions; see Materials and Methods). L1 = layer 1, L2/3 = layers 2 and 3, L4 = layer 4. ROIs were created (yellow line) for transplanted (arrow) and nontransplanted (arrowhead) astrocytes, and for each ROI mean intensity values of GFAP (E) or Kir4.1 (G) were measured. F, H, For each mouse, the mean GFAP (F) or Kir4.1 (H) intensity values per mouse (circles) measured in L1 were compared with those measured in L2/3 (gray lines). F, The mean intensity value for GFAP was significantly higher in L1 compared with L2/3 for both nontransplanted (green, 407.47 ± 53.13 in L1, 199.28 ± 11.5 in L2; p = 0.01, paired Student’s t test) and transplanted astrocytes (magenta, 345.35 ± 42.03 in L1, 251.31 ± 24.09 in L2; p = 0.012, paired Student’s t test). H, The mean intensity value for Kir4.1 was significantly lower in L1 compared with L2/3 for both nontransplanted (green, 582.33 ± 42.48 in L1, 633.49 ± 45.76 in L2; p = 0.013, paired Student’s t test) and transplanted astrocytes (magenta, 592.52 ± 42.23 in L1, 670.18 ± 53.51 in L2; p = 0.028, paired Student’s t test). Scale bar: 100 µm (A) and 50 µm (E, G). Data are mean ± SEM.

Transplanted and nontransplanted astrocytes associate with similar numbers of excitatory and inhibitory presynaptic terminals

In the first three postnatal weeks, astrocytes in the mouse cortex undergo extensive growth and maturation. During this time, they actively participate in the formation, maturation, and maintenance of synapses by signaling to neurons through the release of diffusible factors (Allen and Eroglu, 2017; Tan et al., 2021) and creating tight contacts with presynaptic and postsynaptic sites. Thus, we were interested in determining whether transplanted astrocytes were associated with the same number of cortical synapses as nontransplanted astrocytes. To study this, we visualized presynaptic terminals by immunolabeling for the vesicular glutamate transporters 1 and 2 (vGLUT1 and vGLUT2) to label glutamatergic terminals, and vesicular GABA transporter (vGAT) to label GABAergic terminals. We next selected regions of interest on transplanted or nontransplanted astrocytes (Fig. 7A,B) and calculated the coverage by the synaptic labels (Fig. 7A–C). We then computed the transplanted/nontransplanted particle coverage ratio for each image field. Ratio values lower than one indicated lower density of presynaptic terminals in transplanted astrocytes compared with nontransplanted astrocytes, whereas values higher than one indicate higher presynaptic terminal density in transplanted astrocytes. At 110–130 DPT, the mean transplanted/nontransplanted presynaptic terminal particle ratio was not significantly different from 1 for either vGlut1/2 (1.117 ± 0.111; Fig. 7D) or vGAT particles (1.174 ± 0.09; Fig. 7E), indicating that transplanted and nontransplanted astrocytes were associated with similar numbers of excitatory and inhibitory presynaptic terminals. These results suggested that astrocytic transplantation does not impair the ability of astrocytes to support synapse formation and maintenance.

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Figure 7.

Transplanted and nontransplanted astrocytes are associated with similar numbers of excitatory and inhibitory presynaptic terminals. A, B, To assess presynaptic particle coverage in transplanted (magenta dashed line) and nontransplanted astrocytes (green dashed line), territories occupied by either kind of astrocytes were identified in sections labeled for vGLUT1/2 (excitatory presynaptic terminals) or vGAT (inhibitory presynaptic terminals). Tom = Tomato. C–E, In binary images derived from vGLUT1/2 and VGAT image channels (C), the ratio values between the particle coverage of transplanted and nontransplanted astrocytes (transplanted/nontransplanted particle coverage ratio) were calculated per image fields (gray circles) and per mouse (Ms, magenta lines/circles). The mean transplanted/nontransplanted particle coverage ratio across all mice (black line) was not significantly different from 1 for either vGLUT1/2 (1.117 ± 0,111; p = 0.347, Wilcoxon rank-sum test) or vGAT (1.174 ± 0.09; p = 0.347, Wilcoxon rank-sum test). Scale bar: 5 μm (A, B). Data are mean ± SEM.

Transplanted astrocytes express aquaporin 4 and gap junctional markers at endfeet, and display similar Ca²⁺ transients

Astrocytic endfeet are highly specialized perivascular structural compartments created by astrocytes that closely associate with the basement membrane, pericytes, vascular smooth muscle cells, and endothelial cells. Being located at the interface between the brain parenchyma and vasculature, endfeet mediate communication between these compartments, and play a fundamental role in neurovascular coupling and maintenance of the blood brain barrier (Cohen-Salmon et al., 2021; Stackhouse and Mishra, 2021). A typical feature of astrocytic endfeet is the high concentration of aquaporin 4 (AQP4), a water channel that mediates water exchange and clearance of brain interstitial fluid (Papadopoulos and Verkman, 2013; Mestre et al., 2020). To understand whether transplanted astrocytes concentrate AQP4 at endfeet near vasculature, we compared endfoot expression levels of AQP4 between transplanted and nontransplanted astrocytes within the same image field (Fig. 8A,B, magenta and green dashed lines) and calculated a ratio between these intensity values (transplanted/nontransplanted AQP4 ratio; Fig. 8C). Interestingly, this ratio was significantly lower than 1 at 35–47 DPT (0.812 ± 0.057), indicating a lower level of expression of AQP4 in transplanted astrocytes. However, this lower AQP4 expression normalized by 110–130 DPT (1.006 ± 0.045; Fig. 8C). We also assessed the presence of gap junctional subunits [Connexin 30 (CX30) and 43 (CX43)] that are known to be enriched at endfeet (Rash et al., 2001) and arranged in large puncta (Ezan et al., 2012). Co-immunolabeling for CX30 and CX43 at 35–47 DPT showed large CX30+ and CX43+ puncta at the endfeet of transplanted astrocytes, with the two gap junctional subunits frequently co-localized in these puncta (Fig. 8D). Thus, transplanted astrocytes concentrate AQP4 and gap junctional subunits at their endfeet.

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Figure 8.

Transplanted and nontranspanted astrocytes express aquaporin 4 and gap junction proteins at endfeet and display similar properties of Ca²⁺ events. A–C, The expression level of the water channel aquaporin 4 (AQP4) was compared between transplanted and nontransplanted astrocytes at 35–47 DPT and 110–130 DPT. Tom = Tomato. A, B, AQP4 intensity was measured in endfeet from transplanted astrocytes (magenta dashed line) and adjacent endfeet from nontransplanted astrocytes (green dashed line). C, The ratio for AQP4 intensity at endfeet (transplanted/nontransplanted AQP4 ratio) was calculated for single image fields (gray circles), for each transplanted mouse (Ms, magenta lines/circles), and across all mice in the same experimental group (black lines). The mean value of this ratio was significantly lower than 1 at 35–47 DPT (0.812 ± 0.057; *p = 0.007, Wilcoxon rank-sum test) but was not significantly different from 1 at 110–130 DPT (1.006 ± 0.045; p = 0.4, Wilcoxon rank-sum test). D, The endfoot of a transplanted astrocyte (Tom, left panel) in contact with a blood vessel labeled for Collagen IV (left panel) enriched with large aggregates of Connexin 43 (CX43) and Connexin 30 (CX30; right panel). E, Ca²⁺ events were recorded in cortical slices prepared from transplanted mice in a field containing a transplanted and nontransplanted astrocyte (dashed lines). Ca²⁺ event recordings were processed for noise reduction and analyzed using AQuA. Ca²⁺ events were detected (colored regions) in the soma and processes of both transplanted and nontransplanted astrocytes. F, Ca²⁺ event area, duration, decay time, and maximum deltaF/F were calculated. No significant differences were detected between transplanted and nontransplanted astrocytes (Ca²⁺ event area: 19.53 ± 1.83 for nontransplanted cells, 22.93 ± 2.55 for transplanted cells, p = 0.296, Student’s t test; Duration: 15.22 ± 0.59 for nontransplanted cells, 15.15 ± 1.07 for transplanted cells, p = 0.949 Student’s t test; Decay time constant: 16.65 ± 1.88 for nontransplanted cells, 15.76 ± 2.18 for transplanted cells, p = 0.767 Student’s t test; Max d F/F: 0.242 ± 0.002 for nontransplanted cells, 0.247 ± 0.005 for transplanted cells, p = 0.286, Student’s t test). Scale bar: 5 µm (A, B) and 10 µm (D, E). Data are mean ± SEM.

To determine whether transplanted astrocytes show Ca²⁺ activity similar to nontransplanted astrocytes, we prepared acute cortical slices and monitored Ca²⁺ activity following bulk loading of the Ca²⁺ indicator Fluo-4AM. We analyzed the properties of spontaneous Ca²⁺ events in transplanted and nontransplanted astrocytes and found similar properties with respect to event area, deltaF/F, duration, and decay (Fig. 8E,F). Overall, these results along with those from the endfoot analysis suggest that transplanted astrocytes display molecular and functional properties similar to those of nontransplanted astrocytes.

Cortical astrocytes transplanted to more mature brain survive but are largely retained at the transplantation site

Having found that cortical astrocytes integrate and survive following their transplantation to the immature brain, we next determined whether astrocytes display similar properties when transferred to the more mature brain. To address this, we injected astrocytes into the cortex of young adult mice (P34–P43; Fig. 9A) and tracked astrocyte integration at 35–47 DPT. We found that transplanted astrocytes survived in large numbers and maintained their astrocyte identity as confirmed by immunolabeling for Sox9 (Fig. 9B). However, transplanted astrocytes were largely confined to the transplantation site or at superficial layers of the cortex (Fig. 9B) and in some cases at a small distance from the injection site (Fig. 9B,F). We measured the ability of transplanted astrocytes to migrate away from the injection site and found that the lateral migration of transplanted astrocytes was significantly lower in mice transplanted at P34–P43 (adult transplantation) compared with mice transplanted at P3–P7 (developmental transplantation; Fig. 9G). Astrocytes transplanted at P34–P43 that remained at the injection site commonly displayed elongated shapes and thick processes containing high expression of GFAP consistent with a reactive phenotype (Fig. 9C,F). In some instances, transplanted astrocytes escaped the transplantation site and were found to acquire a protoplasmic morphology (Fig. 9E,F). These cells also showed lower levels of GFAP expression (Fig. 9F). We also analyzed the expression of glutamate transporters EAAT2/GLT1 and EAAT1/GLAST in astrocytes transplanted at P34–P43. We found that transplanted and nontransplanted protoplasmic astrocytes express similar levels of these transporters at 110–130 DPT (transplanted/nontransplanted ratios: 0.937 ± 0.039 for EAAT2/GLT1,> p = 0.282, Wilcoxon rank-sum test; 1.117 ± 0.111 for EAAT1/GLAST, p = 0.282, Wilcoxon rank-sum test). Interestingly, we did not detect significant differences in the expression of cleaved caspase 3 (an indicator of apoptotic cell death) between the adult and developmental transplantation groups, suggesting the absence of increased cell death following transplantation to more mature mice (Fig. 9H). Thus, transplantation of cortical astrocytes into more mature cortex results in heterogenous astrocytic phenotypes with reactive astrocytes and protoplasmic astrocytes found near and distal to the transplantation site, respectively.

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Figure 9.

Cortical astrocytes transplanted to the more mature mouse brain survive but generally fail to escape the injection site. A, Overview of the experimental design for transplantation of cortical astrocytes into the cerebral cortex of young adult mice (P34–P43). Tam = 4-hydroxytamoxifen. Figure created with BioRender. B–E, At 35–47 d post-transplantation (DPT), most transplanted astrocytes were accumulated around the injection site, had elongated morphologies typical of reactive astrocytes, and displayed high expression of GFAP (C). Some of these cells displayed protoplasmic astrocytic morphology (B, arrows; D, E) and displayed low levels of GFAP (F). Tom = Tomato. G, Lateral migration of transplanted astrocytes in mice transplanted at P3–P7 (P7) and analyzed at 35–47 DPT, and mice transplanted at P34–P43 (P34) and analyzed at 35–47 DPT. Gray circles represent mean lateral migration distance (LMD) values per mouse. The mean LMD value for the P7 group was significantly lower than that for the P34 group (magenta circles, P7, 0.676 ± 0.101 mm; P34, 0.222 ± 0.031 mm; *p = 0.009, Student’s t test). H, Expression of activated caspase 3 by transplanted astrocytes in mice transplanted at P3–P7 (P7) and analyzed at 1 DPT and mice transplanted at P34–43 (P34) and analyzed at 1 DPT. Circles and bars represent mean values of active caspase 3 coverage per mouse and per experimental group, respectively. No significant differences were detected between P7 and P34 (P7, 0.55 ± 0.17%; P34, 0.57 ± 0.15%; p = 0.942, Student’s t test). Scale bar: 50 µm (B, C), 10 µm (D, E), and 20 µm (F). Data are mean ± SEM.

Transplanted cortical astrocytes integrate into the developing cerebellar cortex but retain cortical-like astrocytic features

Having shown that cortical astrocytes transplanted into developing cortex establish structural and molecular features similar to nontransplanted cortical astrocytes, we next asked whether cortical astrocytes can acquire the characteristics of nearby nontransplanted astrocytes if transplanted to a mismatched brain region. To answer this question, we transplanted cortical astrocytes into the cerebellum of P5–P8 mice (Fig. 10A) and tracked their development in the molecular layer (ML) of the cerebellar cortex where Bergmann glia (BG) extend highly polarized processes from the Purkinje cell (PC) layer to the pial surface (Yamada and Watanabe, 2002; Kita et al., 2013). In addition to these specific structural features, BG express high levels of the glutamate transporter EAAT1/GLAST (Farmer et al., 2016) and uniquely express the AMPA receptor subunit GluA1 (Saab et al., 2012).

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Figure 10.

Transplanted cortical astrocytes integrate into the cerebellar cortex but retain structural and molecular features of cortical astrocytes. A, Overview of the experimental design for transplantation of cortical astrocytes into the cerebellum of mouse pups. Tam = 4-hydroxytamoxifen. Figure created with BioRender. B, Most astrocytes transplanted in the molecular layer (ML) remained confined in the subpial region of the cerebellar cortex and extended process deeper into the ML. Some transplanted astrocytes showed a stellate shape similar to cortical protoplasmic astrocytes (arrow). C–F, Transplanted cortical astrocytes expressed lower amounts of the AMPA-type glutamate receptor subunit GluA1 and glutamate transporter EAAT1/GLAST compared with nearby Bergmann glia but maintained high levels of EAAT2/GLT-1. Radial lines were drawn in the ML across transplanted astrocytes and adjacent BG cells (white dashed lines, C, E) to identify sites of intensity profile measurments for GluA1 (D), EAAT1 (F, gray), and EAAT2 (F, black). The region occupied by transplanted astrocytes (magenta line D, F, black) was associated with low levels of GluA1 and EAAT1 and high levels of EAAT2, whereas the region occupied by BG displayed complementary intensity profiles for the three markers. G, The presence of cortical astrocytes in the ML did not interfere with the growth or maintenance of Purkinje cell (PC) dendrites. The direction of PC dendrites did not change at the interface between transplanted cortical astrocytes and BG cells (thick red dashed line). Tom = Tomato. B, C, E, G, A thin red dashed line indicates the border between the ML and the PC layer. Scale bar: 50 µm (B) and 20 µm (C, E, G).

At 21 DPT, we found many Tomato+/Sox9+ astrocytes in the cerebellar cortex (Fig. 10B). The cell bodies of these astrocytes were often found positioned superficially in the ML. Despite this abnormal cell body positioning, astrocytes elongated processes deeper within the ML (Fig. 10B). In areas where the density of transplanted astrocytes was lower, we observed that they displayed a less-polarized, stellate shape reminiscent of cortical astroglia (compare Fig. 10B, arrow, with 1G). Interestingly, transplanted astrocytes showed low to undetectable levels of GluA1 and lower levels of EAAT1/GLAST compared with nearby BG cells (Fig. 10C,E). Measuring the signal intensity profiles across the ML revealed a steep increase of GluA1 and EAAT1/GLAST levels at the interface between transplanted cells and nontransplanted BGs (Fig. 10D,F). Transplanted astrocytes also retained higher levels of EAAT2/GLT1 (Fig. 10E). These results suggested that transplanted cortical astrocytes integrate and develop in the cerebellar cortex, but retain structural and molecular features of cortical astrocytes.

BG develop in close association with PCs, and BG processes have been proposed to function as scaffold for the elongation of PC dendrites in the first postnatal weeks (Lordkipanidze and Dunaevsky, 2005). Given that transplanted cortical astrocytes lacked the specialized morphology and molecular phenotype of BG, we wondered whether they could provide a favourable environment for the growth and maintenance of PC dendritic arbors. We visualized PC dendrites through Calbindin immunolabeling, and observed that PC dendrites elongated normally on territories occupied by cortical astrocytes (Fig. 10G). This suggested that astrocytes derived from cortex can replace BG arbors without noticeable effects on Purkinje cell dendritic branching.

Transplanted cerebellar astrocytes develop both Bergmann glia and velate-like astrocytic properties in the cerebellum

Finally, we investigated whether transplanting cerebellar astrocytes into cerebellum can generate appropriate cerebellar astrocytic phenotypes. Astrocyte maturation in the cerebellar cortex is a highly regulated and timed process that gives rise to two main regionally specialized astrocyte types, namely, BG in the ML and velate astrocytes in the granule cell layer (GCL; Farmer et al., 2016). In contrast to BGs, velate astrocytes have more stellate morphology and form sheath-like processes around granule cells. Velate astrocytes, unlike BGs, also have low levels of GluA1 but have high levels of AQP4 (Yamada and Watanabe, 2002; Kita et al., 2013). We transplanted cerebellar astrocytes into the cerebellum of P5–P8 mice (Fig. 11A). Interestingly, at 21 DPT, astrocytes transplanted in the ML showed distinct morphologies with thick radial shafts extending from the cell body toward the pial surface (Fig. 11B,C), mimicking a major structural property of BGs. Cerebellar astrocytes transplanted in the ML also expressed GluA1 and displayed low levels of AQP4 (Fig. 11D,E). Conversely, cerebellar astrocytes transplanted deeper into the GCL showed morphologies similar to velate astrocytes, and did not express noticeable levels of GluA1 (Fig. 11F). However, these cells showed high levels of AQP4 (Fig. 11G). These findings showed that cerebellar astrocytes behave differently than cortical astrocytes when transplanted into the developing cerebellum, acquiring the unique properties of cerebellar astrocytes.

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Figure 11.

Cerebellar astrocytes develop into Bergmann glia-like and velate-like astrocytes when transplanted into the cerebellum. A, Overview of the experimental design for transplantation of cerebellar astrocytes into the cerebellum of mouse pups. Tam = 4-hydroxytamoxifen. Figure created with BioRender. B–G, Cerebellar astrocytes transplanted in the cerebellar cortex acquired region-specific structural and molecular features. B, C, Transplanted cerebellar astrocytes located in the ML display BG-like morphologies. D, E, Astrocytes transplanted in the ML (arrows) express GluA1 and low levels of AQP4, like BG cells. F, G, Astrocytes transplanted in the granule cell layer (GCL; arrowheads) display a velate-like astrocytic morphology and, like velate astrocytes, are negative for GluA1 and express high levels of AQP4. Tom = Tomato. Scale bar: 20 µm (B–G). Dashed yellow line marks the interface between ML and GCL.