Glial progenitor cells (GPCs) were generated in vitro and possess functional properties of primary astrocytes
Glial progenitor cells (GPCs) comprise an already lineage-restricted glial progenitor population, that may be more appropriate for treatment of glial disorders . However, it is difficult to instruct in vivo differentiation of neural stem cells (NSCs) to GPCs . Hence, in order to obtain a stable source of GPCs for the following transplantations, we previously developed a high-efficiency in vitro protocol for generating GPCs from embryonic cortical NSCs  (Supplementary Fig. 1A). According to this protocol, GPCs were generated from NSCs and used for the following transplantation experiments (Supplementary Fig. 1B-D). Further experiments confirmed that these GPCs acquired the astrocytic differentiation potential (Supplementary Fig. 1E and F).
Astrocytic Ca2+ transients relate to a wide variety of significant functions [19, 20]. To determine if in vitro generated GPC-derived astrocytes possess these Ca2+ events, we crossed the Cre-dependent GCaMP5G mouse line, termed PC-G5-tdT (Polr2a, CAG, GCaMP5G, tdTomato) , with the Aldh1l1-Cre/ERT2 mouse line , to obtain a line that expresses the GCaMP5G genetically-encoded Ca2+ indicator specifically in astrocytes (Fig. 1A). It has been shown that following treatment with tamoxifen, almost all in-vitro-generated GPC-derived astrocytes, identified as GFAP positive cells, were labeled by expression of both GCaMP5G and tdTomato (Fig. 1B). To investigate the functionality of these in-vitro-generated GPC-derived astrocytes, we directly activated the astrocytes via focal application of adenosinetriphosphate (ATP), a P2Y agonist known to induce Ca2+ release from the internal stores of primary astrocytes . Focal ATP (200 µmol/L) administration evoked a cytosolic Ca2+ increase in astrocytes that propagated across the field of view as a wave (Fig. 1C). This propagation of Ca2+ waves across astrocytes plays a critical role in glial and neuron-glial cell communication . The mean ATP-evoked peak ΔF/F0 was 185.0 ± 13.8 % (n = 50 cells, Fig. 1D, E). Therefore, similar to primary astrocytes, astrocytes derived from in vitro generated GPCs possess Ca2+ transients and are competent for network communication.
Engrafted GPCs differentiate into astrocytes with younger morphology and maintain long-term integration in the aged neocortex
In our previous study, we found that engrafted GPCs could morphologically and functionally integrate into the adult mammalian neocortex . However, it was not clear whether the engrafted GPCs could migrate, differentiate, and maintain long-term integration in the aged mammalian neocortex. To explore these processes, in-vitro-generated GPCs were transplanted into the somatosensory cortex of 6-8 month old mice, which were sacrificed 12 months after transplantation for histological analysis (Supplementary Fig. 1A).
The dispersal pattern of donor cells is a critical indicator of their integration in the host brain [14, 26]. Our data revealed that 12 months after transplantation the engrafted GPCs had migrated widely in the somatosensory cortex and no signs of tumor formation were observed. They advanced into both the superficial and layers of the cortex (Fig. 2A). Furthermore, the vast majority of engrafted GPCs differentiated into astrocytes with complex star-like morphology and dense processes (Fig. 2B, C).
It was demonstrated that astrocytes display age-dependent morphological changes, including significant reductions in the number and the length of processes, territorial domains, and astrocyte-to-astrocyte coupling in the aged brain . We next examined whether age-dependent structural degeneration would take place in engrafted astrocytes 12 months after transplantation. Consistent with previous studies [1, 2, 4], our data showed that cortical astrocytes of aged-control mice had a flattened shape, reductions in cellular surface area, and morphological complexity compared with those of adult-control ones (Fig. 2D, E, G, J-L). However, 12 months after transplantation the engrafted GPC-derived astrocytes in aged mice remained much younger morphologically and displayed more complex structure compared with the endogenous cortical astrocytes of aged-control mice (Fig. 2E, F). Statistical analysis also indicated that the engrafted GPC-derived astrocytes had more intersections (Fig. 2G, J, K) and primary branches (Fig. 2L). The engrafted GPC-derived astrocytes were also positive for connexin 30 (CX30) (Fig. 2H), a major astrocytic gap junction protein , and D-serine (Fig. 2I), a gliotransmitter . These findings indicated that engrafted GPC-derived astrocytes could form dynamic networks and regulate synaptic plasticity in the same manner as younger cells in the aged neocortex, 12 months after transplantation. These results demonstrate that engrafted GPCs are able to migrate, differentiate, retain a younger morphology, and achieve long-term integration in the aged mammalian brain.
Engrafted GPC-derived astrocytes establish endfeet expressing AQP4 and reverse the depolarization of perivascular AQP4 in the aged neocortex
Ageing causes degeneration of astrocytic endfeet  and depolarization of perivascular AQP4 , resulting in prominent neurovascular dysfunction  and the accumulation of protein waste . Our previous studies demonstrated that engrafted astrocytes could establish endfeet along blood vessel walls . However, it was unknown if the endfeet of engrafted GPC-derived astrocytes would be retained for a long time and express AQP4 in the aged brain. Our histological results revealed that extended endfeet (white arrows, Fig. 3A) from engrafted GPC-derived astrocytes still contiguously arrayed along the vessel wall (outlined with dashes, Fig. 3A, right panel) 12 months after transplantation in the aged brain. Additionally, AQP4 as expressed and remained on the endfeet (white arrows, Fig. 3B). More interestingly, our results revealed that engrafted GPC-derived astrocytes ameliorated AQP4 polarization in the aged mouse cortex (Fig. 3C-E). AQP4 localization became dispersed in the cortex of aged-control mice brains but remained highly polarized in brain regions engrafted with GPC-derived astrocytes (Fig. 3C-E). Ameliorated AQP4 polarization in the aged brain facilitates the clearance of interstitial solutes and contributes to the improvement of neuronal functions .
Engrafted Gpc-derived Astrocytes Reverse Age-induced Sensory Function Deficiency
Our previous work revealed that engrafted GPC-derived astrocytes in the somatosensory cortex are able to respond to sensory stimulation with Ca2+ signals . In addition, it has been reported that the somatosensory cortex experiences age-dependent morphological and functional degeneration [32–36]. We subsequently investigated whether the integration of engrafted GPC-derived astrocytes and their amelioration of AQP4 polarization could yield any potential functional improvement in the aged somatosensory cortex.
Previous studies indicated that the somatosensory cortex is involved in sensorimotor integration and sensory response modulation [37–39]. To assess the functional properties of this brain region, we examined the escape response latencies of the sensory response in aged GPC-transplanted mice 12 months post transplantation (Fig. 4A). Consistent with previous reports [33–36], our study found obvious functional degeneration of the somatosensory cortex of aged-control mice which showed much longer escape response latencies, as compared with adult-control mice (Fig. 4B, C). In contrast, 12 months after transplantation of GCPs in the somatosensory cortex, engrafted aged mice showed an improved sensory response, exhibiting obviously reduced escape response latencies compared with the aged-control mice (Fig. 4B, C). Thus, the engrafted GPC-derived astrocytes not only achieved morphologically long-term integration and ameliorated AQP4 polarization in the aged somatosensory cortex, but also functionally reversed the age-dependent functional degeneration of this brain region.