Phenotypic analysis of astrocytes derived from glial restricted precursors and their impact on axon regeneration

Abstract

Although astrocytes are involved in the production of an inhibitory glial scar following injury, they are also capable of providing neuroprotection and supporting axonal growth. There is growing appreciation for a diverse and dynamic population of astrocytes, specified by a variety of glial precursors, whose function is regulated regionally and temporally. Consequently, the therapeutic application of glial precursors and astrocytes by effective transplantation protocols requires a better understanding of their phenotypic and functional properties and effective protocols for their preparation. We present a systematic analysis of astrocyte differentiation using multiple preparations of glial-restricted precursors (GRP), evaluating their morphological and phenotypic properties following treatment with fetal bovine serum (FBS), bone morphogenetic protein 4 (BMP-4), or ciliary neurotrophic factor (CNTF) in comparison to controls treated with basic fibroblast growth factor (bFGF), which maintains undifferentiated GRP. We found that treatments with FBS or BMP-4 generated similar profiles of highly differentiated astrocytes that were A2B5−/GFAP+. Treatment with FBS generated the most mature astrocytes, with a distinct and nearhomogeneous morphology of fibroblast-like flat cells, whereas BMP-4 derived astrocytes had a stellate, but heterogeneous morphology. Treatment with CNTF induced differentiation of GRP to an intermediate state of GFAP+ cells that maintained immature markers and had relatively long processes. Furthermore, astrocytes generated by BMP-4 or CNTF showed considerable experimental plasticity, and their morphology and phenotypes could be reversed with complementary treatments along a wide range of mature-immature states. Importantly, when GRP or GRP treated with BMP-4 or CNTF were transplanted acutely into a dorsal column lesion of the spinal cord, cells from all 3 groups survived and generated permissive astrocytes that supported axon growth and regeneration of host sensory axons into, but not out of the lesion. Our study underscores the dynamic nature of astrocytes prepared from GRP and their permissive properties, and suggest that future therapeutic applications in restoring connectivity following CNS injury are likely to require a combination of treatments.

Human Astrocytes Derived from Glial Restricted Progenitors Support Regeneration of the Injured Spinal Cord

“…16,17 However, our recent studies have shown that despite the morphological and phenotypic differences between GRP and astrocytes derived from GRP by BMP-4 or CNTF differentiation, they all have permissive properties, highlighting the general therapeutic potential of embryonic astrocytes for SCI. 18,19 In our studies, GRP and GRP-derived astrocytes showed equivalent abilities to support axonal growth and regeneration, without neuropathic pain. 12,19 The various glial phenotypes resulting from the process of limited GRP pre-differentiation 3,[15][16][17]19 underscores the specific properties and functions that these cells serve in the developing and adult CNS-neuronal development, neuronal survival, axonal growth, synapse formation, blood-brain barrier maintenance, and metabolic and trophic support.…”

“…18,19 In our studies, GRP and GRP-derived astrocytes showed equivalent abilities to support axonal growth and regeneration, without neuropathic pain. 12,19 The various glial phenotypes resulting from the process of limited GRP pre-differentiation 3,[15][16][17]19 underscores the specific properties and functions that these cells serve in the developing and adult CNS-neuronal development, neuronal survival, axonal growth, synapse formation, blood-brain barrier maintenance, and metabolic and trophic support. 10,[20][21][22][23] Indeed, there is growing evidence for a diverse population of astrocytes, 20,[23][24][25][26][27][28] particularly in humans, 29,30 capable of performing functions that are temporally, regionally, and contextually specific.…”

“…For comparison and reproducibility between cell batches, hGRP (Batch AE2 080530-01) were differentiated and stained as described above. Immunofluorescent staining was performed as previously described, 19,41 followed by incubation with 4¢ 6¢ diamidino-2-phenyindole (Dapi; Sigma Aldrich, St. Louis, MO). Coverslips were mounted on glass slides using FluoroSave Reagent (Calbiochem, San Diego, CA).…”

“…20,33 This interaction with the external microenvironment allows for a significant degree of morphological, phenotypic, physiological, and functional plasticity, particularly following injury. 22,23,34 Similarly, limited pre-differentiation of GRP into immature, embryonic astrocytes still allows for significant morphological and phenotypic plasticity upon exposure to an alternate environment, 19 suggesting that the therapeutic potential of naïve GRP and embryonic astrocytes following SCI stems from their relatively immature state, 8,19 similar to the therapeutic potential demonstrated by grafts of immature astrocytes. [35][36][37][38][39][40] In this study, we used hGRP and astrocytes derived from hGRP for a detailed morphological and phenotypic analysis.…”

Transplantation of stem cell-derived astrocytes for the treatment of amyotrophic lateral sclerosis and spinal cord injury

“…These studies highlighted that heterogeneous astrocyte populations may have distinct therapeutic potentials. Contrasting results from the Fischer lab indicated that, although heterogeneous, both BMP-4 and CNTF GDAs promoted axon outgrowth to a similar extent [157] . Although it is not possible to compare in detail the precursor populations used by the two labs, the properties of specific glial precursor populations, differences in the culture conditions, and different transplantation and tracing protocols may have led to these contrasting results.…”