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. 2016 Jun;22(11-12):885-98.
doi: 10.1089/ten.TEA.2016.0103.

Three-Dimensional Environment Sustains Morphological Heterogeneity and Promotes Phenotypic Progression During Astrocyte Development

Swarnalatha Balasubramanian  1 , John A Packard  1 , Jennie B Leach  1 , Elizabeth M Powell  2
Affiliations

Three-Dimensional Environment Sustains Morphological Heterogeneity and Promotes Phenotypic Progression During Astrocyte Development

Swarnalatha Balasubramanian et al. Tissue Eng Part A. 2016 Jun.

Abstract

Astrocytes are critical for coordinating normal brain function by regulating brain metabolic homeostasis, synaptogenesis and neurotransmission, and blood-brain barrier permeability and maintenance. Dysregulation of normal astrocyte ontogeny contributes to neurodevelopmental and neurodegenerative disorders, epilepsies, and adverse responses to injury. To achieve these multiple essential roles, astrocyte phenotypes are regionally, morphologically, and functionally heterogeneous. Therefore, the best regenerative medicine strategies may require selective production of distinct astrocyte subpopulations at defined maturation levels. However, little is known about the mechanisms that direct astrocyte diversity or whether heterogeneity is represented in biomaterials. In vitro studies report lack of normal morphologies and overrepresentation of the glial scar type of reactive astrocyte morphology and expression of markers, questioning how well the in vitro astrocytes represent glia in vivo and whether in vitro tissue engineering methods are suitable for regenerative medicine applications. Our previous work with neurons suggests that the three-dimensional (3D) environment, when compared with standard two-dimensional (2D) substrate, yields cellular and molecular behaviors that more closely approximately normal ontogeny. To specifically study the effects of dimensionality, we used purified glial fibrillary acidic protein (GFAP)-expressing primary cerebral cortical astrocyte cultures from single pups and characterized the cellular maturation profiles in 2D and 3D milieu. We identified four morphological groups in vitro: round, bipolar, stellate, and putative perivascular. In the 3D hydrogel culture environment, postnatal astrocytes transitioned from a population of nearly all round cells and very few bipolar cells toward a population with significant fractions of round, stellate, and putative perivascular cells within a few days, following the in vivo ontogeny. In 2D, however, the population shift from round and bipolar to stellate and perivascular was rarely observed. The transition to distinct cellular morphologies in 3D corresponded to the in vivo expression of phenotypic markers, supporting the generation of mature heterogeneous glial populations in vitro. This study presents quantitative data supporting that 3D culture is critical for sustaining the heterogeneity of astrocytes in vitro and for generating a representation of the in vivo portfolio of heterogeneous populations of astrocytes required for therapeutic interventions in neurodevelopmental disorders, epilepsy, and brain injury.

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Figures

<b>FIG. 1.</b>
FIG. 1.
Schematic of experimental strategy. (A) Gray matter cerebral cortical astrocytes were isolated from individual mouse pups at P1–P3. The astrocytes from a single pup are grown and purified in an individual flask and then used to compare the cellular responses to 2D and 3D environments. Each color represents an individual pup and cells obtained from it. Each experiment simultaneously compared astrocytes derived from three to four individual pups grown in 2D and 3D substrates and, in some cases, different medium formulations for each substrate. (B) Cells grown at a plating density of 7.5 × 103 cells/cm2 on a 2D substrate appear as flattened cobblestones and are immunoreactive for GFAP (red fluorophore). (C) Cells grown within the 3D substrate (7.5 × 105 cells/ml) are distributed throughout the scaffold and are observed at multiple focal planes. The cells are immunoreactive for GFAP (red fluorophore) and appear much smaller than those in 2D (B). DAPI (blue) is used to stain cell nuclei. Scale bar: 50 μm. 2D, two-dimensional; 3D, three-dimensional; GFAP, glial fibrillary acidic protein. Color images available online at www.liebertpub.com/tea
<b>FIG. 2.</b>
FIG. 2.
Astrocytes exhibited unique profiles of morphologies dependent upon in vitro growth environment. Representative confocal images of primary astrocytes grown on 2D collagen-coated coverslips (A, C, E) or within 3D collagen gels (B, D, F) for 4 days in serum-containing media show immunoreactivity for the astrocyte marker, GFAP, as shown in red and cell nuclei are labeled with DAPI (blue). Multiple morphologies were observed in the GFAP+ cells: round, without distinct processes or projections (A, B), bipolar cells, which had two opposing processes (C, D), and stellate cells, with multiple processes emerging from a centrally located soma (E, F). The stellate cells resembled mature astrocytes in vivo. (G) Distribution of morphologies in 2D and 3D cultured substrates. Asterisks denote significant differences between the percentages of cells grown in 3D exhibiting each morphology compared with those on 2D substrates (p < 0.001 level). Scale bar: 20 μm. Please note that the cell in (B) is much smaller than the others. Color images available online at www.liebertpub.com/tea
<b>FIG. 3.</b>
FIG. 3.
Growth medium composition and substrate dimensionality contributed to the profiles of astrocyte morphologies. Astrocytes were cultured for 4 days in either serum-containing (A, C, E) or serum-free defined media (B, D, F). Plating density was not a major factor determining the percentage of GFAP+ cells with round (A, B), bipolar (C, D), or stellate morphologies (E, F). Substrate dimension significantly altered each cell morphology category for all plating densities in serum media. Serum-free media induced significant distributions in cell morphologies that were dependent upon plating density and often different from serum-containing media. Dimensionality contributed dramatic shifts in the distributions of cell morphologies, with interactions with plating density and medium composition. For 2D experiments: low density is 5 × 103 cells/cm2, medium density is 7.5 × 103 cells/cm2, and high density is 1.5 × 104 cells/cm2. For 3D experiments, low density is 5 × 105 cells/mL, medium density is 7.5 × 105 cells/mL, and high density is 1.5 × 106 cells/mL. Each bar represents the mean ± SEM of at least n = 3 independent samples of >50 astrocytes per sample. An asterisk denotes a significant difference between cells comparing 2D and 3D substrates at the p < 0.05 level.
<b>FIG. 4.</b>
FIG. 4.
Dimensionality and medium composition interact to alter cell proliferation and lineage. Astrocytes were cultured on 2D collagen-coated coverslips and within 3D collagen gels in serum-containing or serum-free media for 4 days. (A) The overall total number of cells, in equivalent regions, was increased in the 3D serum-free culture condition. (B) EdU was used to label cells that were in S-phase as a measure of cell proliferation. Double immunohistochemistry for GFAP (red) and EdU (green) demonstrates that round and bipolar astrocytes were among the proliferative populations. Bar = 10 μm. (C) Analysis of the percent of the population that had incorporated EdU yielded an effect of dimensionality. (D) The distribution of morphologies of EdU+ cells was dependent upon medium composition and culture substrate dimension. Bars represent mean ± SEM from n > 3 samples; in each sample, n > 50 astrocytes were counted. An asterisk denotes a significant difference at the p < 0.05 level for an effect of substrate dimension. Color images available online at www.liebertpub.com/tea
<b>FIG. 5.</b>
FIG. 5.
Long-term culture in 3D substrates revealed a novel cell morphology. (A) Astrocytes were cultured in 3D substrates for 4 and 10 days in serum-containing and serum-free media. After 10 days in culture, fewer round astrocytes were observed, but the percent of bipolar and stellate cells remained similar to 4 days. However, a new type of cell that resembles the perivascular astrocyte proximal to blood vessels in vivo was observed. (B) The perivascular type of astrocyte is GFAP+ and has a long central process and several fine endfeet. (C) Schematic of the progression of astrocyte phenotypes during perinatal maturation, adapted from Hunter and Hatten. Bars represent mean ± SEM from at least n = 3 samples; in each sample, n > 50 astrocytes were counted. Asterisks denote significant effect of medium composition (p < 0.05), whereas ampersands show significant effects of time in culture (p < 0.05). Scale bar: 20 μm. Color images available online at www.liebertpub.com/tea
<b>FIG. 6.</b>
FIG. 6.
Round GFAP+ cells mainly express the astrocyte marker, S100β. Representative fluorescent images of round astrocytes cultured on 2D collagen-coated coverslips (A, B, E, F) and in 3D collagen gels (C, D, G, H). Cells were colabeled for GFAP (red, A, C, E, G) and either S100β (B, D, green) or RC2 (F, H) or nestin. Cell nuclei were labeled with DAPI (blue). Scale bars: 20 μm. Please note the differences in cell size in 2D (A, B, E, F) and 3D (C, D, G, H). (I) Analysis of immunoreactivity patterns shows that the majority of the round cells express S100β, with only a few that were grown in 3D-expressing RC2 or nestin. Bars represent mean ± SEM from at least n = 3 samples; in each sample, n > 50 astrocytes were counted. An asterisk denotes a significant difference at the p < 0.05 level. Color images available online at www.liebertpub.com/tea
<b>FIG. 7.</b>
FIG. 7.
Bipolar cells express phenotypic markers of radial glia. Representative images of bipolar cells cultured on 2D (A, B, E, F, I, J) or 3D substrates (C, D, G, H, K, M). On 2D, the GFAP+ (red) bipolar cells colabeled with the mature astrocyte marker, S100β (B, green), as well as the radial glial markers, RC2 (F, green) and nestin (J, green). By contrast, in 3D, very few bipolar cells expressed S100β (D), but nearly all expressed RC2 (H) and nestin (M). Scale bar: 20 μm. (N) Expression of markers in bipolar cells. Bars represent mean ± SEM from at least n = 3 samples; in each sample, n > 50 astrocytes were counted. An asterisk denotes a significant difference at the p < 0.05 level. Color images available online at www.liebertpub.com/tea
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