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

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.

Development of a Platform for Studying 3D Astrocyte Mechanobiology: Compression of Astrocytes in Collagen Gels

“…As a complement to in vivo experiments, in vitro studies offer great potential in this regard; however, previous studies of ONH astrocyte mechanobiology have used monolayer-based approaches, which do not accurately represent the physiological environment of the ONH. Notably, monolayer culture induces an artificially reactive phenotype in cultured astrocytes,4,14 making it difficult to interpret the effect of biomechanical strain in such studies.…”

Impact of four common hydrogels on amyloid-β (Aβ) aggregation and cytotoxicity: Implications for 3D models of Alzheimer’s disease

“…In a broad range of contexts, the epigenetics and morphology of cells in vivo can be well-approximated in 3D hydrogel cultures. Though simple hydrogels cannot recapitulate the entire complexity of in vivo tissues, they do provide strikingly similar results compared to 2D cell culture [26–27, 29]. Depending on the application, the optimal physiochemical properties of the hydrogel model will vary.…”

“…Also, we cannot rule out that the attenuation of Aβ toxicity in hydrogels is influenced by confinement of the cells themselves or effects from the stiffness of the cellular microenvironment. However, we hold that these possibilities are unlikely given the evidence that 3D culture allows cells to more closely mimic in vivo phenotypes vs. 2D culture [26–27, 29]. More importantly, we reported in 2002 that agarose itself does not confer a protective effect against the cytotoxicity of the Aβ 1-40 amino acid sequence [43].…”

Collagen hydrogel confinement of amyloid-βaccelerates aggregation and reduces cytotoxic effects

“…There is a growing appreciation that cells grown in vitro , on rigid polystyrene tissue culture surfaces, behave very differently than cells in vivo , and that the stiffness of the surface upon which cells are grown can have profound epigenetic effects on cells that influence their phenotype [80, 81]. In addition, many investigators have observed that morphologies of neuronal cells are strikingly more similar to those expressed in vivo in 3D vs. 2D culture [22, 23, 82, 83]. Thus, the use of 3D cell culture models for AD drug development may lead to a more positive correlation between successes in vitro and successes in clinical outcomes.…”

“…We supposed that this effect could be due to one of two effects, either (1) the 3D environment altered the phenotype of the PC12 cells, making them less susceptible to Aβ, or (2) the 3D environment altered the aggregation of Aβ. Given that PC12 cells and other neuron-like cells take on a phenotype more closely associated with an in vivo neuron in 3D [22, 23, 84], we assumed that our first hypothesis was less probable. Therefore, in this work, we set out to investigate how the 3D collagen hydrogel environment affects Aβ structure and aggregation.…”