Generation of Human iPSC-Derived Astrocytes with a mature star-shaped phenotype for CNS modeling

Abstract

The generation of astrocytes from human induced pluripotent stem cells has been hampered by either prolonged differentiation—spanning over two months—or by shorter protocols that generate immature astrocytes, devoid of salient mature astrocytic traits pivotal for central nervous system (CNS) modeling. We directed stable hiPSC-derived neuroepithelial stem cells to human iPSC-derived Astrocytes (hiAstrocytes) with a high percentage of star-shaped cells by orchestrating an astrocytic-tuned culturing environment in 28 days. We employed RT-qPCR and ICC to validate the astrocytic commitment of the neuroepithelial stem cells. To evaluate the inflammatory phenotype, we challenged the hiAstrocytes with the pro-inflammatory cytokine IL-1β (interleukin 1 beta) and quantitatively assessed the secretion profile of astrocyte-associated cytokines and the expression of intercellular adhesion molecule 1 (ICAM-1). Finally, we quantitatively assessed the capacity of hiAstrocytes to synthesize and export the antioxidant glutathione. In under 28 days, the generated cells express canonical and mature astrocytic markers, denoted by the expression of GFAP, AQP4 and ALDH1L1. In addition, the notion of a mature phenotype is reinforced by the expression of both astrocytic glutamate transporters EAAT1 and EAAT2. Thus, hiAstrocytes have a mature phenotype that encompasses traits critical in CNS modeling, including glutathione synthesis and secretion, upregulation of ICAM-1 and a cytokine secretion profile on a par with human fetal astrocytes. This protocol generates a multifaceted astrocytic model suitable for in vitro CNS disease modeling and personalized medicine. Graphical abstract

Robust induction of functional astrocytes using NGN2 expression in human pluripotent stem cells

“…Recognizing the utility of human in vitro derived astrocytes, several protocols have been developed including those following a protracted developmental time-course in 3-dimensions (up to 20 mos.) (Sloan et al, 2017) as well as more rapid 2-dimensional protocols (Canals et al, 2018; Hedegaard et al, 2020; Santos et al, 2017; Tcw et al, 2017; Voulgaris et al, 2022). While these protocols produce cells expressing canonical astrocyte markers and are capable of recapitulating key functions such as responding to pro-inflammatory stimuli, a tremendous amount remains to be determined regarding: (i) the precise cell types and cell stages being generated and / or how closely they resemble fetal or adult human astrocytes from primary cultures or post-mortem preparations, (ii) the robustness of protocols across individual hPSC lines, and (iii) their utility for disease modeling applications.…”

“…Several astrocyte differentiation protocols have recognized the importance of robust NPC generation as an important foundation for efficient astrocyte differentiation (Tcw et al, 2017;Voulgaris et al, 2022). Here, we leveraged previous studies showing that the combination of NGN2 patterning with dual-SMAD and WNT inhibition can direct hPSCs toward diverse neural fates Lin et al, 2021;Nehme et al, 2018;Wells, 2021) as well as one study identifying optimal media conditions to differentiate neural progenitor-like cells (NPCs) into astrocytes (Tcw et al, 2017).…”

Metabolic Assessment of Human Induced Pluripotent Stem Cells-Derived Astrocytes and Fetal Primary Astrocytes: Lactate and Glucose Turnover

“…hiPSCs were differentiated to astrocytes based on our recent protocol [ 10 ]. Fetal human cortical astrocytes were obtained from ScienCell (#1800).…”

“…As an alternative cell source, cell lines or hiPSC-derived cells can be used to generate functional astrocytes [ 8 ]. The biological relevance of each in vitro model, primary, cell line or stem-cell-derived cells must, however, be validated, as we recently showed with two studies using hiPSC-derived astrocytes [ 9 , 10 ].…”

“…HFAs, as their name implies, are obtained and purified from human fetal samples and can be expanded in vitro for a limited number of passages. The human-induced astrocytes (hiAstrocytes) we use are differentiated from a stable neural stem cell state derived from hiPSC [ 10 ]. hiAstrocytes show astrocyte marker expression, functional glutamate transporters, neuroinflammatory response, and glutathione secretion.…”