High‐throughput identification of factors promoting neuronal differentiation of human neural progenitor cells in microscale 3D cell culture

Abstract

Identification of conditions for guided and specific differentiation of human stem cell and progenitor cells is important for continued development and engineering of in vitro cell culture systems for use in regenerative medicine, drug discovery, and human toxicology. Three-dimensional (3D) and organotypic cell culture models have been used increasingly for in vitro cell culture because they may better model endogenous tissue environments. However, detailed studies of stem cell differentiation within 3D cultures remain limited, particularly with respect to high-throughput screening. Herein, we demonstrate the use of a microarray chip-based platform to screen, in high-throughput, individual and paired effects of 12 soluble factors on the neuronal differentiation of a human neural progenitor cell line (ReNcell VM) encapsulated in microscale 3D Matrigel cultures. Dose-response analysis of selected combinations from the initial combinatorial screen revealed that the combined treatment of all-trans retinoic acid (RA) with the glycogen synthase kinase 3 inhibitor CHIR-99021 (CHIR) enhances neurogenesis while simultaneously decreases astrocyte differentiation, whereas the combined treatment of brain-derived neurotrophic factor and the small azide neuropathiazol enhances the differentiation into neurons and astrocytes. Subtype specification analysis of RA- and CHIR-differentiated cultures revealed that enhanced neurogenesis was not biased toward a specific neuronal subtype. Together, these results demonstrate a high-throughput screening platform for rapid evaluation of differentiation conditions in a 3D environment, which will aid the development and application of 3D stem cell culture models.

Parkinson's disease, aging and adult neurogenesis: Wnt/β‐catenin signalling as the key to unlock the mystery of endogenous brain repair

“…Amongs others, CHIR is a small molecule used to promote neuroprogenitor homeostasis and neural induction. with CHIR significantly enhanced neurogenesis, while simultaneously decreasing astrocyte differentiation (Nierode et al, 2019).…”

“…CHIR was shown to restore WβC‐signalling and to rescue DAergic differentiation in iPSC‐derived NSCs from Gaucher's disease patients, exhibiting developmental defects due to downregulation of canonical WβC‐signalling (Awad et al, ). Recently, Nierode et al () reported high‐throughput identification of factors promoting neuronal differentiation of human neural progenitor cells in microscale 3D cell cultures. In this study, the authors looked at a microarray chip‐based platform to screen the individual and combined effects of 12 soluble factors on the neuronal differentiation of a human neural progenitor cell line (ReNcell VM) encapsulated in microscale 3D Matrigel cultures, revealing that the combined treatment of trans ‐retinoic acid with CHIR significantly enhanced neurogenesis, while simultaneously decreasing astrocyte differentiation (Nierode et al, ).…”

Extracellular vesicle-mediated amyloid transfer to neural progenitor cells: implications for RAGE and HIV infection

“…6 and Additional file 1: Figure S6). We also measured the brain-derived neurotrophic factor (BDNF) and the nerve growth factor-β (NGF-β) because they provide signaling and trophic support during NPC differentiation [48]. The levels of these growth factors were not detectable in NPC culture media 3 days after differentiation.…”

Using stem cell–derived neurons in drug screening for neurological diseases

“…Furthermore, coupling microfluidic systems with 3D culture can improve differentiation and enhance neural migration. Recently a microarray chip-based platform was used to grow 3D cultures of neural progenitor cells and assess the effects of soluble factors on neuronal differentiation (Nierode et al, 2018).…”