Derivation of primitive neural stem cells from human‐induced pluripotent stem cells

Abstract

Human-induced pluripotent stem cells (hiPSCs) have facilitated studies on organ development and differentiation into specific lineages in in vitro systems. Although numerous studies have focused on cellular differentiation into neural lineage using hPSCs, most studies have initially evaluated embryoid body (EB) formation, eventually yielding terminally differentiated neurons with limited proliferation potential. This study aimed to establish human primitive neural stem cells (pNSCs) from exogenefree hiPSCs without EB formation. To derive pNSCs, we optimized N2B27 neural differentiation medium through supplementation of two inhibitors, CHIR99021 (GSK-3 inhibitor) and PD0325901 (MEK inhibitor), and growth factors including basic fibroblast growth factor (bFGF) and human leukemia inhibitory factor (hLIF). Consequently, pNSCs were efficiently derived and cultured over a long term. pNSCs displayed differentiation potential into neurons, astrocytes, and oligodendrocytes.These early NSC types potentially promote the clinical application of hiPSCs to cure human neurological disorders. K E Y W O R D S differentiation, human-induced pluripotent stem cells, neural lineage, primitive neural stem cells

Pharmacological Manipulation of Wnt/β-Catenin Signaling Pathway in Human Neural Precursor Cells Alters Their Differentiation Potential and Neuronal Yield

“…Taking advantage of our in vitro drug screening platform of self-renewable hNPCs, we also asked whether CHIR or XAV might be cytotoxic, by measuring cell proliferation and survival. Both drugs were used in this study at the same concentration or lower as in similar studies employing human or murine ESCs, hiPSCs and other types of stem cells ( De Kumar et al, 2017 ; Malleske et al, 2018 ; Shafa et al, 2018 ; Gomez et al, 2019 ; Hamad et al, 2019 ; Qiu et al, 2019 ; Shin et al, 2019 ; Almasoud et al, 2020 ; Bataille et al, 2020 ; Bejoy et al, 2020 ; Govarthanan et al, 2020 ; Han et al, 2020 ; Kanagaki et al, 2020 ; Leigh et al, 2020 ; Yang J. et al, 2020 ; Yang Y. et al, 2020 ; Ren et al, 2021 ; Wang et al, 2021 ). When stem cells differentiate into more mature cell types, their proliferation potential is gradually reduced, until cells reach a post-mitotic fate.…”

“…Most relevant to the present report, is the use of CHIR and XAV as critical components of chemically defined protocols aiming to guide differentiation of human and murine stem cells into myriad cellular fates. CHIR has been used to promote neural and neuronal differentiation in embryonic stem cells and induced pluripotent stem cells ( De Kumar et al, 2017 ; Shafa et al, 2018 ; Gomez et al, 2019 ; Qiu et al, 2019 ; Shin et al, 2019 ; Bejoy et al, 2020 ) and in several different types of neural precursors ( Yang Y. et al, 2020 ; Ren et al, 2021 ; Wang et al, 2021 ), as well as to induce trans- differentiation into neural lineages from non-neural cell types, such as skin ( Bataille et al, 2020 ; Yang J. et al, 2020 ) and mesenchymal cells ( Govarthanan et al, 2020 ). XAV has been used to promote differentiation of pluripotent stem cells into lung cells ( Malleske et al, 2018 ; Kanagaki et al, 2020 ), to increase osteogenic differentiation of mesenchymal stem cells ( Almasoud et al, 2020 ; Han et al, 2020 ), and to promote generation of cardiomyocytes ( Hamad et al, 2019 ; Leigh et al, 2020 ).…”

Neural Lineage Differentiation From Pluripotent Stem Cells to Mimic Human Brain Tissues

“…These pNSCs showed expression patterns similar to those exhibited by the neural rosette and NSCs in the fetal cortex, as well as differentiation potential to neurons, astrocytes, and oligodendrocytes, in addition to the specialized neuronal subtypes, namely GABAergic, dopaminergic, and motor neurons. We have also, recently, succeeded in deriving pNSCs from human iPSCs in the presence of PD0325901 (MEK inhibitor) and leukemia inhibitory factor (LIF) using simple method that does not require EB formation (Shin et al, 2019). Once NSCs are established, the differentiation potential becomes restricted to neurons and glial cells, regardless of NSC type, except in the case of transdifferentiation—a very rare phenomenon—by which a mature cell directly differentiates into different cell lineages (Wagers and Weissman, 2004).…”

“…In particular, by differentiating patient-derived iPSCs into a neural lineage, study and modeling on a neurological disease which would otherwise be arduous to perform can be easily conducted. iPSCs have been differentiated into neural stem cells (NSCs) in a 3 dimensional (3D) environment, including neurospheres, and 2 dimensional (2D) NSCs, including rosette-types (Elkabetz et al, 2008) and primitive NSCs (Shin et al, 2019). Continuing efforts on the differentiation technology to mimic brain tissue in vitro using pluripotent stem cells have led to technical advances, such as the formation of a mini brain-like structure or brain organoids (Lancaster et al, 2013).…”