Chemical Conversion of Human Fetal Astrocytes into Neurons through Modulation of Multiple Signaling Pathways

Abstract

SummaryWe have previously developed a cocktail of nine small molecules to convert human fetal astrocytes into neurons, but a nine-molecule recipe is difficult for clinical applications. Here, we identify a chemical formula with only three to four small molecules for astrocyte-to-neuron conversion. We demonstrate that modulation of three to four signaling pathways among Notch, glycogen synthase kinase 3, transforming growth factor β, and bone morphogenetic protein pathways is sufficient to change an astrocyte into a neuron. The chemically converted human neurons can survive >7 months in culture, fire repetitive action potentials, and display robust synaptic burst activities. Interestingly, cortical astrocyte-converted neurons are mostly glutamatergic, while midbrain astrocyte-converted neurons can yield some GABAergic neurons in addition to glutamatergic neurons. When administered in vivo through intracranial or intraperitoneal injection, the four-drug combination can significantly increase adult hippocampal neurogenesis. Together, human fetal astrocytes can be chemically converted into functional neurons using three to four small molecules, bringing us one step forward for developing future drug therapy.

In Vivo Neuroregeneration to Treat Ischemic Stroke in Adult Non-Human Primate Brains through NeuroD1 AAV-based Gene Therapy

“…Besides NeuroD1, other groups have reported that expression of transcription factors Ngn2, Ascl1, Sox2, or combinations of factors can also convert internal glial cells into neurons in the brain or spinal cord (Gascon et al, 2016; Grande et al, 2013; Jorstad et al, 2017; Liu et al, 2015; Niu et al, 2015; Niu et al, 2013; Pereira et al, 2017; Rivetti di Val Cervo et al, 2017; Su et al, 2014). In addition to the overexpression of transcription factors, we have also demonstrated that small molecules can directly convert human glial cells into functional neurons (Yin et al, 2019; Zhang et al, 2015) through transcriptome-wide activation of neuronal genes (Ma et al, 2019). Other groups also reported direct chemical reprogramming of glial cells (Gao et al, 2017) or fibroblast cells (Ambasudhan et al, 2011; Hu et al, 2015; Li et al, 2015) into neurons.…”

MiR-124 and small molecules synergistically regulate the direct generation of neuronal cells from rat cortical reactive astrocytes

“…Recently, the use of diverse small molecule cocktails to reprogram astrocytes into neuronal cells has achieved considerable success [13,14]. Yin et al proved that using three to four small molecules (SB431542, CHIR99021, LDN193189, and DAPT) or their respective functional analogues to modulate the TGF-β/SMAD, BMP, NOTCH, and WNT pathways was su cient to reprogram human foetal astrocytes into neurons [14]. However, RA characteristics are also regulated by other signals, such as the JAK/STAT and P38 MAPK pathways and post-transcriptional levels of several non-coding RNAs.…”

“…Alternatively, the biological effects of small molecules are netuneable and reversible; thus, their development into pharmaceuticals can provide broad application prospects for neuronal reprogramming without genetic modi cation [12]. The small molecule cocktail of VPA, CHIR99021, RepSox, forskolin, i-BET151, and ISX-9 reprogrammed adult astrocytes into mainly glutamatergic neurons [13], and foetal astrocyte-derived functional neurons could be obtained via mediation by a small molecule cocktail of SB431542, LDN193189, CHIR99021, and 2,4-diamino-5phenylthiazole (DAPT) [14]. However, since the signals activated or inhibited by each small molecule differ, inconsistent chemical combinations lead to variable neuronal properties and conversion e ciencies.…”

Next‐generation disease modeling with direct conversion: a new path to old neurons

“…Additionally, electrophysiological analysis has proven valuable to define functional neurons, as neurons have very specific electrophysiological features like hyperpolarized membrane potential and spontaneous and triggered action potentials [32,46,49]. In this regard, iNs have been shown to express neuronal markers, show mature neuronal morphology, and possess both spontaneous and induced postsynaptic currents [15,57,81,118,119]. In this regard, iNs have been shown to express neuronal markers, show mature neuronal morphology, and possess both spontaneous and induced postsynaptic currents [15,57,81,118,119].…”

“…For many studies that are not neuronal function-centered, Ca 2+ imaging and multielectrode arrays can be used as surrogates for electrophysiology and can further deliver insights into neuronal network activity in vitro [20,21,119,120]. In this regard, iNs have been shown to express neuronal markers, show mature neuronal morphology, and possess both spontaneous and induced postsynaptic currents [15,57,81,118,119]. Differentiating neurons in the developing brain and in iPSC differentiation pass through stages that specifically allow them to participate in functional circuits [121], while iNs skip these stages.…”