Direct Reprogramming of Human Fetal- and Stem Cell-Derived Glial Progenitor Cells into Midbrain Dopaminergic Neurons

Abstract

Summary Human glial progenitor cells (hGPCs) are promising cellular substrates to explore for the in situ production of new neurons for brain repair. Proof of concept for direct neuronal reprogramming of glial progenitors has been obtained in mouse models in vivo, but conversion using human cells has not yet been demonstrated. Such studies have been difficult to perform since hGPCs are born late during human fetal development, with limited accessibility for in vitro culture. In this study, we show proof of concept of hGPC conversion using fetal cells and also establish a renewable and reproducible stem cell-based hGPC system for direct neural conversion in vitro . Using this system, we have identified optimal combinations of fate determinants for the efficient dopaminergic (DA) conversion of hGPCs, thereby yielding a therapeutically relevant cell type that selectively degenerates in Parkinson's disease. The induced DA neurons show a progressive, subtype-specific phenotypic maturation and acquire functional electrophysiological properties indicative of DA phenotype.

Direct Conversion of Human Stem Cell-Derived Glial Progenitor Cells into GABAergic Interneurons

“…Previous data from our lab have demonstrated that PV interneurons can be generated by direct in vivo reprogramming of mouse GPCs using the transcription factors Ascl1, Lmx1a, and Nurr1 (together referred as to ALN) [ 17 ], whereas this combination with or without REST knockdown gives rise to DA neurons when fibroblasts or glia are converted in vitro [ 28 , 34 , 35 ]. Here, we evaluated if ALN + shREST could also convert hGPCs into PV + GABAergic interneurons and improve the PV yield compared to ADLSF.…”

“…We have recently developed a stem cell-based system that can model hGPC-conversion in vitro [ 25 , 28 ]. In this study, we used these hESC-derived GPCs for neuronal conversion with a focus on GABAergic interneurons.…”

“…The hESCs were expanded in IPS-Brew XF medium (StemMACS, Miltenyi, Bergisch Gladbach, Germany) on LN521 (0.5 µg/cm 2 , Biolamina, Sundbyberg, Sweden) coated tissue plates at 37 °C in 5% CO 2 and passaged weekly with ethylenediaminetetraacetic acid (EDTA, 0.5 mM, Gibco, Thermo Fisher; Waltham, MA, USA). For details on the glial differentiation protocol and cryopreservation, see Nolbrant et al [ 28 ].…”

“…For monitoring the neuronal maturation and labeling cells in whole cell patch-clamp recordings, we used a lentivirus expressing red fluorescent protein (RFP) under the control of the human Synapsin 1 promoter (hSyn-RFP; Addgene, Watertown, MA, USA). Doxycycline-regulated LVs expressing mouse Ascl1, Lmx1a, and Nurr1 cDNAs have been previously described [ 28 ]. LVs were produced as previously described [ 29 ] and titrated by quantitative PCR analysis [ 30 ].…”

Induced Neurons for Disease Modeling and Repair: A Focus on Non-fibroblastic Cell Sources in Direct Reprogramming

“…Recent studies have shown that the use of Ascl1 alone did not induce any neurogenesis; however, NeuroD1 or Sox2 alone is sufficient to reprogram into iN (Guo et al, 2014;Heinrich et al, 2014). Recently, human glial progenitor cells (NG2 glial, fetal, or stem cell derived) were able to be reprogrammed into midbrain dopaminergic neurons by the expression of transcription factors Ascl1, Lmx1a, and Nr4a2 (Nurr1) along with inhibition of the RNA against the RE1-silencing transcription factor (REST) complex (Nolbrant et al, 2020).…”