Direct lineage conversion of postnatal mouse cortical astrocytes to oligodendrocyte lineage cells

Reviewer #1 (Public Review):

Faiz et al. investigate small molecule-driven direct lineage reprogramming of mouse postnatal mouse astrocytes to oligodendrocyte lineage cells (OLCs). They use a combination of in vitro, in vivo, and computational approaches to confirm lineage conversion and to examine the key underlying transcription factors and signaling pathways. Lentiviral delivery of transcription factors previously reported to be essential in OLC fate determination-Sox10, Olig2, and Nkx2.2-to astrocytes allows for lineage tracing. They found that these transcription factors are sufficient in reprogramming astrocytes to iOLCs, but that the OLCs range in maturity level depending on which factor they are transfected with. They followed up with scRNA-seq analysis of transfected and control cultures 14DPT, confirming that TF-induced astrocytes take on canonical OLC gene signatures. By performing astrocyte lineage fate mapping, they further confirmed that TF-induced astrocytes give rise to iOLCs. Finally, they examined the distinct genetic drivers of this fate conversion using scRNA-seq and deep learning models of Sox10- astrocytes at multiple time points throughout the reprogramming. These findings are certainly relevant to diseases characterized by the perturbation of OLC maturation and/or myelination, such as Multiple Sclerosis and Alzheimer's Disease. Their application of such a wide array of experimental approaches gives more weight to their findings and allows for the identification of additional genetic drivers of astrocyte to iOLC conversion that could be explored in future studies. Overall, I find this manuscript thoughtfully constructed and only have a few questions to be addressed.

(1) The authors suggest that Sox10- and Olig2- transduced astrocytes result in distinct subpopulations iOLCs. Considering it was discussed in the introduction that these TFs cyclically regulate one another throughout differentiation, could they speculate as to why such varying iOLCs resulted from the induction of these two TFs?

(2) In Figure 1B it appears that the Sox10- MBP+ tdTomato+ cells decreases from D12 to D14. Does this make sense considering MBP is a marker of more mature OLCs?

(3) Previous studies have shown that MBP expression and myelination in vitro occurs at the earliest around 4-6 weeks of culturing. When assessing whether further maturation would increase MBP positivity, authors only cultured cells up to 22 DPT and saw no significant increase. Has a lengthier culture timeline been attempted?

(4) Figure S4D is described as "examples of tdTomatonegzsGreen+OLCmarker+ cells that arose from a tdTomatoneg cell with an astrocyte morphology." The zsGreen+ tdTomato- cell is not convincingly of "astrocyte morphology"; it could be a bipolar OLC. To strengthen the conclusions and remove this subjectivity, more extensive characterizations of astrocyte versus OLC morphology in the introduction or results are warranted. This would make this observation more convincing since there is clearly an overlap in the characteristics of these cell types.

Reviewer #2 (Public Review):

The study by Bajohr investigates the important question of whether astrocytes can generate oligodendrocytes by direct lineage conversion (DLR). The authors ectopically express three transcription factors - Sox10, Olig2 and Nkx6.2 - in cultured postnatal mouse astrocytes and use a combination of Aldh1|1-astrocyte fate mapping and live cell imaging to demonstrate that Sox10 converts astrocytes to MBP+ oligodendrocytes, whereas Olig2 expression converts astrocytes to PDFRalpha+ oligodendrocyte progenitor cells. Nkx6.2 does not induce lineage conversion. The authors use single-cell RNAseq over 14 days post-transduction to uncover molecular signatures of newly generated iOLs.

The potential to convert astrocytes to oligodendrocytes has been previously analyzed and demonstrated. Despite the extensive molecular characterization of the direct astrocyte-oligodendrocyte lineage conversion, the paper by Bajohr et al. does not represent significant progress. The entire study is performed in cultured cells, and it is not demonstrated whether this lineage conversion can be induced in astrocytes in vivo, particularly at which developmental stage (postnatal, adult?) and in which brain region. The authors also state that generating oligodendrocytes from astrocytes could be relevant for oligodendrocyte regeneration and myelin repair, but they don't demonstrate that lineage conversion can be induced under pathological conditions, particularly after white matter demyelination. Specific issues are outlined below.

(1) The authors perform an extensive characterization of Sox10-mediated DLR by scRNAseq and demonstrate a clear trajectory of lineage conversion from astrocytes to terminally differentiated MBP+ iOLCs. A similar type of analysis should be performed after Olig2 transduction, to determine whether transcriptomics of OPC induction overlaps with any phase of MBP+ oligodendrocyte induction.

(2) A complete immunohistochemical characterization of the cultures should be performed at different time points after Sox10 and Olig2 transduction to confirm OL lineage cell phenotypes.