Lesion environments direct transplanted neural progenitors towards a wound repair astroglial phenotype in mice

Article https://doi.org/10.1038/s41467-022-33382-x Lesion environments direct transplanted neural progenitors towards a wound repair astroglial phenotype in mice Received: 11 January 2022 Check for updates 1234567890():,; 1234567890():,; Accepted: 14 September 2022 T. M. O’Shea 1,2 , Y. Ao1, S. Wang1, A. L. Wollenberg3,4, J. H. Kim 1, R. A. Ramos Espinoza2, A. Czechanski5, L. G. Reinholdt5, T. J. Deming3,4 & M. V. Sofroniew 1 Neural progenitor cells (NPC) represent potential cell transplantation therapies for CNS injuries. To understand how lesion environments influence transplanted NPC fate in vivo, we derived NPC expressing a ribosomal proteinhemagglutinin tag (RiboTag) for transcriptional profiling of transplanted NPC. Here, we show that NPC grafted into uninjured mouse CNS generate cells that are transcriptionally similar to healthy astrocytes and oligodendrocyte lineages. In striking contrast, NPC transplanted into subacute CNS lesions after stroke or spinal cord injury in mice generate cells that share transcriptional, morphological and functional features with newly proliferated host astroglia that restrict inflammation and fibrosis and isolate lesions from adjacent viable neural tissue. Our findings reveal overlapping differentiation potentials of grafted NPC and proliferating host astrocytes; and show that in the absence of other interventions, non-cell autonomous cues in subacute CNS lesions direct the differentiation of grafted NPC towards a naturally occurring wound repair astroglial phenotype. Neural tissue that is lost to injury or disease in the mature mammalian central nervous system (CNS) is not spontaneously replaced. Instead, naturally occurring and conserved CNS wound repair mechanisms generate lesions in which non-neural lesion cores of fibrotic and inflammatory cells are partitioned from adjacent preserved neural tissue by newly formed astroglial borders1–10. Although this wound repair response is effective in clearing debris, limiting infection, and protecting nearby viable neural tissue, the resulting lesions often contain large volumes of non-neural fibrotic scar tissue that lacks the specialized neural cells necessary to support axon regeneration or the remodeling of neural circuits10–14. Neural cell transplantation represents one potential therapeutic strategy for replacing lost neural tissue and improving outcome after CNS insults15–18. Different types of cell transplantation are being explored for this purpose, including fetal cell grafts19–24, adult neural progenitor cells (NPC)25–27, and NPC derived from lines of embryonic stem cells (ESC) or induced pluripotent stem cells (iPSC)28–32. Despite considerable progress in the derivation, production and transplantation of NPC into CNS injuries, many questions remain about the roles of cell autonomous versus non-cell autonomous factors in determining NPC differentiation and their neural repair support functions after grafting in vivo33. Here, we examined how transplantation of NPC into different CNS environments altered their gene expression and differentiation fate in vivo. To do so we derived NPC from mouse embryonic stem cells (ESC) that constitutively express the ribosomal protein Rpl22 with a hemagglutinin (HA) tag (Rpl22-HA), also known as RiboTag, which permits cell-specific transcriptional profiling by RNA sequencing 1 Department of Neurobiology, David Geffen School of Medicine, University of California, Los Angeles, CA 90095-1763, USA. 2Department of Biomedical Engineering, Boston University, Boston, MA 02215-2407, USA. 3Department of Chemistry and Biochemistry, University of California Los Angeles, Los Angeles, CA 90095-1600, USA. 4Department of Bioengineering, University of California Los Angeles, Los Angeles, CA 90095-1600, USA. 5The Jackson Laboratory, Bar e-mail: ; Harbor, ME 04609, USA. Nature Communications | (2022)13:5702 1 Article (RNAseq) and immunohistochemical characterization of HA-positive cells34,35. These RiboTag-NPC permit the selective transcriptomic analysis of NPC and their progeny after transplantation into host tissue in vivo even when the NPC are present in relatively small numbers compared with host cell numbers, and without the need for mechanical tissue dissociation and cell sorting that have the potential to induce transcriptional changes. We first extensively characterized these Rpl22-HA-expressing NPC (RiboTag-NPC), in vitro, including determining their transcriptional responses to factors known to induce different types of differentiation. We then used these wellcharacterized RiboTag-NPC to selectively evaluate NPC transcriptional profiles and differentiation fates following transplantation into uninjured CNS or into CNS lesions after forebrain stroke or spinal cord injury. We compared NPC transcriptional responses in vivo with NPC transcriptional responses in vitro to specific non-cell autonomous molecular cues that modified their differentiation; and we compared NPC differentiation phenotypes in vivo with the profiles of newly proliferated host astroglia that naturally adopt wound repair functions. We found that non-cell autonomous cues powerfully modify NPC transcription and can instruct different differentiation fates both in vitro and in vivo, and that grafted NPC are directed towards different cell fates by non-cell autonomous cues in uninjured or lesioned CNS tissue. Our findings reveal similarities between the transcriptional profiles, cellular morphologies, and certain functional features of cells derived from NPC transplanted into subacute CNS lesions and host astroglia that are stimulated by CNS injuries to proliferate and adopt a naturally occurring, border-forming wound repair astroglial phenotype. Results Neural induction and expansion of RiboTag mESC derives reproducible and stable NPC lines Mouse ESC expressing RiboTag through Cre-Lox recombination (Fig. 1a) was used to generate NPC by neural induction and expansion35,36 (Fig. 1b). A single female mESC line that expressed RiboTag was used to generate NPC for all experiments in this study. Discrete multicellular ESC colonies (Fig. 1c) were transitioned into spindle-shaped NPC and expanded as adherent monolayer cultures rather than as floating neurospheres (Fig.1d). Transcriptome profiling by bulk RiboTag RNA Sequencing (RNA-Seq) showed gain of NPC phenotype and loss of ESC characteristics assessed using defined panels of canonical genes for each cell type37. NPC generation markedly reduced ESC gene expression with a median log2Fold Change (FC) of approximately −10 (Fig. 1e, f, h). Concurrently, NPC derivation induced increased expression of canonical neural stem cell genes with a median log2FC of approximately +5 (Fig. 1h). Loss of protein expression of ES markers Dppa4, Oct4, Nanog as well as emergence of NPC markers Nestin, Sox9 and Fabp7 by immunocytochemistry (ICC) and quantitative western blotting (WB) further supported successful NPC generation (Fig. 1g, Supplementary Fig. 1h, i). RiboT (...truncated)