Waye, S. E. | Search bioRxiv

bioRxiv2019

Single-Cell Analysis Reveals Regional Reprogramming during Adaptation to Massive Small Bowel Resection in Mice

Background & AimsThe small intestine (SI) displays regionality in nutrient and immunological function. Following SI tissue loss (as occurs in short gut syndrome, or SGS), remaining SI must compensate, or adapt; the capacity of SI epithelium to reprogram its regional identity has not been described. Here, we apply single-cell resolution analyses to characterize molecular changes underpinning adaptation to SGS.\n\nMethodsSingle-cell RNA-sequencing was performed on epithelial cells isolated from distal SI of mice following 50% proximal small bowel resection (SBR) vs. sham surgery. Single-cell profiles were clustered based on transcriptional similarity, reconstructing differentiation events from intestinal stem cells (ISCs) through to mature enterocytes. An unsupervised computational approach to score cell identity was used to quantify changes in regional (proximal vs distal) SI identity, validated using immunofluorescence, immunohistochemistry, qPCR, western blotting, and RNA-FISH.\n\nResultsUniform Manifold Approximation and Projection-based clustering and visualization revealed differentiation trajectories from ISCs to mature enterocytes in sham and SBR. Cell identity scoring demonstrated segregation of enterocytes by regional SI identity: SBR enterocytes assumed more mature proximal identities. This was associated with significant upregulation of lipid metabolism and oxidative stress gene expression, which was validated via orthogonal analyses. Observed upstream transcriptional changes suggest retinoid metabolism and proximal transcription factor Creb3l3 drive proximalization of cell identity in response to SBR.\n\nConclusionsAdaptation to proximal SBR involves regional reprogramming of ileal enterocytes toward a proximal identity. Interventions bolstering the endogenous reprogramming capacity of SI enterocytes--conceivably by engaging the retinoid metabolism pathway--merit further investigation, as they may increase enteral feeding tolerance, and obviate intestinal failure, in SGS.\n\n\n\nO_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=83 SRC=\"FIGDIR/small/615054v1_ufig1.gif\" ALT=\"Figure 1\">\nView larger version (26K):\norg.highwire.dtl.DTLVardef@156d1b4org.highwire.dtl.DTLVardef@e1a3fcorg.highwire.dtl.DTLVardef@1d519bforg.highwire.dtl.DTLVardef@18d4e9d_HPS_FORMAT_FIGEXP M_FIG C_FIG SynopsisHere, single-cell RNA sequencing reveals interactions between the retinoid metabolism pathway and regional reprogramming of distal small intestinal epithelium to a proximal identity following proximal small bowel resection. This provides novel insight into physiological adaptation to short gut syndrome.

genomics↗

bioRxiv2017

Single-cell analysis of clonal dynamics in direct lineage reprogramming: a combinatorial indexing method for lineage tracing

Single-cell technologies are offering unprecedented insight into complex biology, revealing the behavior of rare cell populations that are typically masked in bulk population analyses. The application of these methodologies to cell fate reprogramming holds particular promise as the manipulation of cell identity is typically inefficient, generating heterogeneous cell populations. One current limitation of single-cell approaches is that lineage relationships are lost as a result of cell processing, restricting interpretations of the data collected. Here, we present a single-cell resolution lineage-tracing approach based on the combinatorial indexing of cells, CellTagging. Application of this method, in concert with high-throughput single-cell RNA-sequencing, reveals the transcriptional dynamics of direct reprogramming from fibroblasts to induced endoderm progenitors. These analyses demonstrate that while many cells initiate reprogramming, complete silencing of fibroblast identity and transition to a progenitor-like state represents a rare event. Clonal analyses uncover a remarkable degree of heterogeneity arising from individual cells. Overall, very few cells fully reprogram to generate expanded populations with a low degree of clonal diversity. Extended culture of these engineered cells reveals an instability of the reprogrammed state and reversion to a fibroblast-like phenotype. Together, these results demonstrate the utility of our lineage-tracing approach to reveal dynamics of lineage reprogramming, and will be of broad utility in many cell biological applications.

genomics↗