Stem cell-derived tissues that recap endogenous physiology are key for regenerative medicine. Yet, most methods yield products that function like fetal, not adult tissues. Organoids are typically grown in constant environments, while our tissues mature along with behavioral cycles. Here, we show that inducing circadian rhythms in pancreatic islet organoids, by entraining them to daily feeding-fasting cycles, elicits their metabolic maturation. Our results show that rhythms can be harnessed to further functional maturation of organoids destined for human therapeutics.
Islet transplantation can cure insulin-dependent diabetes, but is curbed by scarcity of acceptable islets . Stem cell-derived islet organoids offer an unlimited islet supply, yet an immature physiology limits their therapeutic use . To find maturation-driving mechanisms, we studied regulatory dynamics during their stepwise formation from human pluripotent stem cells .
MATERIALS AND METHODS
Human cell lines, primary samples, and rodent strains are detailed in . We devised flow sorting strategies to purify cells at successive stages of differentiation into the islet lineage . These included stem cell-derived β (SC-β), insulin+ glucagon+ polyhormonal (PH) cells, and their progenitors. Cells were subject to whole-genome bisulfite sequencing (WGBS), assay for transposase-accessible chromatin by sequencing (ATAC-seq), chromatin immunoprecipitation sequencing for two histone marks (H3K27ac and H3K4me1), and directional total RNA sequencing (RNA-seq), as detailed in . Computational methods are described elsewhere [3, 5–7]. Animal studies were conducted as described in . Cell culture methods and assays are detailed in .
Our studies reveal mechanisms controlling cell fate during islet organoid development and show that organoids are amenable to functional improvement by circadian modulation.
This work provides a proof-of-principle that circadian control can drive functional maturation of stem cell-derived organoids destined for human therapeutics. Islet maturity develops between birth and weaning, along with onset of circadian behavioral (sleep, feeding) cycles. We show that recreating fasting/feeding cycles recapitulates metabolic maturation in islet organoids. Entrained organoids develop a capacity to anticipate diurnal changes in insulin demand, and are functional within days—rather than weeks—of transplantation. Circadian entrainment may be harnessed to further functional maturation of other stem cell-derived products, consistent with the ability of clock regulators to bind distinct targets in distinct tissues. Thus, our general approach may inform attempts to control the fate and function of any human cell type.
• Epigenome dynamics show how epigenetic priming steers endocrine cell fates.
• Modeling maturation regulatory circuits uncovers roles for circadian controllers.
• Circadian entrainment triggers organoid maturation via clock-controlled metabolic cycles.
• Entrained organoids gain stable genomic changes and function within days of transplant.
The authors thank Deanne Watson, Dani Swain, Jeff Davis, Ramona Pop, and Samantha Collins for reagents and assistance with experiments; the HSCRB histology and flow cytometry, BPF next-gen sequencing, and Bauer flow cytometry and sequencing core facilities at Harvard University for technical support and critical discussions. J.R.A-D. is a Howard Hughes Medical Institute Fellow of the Life Sciences Research Foundation. D.A.M. is an investigator of the Howard Hughes Medical Institute. This work was supported by the Max Planck Society and NIH grants DP3K111898 and P01GM099117 (A.M.) and by grants from the Juvenile Diabetes Research Foundation, Helmsley Charitable Trust, and the JPB Foundation.
J.R.A.-D., J.D., N.R., J.H.R.K., and A.H. performed experiments. J.R.A.-D., J.D., J.C., and J.R.S. conducted bioinformatics analyses. J.R.A.-D., J.D., J.H.R.K., A.M., and D.A.M. designed the research, interpreted results, and wrote the manuscript.
CONFLICT OF INTEREST/DISCLOSURE STATEMENT
D.A.M. is a founder and scientific advisory board member for Semma Therapeutics. All other authors have no conflict of interest to report.
15th Annual Harvard Stem Cell Institute Retreat: Translating Science to the Clinic, Virtual (Wednesday May 20, 2020).
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