Journal: bioRxiv
Article Title: Transcription factor dynamics, oscillation, and functions in human enteroendocrine cell differentiation
doi: 10.1101/2024.01.09.574746
Figure Lengend Snippet: (A) Human intestinal biopsies, first cultured as 3D organoids in Matrigel, were subsequently expanded as 2D colonies on tissue culture dishes coated with a feeder layer of mitotically inactive mouse embryonic fibroblasts (MEFs). Scale bars 100 µm. (B) Ileal hISCs express SOX9 (an intestinal stem-cell marker), KI67 (a proliferative cell marker), GATA6 (a distal intestine marker), and E-cadherin (Ecad, an epithelial marker) but they lack proximal intestinal markers PDX1 and GATA4. Gene expression, determined by qRT-PCR and displayed relative to human fibroblasts, shows enrichment of broad intestinal markers GATA6 and CDX2 and of stem cell markers LGR5 and OLFM4 in both duodenal and ileal cells, and of proximal intestinal markers PDX1 and GATA4 only in duodenal cells. CHGA , a marker of differentiated EECs, was absent from uninduced hISCs. (C) Transcript abundance (qRT-PCR, shown relative to GAPDH ) reveals uniform expression of ý-actin and mCherry across time. Stem-cell markers GPRC5A and LGR5 were abundant in untreated cells (0 h) and EEC markers (native NEUROG3, CHGA, NEUROD1, SSTR2, EPHA4, CD200 and GNG2 ) were present by 96 h. Late EEC markers ( DSCAML1, TPH1, TAC1, GPR119 and ABCC8 ) were enriched at 192 h. (D) Bright-field and fluorescence microscopy of uninduced hISCs reveal mCherry expression in compact colonies that lack CHGA (scale bars 100 µm). Following 48 h of Tam exposure, mCherry expressing hISC NEUROG cells were dispersed and expressed CHGA by 96 h. Induced cells expressed serotonin (SER, 5HT, marks EC cells), SST (somatostatin, D cells) and GLP-1 (glucagon-like peptide 1, L cells). (E) Tam-induced hISC NEUROG cells were infused into the portal veins of immunodeficient NSG mice. Two months later, recipient livers contained numerous clusters of non-proliferating mCherry + cells that expressed 5HT and GLP-1. (F) Multi-pairwise analysis of bulk RNA-seq samples at 0, 24, 48, 72, 96 and 192 h after initial Tam or doxycycline (Dox) exposure revealed 4,002 genes differentially expressed between any two time points in two independent ileal hISC NEUROG lines (n=2 replicates each), one dependent on tamoxifen and other dependent on doxycycline to activate NEUROG3. (G) Dynamic expression of 4,002 genes over the course of EEC differentiation and maturation. Intestinal stem cell (ISC) markers, e.g., FOXQ1, SOX9, MYC and KLF9 , decline soon after NEUROG3 activation, giving way to EEC markers such as SOX4, PROX1, NEUROD1, CHGA and SYP between 72 h and 96 h, followed by enrichment of hormone genes such as SST, GHRL, GIP and TPH1 at 192 h. Selected Gene Ontology (GO) terms enriched among genes differentially enriched on different days are listed to the right. (H) Integrative Genomics Viewer (IGV) tracks showing bulk RNA-seq reads from representative ISC ( MYC ), intermediate cell ( SERPINA1 ), and mature EEC ( CHGA and SST ) genes. (I) Over the course of EEC differentiation, GO terms representing differentially expressed genes show rapid loss of proliferative cell properties, early interim immune signaling, and subsequent acquisition of neurosecretory features. (J) Mature EEC types and their known specific hormones.
Article Snippet: When organoid growth had slowed after a few passages, cells were pelleted, washed with F12K media, resuspended in hISC culture medium, (90% basal media - containing 60% Dulbecco’s Modified Eagle Medium (DMEM, high glucose, Thermo Fisher Scientific, 11965092), 20% F12K (Thermo Fisher Scientific, 21-127-022), and 20% fetal bovine serum (FBS, Corning 35-010-CV); supplemented with 10% Rspo2 conditioned medium, 10 mM nicotinamide (Sigma, N5535), 25 μM Primocin (Invivogen, ant-pm-1), 1 μM A8301 (Sigma, SML0788), 5 μg/ml Insulin (Sigma I0516), 10 μM Y27632 (LC Laboratories Y5301), 1μM DMH1 (Sigma D8946), 50 ng/ml EGF (R&D, 236EG200) and 2 μM T3 (Sigma, T3697)), seeded on dishes coated with mitomycin C (Cayman, 11435) inactivated DR4 mouse embryonic fibroblasts (MEFs, American Type Culture Collection, SCRC-1045), and expanded and maintained as 2D colonies at 37°C in a 7.5% CO 2 incubator.
Techniques: Cell Culture, Marker, Gene Expression, Quantitative RT-PCR, Expressing, Fluorescence, Microscopy, RNA Sequencing, Activation Assay
