Genetically engineering self-organization of human pluripotent stem cells into a liver bud-like tissue using Gata6

Abstract

Human induced pluripotent stem cells (hiPSCs) have potential for personalized and regenerative medicine. While most of the methods using these cells have focused on deriving homogenous populations of specialized cells, there has been modest success in producing hiPSC-derived organotypic tissues or organoids. Here we present a novel approach for generating and then co-differentiating hiPSC-derived progenitors. With a genetically engineered pulse of GATA-binding protein 6 (GATA6) expression, we initiate rapid emergence of all three germ layers as a complex function of GATA6 expression levels and tissue context. Within 2 weeks we obtain a complex tissue that recapitulates early developmental processes and exhibits a liver bud-like phenotype, including haematopoietic and stromal cells as well as a neuronal niche. Collectively, our approach demonstrates derivation of complex tissues from hiPSCs using a single autologous hiPSCs as source and generates a range of stromal cells that co-develop with parenchymal cells to form tissues.

Figure 1. System overview.

(a) Lentiviral constructs used to generate stable cells lines. (b) General timeline for experiments, media conditions and cell extractions for exome microarray analysis. Y, rock-inhibitor; Dox, doxycycline; PP-Med., pluripotency supporting medium. Basal medium: medium without additional growth factors or serum. Filled arrows: RNA isolation and microarray analysis from total cells. Open arrows: RNA isolation and microarray analysis from enriched cells (CXCR4, CD34: enrichment using MACS beads; man. extr., manual extraction of early neuronal cell clusters). (c) Process overview (left) and a model of cell types generated during experiment (right). hiPSC containing an inducible GATA6 transgene are seeded in a monolayer. Transgene expression is then triggered with a small inducer molecule (Dox) causing the cells to co-differentiate. Starting with an undifferentiated monolayer of hiPSCs, we obtain complex tissue after 15 days. LBLT, liver bud-like tissue; NPL, neuronal progenitor-like; HPN, haematopoietic niche. HPN includes all cellular components developed within the system, which directly or indirectly support emergence of haematopoietic-like processes. For more information refer to the text. ME, mesendoderm, PP: pluripotent cells (expressing pluripotency markers, not induced to mesendoderm), En, endoderm; Me, mesoderm; Ec, ectoderm; HpEn, hepatic endoderm; EP, endothelial progenitors; MP, mesenchymal progenitors; Ec, ectoderm; NEc, neurectoderm; HpLC, hepatocyte-like cells; ChLC, cholangiocyte-like cells; EnLC, endothelial-like cells; HE, haemangioblast-like cells; ErLC, erythrocyte-like cells; HmLC, haematopoietic progenitor-like cells; StLC, stellate-like cells; MsLC, mesenchyme/pericyte-like cells; NEp, neuronal progenitors.

Figure 2. Ectopic expression of Gata6 in hiPSCs induces a heterogeneous multi population niche including CXCR4⁺ definitive endoderm.

(a) Cell line A expresses EBFP2 (blue fluorescent protein) constitutively and GATA6-2A-EGFP (GATA6 and green fluorescent protein) in a Dox-inducible fashion. Cell line B expresses mKate2 (red fluorescent colour), constitutively. (b) Cell line A and B were mixed and seeded at a 9:1 ratio and induced with Dox (1,000 ng ml⁻¹) (c). By day 5, the two cell lines proliferate and segregate to distinct high EGFP⁺(also EBFP⁺) and mKate2⁺ subpopulations. When the blue cells express high EGFP (high GATA6), they look blue and green. If they express low amounts of EGFP they look blue (very weakly green) and stay together with mKate⁺ population. (d) Undifferentiated uninduced hiPSCs at day 3 are enriched in Nanog. (e) Dox-induced ectopic expression of lentivirally delivered GATA6 leads to segregation into GATA6⁺ or Nanog⁺ subpopulations. hiPSCs were seeded as single-cell suspension(+Dox, 1,000 ng ml⁻¹). Insets show that Nanog⁺ are weakly GATA6⁺. (f) Brachyury⁺ (also known as T) cells are interspersed with FoxA2⁺ endodermal cells at day 3 for a Dox-induced experiment. (g) Immunostaining for HA, FoxA2 and Nanog. (h) Analysis of the immunostaining: GATA6-HA induces endoderm, but is suppressed in the Nanog⁺ cluster. Cells with low Nanog levels require less GATA6 (HA epitope) to differentiate into endoderm (FoxA2). Scale bars, 200 μm.

Figure 3. Characterization of CXCR4⁺ cells.

(a) GATA6-expressing cells upregulate expression of CXCR4 (left). CXCR4-expressing cells surround TRA-1-81 compact cellular clusters at day 5 (right). TRA-1-81 recognizes hiPSC compact clusters at undifferentiated state. Scale bars, 200 μm. (b) Single-cell analysis of Dox-induced cells (1,000 ng ml⁻¹) at day 5 shows ∼93% of CXCR4-expressing cells are also GATA6⁺. (c) CellTrace fluorescent stain labelled hiPSCs on day 0 before Dox (P₀). The cells are transduced with Dox-inducible GATA6-2A-EGFP vector. (d) After 4 days of Dox (1,000 ng ml⁻¹) treatment, GATA6-induced cells express EGFP (P₁), proliferate and dilute the fluorescent dye through subsequent divisions to levels comparable to GATA6 non-expressing cells (P₂) (e) CXCR4⁺ transcriptional profiling, D, Day of the experiment (D0=Addition of Dox to the cell culture medium). Log₂ NI, log 2 normalized intensities. Scale bars, 200 μm.

Figure 4. Development of endothelial and mesenchymal like cells and maturation of hepatic endoderm.

(a) CD34 and CD93 were detected in endothelial progenitor-like cells on day 7. CEBPα⁺marks the hepatic endoderm in the background (scale bars, 200 μm). (b) Development of endothelial-like cells (arrows indicate CD31). CD34 is present in the hepatocyte-like cell fraction (CEBPα⁺, CD146⁻) and in the endothelial-like cells (CEBPα⁻, CD146⁺). (c) CD51⁺, NES⁺, PDGFRα⁺ mesenchymal stem cell-like cells develop in conjunction with hepatoblasts. (d) AAT and CEBPα on day 8 and (e) 10 show maturation of hepatic endoderm. (f) Upregulation of AAT and fibrinogen (Fib) synthesis (ELISA). Data are mean±s.e.m., n=3 per group. Scale bars: 200 μm.

Figure 5. Development of fetal liver parenchymal and non-parenchymal cells.

(a) CEBPα⁺ hepatocyte-like cells and CD31⁺ CD34⁺ vascular networks on day 14. (b) DES⁺ stellate-like cells (c) CK19⁺ AAT⁺ hepatocyte-like cells with CD146⁺ vascular-like structures. Scale bar, 200 μm. (d) Heatmap shows upregulation of hepatic genes between days 5 and 10. (UBC and EEF1A1 as used as control/housekeeping genes). (e) CK7⁺ bile duct-like channels develop within fetal hepatocyte-like cells; xz and yz slices on the left and bottom. Scale bar, 200 μm. (f) Further maturation of liver-like phenotype as evidenced by increasing albumin production (ELISA) and (g) albumin⁺ cells. Data are mean±s.e.m., n=3 per group. phAlb_mKate2 is a lentivirally integrated reporter where a short human albumin promoter drives expression of a red fluorescent protein (mKate2). Scale bar, 200 μm. (h) Confocal imaging and three-dimensional reconstruction of AAT⁺ hepatocyte-like cells (red) with an apical layer of CD34⁺ endothelial-like cells (green). Scale bar, 50 μm.

Figure 6. The emergence of fetal haematopoiesis after day 14.

(a) Heatmap shows temporal upregulation of markers associated with endothelial and haematopoietic cells in CD34⁺ isolated cells on day 10. (b) Black arrow: endothelial-like cell tube embedded in hepatocyte-like cells and filled with CD34⁺ cells. (c) Immunostaining shows expression of CD45 and HG. HG, pan-haemoglobin. Scale bar, 200 μm. (d) Microarray analysis shows haemoglobin γ expression is strongly upregulated by day 15. The Greek letters are representative of different types of globin chain of human haemoglobin expressed at different developmental stages. Scale bar, 200 μm. (e) Development of TAL1⁺ CD43⁺ cells among CD34+ population on day 8. (f) Left: close-up of a CD34⁺ tube-like structure covered by pericyte-like cells (NES⁺) that contains CD45⁺ haematopoietic-like cells on day 17. Scale bar, 200 μm. (g) Multipotency of isolated CD34⁺ population assessed by the Methocult CFU assay, showing generation of (h) erythrocyte (BFU-E) (i) granulocyte, macrophage (GM) (j) macrophage (M), and (k) granulocyte, erythrocyte, monocyte, megakaryocyte (GEMM) multicellular colonies. Scale bars in h–k, 100 μm. Data are mean±s.e.m. and representative of at least three cultures per group.