Generation of multipotent foregut stem cells from human pluripotent stem cells - PubMed (original) (raw)
Generation of multipotent foregut stem cells from human pluripotent stem cells
Nicholas R F Hannan et al. Stem Cell Reports. 2013.
Abstract
Human pluripotent stem cells (hPSCs) could provide an infinite source of clinically relevant cells with potential applications in regenerative medicine. However, hPSC lines vary in their capacity to generate specialized cells, and the development of universal protocols for the production of tissue-specific cells remains a major challenge. Here, we have addressed this limitation for the endodermal lineage by developing a defined culture system to expand and differentiate human foregut stem cells (hFSCs) derived from hPSCs. hFSCs can self-renew while maintaining their capacity to differentiate into pancreatic and hepatic cells. Furthermore, near-homogenous populations of hFSCs can be obtained from hPSC lines which are normally refractory to endodermal differentiation. Therefore, hFSCs provide a unique approach to bypass variability between pluripotent lines in order to obtain a sustainable source of multipotent endoderm stem cells for basic studies and to produce a diversity of endodermal derivatives with a clinical value.
Figures
Figure 1
Activin and GSK3/WNT Control Anteroposterior Patterning of Definitive Endoderm In Vitro (A and B) Quantitative PCR (qPCR) and immunocytochemistry analyses show that DE cells grown for 5 days in the presence of activin-A express foregut anterior endoderm markers (SOX2 and HHEX) without expressing hindgut markers (CDX2 and HOXC5). (C and D) qPCR and immunocytochemistry analyses show that DE cells grown for 5 days in the presence of GSK3-beta inhibitor CHIR99021 express posterior endoderm markers (CDX2 and HOXC5) without expressing anterior markers (SOX2 and HHEX). (E and F) Fluorescence-activated cell sorting (FACS) analyses showing that DE cells derived from hESCs and grown for 5 days in the presence of activin express the anterior marker SOX2, while DE cells grown in the presence of CHIR99021 express the hindgut marker CDX2. Scale bars, 100 μm. Error bars represent SEM. See also Figure S1.
Figure 2
Hindgut Endoderm Generated from hPSCs Can Differentiate into Intestinal Derivatives When Grown in 3D Conditions (A and B) Hindgut CDX2-positive cells generated by growing DE cells in the presence of CHIR99021 for 5 days were grown in 3D cultures conditions to generate highly folded spheres resembling mature (A) and immature (B) gut organoids. (C) Accordingly, the resulting organoids could be grown for 2 months while progressively increasing the expression of gut markers (OLFM4, CHGA, MUC2, Villin, and KRT19). (D) Morphological similarities between gut organoid derived from hPSCs and primary mouse intestinal epithelial organoid cultures. Scale bars, 100 μm. Error bars represent SEM. See also Figure S1.
Figure 3
hPSC-Derived Foregut Can Self-Renew In Vitro (A) Method to differentiate and to propagate Foregut Stem Cells derived from hPSCs. (B) Bright-field image of foregut stem cells growing as an epithelium. (C and D) After ten passages, approximately 80% of hFSCs express Ki67. (E) CGH profile of late-passage hFSCs showing no change in copy number. (F) Growth curve of hFSCs. (G and H) qPCR and immunocytochemistry analyses showing that hFSCs derived from hESCs (H9) and hIPSCs (BBHX8 and A1ATD-1) can be grown for more than ten passages while maintaining the expression of foregut markers (CERB, HNF1β, HNF6, CXCR4, GATA4, HHEX, HNF4α, and SOX17). The expression of pluripotency (POU5F1, NANOG), pancreatic (PDX1), hepatic (AFP), lung (NKX2.1), and gut (CDX2) was not observed during propagation. (I) A near-homogenous population of hFSCs derived from both hESC- and hIPSC-coexpressing foregut genes was evident after ten passages. (C), (D), and (I) represent the number of positive cells counted from ten random immunocytochemistry fields in three experiments with a minimum of 100 cells per field normalized to the DAPI count. Scale bars, 100 μm. Error bars represent SEM. See also Figure S3.
Figure 4
hFSCs Do Not Form Teratomas and Differentiate into Cells of the Foregut Only (A) Large cystic hFSC outgrowth under the kidney capsule of a NOD-SCID mouse. (B) Cryosection of a hFSC outgrowth showing large cystic structures lined with epithelial cells. (C) Immunocytochemistry showing foregut outgrowths expressing EpCAM, PDX1, AFP, and NKX2.1. Scale bars, 100 μm or 50 μm as shown. See also Figure S4.
Figure 5
hFSCs Have the Capacity to Differentiate into Multiple Derivatives of the Foregut (A) Method to differentiate hFSCs into pancreatic cells. (B) hFSCs grown in these culture conditions progressively express pancreatic bud markers (PDX1 and HLXB9) and then endocrine markers (INS and NGN3). F, fetal pancreas; A, adult pancreas. (C) C-peptide and PDX1 expression was confirmed by immunocytochemistry of cells differentiated for 25 days. (D) Day 25 pancreatic cells released insulin into the medium when treated with high-glucose Dulbecco’s modified Eagle’s medium over 30 min. (E) Method used to differentiate hFSCs into hepatic cells. (F) After 25 days of differentiation, cells expressed hepatic markers (ALB, AFP, and A1AT). C, undifferentiated hESC; Hep, hFSC-derived hepatocytes; F, fetal liver; A, adult liver). (G) Expression of mature hepatocyte markers (ALB, A1AT, CK18, and HNF4α) was confirmed by immunocytochemistry. (H and I) Hepatocytes were capable of uptaking LDL from the medium (H) and secreted albumin (I). C, medium with no cells. Scale bars, 100 μm. ∗∗∗∗p < 0.0001. Error bars represent SEM. See also Figures S4–S6.
Figure 6
Single hFSCs Are Multipotent (A) GFP-expressing hPSCs were differentiated into hFSCs. (B) Single GFP-positive hFSCs were seeded onto a layer of non-GFP hFSCs and then expanded for five passages. The resulting population was then split into culture conditions inductive for liver or pancreatic differentiation. (C and D) GFP-hFSCs differentiated for 25 days were found to respectively generate cells expressing liver markers (ALB, LDL-uptake) and pancreatic markers (PDX1, C-peptide) from both hESC-derived (C) and hIPSC-derived (D) hFSCs. Scale bars, 100 μm.
Figure 7
Generation of hFSCs Overcomes Variability between hIPSC Lines (A) FACS analyses showing the fraction of cells coexpressing the endoderm/foregut markers SOX17/CXCR4 before isolation (colored plot) and after expansion (passage 5, black plot) of hFSCs generated from hIPSC lines with high (BBHX8) and low (CoxS8, CoxV3, and Line4) endoderm differentiation capacity. Secondary only antibody only control, gray population. (B and C) hIPSCs with low endoderm capacity of differentiation cannot differentiate into liver or pancreatic cells [COXS8(B), COXV3(B), 4(B)], while hFSCs generated from the same cells seven passages later can differentiate into cells expressing markers for hepatocytes (A1AT, AFP, and ALB HNF4a) and pancreatic cells (GCG, PDX1, INS, NGN3) at levels comparable to positive control (BBHX8). Error bars represent SEM.
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