Patent Application
20200199538
The Sequence Listing written in file 1012196_ST25.txt, created on Sep. 14, 2016, 465,073 bytes, machine format IBM-PC, MS-Windows operating system, is hereby incorporated by reference in its entirety for all purposes.
The liver is a major organ that is responsible for regulating a multitude of complex metabolic processes. Defects in the liver present a significant burden today, but the management of liver diseases remains inadequate due to two maj or factors: a shortage of liver donors (Vilarinho and Lifton, 2012) and an incomplete understanding of the underlying mechanisms of liver pathology. Research on liver diseases is hampered by the lack of faithful models for metabolic diseases of hepatic origin. There is still a heavy reliance on mouse models, which cannot fully depict human disease pathogenesis and response to drugs (Grompe and Strom, 2013; Seok et al., 2013; van der Worp et al., 2010). On the other hand, disease modelling in human hepatocyte cultures fails to recapitulate complex diseases that involve interactions between multiple cell types within an organ. Furthermore, primary human hepatocytes are defiant to long-term expansion in culture (Mitaka, 1998; Shan et al., 2013), while hepatocyte-like cells differentiated from pluripotent stem cells (PSCs) (Gieseck et al., 2014; Si-Tayeb et al., 2010b) are limited with low differentiation efficiencies and a lack of mature functional capabilities found in the liver. There is a substantial need for high quality liver model that resembles the in vivo liver tissue from basic research to industrial and medical applications.
These issues can potentially be overcomed using 3 dimensional in vitro culture systems to generate tissue-like organoids. Organoids are refered to as “mini-organ” defined by three specific criteria 1) Having at least 2 organ-specific cell types, 2) Cells self-organize in 3D to form structure resembling tissues in the organ and 3) is capable of manifesting organ-specific functionality (Lancaster and Knoblich, 2014). They can be created from cells of human origin to avoid surreptitious species-specific differences in disease pathogenesis and drug response, while mimicking in vivo disease presentation including interactions between various cell types involved in 3D space (Matano et al., 2015; van de Wetering et al., 2015); these properties make them ideal for the study of liver function and diseases in vitro. While no hepatic organoids with the above criteria have been describe, complex 3D liver cultures have been derived from human induced pluripotent stem cells (iPSCs) (Takebe et al, 2013) and adult liver stem cells (Huch et al., 2015). These technologies have not been shown to harbor functional interactions between the two major hepatic cell types: hepatocytes and cholangiocytes and do not exhibit any of liver specific function in vitro. In addition, no liver tissue structures have been observed in these 3D liver cultures in vitro. Current organoid generation protocols are also limited by extremely high costs for large scale expansion (Spence et al 2011, Lancaster et al 2013, Takasato et al 2016), which is necessary for downstream applications requiring a large number of cells such as engraftment (Fisher and Strom, 2006) and high-throughput screening. Scalability is dependent on the ability to propagate culture to large quantities via the proliferation of itself or its precursors. As such, self-renewing PSCs have been regarded as a promising source for terminally differentiated cells.
However, PSC generation of organoids is complex and requires differentiation across multiple intermediate states to generate specific somatic cell types. For endodermal tissues such as the liver, mimicking human embryonic morphogenesis through the sequential exposure of PSCs to cytokines enables their derivation in vitro (Basma et al., 2009; D'Amour et al., 2006; Spence et al., 2011). However, these methods (1) utilize complex and lengthy differentiation protocols often with only low to moderate efficiency (Murry and Keller, 2008), (2) tend to yield cells with immature properties and incomplete functionality, and (3) harbour a risk for teratoma formation (Hentze et al., 2009). The number of intermediate states (steps) required in a protocol is an important factor for the overall efficiency of differentiation. At 80% differentiation efficiency for each step, a 2 step differentiation protocol would have 64% overall efficacy and 3 step protocol would be 51.2%. The low efficacy of 50% differentiation efficacy would also mean that 1 out of 2 cells are not desired and these contaminating cells results in many complications in downstream applications. In order to overcome this issues, several groups have created self-renewing endoderm progenitors that can be used as an alternative cell source (Cheng et al., 2012; Hannan et al., 2013), but these early endoderm progenitors remain relatively naive in the differentiation landscape and still requires much differentiation steps to generate desired endoderm cell types. There is a need to generate later endoderm progenitors that can give rise to organ cell type of interest in shorter time and lesser steps. Beside late endoderm progenitors, adult stem cells which are already committed to form specific organ lineage are desirable cell sources. In addition, these adult stem cells would generate cell types of adult phenotype compared to cells generated from PSC origins.
Described herein are multipotent endoderm spheroid progenitor cells (MESPs), a human pluripotent stem cell (hPSC)-derived self-renewing progenitor population that can serve as a source of human hepatic cells as well as other lineages from the posterior foregut such as the intestine and pancreas. By recapitulating the stepwise process of liver differentiation during development, the methods described herein enable the scalable production of MESP-derived hepatic organoids that contain the major parenchyma hepatic liver cell types, hepatocytes and cholangiocytes. These two cell types self-organize into structures resembling the human liver unit and possess many liver specific functions. Using Genome editing CRISPR/Cas technology, the instant inventors developed a liver organoid model of familiar hypercholesterolemia, and demonstrated the response of the diseased liver organoid to statins. In addition, the inventors generated the organoids in a high throughput manner which can be adapted for large scale screenings, demonstrating the applicability of the technology for both research and industrial applications. In addition, employing similar technology, liver organoids were generated from adult stem cells and these organoids exhibit similar structures and liver specific functions as organoids generated from MESP cells.
The inventors have surprisingly discovered that a single media can be used to generate both hepatocytes and cholangiocytes. This is contrary to what is known in the art. For example, TGFß signaling promotes bile duct cell formation but inhibits hepatocyte formation. TGFß signaling molecules are typically added to bile duct cell cultures, but are excluded from culture media used to generate hepatocytes, and in many methods inhibitors of the TGFß pathway are added to the media used to generate hepatocytes.
In one aspect, a liver organoid is provided, the liver organoid comprising at least two cell types selected from the group consisting of hepatocytes, cholangiocytes, liver specific endothelial cells (LSEC), stellate cells, hepatic myofibroblast and hepatoblasts. In some embodiments, the hepatocytes
In some embodiments, the hepatocyte markers comprise or consist of HNF4a (NCBI: 3172), FAH (NCBI: 2184), TAT (NCBI: 6898), GCK (NCBI: 2645), TTR (NCBI: 7276), MET (NCBI: 4233), GLU1/MGAM (NCBI: 8972), FAHD2A (NCBI: 51011), HNF1B (NCBI: 6928), HNF1A (NCBI: 6927), CYP3A4 (NCBI: 1576), CYP2C9 (NCBI: 1559), CYP2C19 (NCBI: 1557), CYP1A2 (NCBI: 1544), CYP2E1 (NCBI: 1571), CYP2D6 (NCBI: 1565), CYP3A7 (NCBI: 1551), CYP1A1 (NCBI: 1543), CYP3A5 (NCBI: 1577), CYP27A1 (NCBI: 1593), MRP2 (NCBI:1244), NTCP (NCBI: 6554), OATP1B3 (NCBI: 28234), UGT2B7 (NCBI: 7364), UGT2B15 (NCBI: 7366), UGT1A1 (NCBI: 54658), CEBP (NCBI: 1050), KRT8 (NCBI: 3856), NOTCH2 (NCBI: 4853) and CYP2B6 (NCBI: 1555).
In some embodiments, the cholangiocytes express CK7 but do not express albumin (ALB). In some embodiments, the cholangiocytes further express a marker selected from CFTR (NCBI: 1080), CK19 (NCBI: 3880), HNF1B (NCBI: 6928) or SOX9 (NCBI: 6662).
In some embodiments, the hepatoblasts express at least one marker selected from the group consisting of SOX9 (NCBI: 6662), CK19 (NCBI: 3880), CK18 (NCBI: 3875), HNF4a (NCBI: 3172), PROX1 (NCBI: 5629), ONECUT1 (NCBI: 3175), AFP (NCBI: 174), and ALB (NCBI: 213).
In some embodiments, the liver specific endothelial cells (LSEC) express at least one marker selected from the group consisting of CD45, CD80, CD86, CD11c, VAP1, STAB1 and CD31, wherein the CD31 expression that is mainly expressed in the cytoplasm and not on the cell surface.
In some embodiments, the stellate cells express at least one marker selected from the group consisting of GFAP, VIM, LHX2, LRAT, PDGFRb, HAND2, ICAM-1, VCAM-1, and N-CAM-1.
In some embodiments, the hepatic myofibroblast express a marker selected from the group consisting of COL1A1 and α-SMA.
In some embodiments, the parenchymal cell types originate from the same stem cell.
In some embodiments, the liver organoid cells are cultured in suspension without the use of extracellular matrices.
In some embodiments, the organoids are capable of performing liver functions and exhibit a spatially organized structure observed in liver. In some embodiments, the liver functions are selected from the group consisting of liver specific metabolic activities, albumin secretion, glycogen storage, low density lipo-protein uptake, bile acid production, drug metabolism, and cytochrome enzymatic activities. In some embodiments, the spatially organized structure comprises a core of hepatocytes and peripheral bile duct-like structures formed by cholangiocytes around the core of hepatocytes. In some embodiments, the spatially organized structure comprises endogenous extracellular matrix adhesion molecules. In some cases, the spatially organized structure comprises liver parenchymal cells in both the interior and exterior of the organoid. In some embodiments, the hepatocytes are connected by a network of bile canaliculi to the cholangiocyte bile duct-like structures.
In another aspect, a media for generating hepatic organoids is provided, the media comprising:
In some embodiments, the media further comprising a WNT-signaling activator.
In another aspect, a media for generating hepatic organoids is provided, the media comprising:
In another aspect, a method of deriving and maintaining a hepatic (liver) organoid is provided, the method comprising:
In some embodiments, the endoderm stem cell is an early endoderm progenitor cell, a pluripotent stem cell, an induced pluripotent stem cell, a human embryonic stem cell, an MESP, or an adult liver stem cell. In some embodiments, the endoderm stem cell is an MESP or an adult liver stem cell.
In some embodiments, the first medium comprises:
In some embodiments, the culturing under a) is carried out together with a cellular support or an extracellular matrix. In some embodiments, the extracellular matrix promotes cell differentiation and is made of a material selected from the group consisting of matrigel, gelatine, methylcellulose, collagen, alginate, alginate beads, agarose, fibrin, fibrin glue, fibrinogen, blood plasma fibrin beads, whole plasma or components thereof, laminins, fibronectins, protecogylcans, HSP, chitosan, heparin, other synthetic polymer or polymer scaffolds and solid support materials.
In some embodiments, the culturing under a) is for 1 to 10 days or 1 to 8 days or 1 to 6 days. In some embodiments, the culturing under b) is for 6 to 12 days or 4 to 10 days or 6 to 8 days. In some embodiments, the culturing under c) is for 18 to 26 days or 20 to 24 days or 19 to 22 days.
In some embodiments, the second cell culture medium comprises:
In some embodiments, the second cell culture medium further comprises a component to promote survival of late hepatic progenitors, wherein the component is selected from one or two or three or all of the following components:
In some embodiments, the component for inducing late hepatic progenitor formation is a WNT-signaling activator, an inhibitor of γ-secretase; and/or a YAP inhibitor.
In some embodiments, the third cell culture medium comprises:
In some embodiments, the third cell culture medium further comprises:
In another aspect, a multipotent endoderm spheroid progenitor (MESP) cell is provided. In some embodiments, the MESP expresses one, two, three, four, five, six, seven, or more or all of the markers selected from the group consisting of HNF4A (NCBI Gene: 3172), PDX1 (NCBI Gene: 3651), CDX2 (NCBI Gene: 1045), SOX9 (NCBI Gene: 6662), KRT19 (NCBI Gene: 3880), AFP (NCBI Gene: 174), ONECUT2 (NCBI Gene: 9480), LGR5 (NCBI Gene: 8549), EPHB2 (NCBI Gene: 2048), LGR4 (NCBI Gene: 55366), NR5A2 (NCBI Gene: 2494), CDH1 (NCBI Gene: 999), KRT7 (NCBI Gene: 3855), ZNF503 (NCBI Gene: 84858), MSX2 (NCBI Gene: 4488), TRPS1 (NCBI Gene: 7227), ASCL2 (NCBI Gene: 430), IRF8 (NCBI Gene: 3394), HNF4G (NCBI Gene: 3174), ID2 (NCBI Gene: 3398), CD44 (NCBI Gene: 960), EPCAM (NCBI Gene: 4072), MET (NCBI Gene: 4233), IHH (NCBI Gene: 3549) and CLDN3 (NCBI Gene: 1365). In some embodiments, the MESP does not express a marker selected from the group consisting of SOX2 (NCBI Gene: 6657), CER1 (NCBI Gene: 9350), GATA4 (NCBI Gene: 2626), SOX17 (NCBI Gene: 64321), FOXA2 (NCBI Gene: 3170) and CXCR4 (NCBI Gene: 7852). In some embodiments, the karyotype of the MESP is normal for at least 10 passages in culture. In some embodiments, the MESP cells are polarized.
In another aspect, a culture medium for deriving and maintaining endoderm spheroid progenitor cells is provided, the medium comprising:
In some embodiments, the medium further comprises a steroid. In some embodiments, the WNT-signaling activator is a GSK3 inhibitor. In some embodiments, the medium further comprises an activator of AKT/PI3K signaling pathway and MAPK signaling pathway; an activator of STAT3, GAB1 mediated cell adhesion and AKT/PI3K signaling pathway; an activator of cAMP-dependent pathways and/or Protein Kinase A signaling pathway; a compound that activates the Notch receptor; a molecule which is an repressor of NFκB activity and activator of mitogen-activated protein (MAP) kinase ERK, p38 and JNK; a compound selected from the group consisting of nicotinamide, nicotinic acid, 5-fluoronicotinamide, isonicotinic acid hydrazide, and nikethamide; and an inhibitor of histone deacetylase.
In another aspect, a method for producing a multipotent spheroid progenitor (MESP) cell is provided, the method comprising:
In some embodiments, the conditions suitable to differentiate the endoderm progenitor cell into a definitive endoderm (DE) cell, and the conditions suitable to differentiate the definitive endoderm cell into a primitive gut cell comprise two-dimensional monolayer culture.
In some embodiments, the conditions suitable to differentiate the endoderm progenitor cell into a definitive endoderm (DE) cell, and the conditions suitable to differentiate the definitive endoderm cell into a primitive gut cell comprise three-dimensional monolayer culture. In some embodiments, the conditions suitable to differentiate the primitive gut cell into a MESP cell comprise three-dimensional culture.
In some embodiments, the first medium comprises an activator of TGF-β signaling pathway. In some embodiments, the second medium comprises an activator of BMP signaling pathway and an activator of FGF signaling pathway. In some embodiments, the third medium comprises an inhibitor of TGF-β signaling pathway, an activator of WNT signaling pathway, and an activator of Notch signaling pathway. In some embodiments, the third medium further comprises:
In some embodiments, the cellular support comprises a material selected from the group consisting of matrigel, gelatine, methylcellulose, collagen, alginate, alginate beads, agarose, fibrin, fibrin glue, fibrinogen, blood plasma fibrin beads, whole plasma or components thereof, laminins, fibronectins, protecogylcans, HSP, chitosan, heparin, and synthetic polymers or polymer scaffolds.
In another aspect, a pancreatic spheroid is provided. In some embodiments, the pancreatic spheroid comprises cells that express the markers PDX1 (NCBI 3651) and NKX6.1 (NCBI 4825). In some embodiments, the pancreatic spheroid comprises pancreatic exocrine and endocrine cells. In some embodiments, the pancreatic spheroid comprises cells that express:
In some embodiments, the pancreatic spheroid cells secrete one or more hormones or enzymes selected from INS(NCBI 3630), GCG (NCBI 2641), SST (NCBI 6750) or PRSS1 (NCBI 5644). In some embodiments, the pancreatic spheroid cells do not express EPCAM and SOX9.
In another aspect, a method of manufacturing a pancreatic spheroid is described, wherein the method comprises:
In some embodiments, the cells in a) are cultured together with an extracellular matrix. In some embodiments, the culturing under a) is for 1 to 10 days or 1 to 8 days or 1 to 6 days or 1 to 4 days. In some embodiments, the culturing under b) is for 8 to 16 days or 6 to 14 days or 4 to 12 days or 5 to 10 days. In some embodiments, the culturing under c) is for 18 to 26 days or 20 to 24 days or 19 to 22 days or 16 to 20 days.
In some embodiments, the early pancreatic endoderm progenitor expresses one or more markers selected from: SOX9 (NCBI: 6662), PDX1 (NCBI: 3651), NKX6.1 (NCBI: 4825), and CK19 (3880). In some embodiments, the late pancreatic endoderm progenitor express one or more markers selected from: PDX1 (NCBI: 3651), NKX6.1 (NCBI: 4825), NEUROG3 (NCBI: 50674), NKX2.2 (NCBI: 4821), NEUROD1 (NCBI: 4760), and PAX6 (NCBI: 5080).
In another aspect, a medium for early pancreatic endoderm progenitor formation is provided, wherein the medium comprises:
In some embodiments, the medium further comprises a molecule which is an repressor of NFκB activity and/or an activator of mitogen-activated protein (MAP) kinase ERK, p38 and JNK. In some embodiments, the medium further comprises:
In another aspect, a medium for late pancreatic endoderm progenitor formation is provided, wherein the medium comprises:
In some embodiments, the medium further comprises an inhibitor of γ-secretase; an activator of AKT/PI3K signaling pathway and MAPK signaling pathway; a molecule which is an repressor of NFκB activity and activator of mitogen-activated protein (MAP) kinase ERK, p38 and JNK; and/or a compound selected from the group consisting of nicotinamide, nicotinic acid, 5-fluoronicotinamide, isonicotinic acid hydrazide, and nikethamide.
In another aspect, a medium for deriving and maintaining a pancreatic spheroid, wherein the medium comprises:
In some embodiments, the medium further comprises an inhibitor of γ-secretase; an activator of AKT/PI3K signaling pathway and MAPK signaling pathway; a molecule which is an repressor of NFκB activity and activator of mitogen-activated protein (MAP) kinase ERK, p38 and JNK; and/or a compound selected from the group consisting of nicotinamide, nicotinic acid, 5-fluoronicotinamide, isonicotinic acid hydrazide, and nikethamide.
In another aspect, an intestinal organoid is provided. In some embodiments, the intestinal organoid comprises cells that express the intestinal markers Villin and CDX2. In some embodiments, the intestinal organoid comprises coiled structures having a lumen. In some embodiments, the intestinal organoid comprises cells are polarized and comprise an asymmetrical distribution of Villin.
In another aspect, a method for screening a compound for a biological effect is provided. In some embodiments, the method comprises contacting a liver or intestinal organoid described herein with a compound (e.g., a test compound), and determing the biological effect.
In some embodiments, the compound is a small molecule, such as an organic molecule having a molecular weight of less than about 50 kDa, less than about 10 kDa, less than about 1 kDa, less than about 900 daltons, or less than about 500 daltons. In some embodiments, the biological effect is toxicity. In some embodiments, the expression or activity of a marker is determined after contacting the organoid with the test compound.
The term “about,” when modifying any amount, refers to the variation in that amount typically encountered by one of skill in the art, i.e., in the field of stem cell and organoid derivation and differentiation. For example, the term “about” refers to the normal variation encountered in measurements for a given analytical technique, both within and between batches or samples. Thus, the term about can include variation of 1-10% of the measured amount or value, such as +/−1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9% or 10% variation. The amounts disclosed herein include equivalents to those amounts, including amounts modified or not modified by the term “about.”
All numerical ranges disclosed herein include the lower and upper end points of the range, and all numerical values in between the end points, to the significant digit. For example, a range of 1 to 10 includes 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10. A range of 0.1 to 5.0 includes 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, . . . 4.8, 4.9, and 5.0.
The term “substantially” when referring to expression of a gene, protein or cellular marker refers to the complete or nearly complete extent or degree of expression. For example, a cell population that is “substantially” negative of a particular cellular marker is either completely negative for the particular cellular marker or at least 95%, or at least 96%, or at least 97%, or at least 98%, or at least 99%, or at least 99.9% of the cell population is negative for the particular cellular marker. A cell culture system that is “substantially” free of a particular agent would mean that the cell culture system is either completely free of the agent or is at least 95%, or at least 96%, or at least 97%, or at least 98%, or at least 99%, or at least 99.9% free of the particular agent.
The term “organoids” refers to an in vitro 3 dimensional population of cells which resemble the vertebrate, mammalian or human organ. An organoid satisfies the following criteria; 1) contains multiple cell types of the organ, 2) different cell types are spatially organized into structures that resemble the organ tissue, 3) organoids should perform organ specific functions in vitro.
The term “spheroids” refers to an in vitro three-dimensional population of cells which form sphere-like structures. Unlike organoids, spheroids do not have multiple organ cell types, consist mainly of stem cells and do not form structures resembling the organs.
The term “parenchyma” refers to the functional cell types that compose the organ as compared to the connective and vascular supportive tissues.
The term “progenitor cell” refers to a cell state which has the ability to give rise to one or more daughter cells of a different cell state.
The term “early endoderm progenitor cell” refers to a cell that has ability to generate endodermal cell types in the organs of the human gut system. The early endoderm progenitor cell typically expresses early endoderm markers SOX17 and CXCR4.
The term “late endoderm progenitor cell” refers to a cell that has ability to generate endodermal cell types in the organs of the human gut system. The late endoderm progenitor cell typically expresses SOX9 but does not express early endoderm markers SOX17 and CXCR4.
The term “endoderm progenitor cell” refers to a cell that has the potential to generate all cell types found in the differentiated or adult liver tissue.
The term “endoderm spheroid progenitor cell” refers to a cell that is maintained in a spheroid culture system and has the potential to generate generate all cell types found in the differentiated or adult liver, intestine and pancreatic tissue.
The term “early hepatic progenitor” refers to a cell that has the potential to generate cell types found in the differentiated or adult liver tissue and expresses early hepatic progenitor markers such as AFP.
The term “late hepatic progenitor” refers to a cell that has the potential to generate cell types found in the differentiated or adult liver tissue and expresses late hepatic progenitor markers such as ALB.
The term “adult liver stem cell” refers to a cell that is isolated from adult liver and has the capacity to produce different cell types of the liver. In some embodiments, the adult liver stem cell is isolated from a mammal such as a rodent (e.g., mice or rats), bovine, porcine, or human.
The term “stem cell” refers to a cell state which can stably proliferate and maintain its cell state. A stem cell can undergo symmetrical cell division to give rise to 2 daughter cells of similar cell state or asymmetrical division to give rise to 1 daughter cell of similar cell state and 1 daughter cell of different cell state. The term includes an undifferentiated or unspecialized cell capable of perpetuating itself through cell division and having the potential to give rise to differentiated cells with specialized functions, such as liver cells, pancreatic cells, and intestinal cells.
The term “bile duct-like” refers to structures that resemble the bile ducts in a liver. The bile duct is formed by cholangiocytes which are organized to envelope a lumen.
The term “functionally connected” refers to a structural connection between two separate cell types, which, for example, facilitates the transportation of molecules between the two separate cell types, or provides conditions that promote maturation and differentiation of one or more cell types described herein. In some embodiments, the functional connection refers to transport of molecules between cells by diffusion, by active transport, or through a physical cellular structure such as a bile duct-like structure or bile canaliculi.
The term “not expressed” or “undetectable” refers to marker expression that is not more than 1.5 fold greater than the background expression or expression by a negative control. For example, if the assay is an immunofluorescence (IF) staining assay, then the protein is considered “not expressed” if the fluorescent signal is not greater than 1.5 fold the background signal when omitting the primary detection antibody, or is not greater than 1.5 fold the fluorescence signal of a control cell that does not express the marker (i.e., is negative for the marker). In microarray assays and quantitative RT-PCR assays, the transcript is not expressed when the RNA expression or relative intensity is less than 1.5 fold higher than a control cell that does not express the transcript.
The term “expressed” or “enriched” refers to the presence of more than 1.5-fold greater detectable marker expression when compared to background expression or expression by a negative control. For example, if the assay is quantitative PCR assay, then a marker is considered to be “expressed” or “enriched” if the expression level is greater than 1.5-fold the expression of a negative control sample. If the assay is an immunofluorescence (IF) staining assay, then the marker protein is considered “expressed” or “enriched” if the fluorescent signal is greater than 1.5 fold the background signal when omitting the primary detection antibody, or is greater than 1.5 fold the fluorescence signal of a control cell that does not express the marker (i.e., is negative for the marker).
The term “suspension culture” or “suspension culture system” refers to any culture conditions or system in which the cells are not embedded in a solid or semi-solid matrix and are free floating in the culture apparatus without resting on the bottom of the apparatus, or are not attached to a cellular feeder layer or cellular support layer.
The term “solid support materials” refers to solid or semi solid materials used in supporting cell growth where the cells are not in suspension culture.
The term “cellular support” refers a material that provides structural and nutritional support to cells in culture. The cellular support can provide both structural support and cytokines that play a part in maintaining liver stem cells in the undifferentiated state. The cellular support can comprise a material selected from the group consisting of matrigel, gelatine, methylcellulose, collagen, alginate, alginate beads, agarose, fibrin, fibrin glue, fibrinogen, blood plasma fibrin beads, whole plasma or components thereof, laminins, fibronectins, protecogylcans, HSP, chitosan, heparin, and synthetic polymers or polymer scaffolds. In some embodiments, the cellular support maintains the stem cells in a 3D structure such as a spheroid or organoid. In some embodiments, the cellular support comprises an extracellular matrix as further described herein.
The term “endogenous” refers to a component or molecule of a biological system that is produced by or synthesized by the cells or organoids described herein. The term “exogenous” refers to a component or molecule of a biological system that orginates from, or is produced or synthesized by an agent outside the biological system, for example, a molecule that is not produced by or synthesized by the cells or organoids described herein.
The term “genetically modified” refers to a cell that comprises an exogenouos nucleic acid that is not present in the unmodified cell, or that does not have the same structure as an endogenous nucleic acid or gene.
Described herein is a method to derive spheroid progenitor cells of the endoderm lineage from pluripotent stem cells (PSCs). The progenitor cells can be stably propagated and expanded in culture. These spheroid progenitor cells exhibit potential to generate multiple organ cell types of the endoderm lineage, including the intestine, liver and pancreas. Hence, this spheroid progenitor is hereby described as Multipotent Endoderm Spheroid Progenitor (MESP). The MESPs can be stably propagated, do not exhibit signs of senescence, and maintain homogenous expression of stem cell markers. The cells also maintain a normal karyotype of 23 pairs of chromosome without major chromosomal mutations even after long term culture. This stable progenitor culture system is scalable and more cells can be generated with larger culture vessels, making these cells suitable for large scale production of downstream organ cell types for various applications including regenerative therapy and industrial applications.
The MESP represents a different endoderm progenitor stem cell state that differs from endoderm progenitor cells reported by Cheng et al. (Cell Stem Cell, 2012) and Hannan et al. (Stem Cell Reports, 2013). Cheng et al (Cell Stem Cell, 2012) report a progenitor stem cell that resembles early definitive endoderm stem cells, which is the earliest stem cell stage of the endoderm lineage development. Hannan et al (Stem Cell Reports, 2013) describe a progenitor stem cell that resembles the foregut progenitor during endoderm development. The culture conditions and stem cell markers of these two reports are highly similar. On the other hand, the culture conditions, media and stem cell markers of MESP are different from those described in the above references (see Table 1). MESP cells express markers similar to those expressed by cells of the posterior foregut during late endoderm lineage development (
In certain embodiments, the pluripotent stem cells are embryonic stem cells (hESCs).
In certain embodiments, the pluripotent stem cells are induced pluripotent stem cells (iPSCs). The iPSCs can be generated from any human adult tissues using the iPSCs reprogramming technology into cells with pluripotent capacity. MESP can then be generated from these pluripotent stem cells.
In certain aspects, MESP can also be generated from early endoderm progenitors reflecting early endoderm lineage development. The current methods generate MESP via stepwise differentiation along the endoderm lineage, where the PSCs first become definitive endoderm cells and subsequently differentiate into primitive gut endoderm cells.
In certain embodiments, the pluripotent stem cells can be genetically modified by genome editing tools such as the Clustered regularly interspaced short palindromic repeats (CRISPR)/Cas system. These pluripotent stem cells maintain their pluripotential capacity and MESP can be generated from these genetically modified PSCs.
In certain embodiments, the pluripotent stem cells can be induced pluripotent stem cells (iPSCs) from human tissues with specific genetic diseases. The disease-specific human iPSCs maintains their pluripotential capacity to give rise to endoderm lineage tissues. MESP can be generated from these disease-specific iPSCs.
The Multi Endodermal Spheroid Progenitor (MESP) culture system described herein comprises a plurality of soluble agents in a stem cell culture media and a cellular support capable of providing structural and nutritional support. The cellular support maintains the progenitor cells in a 3D structure such as a spheroid or organoid. The cellular support provides both structural support and cytokines that plays a part in maintaining liver stem cells in the undifferentiated state. In some embodiments, the plurality of soluble agents comprises one or more growth factors, an enhancer of the (canonical) WNT pathway, and a stem cell differentiation inhibitor.
In some embodiments, a method for producing a multipotent spheroid progenitor (MESP) cell is provided, the method comprising:
In some embodiments, the conditions suitable to differentiate the endoderm progenitor cell into a definitive endoderm (DE) cell, and/or the conditions suitable to differentiate the definitive endoderm cell into a primitive gut cell comprise two-dimensional or monolayer culture. In some embodiments, the conditions suitable to differentiate the endoderm progenitor cell into a definitive endoderm (DE) cell, and/or the conditions suitable to differentiate the definitive endoderm cell into a primitive gut cell comprise three-dimensional culture. In some embodiments, the conditions suitable to differentiate the primitive gut (GUT) cell into a MESP cell comprise three-dimensional culture. In some embodiments, the endoderm progenitor cell can be cultured in the first or second medium to differentiate the endoderm progenitor cell into a DE cell and a primitive gut cell, which can subsequently be cultured in the third medium to generate MESP cells.
In some embodiments, the cellular support comprises a material selected from the group consisting of matrigel, gelatine, methylcellulose, collagen, alginate, alginate beads, agarose, fibrin, fibrin glue, fibrinogen, blood plasma fibrin beads, whole plasma or components thereof, laminins, fibronectins, protecogylcans, HSP, chitosan, heparin, and synthetic polymers or polymer scaffolds.
In some embodiments, the first culture medium comprises an activator of the TGF-β signaling pathway, such as Activin (e.g., Activin A, B or AB) or TGF-3. In some embodiments, the first culture medium futher comprises an activator of the BMP signaling pathway and an activator of the FGF signaling pathway.
In some embodiments, the second medium comprises an activator of the BMP signaling pathway and an activator of the FGF signaling pathway.
In some embodiments, the third medium comprises an inhibitor of the TGF-β signaling pathway, an activator of the WNT signaling pathway, and an activator of the Notch signaling pathway. In some embodiments, the third culture medium futher comprises a steroid, an activator of cAMP-dependent pathways, such as an activator of Protein Kinase A signaling pathway, an activator of the AKT/PI3K signaling pathway, and an inhibitor of histone deacetylase (HDAC), as described herein.
In some embodiments, the culture medium for deriving and maintaining endoderm spheroid progenitor cells comprises or consists of at least one, two, three, four, five, six, seven, eight or all of the following:
In some embodiments, the stem cell differentiation inhibitor is a TGF-beta signaling inhibitor, wherein the TGF-β inhibitor is characterized by any one of the following:
The TGF-beta inhibitor can block activation of the TGF-beta pathway, which induces stem cell differentiation, whereas inactivation of the TGF-beta pathway can maintain proliferation of endodermal stem cells. In some embodiments, the TGF-beta inhibitor is selected from the group consisting of:
In some embodiments, the TGF-beta inhibitor is used at a concentration of between about 0.5 nM to 20 μM, or 100 nM to 10 μM, or 250 nM to 5 μM, or 400 nM to 2.5 μM, or 0.5 nM to 1 μM, or 0.5 nM to 0.5 μM, or between about 1.5 nM to 0.4 μM, or between about 10 nM to 0.3 μM, or between about 30 nM to 0.2 μM, or between about 40 nM to 0.1 μM, or between about 50 nM to 85 nM, or about 1, 5, 15, 25, 30, 35, 45, 50, 65, 75, 130, 150, 170, 250, 350 or 450 nM; or about 0.5, 0.7, 0.9, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 8, 9, 10, 12, 14, 16, 18, or 20 μM. In some embodiments, the TGF-beta inhibitor selected from the group consisting of (i) to (x) above is used at a concentration of between about 0.5 nM to 20 μM, or 100 nM to 10 μM, or 250 nM to 5 μM, or 400 nM to 2.5 μM, or 0.5 nM to 1 μM, or 0.5 nM to 0.5 μM, or between about 1.5 nM to 0.4 μM, or between about 10 nM to 0.3 μM, or between about 30 nM to 0.2 μM, or between about 40 nM to 0.1 μM, or between about 50 nM to 85 nM, or about 1, 5, 15, 25, 30, 35, 45, 50, 65, 75, 130, 150, 170, 250, 350 or 450 nM; or about 0.5, 0.7, 0.9, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 8, 9, 10, 12, 14, 16, 18, or 20 μM.
In some embodiments, the steroid is capable of inhibiting the NF-κB pathway, activating the PI3K/AKT/mTOR pathway, inhibiting the TGF-β signaling pathway and/or inhibiting the IGF signaling pathway. In some embodiments, the steroid is a corticosteroid such as a glucocorticoid or an anti-inflammatory glucocorticoid which improves maintenance of endodermal stem cells.
In some embodiments, the glucocorticoid is selected from the group consisting of:
In some embodiments, the steroid, corticosteroid or glucocorticoid (such as dexamethasone) is used at a concentration of between about 0.5 μM to 200 μM, or between about 1.5 μM to 150 μM, or between about 5 μM to 100 μM, or between about 10 μM to 90 μM, or between about 20 μM to 80 μM, or between about 30 μM to 70 μM, or between about 40 μM to 60 μM, or about 2, 8, 15, 25, 30, 35, 45, 65, 75, 110, 130, 140, 160, 170 or 190 μM.
In some embodiments, the WNT-signaling activator is a Glycogen synthase kinase 3 (GSK3) inhibitor.
In some embodiments, the GSK3 inhibitor is selected from the group consisting of:
In some embodiments, the WNT-signaling activator is used at a concentration of between about 0.1 μM to 10 μM, or between about 0.5 μM to 8 μM, or between about 1 μM to 7 μM, or between about 2 μM to 6 μM, or between about 3 μM to 5 μM, or about 0.2, 0.3, 0.4, 3, 4, 4.5, 5, 6, 7.5, 8.5, 9, 9.5 or 10 μM.
In some embodiments, the GSK3 inhibitor (e.g., CHIR-09921) is used at a concentration of between about 0.1 M to 10 μM, or between about 0.5 M to 8 μM, or between about 1 M to 7 μM, or between about 2 M to 6 μM, or between about 3 M to 5 μM, or about 0.2, 0.3, 0.4,3, 4, 4.5, 5, 6, 7.5, 8.5, 9, 9.5 or 10 μM.
In some embodiments, the medium further comprises an activator of the AKT/PI3K signaling pathway and/or MAPK signaling pathway; such as but not limited to a compound selected from the group consisting of an epidermal growth factor (EGF), amphiregulin (AR), epigen (EPG), transforming growth factor alpha (TGFα), betacellulin (BTC), epiregulin (EPR), heparin-binding EGF-like growth factor (HB-EGF), and Neuregulin (NRG). In some embodiments, the activator of the AKT/PI3K signaling pathway and/or MAPK signaling pathway is used at a concentration of between about 5 ng/ml to 5 μg/ml, or between about 20 ng/ml to 4 μg/ml, or between about 30 ng/ml to 3 μg/ml, or between about 40 ng/ml to 2 μg/ml, or between about 45 ng/ml to 500 ng/ml, or between about 50 ng/ml to 300 ng/ml, or about 35, 40, 45, 50, 60, 70, 90, 100, 150, 200, 250, 300, 400, 450, 600, 700 or 800 ng/ml. In some embodiments, the epidermal growth factor (EGF), amphiregulin (AR), epigen (EPG), transforming growth factor alpha (TGFα), betacellulin (BTC), epiregulin (EPR), heparin-binding EGF-like growth factor (HB-EGF), and/or Neuregulin (NRG) is used at a concentration of between about 5 ng/ml to 5 μg/ml, or between about 20 ng/ml to 4 μg/ml, or between about 30 ng/ml to 3 μg/ml, or between about 40 ng/ml to 2 μg/ml, or between about 45 ng/ml to 500 ng/ml, or between about 50 ng/ml to 300 ng/ml, or about 35, 40, 45, 50, 60, 70, 90, 100, 150, 200, 250, 300, 400, 450, 600, 700 or 800 ng/ml.
In some embodiments, the medium further comprises an activator of STAT3, an activator of GAB1 mediated cell adhesion and/or an activator of the AKT/PI3K signaling pathway. In some embodiments, the activator of STAT3, activator of GAB1 mediated cell adhesion and/or activator of the AKT/PI3K signaling pathway is used at a concentration of between about 2 ng/ml to 5 μg/ml, or between about 5 ng/ml to 5 μg/ml, or between about 10 ng/ml to 4 μg/ml, or between about 15 ng/ml to 3 μg/ml, or between about 20 ng/ml to 2 μg/ml, or about 5, 18, 20, 25, 28, 30, 50, 60, or 70 ng/ml, or about 1, 2.5, 3.5 or 4.5 μg/ml. In some embodiments, the activator of the AKT/PI3K signaling pathway is a hepatocyte growth factor (HGF). In some embodiments, HGF is used at a concentration of between about 2 ng/ml to 5 μg/ml, or between about 5 ng/ml to 5 μg/ml, or between about 10 ng/ml to 4 μg/ml, or between about 15 ng/ml to 3 μg/ml, or between about 20 ng/ml to 2 μg/ml, or about 5, 18, 20, 25, 28, 30, 50, 60, or 70 ng/ml, or about 1, 2.5, 3.5 or 4.5 μg/ml.
In some embodiments, the medium further comprises an activator of cAMP-dependent pathways, such as an activator of the Protein Kinase A signaling pathway, which induces proliferation of epithelial cell types. In some embodiments, the activator of the cAMP-dependent pathway is a compound selected from the group consisting of dibutyryl-cAMP(dbCAMP), forskolin ((3R,4aR,5 S,6S,6aS, 10S,10aR, 10bS)-6,10,10b-trihydroxy-3,4a,7,7,10a-pentamethyl-1-oxo-3-vinyldodecahydro-1H-benzo[f]chromen-5-yl acetate), caffeine, theophylline, cholera toxin and pertussis toxin. In some embodiments, the activator of cAMP-dependent pathways is used at a concentration of between about 20 ng/ml to 1 μg/ml, or between about 10 ng/ml to 0.8 μg/ml, or between about 15 ng/ml to 0.6 μg/ml, or between about 20 ng/ml to 2 μg/ml, or about 5, 18, 20, 25, 28, 30, 50, 60, or 70 ng/ml, or about 1, 2.5, 3.5 or 4.5 μg/ml. In some embodiments, a compound selected from the group consisting of dibutyryl-cAMP(dbCAMP), forskolin ((3R,4aR,5 S,6S,6aS, 10S,10aR, 10bS)-6,10,10b-trihydroxy-3,4a,7,7,10a-pentamethyl-1-oxo-3-vinyldodecahydro-1H-benzo[f]chromen-5-yl acetate), caffeine, theophylline, cholera toxin and pertussis toxin is used at a concentration of between about 20 ng/ml to 1 μg/ml, or between about 10 ng/ml to 0.8 μg/ml, or between about 15 ng/ml to 0.6 μg/ml, or between about 20 ng/ml to 2 μg/ml, or about 5, 18, 20, 25, 28, 30, 50, 60, or 70 ng/ml, or about 1, 2.5, 3.5 or 4.5 μg/ml.
In some cases, the medium further comprises an activator of the Notch receptor. In some embodiments, the activator of the Notch receptor is used at a concentration of between about 10 nM to 100 μM, or between about 50 nM to 80 μM, or between about 100 nM to 60 μM, or between about 500 nM to 40 μM, or between about 800 nM to 20 μM, or between about 900 nM to 10 μM, or about 20, 40, 60, or 80 nM or about 1, 1.5, 15, 30, 50, 60, 90 or 100 μM. In some embodiments, the activator of the Notch receptor is a compound selected from the group consisting of Jagged1 protein (Homo sapiens, also known as AGS; AHD; AWS; HJ1; CD339; JAGL1; JAG1), Jagged2 (NCBI 3714), Delta-like1 (NCBI 28514), Delta-like3 (NCBI 10683), and Delta-like4 (NCBI 54567). In some embodiments, the Jagged1 protein (Homo sapiens, also known as AGS; AHD; AWS; HJ1; CD339; JAGL1; JAG1), Jagged2 (NCBI 3714), Delta-like1 (NCBI 28514), Delta-like3 (NCBI 10683), and/or Delta-like4 (NCBI 54567) is used at a concentration of between about 10 nM to 100 μM, or between about 50 nM to 80 μM, or between about 100 nM to 60 μM, or between about 500 nM to 40 μM, or between about 800 nM to 20 μM, or between about 900 nM to 10 μM, or about 20, 40, 60, or 80 nM or about 1, 1.5, 15, 30, 50, 60, 90 or 100 μM.
In some embodiments, the medium further comprises a molecule which is a repressor of NFκB activity and/or activator of mitogen-activated protein (MAP) kinase ERK, p38 and JNK. In some embodiments, the molecule which is a repressor of NFκB activity and/or activator of mitogen-activated protein (MAP) kinase ERK, p38 and JNK is used at a concentration of between about 0.1 mM to 1 M, or between about 2 mM to 0.8 M, or between about 4 mM to 0.6 μM, or between about 6 mM to 0.4 M, or between about 8 mM to 0.2 M, or between about 10 mM to 800 mM, or between about 50 mM to 500 mM, or about 3, 5, 9, 15, 20, 30, 50, 80, 100, 120, 150, 200, 250, 300, 350, 400 or 450 mM. In some embodiments, the medium further comprises a compound selected from the group consisting of nicotinamide, nicotinic acid, 5-fluoronicotinamide, isonicotinic acid hydrazide, and nikethamide. In some embodiments, nicotinamide, nicotinic acid, 5-fluoronicotinamide, isonicotinic acid hydrazide, and/or nikethamide is used at a concentration of between about 0.1 mM to 1 M, or between about 2 mM to 0.8 M, or between about 4 mM to 0.6 M, or between about 6 mM to 0.4 M, or between about 8 mM to 0.2 M, or between about 10 mM to 800 mM, or between about 50 mM to 500 mM, or about 3, 5, 9, 15, 20, 30, 50, 80, 100, 120, 150, 200, 250, 300, 350, 400 or 450 mM.
In some embodiments, the medium further comprises an inhibitor of histone deacetylase (HDACs). In some embodiments, the inhibitor of histone deacetylase is a compound selected from the group consisting of valporic acid (VPA) (2-propylpentanoic acid), sodium butyrate (sodium;4-hydroxybutanoate), vorinotstat (N′-hydroxy-N-phenyloctanediamide), panobinostat ((E)-N-hydroxy-3-[4-[[2-(2-methyl-1H-indol-3-yl)ethylamino]methyl]phenyl]prop-2-enamide), trichostatin A ((2E,4E,6R)-7-[4-(dimethylamino)phenyl]-N-hydroxy-4,6-dimethyl-7-oxohepta-2,4-dienamide), mocetinostat (N-(2-aminophenyl)-4-[[(4-pyridin-3-ylpyrimidin-2-yl)amino]methyl]benzamide), BG45 (N-(2-aminophenyl)-2-pyrazinecarboxamide), 4SC-202 ((E)-N-(2-aminophenyl)-3-(1-((4-(1-methyl-1H-pyrazol-4-yl)phenyl)sulfonyl)-1H-pyrrol-3-yl)acrylamide), belinostat, scriptaid (6-(1,3-Dioxo-1H-benzo[de]isoquinolin-2(3H)-yl)-N-hydroxyhexanamide), M344 (4-(dimethylamino)-N-[7-(hydroxyamino)-7-oxoheptyl]benzamide), dacinostat ((E)-N-hydroxy-3-[4-[[2-hydroxyethyl-[2-(1H-indol-3-yl)ethyl]amino]methyl]phenyl]prop-2-enamide), abexinostat, CUDC-101 (7-(4-(3-ethynylphenylamino)-7-methoxyquinazolin-6-yloxy)-N-hydroxyheptanamide), CUDC-907 (N-hydroxy-2-(((2-(6-methoxypyridin-3-yl)-4-morpholinothieno[3,2-d]pyrimidin-6-yl)methyl)(methyl)amino)pyrimidine-5-carboxamide), and AR-42 ((S)—N-hydroxy-4-(3-methyl-2-phenylbutanamido)benzamide). In some embodiments, the inhibitor of histone deacetylase (including but not limited to the compounds listed above) is used at a concentration of between about 0.1 μM to 5 mM, or between about 0.3 μM to 4 mM, or between about 0.6 μM to 3 mM, or between about 0.8 μM to 2 mM, or between about 1 mM to 1.5 mM, or about 0.2, 0.4, 0.7, 0.9, 5, 10, 20, 50, 70 or 90 μM, or about 1.5, 2.5, 3.5 mM.
In some embodiments, stem cells are cultured in a microenvironment that mimics at least in part a cellular niche in which said stem cells naturally reside. This cellular niche may be mimicked by culturing said stem cells in the presence of biomaterials, such as matrices, scaffolds, and culture substrates that represent key regulatory signals controlling stem cell fate. Such biomaterials comprise natural, semi-synthetic and synthetic biomaterials, and/or mixtures thereof. A scaffold provides a two-dimensional or three dimensional network. Suitable synthetic materials for such a scaffold comprise polymers selected from porous solids, nanofibers, and hydrogels such as, for example, peptides including self-assembling peptides, hydrogels composed of polyethylene glycol phosphate, polyethylene glycol fumarate, polyacrylamide, polyhydroxyethyl methacrylate, polycellulose acetate, and/or co-polymers thereof (see, for example, Saha et al., 2007. Curr Opin Chem. Biol. 11(4): 381-387; Saha et al., 2008. Biophysical Journal 95: 4426-4438; Little et al., 2008. Chem. Rev 108, 1787-1796). As is known to a skilled person, the mechanical properties such as, for example, the elasticity of the scaffold influences proliferation, differentiation and migration of stem cells. In some embodiments, the scaffold comprises biodegradable (co)polymers that are replaced by natural occurring components after transplantation in a subject, for example to promote tissue regeneration and/or wound healing. In some embodiments, said scaffold does not substantially induce an immunogenic response after transplantation in a subject. Said scaffold is supplemented with natural, semi-synthetic or synthetic ligands, which provide the signals that are required for proliferation and/or differentiation, and/or migration of stem cells. In one embodiment, said ligands comprise defined amino acid fragments. Examples of said synthetic polymers comprise Pluronic® F127 block copolymer surfactant (BASF), and Ethisorb (Johnson and Johnson).
A cellular niche is in part determined by the stem cells and surrounding cells, and the extracellular matrix (ECM) that is produced by the cells in said niche. In some embodiments, MESP are attached to an ECM. ECM is composed of a variety of polysaccharides, water, elastin, and glycoproteins, wherein the glycoproteins comprise collagen, entactin (nidogen), fibronectin, and laminin. ECM is secreted by connective tissue cells. Different types of ECM are known, comprising different compositions including different types of glycoproteins and/or different combination of glycoproteins. Said ECM can be provided by culturing ECM-producing cells, such as for example fibroblast cells, in a receptacle, prior to the removal of these cells and the addition of isolated liver fragments or isolated biliary duct or isolated epithelial stem cells. Examples of extracellular matrix-producing cells are chondrocytes, producing mainly collagen and proteoglycans, fibroblast cells, producing mainly type IV collagen, laminin, interstitial procollagens, and fibronectin, and colonic myofibroblasts producing mainly collagens (type I, III, and V), chondroitin sulfate proteoglycan, hyaluronic acid, fibronectin, and tenascin-C. Alternatively, said ECM is commercially provided. Examples of commercially available extracellular matrices are extracellular matrix proteins (Invitrogen) and basement membrane preparations from Engelbreth-Holm-Swarm (EHS) mouse sarcoma cells (e.g. Matrigel™ (BD Biosciences)). A synthetic extracellular matrix material, such as ProNectin (Sigma Z378666) may be used. Mixtures of extracellular matrix materials may be used, if desired. The use of an ECM for culturing stem cells enhanced long-term survival of the stem cells and the continued presence of undifferentiated stem cells. In the absence of an ECM, stem cell cultures could not be cultured for longer periods and no continued presence of undifferentiated stem cells was observed. In addition, the presence of an ECM allowed culturing of three-dimensional tissue organoids, which could not be cultured in the absence of an ECM. The extracellular matrix material will normally be coated onto a cell culture vessel, but may (in addition or alternatively) be supplied in solution. A fibronectin solution of about 1 mg/ml may be used to coat a cell culture vessel, or between about 1 μg/cm2 to about 250 μg/cm2, or at about 1 μg/cm2 to about 150 ag/cm2. In some embodiments, a cell culture vessel is coated with fibronectin at between 8 ag/cm2 and 125 ag/cm2. In some embodiments, the ECM comprises at least two distinct glycoproteins, such as two different types of collagen or a collagen and laminin. The ECM can be a synthetic hydrogel extracellular matrix or a naturally occurring ECM. AnotherECM is provided by Matrigel™ (BD Biosciences), which comprises laminin, entactin, and collagen IV.
The endoderm spheroid progenitor cells described herein are characterized by expression of any one or more, or at least two, or at least three, or at least four, or at least five, or at least six, or 1 or 2 or 3 or 4 or 5 or 6 or 7 or all of the following markers: HNF4A (NCBI Gene: 3172), PDX1 (NCBI Gene: 3651), CDX2 (NCBI Gene: 1045), SOX9 (NCBI Gene: 6662), KRT19 (NCBI Gene: 3880), AFP (NCBI Gene: 174), ONECUT2 (NCBI Gene: 9480), LGR5 (NCBI Gene: 8549), EPHB2 (NCBI Gene: 2048), LGR4 (NCBI Gene: 55366), NR5A2 (NCBI Gene: 2494), CDH1 (NCBI Gene: 999), KRT7 (NCBI Gene: 3855), ZNF503 (NCBI Gene: 84858), MSX2 (NCBI Gene: 4488), TRPS1 (NCBI Gene: 7227), ASCL2 (NCBI Gene: 430), IRF8 (NCBI Gene: 3394), HNF4G (NCBI Gene: 3174), ID2 (NCBI Gene: 3398), CD44 (NCBI Gene: 960), EPCAM (NCBI Gene: 4072), MET (NCBI Gene: 4233), IHH (NCBI Gene: 3549) and CLDN3 (NCBI Gene: 1365).
The endoderm spheroid progenitor cells do not express or essentially do not express any one or two or three or four or five or all of the following markers SOX2 (NCBI Gene: 6657), CER1 (NCBI Gene: 9350), GATA4 (NCBI Gene: 2626), SOX17 (NCBI Gene: 64321), FOXA2 (NCBI Gene: 3170) and CXCR4 (NCBI Gene: 7852).
The endoderm spheroid progenitor cells display polarity (herein, polarity of cells refers to the unique expression of proteins in specific membrane regions of the cells that is in contact with different environment. Polarity in endoderm spheroid progenitor cells is evident from the uneven distribution of E-cadherin protein on the cells. Regions enriched with E-Cadherin marks the apical and lateral membrane of the cells (
MESP is a unique stem cell that expresses many markers of the posterior foregut, including the HNF4A, PDX1 and CDX2 (
Unlike reported endoderm progenitor stem cells, MESP is cultured in 3D and forms a spheroid structure compared to the 2D monolayer cells (Table 1). Cells cultured in spheroid are arranged spatially to generate a lumen within. The 2 surface of the cells are thus exposed to 2 different environments, adding to the complexity of cell state regulation in the spheroid. The uneven distribution of the adhesion molecule E-cadherin further supports that the cells expose to 2 different environments exhibit polarity. The endoderm stem cell state maintained in the MESPs is different from the other endoderm stem cell reported. This difference is also evident in the signaling requirement of the MESP. TGFβ signaling plays a role in maintaining early endoderm stem cell state (Table 1) and has been widely used in many PSCs differentiation protocol to induce early endoderm development (
As a stem cell, MESP can be propagated for 19 passages (
As MESP can be efficiently derived by pluripotent stem cells including iPSCs (
Exemplary methods for deriving and maintaining MESP from endoderm cells are described in the Examples. The steps are illustrated in
Described herein is a method to generate liver organoids from progenitors and stem cells. The liver organoid fulfills key criteria's of a mini-organ which contain multiple cell types of the organ, spatially organized into structures that resembles organ tissues and performing organ specific functions. The liver organoid described herein contain at least the two major cells types of the liver, the hepatocytes and the cholangiocytes. The core of the organoids is formed by the hepatocytes and the cholangiocytes form bile duct-like structures around the core of hepatocytes. The hepatocytes form a network of bile canaliculi which connect to the cholangiocyte bile duct-like structures at the periphery, resembling the hepatocytes arrangement in the liver lobule which similarly connects to the bile duct via the bile canaliculi network (
One advantage of generating liver organoids from stem cells is scalability and an amendable system for modeling diseases. The stem cells can be expanded in large scale to allow production of large number of organoids. The self-renewing ability of the stem cells allows continuous generation of liver organoids from the stem cell population. Genetic modifications using genome editing tools such as CRISPR/Cas system or iPSC reprogramming would facilitate the generation disease-specific stem cells and liver organoids that exhibit various disease phenotypes. These disease specific organoids would be highly useful for modeling disease in vitro (
Liver organoid-like structures have also been reported; however, they do not consist of somatic liver cell types and do not perform liver specific functions. A liver epithelial organoid has been derived from the adult liver (PCT/IB 11/02167). The ‘organoid’ describe in the PCT/IB 1/02167 application consists largely of liver epithelial stem cells. These stem cells are used to generate either hepatocytes or cholangiocytes. These epithelial organoid stem cells do not contain multiple liver cell types, nor structures that resemble human liver tissue, and do not exhibit liver functions. A liver bud consisting of multiple cells types including liver hepatoblast have been generated (WO2013047639 A1). The liver bud consists of mesenchymal cells, endothelial cells and liver hepatoblast which are aggregated on a gel. While the three cell types aggregate to form a mass on a dish, this liver bud does not form organized structures and does not exhibit liver specific functions. The liver bud has to be transplanted into a host for further maturation to functional liver tissues. In contrast, we describe herein the first human liver organoid comprising multiple, functional liver cell types, which has liver tissue organization and performs organ level functions such as bile secretion and transport (Table 2 and Table 3).
Liver tissues have also been engineered in vitro using 3D printing technology (US 2014/0287960 A1). In contrast with 3D printing technology, the instant methods employ the self-organizing capacity of stem cells during differentiation. 3D printed liver tissues employ extracellular matrices as gels to adhere cells in layers at precise locations. The resulting liver tissue structure is predetermined and cells are printed to desired configurations. In constrast, the stem cell derived organoids described herein provide conditions for the cells to interact and self-organize into structures resembling the liver tissue. The cells in the organoid interact and adhere without the need for addition of extracellular matrix. For example, the organoids described herein comprise endogenous extracellular matrix adhesion molecules produced by the cells in the organoid, compared to previous methods that use an exogenous matrigel or other extracellular matrix to adhere the cells together in the structure. Table 4 summarizes important differences between the liver organoids described herein and 3D printed liver tissues. For example, in the liver organoids described herein, the parenchymal and non-parachymal cells are derived from primary stem cells, whereas previous methods (e.g., 3D printing and cell aggregation methods) use parenchymal and non-parachymal cell types from different stem cell origins or immortalized cell lines. Further, the liver organoids described herein comprise functional bile canaliculi, which were not produced using previous methods.
In certain embodiments, the stem cells are the MESP.
In certain embodiments, the stem cells are adult liver stem cells.
In certain embodiments, the stem cells can be endoderm lineage progenitors that have the potential to give rise to liver tissue cell types.
Thus, in some embodiments, the method of producing a liver organoid comprises culturing an endoderm stem cell in a first cell culture medium to obtain an early hepatic progenitor. In some cases, the endoderm stem cell is an early endoderm progenitor cell, a pluripotent stem cell, an induced pluripotent stem cell, a human embryonic stem cell, an MESP, or an adult liver stem cell.
In certain embodiments, the organoids can consist of hepatocytes and cholangiocytes with at least one other liver cell types including stellate cells, Kupffer cells, hepatic progenitor cells and liver endothelial sinusoidal stem cells.
In some embodiments, the organoids do not comprise genetically engineered cells, such as recombinantly modified cells. In some embodiments, the organoids do not comprise cells that are genetically engineered to express gene products such as RNA and/or proteins that regulate the proliferation of the cells.
As shown in Table 5, the liver organoids described herein differ in certain aspects from primary liver tissue. For example, the hepatocytes in primary liver tissue are larger in size, comprise a double nucleus and exhibit polyploid chromosome number, whereas the hepatocytes in the liver organoids are about half the size of hepatocytes in primary liver, and comprise a single nucleus containing diploid chromosome number. In addition, primary hepatocytes show a rapid decline in CYP functions after 24 hours in culture, whereas CYP function in the organoid hepatocytes is stable and maintained for weeks in culture. Primary cholangiocytes form long branching tubular structures and proliferate in culture, whereas organoid cholangiocytes form large cysts in culture, and do not proliferate.
Derivation of Hepatic Organoids from MESP
The Hepatic organoid culture system described herein comprises a plurality of soluble agents in three different hepatic culture media, a cellular support and suspension culture system. The cellular support provides culture conditions suitable for differentiation of MESP to early hepatic progenitors, and the suspension culture system provides culture conditions suitable for formation of late hepatic progenitors and subsequently organoids. In some embodiments, the plurality of soluble agents comprises one or more growth factors, an enhancer of the (canonical) WNT pathway, an inhibitor of TGF-β signaling, and an inhibitor of Notch signaling.
Media Components:
In some embodiments, H1 media comprises:
In some embodiments, molecule for inducing hepatic specification is a TGF-beta signaling inhibitor, wherein the TGF-β inhibitor is characterized by any one of the following:
The TGF-beta inhibitor can block activation of TGF-beta pathway, inducing hepatic lineage specification. In some embodiments, the TGF-beta inhibitor is selected from the group consisting of:
In some embodiments, the TGF-beta inhibitor is used at a concentration of between about 0.5 nM to 20 μM, or 100 nM to 10 μM, or 250 nM to 5 μM, or 400 nM to 2.5 μM, or 0.5 nM to 1 μM, or 0.5 nM to 0.5 μM, or between about 1.5 nM to 0.4 μM, or between about 10 nM to 0.3 μM, or between about 30 nM to 0.2 μM, or between about 40 nM to 0.1 μM, or between about 50 nM to 85 nM, or about 1, 5, 15, 25, 30, 35, 45, 50, 65, 75, 130, 150, 170, 250, 350 or 450 nM; or about 0.5, 0.7, 0.9, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 8, 9, 10, 12, 14, 16, 18, or 20 μM. In some embodiments, the TGF-beta inhibitor selected from the group consisting of (i) to (x) above is used at a concentration of between about 0.5 nM to 20 μM, or 100 nM to 10 μM, or 250 nM to 5 μM, or 400 nM to 2.5 μM, or 0.5 nM to 1 μM, or 0.5 nM to 0.5 μM, or between about 1.5 nM to 0.4 μM, or between about 10 nM to 0.3 μM, or between about 30 nM to 0.2 μM, or between about 40 nM to 0.1 μM, or between about 50 nM to 85 nM, or about 1, 5, 15, 25, 30, 35, 45, 50, 65, 75, 130, 150, 170, 250, 350 or 450 nM; or about 0.5, 0.7, 0.9, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 8, 9, 10, 12, 14, 16, 18, or 20 μM.
In some embodiments, the steroid is capable of inhibiting the NF-κB pathway, activating the PI3K/AKT/mTOR pathway, inhibiting the TGF-β signaling pathway and/or inhibiting the IGF signaling pathway. In some embodiments, the steroid is a corticosteroid such as a glucocorticoid or an anti-inflammatory glucocorticoid which improves maintenance of endodermal stem cells.
In some embodiments, the glucocorticoid is selected from the group consisting of:
In some embodiments, the steroid, corticosteroid or glucocorticoid (such as dexamethasone) is used at a concentration of between about 0.5 μM to 200 μM, or between about 1.5 μM to 150 μM, or between about 5 μM to 100 μM, or between about 10 μM to 90 μM, or between about 20 μM to 80 μM, or between about 30 μM to 70 μM, or between about 40 μM to 60 μM, or about 2, 8, 15, 25, 30, 35, 45, 65, 75, 110, 130, 140, 160, 170 or 190 μM.
In some embodiments, the WNT-signaling activator is a Glycogen synthase kinase 3 (GSK3) inhibitor. In some embodiments, the GSK3 inhibitor is selected from the group consisting of:
In some embodiments, the WNT-signaling activator is used at a concentration of between about 0.1 μM to 10 μM, or between about 0.5 μM to 8 μM, or between about 1 μM to 7 μM, or between about 2 μM to 6 μM, or between about 3 μM to 5 μM, or about 0.2, 0.3, 0.4, 3, 4, 4.5, 5, 6, 7.5, 8.5, 9, 9.5 or 10 μM.
In some embodiments, the GSK3 inhibitor (e.g., CHIR-09921) is used at a concentration of between about 0.1 M to 10 μM, or between about 0.5 M to 8 μM, or between about 1 M to 7 μM, or between about 2 M to 6 μM, or between about 3 M to 5 μM, or about 0.2, 0.3, 0.4,3, 4, 4.5, 5, 6, 7.5, 8.5, 9, 9.5 or 10 μM.
In some embodiments, the medium further comprises an activator of the AKT/PI3K signaling pathway and/or MAPK signaling pathway; such as but not limited to a compound selected from the group consisting of an epidermal growth factor (EGF), amphiregulin (AR), epigen (EPG), transforming growth factor alpha (TGFα), betacellulin (BTC), epiregulin (EPR), heparin-binding EGF-like growth factor (HB-EGF), and Neuregulin (NRG). In some embodiments, the activator of the AKT/PI3K signaling pathway and/or MAPK signaling pathway is used at a concentration of between about 5 ng/ml to 5 μg/ml, or between about 20 ng/ml to 4 μg/ml, or between about 30 ng/ml to 3 μg/ml, or between about 40 ng/ml to 2 μg/ml, or between about 45 ng/ml to 500 ng/ml, or between about 50 ng/ml to 300 ng/ml, or about 35, 40, 45, 50, 60, 70, 90, 100, 150, 200, 250, 300, 400, 450, 600, 700 or 800 ng/ml. In some embodiments, the epidermal growth factor (EGF), amphiregulin (AR), epigen (EPG), transforming growth factor alpha (TGFα), betacellulin (BTC), epiregulin (EPR), heparin-binding EGF-like growth factor (HB-EGF), and/or Neuregulin (NRG) is used at a concentration of between about 5 ng/ml to 5 μg/ml, or between about 20 ng/ml to 4 μg/ml, or between about 30 ng/ml to 3 μg/ml, or between about 40 ng/ml to 2 μg/ml, or between about 45 ng/ml to 500 ng/ml, or between about 50 ng/ml to 300 ng/ml, or about 35, 40, 45, 50, 60, 70, 90, 100, 150, 200, 250, 300, 400, 450, 600, 700 or 800 ng/ml.
In some embodiments, the medium further comprises an activator of STAT3, an activator of GAB1 mediated cell adhesion and/or an activator of the AKT/PI3K signaling pathway. In some embodiments, the activator of STAT3, activator of GAB1 mediated cell adhesion and/or activator of the AKT/PI3K signaling pathway is used at a concentration of between about 2 ng/ml to 5 μg/ml, or between about 5 ng/ml to 5 μg/ml, or between about 10 ng/ml to 4 μg/ml, or between about 15 ng/ml to 3 μg/ml, or between about 20 ng/ml to 2 μg/ml, or about 5, 18, 20, 25, 28, 30, 50, 60, or 70 ng/ml, or about 1, 2.5, 3.5 or 4.5 μg/ml. In some embodiments, the activator of the AKT/PI3K signaling pathway is a hepatocyte growth factor (HGF). In some embodiments, HGF is used at a concentration of between about 2 ng/ml to 5 μg/ml, or between about 5 ng/ml to 5 μg/ml, or between about 10 ng/ml to 4 μg/ml, or between about 15 ng/ml to 3 μg/ml, or between about 20 ng/ml to 2 μg/ml, or about 5, 18, 20, 25, 28, 30, 50, 60, or 70 ng/ml, or about 1, 2.5, 3.5 or 4.5 μg/ml.
In some embodiments, the medium further comprises a molecule which is a repressor of NFκB activity and/or activator of mitogen-activated protein (MAP) kinase ERK, p38 and JNK. In some embodiments, the molecule which is a repressor of NFκB activity and/or activator of mitogen-activated protein (MAP) kinase ERK, p38 and JNK is used at a concentration of between about 0.1 mM to 1 M, or between about 2 mM to 0.8 M, or between about 4 mM to 0.6 M, or between about 6 mM to 0.4 M, or between about 8 mM to 0.2 M, or between about 10 mM to 800 mM, or between about 50 mM to 500 mM, or about 3, 5, 9, 15, 20, 30, 50, 80, 100, 120, 150, 200, 250, 300, 350, 400 or 450 mM. In some embodiments, the medium further comprises a compound selected from the group consisting of nicotinamide, nicotinic acid, 5-fluoronicotinamide, isonicotinic acid hydrazide, and nikethamide. In some embodiments, nicotinamide, nicotinic acid, 5-fluoronicotinamide, isonicotinic acid hydrazide, and/or nikethamide is used at a concentration of between about 0.1 mM to 1 M, or between about 2 mM to 0.8 M, or between about 4 mM to 0.6 M, or between about 6 mM to 0.4 M, or between about 8 mM to 0.2 M, or between about 10 mM to 800 mM, or between about 50 mM to 500 mM, or about 3, 5, 9, 15, 20, 30, 50, 80, 100, 120, 150, 200, 250, 300, 350, 400 or 450 mM.
In some embodiments, the medium further comprises at least one, at least two, or at least three molecules inducing phosphorylation of SMAD1, SMAD5 and SMAD8 and activating MAPK signaling. In some embodiments, the molecule(s) inducing phosphorylation of SMAD1, SMAD5 and SMAD8 and activating MAPK signaling is used at a concentration of between about 2 ng/ml to 5 μg/ml, or between about 5 ng/ml to 5 μg/ml, or between about 10 ng/ml to 4 μg/ml, or between about 15 ng/ml to 3 μg/ml, or between about 20 ng/ml to 2 μg/ml, or about 5, 18, 20, 25, 28, 30, 50, 60, 70 ng/ml, or about 1, 2.5, 3.5 or 4.5 μg/ml. In some embodiments, the medium further comprises a molecule selected from the group consisting of BMP4, BMP2, BMP3, BMP5, BMP6, and BMP7. In some embodiments, the BMP family molecule(s) is/are used at a concentration of between about 2 ng/ml to 5 μg/ml, or between about 5 ng/ml to 5 μg/ml, or between about 10 ng/ml to 4 μg/ml, or between about 15 ng/ml to 3 μg/ml, or between about 20 ng/ml to 2 μg/ml, or about 5, 18, 20, 25, 28, 30, 50, 60, 70 ng/ml, or about 1, 2.5, 3.5 or 4.5 μg/ml.
In some embodiments, the medium further comprises a molecule which is an activator of the FGF and MAPK pathway. In some embodiments, the medium further comprises a molecule selected from the group consisting of FGF7, FGF1, FGF3, FGF10, and FGF22. In some embodiments, the activator of the FGF and MAPK pathway, or FGF family molecule is used at a concentration of between about 2 ng/ml to 5 μg/ml, or between about 5 ng/ml to 5 μg/ml, or between about 10 ng/ml to 4 μg/ml, or between about 15 ng/ml to 3 μg/ml, or between about 20 ng/ml to 2 μg/ml, or about 5, 18, 20, 25, 28, 30, 50, 60, 70 ng/ml, or about 1, 2.5, 3.5 or 4.5 μg/ml.
Additional Components Found in Some Embodiments:
In some embodiments, the medium further comprises an activator of cAMP-dependent pathways, such as an activator of the Protein Kinase A signaling pathway, which induces proliferation of epithelial cell types. In some embodiments, the activator of the cAMP-dependent pathway is a compound selected from the group consisting of dibutyryl-cAMP(dbCAMP), forskolin ((3R,4aR,5 S,6S,6aS, 10S,10aR, 10bS)-6,10,10b-trihydroxy-3,4a,7,7,10a-pentamethyl-1-oxo-3-vinyldodecahydro-1H-benzo[f]chromen-5-yl acetate), caffeine, theophylline, cholera toxin and pertussis toxin. In some embodiments, the activator of cAMP-dependent pathways is used at a concentration of between about 20 ng/ml to 1 μg/ml, or between about 10 ng/ml to 0.8 μg/ml, or between about 15 ng/ml to 0.6 μg/ml, or between about 20 ng/ml to 2 μg/ml, or about 5, 18, 20, 25, 28, 30, 50, 60, or 70 ng/ml, or about 1, 2.5, 3.5 or 4.5 μg/ml. In some embodiments, a compound selected from the group consisting of dibutyryl-cAMP(dbCAMP), forskolin ((3R,4aR,5 S,6S,6aS, 10S,10aR, 10bS)-6,10,10b-trihydroxy-3,4a,7,7,10a-pentamethyl-1-oxo-3-vinyldodecahydro-1H-benzo[f]chromen-5-yl acetate), caffeine, theophylline, cholera toxin and pertussis toxin is used at a concentration of between about 20 ng/ml to 1 μg/ml, or between about 10 ng/ml to 0.8 μg/ml, or between about 15 ng/ml to 0.6 μg/ml, or between about 20 ng/ml to 2 μg/ml, or about 5, 18, 20, 25, 28, 30, 50, 60, or 70 ng/ml, or about 1, 2.5, 3.5 or 4.5 μg/ml.
In some cases, the medium further comprises an activator of the Notch receptor. In some embodiments, the activator of the Notch receptor is used at a concentration of between about 10 nM to 100 μM, or between about 50 nM to 80 μM, or between about 100 nM to 60 μM, or between about 500 nM to 40 μM, or between about 800 nM to 20 μM, or between about 900 nM to 10 μM, or about 20, 40, 60, or 80 nM or about 1, 1.5, 15, 30, 50, 60, 90 or 100 μM. In some embodiments, the activator of the Notch receptor is a compound selected from the group consisting of Jagged1 protein (Homo sapiens, also known as AGS; AHD; AWS; HJ1; CD339; JAGL1; JAG1), Jagged2 (NCBI 3714), Delta-like1 (NCBI 28514), Delta-like3 (NCBI 10683), and Delta-like4 (NCBI 54567). In some embodiments, the Jagged1 protein (Homo sapiens, also known as AGS; AHD; AWS; HJ1; CD339; JAGL1; JAG1), Jagged2 (NCBI 3714), Delta-like1 (NCBI 28514), Delta-like3 (NCBI 10683), and/or Delta-like4 (NCBI 54567) is used at a concentration of between about 10 nM to 100 μM, or between about 50 nM to 80 μM, or between about 100 nM to 60 μM, or between about 500 nM to 40 μM, or between about 800 nM to 20 μM, or between about 900 nM to 10 μM, or about 20, 40, 60, or 80 nM or about 1, 1.5, 15, 30, 50, 60, 90 or 100 μM.
In some embodiments, the medium further comprises an inhibitor of histone deacetylase (HDACs). In some embodiments, the inhibitor of histone deacetylase is a compound selected from the group consisting of valporic acid (VPA) (2-propylpentanoic acid), sodium butyrate (sodium;4-hydroxybutanoate), vorinotstat (N′-hydroxy-N-phenyloctanediamide), panobinostat ((E)-N-hydroxy-3-[4-[[2-(2-methyl-1H-indol-3-yl)ethylamino]methyl]phenyl]prop-2-enamide), trichostatin A ((2E,4E,6R)-7-[4-(dimethylamino)phenyl]-N-hydroxy-4,6-dimethyl-7-oxohepta-2,4-dienamide), mocetinostat (N-(2-aminophenyl)-4-[[(4-pyridin-3-ylpyrimidin-2-yl)amino]methyl]benzamide), BG45 (N-(2-aminophenyl)-2-pyrazinecarboxamide), 4SC-202 ((E)-N-(2-aminophenyl)-3-(1-((4-(1-methyl-1H-pyrazol-4-yl)phenyl)sulfonyl)-1H-pyrrol-3-yl)acrylamide), belinostat, scriptaid (6-(1,3-Dioxo-1H-benzo[de]isoquinolin-2(3H)-yl)-N-hydroxyhexanamide), M344 (4-(dimethylamino)-N-[7-(hydroxyamino)-7-oxoheptyl]benzamide), dacinostat ((E)-N-hydroxy-3-[4-[[2-hydroxyethyl-[2-(1H-indol-3-yl)ethyl]amino]methyl]phenyl]prop-2-enamide), abexinostat, CUDC-101 (7-(4-(3-ethynylphenylamino)-7-methoxyquinazolin-6-yloxy)-N-hydroxyheptanamide), CUDC-907 (N-hydroxy-2-(((2-(6-methoxypyridin-3-yl)-4-morpholinothieno[3,2-d]pyrimidin-6-yl)methyl)(methyl)amino)pyrimidine-5-carboxamide), and AR-42 ((S)—N-hydroxy-4-(3-methyl-2-phenylbutanamido)benzamide). In some embodiments, the inhibitor of histone deacetylase (including but not limited to the compounds listed above) is used at a concentration of between about 0.1 μM to 5 mM, or between about 0.3 μM to 4 mM, or between about 0.6 μM to 3 mM, or between about 0.8 μM to 2 mM, or between about 1 mM to 1.5 mM, or about 0.2, 0.4, 0.7, 0.9, 5, 10, 20, 50, 70 or 90 μM, or about 1.5, 2.5, or 3.5 mM.
In some embodiments, culture using the first media H1 is for 1 to 10 days or 1 to 8 days or 1 to 6 days.
In some embodiments, culture using second media H2 is for 6 to 12 days or 4 to 10 days or 6 to 8 days.
In some embodiments, culture using third media H3 is for 18 to 26 days or 20 to 24 days or 19 to 22 days.
In some embodiments, H2 medium comprises:
In some embodiments, the TGF-beta signaling inhibitor is as described and used at the concentrations described in media H1.
In some embodiments, the steroid and concentrations are as described above for media H1.
In some embodiments, the medium further comprises an activator of AKT/PI3K signaling pathway and MAPK signaling pathway as described and used at the concentrations described in media H1. In some embodiments, the medium further comprises a compound selected from the group consisting of an epidermal growth factor (EGF), amphiregulin (AR), epigen (EPG), transforming growth factor alpha (TGFα), betacellulin (BTC), epiregulin (EPR), heparin-binding EGF-like growth factor (HB-EGF), and Neuregulin (NRG). In some embodiments, the compound (e.g., EGF) is used at a concentration of between about 5 ng/ml to 5 μg/ml, or between about 20 ng/ml to 4 μg/ml, or between about 30 ng/ml to 3 μg/ml, or between about 40 ng/ml to 2 μg/ml, or between about 45 ng/ml to 500 ng/ml, or between about 50 ng/ml to 300 ng/ml, or about 35, 40, 45, 50, 60, 70, 90, 100, 150, 200, 250, 300, 400, 450, 600, 700 or 800 ng/ml.
In some embodiments, the medium further comprises an activator of STAT3, GAB1 mediated cell adhesion and AKT/PI3K signaling pathway as described and used at the concentrations described in media H1. In some embodiments, the medium further comprises a hepatocyte growth factor (HGF). In some embodiments, HGF is used at a concentration of between about 2 ng/ml to 5 μg/ml, or between about 5 ng/ml to 5 μg/ml, or between about 10 ng/ml to 4 μg/ml, or between about 15 ng/ml to 3 μg/ml, or between about 20 ng/ml to 2 μg/ml, or about 5, 18, 20, 25, 28, 30, 50, 60, 70 ng/ml, or about 1, 2.5, 3.5 or 4.5 μg/ml.
In some embodiments, the medium further comprises a molecule which is an repressor of NFκB activity and activator of mitogen-activated protein (MAP) kinase ERK, p38 and JNK as described and used at the concentrations described in media H1. In some embodiments, the medium further comprises a compound selected from the group consisting of nicotinamide, nicotinic acid, 5-fluoronicotinamide, isonicotinic acid hydrazide, and nikethamide. In some embodiments, the compound (e.g., Nicotinamide) is used at a concentration of between about 0.1 mM to 1 M, or between about 2 mM to 0.8 M, or between about 4 mM to 0.6 M, or between about 6 mM to 0.4 M, or between about 8 mM to 0.2 M, or between about 10 mM to 800 mM, or between about 50 mM to 500 mM, or about 3, 5, 9, 15, 20, 30, 50, 80, 100, 120, 150, 200, 250, 300, 350, 400 or 450 mM.
In some embodiments, the medium further comprises at least one, at least two, or at least three molecule(s) inducing phosphorylation of SMAD1, SMAD5 and SMAD8 and activating MAPK signaling as described and used at the concentrations described in media H1.
In some embodiments, the medium further comprises a molecule regulating bile acid synthesis and activates FGF and MAPK pathway as described and used at the concentrations described in media H1. In some embodiments, the molecule is selected from the group consisting of FGF 19, FGF1, FGF2, FGF4, FGF6, FGF8, FGF9, FGF 16, FGF17, FGF18, FGF20, and FGF23. In some embodiments, the FGF family member is used at a concentration of 5 ng/ml to 0.8 μg/ml, or between about 10 ng/ml to 0.6 μg/ml, or between about 50 ng/ml to 0.5 μg/ml, or between about 150 ng/ml to 1 μg/ml, or about 5, 20, 50, 100, 200, 250, 300, 400, 500 ng/ml, or about 1, 0.8, 0.7 or 0.9 μg/ml.
H2 media can further comprises a component for inducing late hepatic progenitor differentiation, wherein the component is any one or two of the following components:
In some embodiments, the inhibitor of γ-secretase is selected from the group consisting of Compound E (C-E) (2S)-2-[[2-(3,5-difluorophenyl)acetyl]amino]-N-[(3S)-1-methyl-2-oxo-5-phenyl-3H-1,4-benzodiazepin-3-yl]propanamide, Dibenzazepine (DBZ): (2S)-2-[[2-(3,5-difluorophenyl)acetyl]amino]-N-[(7 S)-5-methyl-6-oxo-7H-benzo[d][1]benzazepin-7-yl]propanamide, DAPT:tert-butyl (2S)-2-[[(2S)-2-[[2-(3,5-difluorophenyl)acetyl]amino]propanoyl]amino]-2-phenylacetate, Begacestat: 5-chloro-N-[(2S)-4,4,4-trifluoro-1-hydroxy-3-(trifluoromethyl)butan-2-yl]thiophene-2-sulfonamide, and Flurizan: (2R)-2-(3-fluoro-4-phenylphenyl)propanoic acid. In some embodiments, the inhibitor of γ-secretase is used at a concentration of between about 10 nM to 5 μM, or between about 100 nM to 4 μM, or between about 200 nM to 3.5 μM, or between about 300 nM to 3 μM, or between about 400 nM to 2.5 μM, or between about 450 nM to 2 μM, or between about 500 nM to 1.5 μM, or about 50, 90, 150, 250, 350, 450, 480, 500, 650, or 700 nM.
In some embodiments, the YAP inhibitor is selected from the group consisting of:
In some embodiments, H3 medium comprises:
The pleiotropic cytokine that belongs to the interleukin 6 group of cytokines is capable of activating JAK-STAT, MAPK and AKT/PI3K signaling. In some embodiments, the pleiotropic cytokine is oncostatin M (OSM) or leukemia inhibitory factor (LIF; NCBI: 3976), or Cardiotrophin-1/CT-1 (NCBI: 1489), or ciliary neurotrophic factor receptor (CNTF; NCBI: 1271), IL-11 or IL-31. In some embodiments, the pleiotropic cytokine (e.g., OSM) is used at a concentration of between about 0.1 ng/ml to 1 μg/ml, or between about 10 ng/ml to 0.8 μg/ml, or between about 15 ng/ml to 0.6 μg/ml, or between about 20 ng/ml to 2 μg/ml, or about 5, 18, 20, 25, 28, 30, 50, 60, 70 ng/ml, or about 1, 2.5, 3.5 or 4.5 μg/ml.
In some embodiments, the inhibitor of γ-secretase is selected from the group consisting of Compound E (C-E) (2S)-2-[[2-(3,5-difluorophenyl)acetyl]amino]-N-[(3S)-1-methyl-2-oxo-5-phenyl-3H-1,4-benzodiazepin-3-yl]propanamide, Dibenzazepine (DBZ): (2S)-2-[[2-(3,5-difluorophenyl)acetyl] amino]-N-[(7 S)-5-methyl-6-oxo-7H-benzo[d][1]benzazepin-7-yl]propanamide, DAPT:tert-butyl (2S)-2-[[(2S)-2-[[2-(3,5-difluorophenyl)acetyl]amino]propanoyl]amino]-2-phenylacetate, Begacestat: 5-chloro-N-[(2S)-4,4,4-trifluoro-1-hydroxy-3-(trifluoromethyl)butan-2-yl]thiophene-2-sulfonamide, and Flurizan: (2R)-2-(3-fluoro-4-phenylphenyl)propanoic acid. In some embodiments, the inhibitor of γ-secretase (e.g., Compound E (C-E)) is used at a concentration of between about 10 nM to 5 μM, or between about 100 nM to 4 μM, or between about 200 nM to 3.5 μM, or between about 300 nM to 3 μM, or between about 400 nM to 2.5 μM, or between about 450 nM to 2 μM, or between about 500 nM to 1.5 μM, or about 50, 90, 150, 250, 350, 450, 480, 500, 650, or 700 nM.
In some embodiments, the TGF-beta signaling inhibitor is as described and is used at the concentrations described above for media H1.
In some embodiments, the steroid is as described and is used at the concentrations described above for media H1.
H3 medium can further comprises at least one or two or three or four or five or six component(s) promoting maturation of hepatic organoid and/or at least one or two or three component(s) promoting survival of hepatic organoids.
The component(s) promoting maturation of the hepatic organoid is selected from the group consisting of:
In some embodiments, the above components that promote maturation of the hepatic organoid are as described and are used at the concentrations described above for media H1 and H2.
The interleukin that acts as both a pro-inflammatory cytokine and an anti-inflammatory myokine activates JAK-STAT, MAPK and AKT/PI3K signaling. In some embodiments, the interleukin is IL-6. In some embodiments, the interleukin is at a concentration of between about 0.1 ng/ml to 1 μg/ml, or between about 5 ng/ml to 0.5 μg/ml or between about 10 ng/ml to 0.8 μg/ml, or between about 15 ng/ml to 0.6 μg/ml, or between about 20 ng/ml to 2 μg/ml, or about 5, 18, 20, 25, 28, 30, 50, 60, 70 ng/ml, or about 1, 2.5, 3.5 or 4.5 μg/ml.
In some embodiments, the compound with biliary acid potency is characterized by any one or more or all of the following capabilities: activating the nuclear farnesoid X receptor, increasing cAMP and thus activating the PKC signaling pathway. In some embodiments, the compound with biliary acid potency is selected from the group consisting of:
In some embodiments, the compound with biliary acid potency is used at a concentration of between about 1 μM to 1 mM, or about 10 μM to 0.8 mM, or about 50 μM to 0.6 mM, or about 100 μM to 0.4 mM, or about 150 μM to 0.2 mM, or about 5, 15, 20, 40, 60, 80, 90, 100, 150, 250, 350, 450, 550, 650, 750, or 850 μM.
In some embodiments, the component(s) promoting survivability of the hepatic organoid is selected from the group consisting of:
In some embodiments, the activator of the STAT3, GAB1 mediated cell adhesion and/or AKT/PI3K signaling pathway, and the activator of AKT/PI3K signaling pathway and MAPK signaling pathway are as described and are used at the concentrations described above for media H1 and H2.
In some embodiments, the glycosaminoglycan is used at a concentration of between about 100 ng/ml to 1 mg/ml, or between about 100 ng/ml to 1 mg/ml, or between about 500 ng/ml to 0.5 mg/ml, or between about 1 μg/ml to 0.1 mg/ml, or between about 5 μg/ml to 500 μg/ml, or about 300, 500, 700 or 800 ng/ml, or about 1, 2, 3, 10, 20, 40, 50, 60, 100, 500, 700 μg/ml. In some embodiments, the glycosaminoglycan is heparin, and is used at a concentration of between about 100 ng/ml to 1 mg/ml, or between about 100 ng/ml to 1 mg/ml, or between about 500 ng/ml to 0.5 mg/ml, or between about 1 μg/ml to 0.1 mg/ml, or between about 5 μg/ml to 500 μg/ml, or about 300, 500, 700 or 800 ng/ml, or about 1, 2, 3, 10, 20, 40, 50, 60, 100, 500, 700 μg/ml
Extracellular Matrix:
In some embodiments, Stem cells are cultured in a microenvironment that mimics at least in part a cellular niche in which said stem cells naturally reside. This cellular niche may be mimicked by culturing said stem cells in the presence of biomaterials, such as matrices, scaffolds, and culture substrates that represent key regulatory signals controlling stem cell fate. Such biomaterials comprise natural, semi-synthetic and synthetic biomaterials, and/or mixtures thereof. A scaffold provides a two-dimensional or three dimensional network. Suitable synthetic materials for such a scaffold comprise polymers selected from porous solids, nanofibers, and hydrogels such as, for example, peptides including self-assembling peptides, hydrogels composed of polyethylene glycol phosphate, polyethylene glycol fumarate, polyacrylamide, polyhydroxyethyl methacrylate, polycellulose acetate, and/or co-polymers thereof (see, for example, Saha et al., 2007. Curr Opin Chem. Biol. 11(4): 381-387; Saha et al., 2008. Biophysical Journal 95: 4426-4438; Little et al., 2008. Chem. Rev 108, 1787-1796). As is known to a skilled person, the mechanical properties such as, for example, the elasticity of the scaffold influences proliferation, differentiation and migration of stem cells. In some embodiments, the scaffold comprises biodegradable (co)polymers that are replaced by natural occurring components after transplantation in a subject, for example to promote tissue regeneration and/or wound healing. In some embodiments, said scaffold does not substantially induce an immunogenic response after transplantation in a subject. Said scaffold is supplemented with natural, semi-synthetic or synthetic ligands, which provide the signals that are required for proliferation and/or differentiation, and/or migration of stem cells. In one embodiment, said ligands comprise defined amino acid fragments. Examples of said synthetic polymers comprise Pluronic® F127 block copolymer surfactant (BASF), and Ethisorb (Johnson and Johnson). A cellular niche is in part determined by the stem cells and surrounding cells, and the extracellular matrix (ECM) that is produced by the cells in said niche. In some embodiments, MESP are attached to an ECM. ECM is composed of a variety of polysaccharides, water, elastin, and glycoproteins, wherein the glycoproteins comprise collagen, entactin (nidogen), fibronectin, and laminin. ECM is secreted by connective tissue cells. Different types of ECM are known, comprising different compositions including different types of glycoproteins and/or different combination of glycoproteins. Said ECM can be provided by culturing ECM-producing cells, such as for example fibroblast cells, in a receptacle, prior to the removal of these cells and the addition of isolated liver fragments or isolated biliary duct or isolated epithelial stem cells. Examples of extracellular matrix-producing cells are chondrocytes, producing mainly collagen and proteoglycans, fibroblast cells, producing mainly type IV collagen, laminin, interstitial procollagens, and fibronectin, and colonic myofibroblasts producing mainly collagens (type I, III, and V), chondroitin sulfate proteoglycan, hyaluronic acid, fibronectin, and tenascin-C. Alternatively, said ECM is commercially provided. Examples of commercially available extracellular matrices are extracellular matrix proteins (Invitrogen) and basement membrane preparations from Engelbreth-Holm-Swarm (EHS) mouse sarcoma cells (e.g. Matrigel™ (BD Biosciences)). A synthetic extracellular matrix material, such as ProNectin (Sigma Z378666) may be used. Mixtures of extracellular matrix materials may be used, if desired. The use of an ECM for culturing stem cells enhanced long-term survival of the stem cells and the continued presence of undifferentiated stem cells. In the absence of an ECM, stem cell cultures could not be cultured for longer periods and no continued presence of undifferentiated stem cells was observed. In addition, the presence of an ECM allowed culturing of three-dimensional tissue organoids, which could not be cultured in the absence of an ECM. The extracellular matrix material will normally be coated onto a cell culture vessel, but may (in addition or alternatively) be supplied in solution. A fibronectin solution of about 1 mg/ml may be used to coat a cell culture vessel, or between about 1 μg/cm2 to about 250 μg/cm2, or at about 1 jag/cm2 to about 150 μg/cm2. In some embodiments, a cell culture vessel is coated with fibronectin at between 8 μg/cm2 and 125 μg/cm2. One ECM for use in the methods described herein comprises at least two distinct glycoproteins, such as two different types of collagen or a collagen and laminin. The ECM can be a synthetic hydrogel extracellular matrix or a naturally occurring ECM. Another ECM is provided by Matrigel™ (BD Biosciences), which comprises laminin, entactin, and collagen IV.
The suspension culture system refers to any culture system, in which the cells are not embedded in a solid or semi-solid matrix in the culture and are free floating in the culture apparatus without resting on the bottom of the apparatus.
In some embodiments, the early hepatic progenitor is characterized by any one or more or at least two, or at least three, or at least four, or at least five, or at least six, or between 1 or 2 or 3 to 4 or 5 or 6 or 7 or all, or all of the following markers: SOX9 (NCBI: 6662), CK19 (NCBI: 3880), CK18 (NCBI: 3875), HNF4a (NCBI: 3172), PROX1 (NCBI: 5629), ONECUT1 (NCBI: 3175), AFP (NCBI: 174), TBX3 (NCBI:6926).
In some embodiments, the late hepatic progenitor is characterized by any one or more or at least two, or at least three, or at least four, or at least five, or at least six, or between 1 or 2 or 3 to 4 or 5 or 6 or 7 or all, or all of the following markers: CK19 (NCBI: 3880), CK18 (NCBI: 3875), HNF4a (NCBI: 3172), PROX1 (NCBI: 5629), ONECUT1 (NCBI: 3175), AFP (NCBI: 174), TBX3 (NCBI:6926), ALB (NCBI: 213).
In some embodiments, the hepatic (liver) organoids comprise more than one liver specific cell type selected from the group consisting of hepatocytes, cholangiocytes, liver specific endothelial cells (LSEC), stellate cells, hepatic myofibroblast and hepatoblasts.
In some embodiments, the hepatocytes are characterized by their expression of albumin (ALB) and not cholangiocytes marker, such as Cytokeratin 7 (CK7). In some embodiments, the hepatocytes express any one or more, or at least two, three, four, five, six, seven, eight, nine, ten or all of the following hepatocyte markers: HNF4a (NCBI: 3172), FAH (NCBI: 2184), TAT (NCBI: 6898), GCK (NCBI: 2645), TTR (NCBI: 7276), MET (NCBI: 4233), GLU1/MGAM (NCBI: 8972), FAHD2A (NCBI: 51011), HNF1B (NCBI: 6928), HNF1A (NCBI: 6927), CYP3A4 (NCBI: 1576), CYP2C9 (NCBI: 1559), CYP2C19 (NCBI: 1557), CYP1A2 (NCBI: 1544), CYP2E1 (NCBI: 1571), CYP2D6 (NCBI: 1565), CYP3A7 (NCBI: 1551), CYP1A1 (NCBI: 1543), CYP3A5 (NCBI: 1577), CYP27A1 (NCBI: 1593) and CYP2B6 (NCBI: 1555).
In some embodiments, the cholangiocytes are characterized by their expression of CK7 but not albumin (ALB) and optionally by their expression of other cholangiocytes markers, such as CK19 (NCBI: 3880), HNF1B (NCBI: 6928) and SOX9 (NCBI: 6662).
In some embodiments, the hepatoblasts are characterized by expression of any one or more markers selected from the group consisting of SOX9 (NCBI: 6662), CK19 (NCBI: 3880), CK18 (NCBI: 3875), HNF4a (NCBI: 3172), PROX1 (NCBI: 5629), ONECUT1 (NCBI: 3175), AFP (NCBI: 174), and ALB (NCBI: 213).
In some embodiments, the liver specific endothelial cells (LSEC) are characterized by expression of any one or more markers selected from the group consisting of CD45, CD80, CD86, CD11c, VAP1, STAB1 and CD31 that is mainly expressed in the cytoplasm and not on the cell surface.
In some embodiments, the stellate cells are characterized by expression of any one or more markers selected from the group consisting of GFAP, VIM, LHX2, LRAT, PDGFRb, HAND2, ICAM-1, VCAM-1, and N-CAM
In some embodiments, the hepatic myofibroblast are characterized by expression of any one or more markers selected from the group consisting of COL1A1 and α-SMA.
The hepatic (liver) organoids described herein are capable of performing liver functions and exhibit a structural composition observed in liver.
In some embodiments, the liver functions are selected from the group consisting of albumin secretion, cytochrome enzymatic activities, glycogen storage, low density lipo-protein uptake, bile acid production and drug metabolism.
The structural composition observed in liver that is found in the hepatic (liver) organoid described herein is characterized by the non-random distribution of the different liver cell types of which the liver is composed.
Liver organoids can be generated from the MESP via a step wise induction method where the stem cells first commit to early hepatic progenitors expressing AFP but not ALB (
The liver organoids generated from MESP also expresses many of the functional metabolic enzymes found in the liver (
The liver tissue structures form by multiple cell types observed in the liver organoid would allow us to model liver organ function which is not possible using pure 2D and 3D hepatocyte cultures. Herein, the liver organ is shown to exhibit liver specific organ-level functions such as the bile secretion and transport to the bile duct. Bile secretion is an important unique function of the liver (Boyer et al, 2013). The hepatocytes secrete bile which contains many important components such as bile salts, cholesterol and metabolized exogenous drugs, xenobiotics and toxins. Bile plays important physiological functions such as the removal of the harmful lipophilic substances, digestion and absorption of fats in the small intestine, elimination of cholesterols and regulation of many hormones and pheromones which aids in the development of the intestine. Bile secretion and transport in the liver cannot be modeled with 2D and 3D and hepatocytes as there are no functional network of bile canaliculi that connects to the bile duct. In contrast, the liver organoid described herein contains an extensive network of bile canaliculi in the core of hepatocytes which connects to the bile duct-like cyst (
The successful generation of liver organoids from MESP enables the modeling of genetic diseases of the liver. The LDLR−/− MESP can be differentiated to form liver organoids. These liver organoids produces an elevated level of cholesterol which reflects the pathological conditions of Familial hypercholesterolemia (
While organoids that mimic different human organs have been generated, a key hurdle is to produce homogenous organoids in a high throughput manner to allow large scale drug screening (Spence et al 2011, Lancaster et al 2013, Takasato et al 2016). The complex culture conditions and reliance on the self-organizing capacity of stem cells for organoid generation make it hard to generate a dish of organoids of similar size, structure and function. This is a challenging hurdle to overcome, towards the use of organoids in industrial applications. As described herein, the organoids are optimized for generation in a high throughput manner where each single 96 well contains a single organoid of similar size and structure (
Derivation of Hepatic Organoids from MESP
Exemplary methods for producing hepatic organoids from MESP are described in the Examples and illustrated schematically in
The successful step wise generation of the liver organoids from the posterior foregut-like MESP suggests that this methodology can also be used to generate liver organoids from stem cells of the endoderm lineage that is developmentally in line with liver organ development. This would include early hepatic progenitor stem cells arising from the MESP or stem cells existing in the adult liver stem cells. Using stem cells derived from human adult liver (as described in PCT/SG2016/050270) the organoid generation methodology described herein can similarly be employed to generate liver organoids from the adult liver stem cells (ALSC) (
Both MESP and ALSC generated similar organoids consisting of a hepatocyte core with cholangiocyte forming ductal-like structures in the periphery of organoids. Structural differences can be observed in the ductal structure formed by the cholangiocytes in both organoids. The cholangiocytes of the MESP derived organoid forms a spherical cyst structure whereas the cholangiocytes of the ALSC derived organoids arrange into a ring with a lumen in the center. Such differences can be expected as the stem cells are of different developmental potential. MESP derived from embryonic stem cells are fetal in nature and similarly the liver organoids derived MESP reflects fetal liver tissue. On the other hand, the liver organoids derived from ALSC reflect the adult liver tissue. In light of the differences in the developmental stages of both organoids, the overall structure and cell organization of the organoids are similar; Hepatocytes are found in the core of the organoids and cholangiocytes are in the periphery of the organoids. More remarkably, the multi-step methodology described herein can be differentially employed according to the initial endoderm stem cell state to generate liver organoids. As such, it is obvious that any early endoderm progenitors such as definitive endoderm or foregut can be first differentiated to MESP and generating organoids subsequently using methods described herein. Similarly, protocol can be employed by someone skilled in the art on stem cells or progenitors that arise during the development of posterior foregut to the adult liver.
Derivation of Hepatic Organoids from Adult Liver Stem Cells
The Hepatic organoid culture system described herein comprises a plurality of soluble agents in two different hepatic culture media and suspension culture system. The suspension culture system provides conditions for formation of late hepatic progenitors and subsequently organoids. In some embodiments, the plurality of soluble agents comprises one or more growth factors, an enhancer of the (canonical) WNT pathway, a TGF-β inducer and an inhibitor of Notch signaling.
Characterization of adult late hepatic progenitors and adult hepatic organoids
In some embodiments, the late hepatic progenitor is characterized by any one or more or at least two, or at least three, or at least four, or at least five, or at least six, or 1 or 2 or 3 or 4 or 5 or 6 or all, or all of the following markers: CK19 (NCBI: 3880), CK18 (NCBI: 3875), HNF4a (NCBI: 3172), ALB (NCBI: 213), HNF1B (NCBI: 6928) and SOX9 (NCBI).
In some embodiments, the hepatic (liver) organoids comprise more than one liver specific cell type selected from the group consisting of hepatocytes, cholangiocytes, liver specific endothelial cells (LSEC), stellate cells, hepatic myofibroblast and hepatoblasts.
In some embodiments, the hepatocytes are characterized by
In some embodiments, the cholangiocytes are characterized by their expression of CK7 but not albumin (ALB) and optionally by their expression of other cholangiocytes markers, such as CK19 (NCBI: 3880), HNF1B (NCBI: 6928) and SOX9 (NCBI: 6662).
In some embodiments, the liver specific endothelial cells (LSEC) are characterized by expression of any one or more markers selected from the group consisting of CD45, CD80, CD86, CD11c, VAP1, STAB1 and CD31, wherein the CD31 is predominantly expressed in the cytoplasm and not on the cell surface.
In some embodiments, the stellate cells are characterized by expression of any one or more markers selected from the group consisting of GFAP, VIM, LHX2, LRAT, PDGFRb, HAND2, ICAM-1, VCAM-1, and N-CAM
In some embodiments, the hepatic myofibroblast are characterized by expression of any one or more markers selected from the group consisting of COL1A1 and α-SMA.
In some embodiments, the hepatoblasts are characterized by expression of any one or more markers selected from the group consisting of SOX9 (NCBI: 6662), CK19 (NCBI: 3880), CK18 (NCBI: 3875), HNF4a (NCBI: 3172), HNF1B (NCBI: 6928) and ALB (NCBI: 213).
The hepatic (liver) organoids derived from ALSC are capable of performing liver functions and exhibit a structural composition observed in liver.
The liver functions are selected from the group consisting of albumin secretion, cytochrome enzymatic activities, glycogen storage, low density lipo-protein uptake, bile acid production and drug metabolism.
The structural composition observed in liver that is found in the hepatic (liver) organoid is characterized by the non-random distribution of the different liver cell types of which the liver is composed.
An exemplary method for generating hepatic organoids from adult liver stem cells is described in the Examples and illustrated schematically in
3. Intestinal Organoid and Pancreatic Spheroid from MESP
Provided herein are methods to generate intestinal organoids and pancreatic spheroids from MESP. MESP expresses markers PDX1, HNF4A and CDX2 which are important developmental regulators of the organs generated by the posterior foregut lineage, namely the liver, intestine and pancreas. MESP generated intestinal organoids resembles intestinal-like coiled structures with a lumen. The cells in the organoid express key intestinal markers such as CDX2 and Villin and the asymmetrical distribution of Villin suggest the cells are highly matured. The gut-like structures are envelope in a layer of mesenchymal tissues. This resembles the small intestine in vivo which is envelope by muscle tissues important for peristalsis.
Described herein is the first derivation of pancreatic spheroids from PSC via the use of MESP. The pancreatic spheroid expresses markers of pancreatic progenitors such as PDX1 and NKX6.1. The pancreatic spheroid progenitors described herein have the potential to give rise to all cells type of the pancreatic organs. Pancreatic spheroids have been generated from adult pancreas (see U.S. Pat. No. 8,642,339 B2). However, these adult pancreatic spheroid consist of epithelial progenitorsand do not express PDX1 and NKX6. 1, which are expressed by almost all pancreatic cell types. The pancreatic spheroids described herein have the potential to further generated pancreatic organoids containing multiple pancreatic cell types.
Derivation of Pancreatic Spheroid Progenitors from MESP
The pancreatic spheroid culture system described herein comprises a plurality of soluble agents in three different pancreatic culture media, a cellular support and suspension culture system. The cellular support provides culture conditions suitable for differentiation of MESP to early pancreatic progenitors and the suspension culture systemprovides culture conditions suitable for formation of late pancreatic progenitors. In some embodiments, the plurality of soluble agents comprises one or more growth factors, an enhancer of the (canonical) WNT pathway, a TGF-β inducer and an inhibitor of Notch signaling.
In some embodiments, a medium for early pancreatic endoderm progenitor formation is provided, wherein the medium comprises:
In some embodiments, the activator of AKT/PI3K signaling pathway and MAPK signaling pathway; TGF-β inhibitor and/or SMAD2/3 inhibitor; WNT-signaling activator and GSK3 inhibitor; FGF and MAPK pathway activator; and the molecule which is an repressor of NFκB activity and activator of mitogen-activated protein (MAP) kinase ERK, p38 and JNK, are as described and are used at the concentrations described herein above.
In some embodiments, the medium further comprises a molecule which is an activator of the FGF and MAPK pathway. In some embodiments, the activator of the FGF and MAPK pathway is used at a concentration of between about 2 ng/ml to 5 μg/ml, or between about 5 ng/ml to 5 μg/ml, or between about 10 ng/ml to 4 μg/ml, or between about 15 ng/ml to 3 μg/ml, or between about 20 ng/ml to 2 μg/ml, or about 5, 18, 20, 25, 28, 30, 50, 60, 70 ng/ml, or about 1, 2.5, 3.5 or 4.5 μg/ml. In some embodiments, the activator of the FGF and MAPK pathway is selected from the group consisting of FGF7, FGF1, FGF3, FGF10, and FGF22. In some embodiments, the FGF selected from the group consisting of FGF7, FGF1, FGF3, FGF10, and FGF22 is used at a concentration of between about 2 ng/ml to 5 μg/ml, or between about 5 ng/ml to 5 μg/ml, or between about 10 ng/ml to 4 μg/ml, or between about 15 ng/ml to 3 μg/ml, or between about 20 ng/ml to 2 μg/ml, or about 5, 18, 20, 25, 28, 30, 50, 60, 70 ng/ml, or about 1, 2.5, 3.5 or 4.5 μg/ml.
In some embodiments, media P1 comprises one, two, three, four or all of the following additional components that promote survivability and proliferation of pancreatic endoderm:
(i) A steroid as described above;
(ii) an activator of cAMP-dependent pathways, such as an activator of Protein Kinase A signaling pathway, as described above;
(iii) an activator of the Notch receptor as decribed above; and/or
(iv) an inhibitor of histone deacetylase (HDAC) as decribed above.
The additional components of media P1 can be used at the concentrations previously described herein.
In some embodiments, the medium for late pancreatic endoderm progenitor formation comprises:
In some embodiments, the metabolite of retinol is selected from the group consisting of retinoic acid, 9-cis-retinoic acid, isotretinoin, acitretin, bexarotene and alitretinoin. In some embodiments, the metabolite of retinol is used at a concentration of between about 0.2 μM to 5 μM, or between about 0.4 to 4 μM, or between about 0.6 to 3 μM, or between about 0.8 to 2 μM, or between about 1 to 1.5 μM, or about 0.3, 0.5, 0.7, 0.9, 1, 2, 2.5, 3.5, 4.5 μM.
In some embodiments, the Smoothened (Smo) and Sonic hedgehog (Shh) inhibitor is selected from the group consisting of N-[2-[(3′R,7′aR)-3′,6′,10,11b-tetramethyl-3-oxospiro[1,2,4,6,6a,6b,7,8,11,11a-decahydrobenzo[a]fluorene-9,2′-3,3a,5,6,7,7a-hexahydrofuro[3,2-b]pyridine]-4′-yl]ethyl]-6-(3-phenylpropanoylamino)hexanamide (KAAD-cyclopamine), (E)-N-(4-benzylpiperazin-1-yl)-1-(3,5-dimethyl-1-phenylpyrazol-4-yl)methanimine (SANT-1), and (3 S,3′R,3′aS,6'S,6aS,6bS,7′aR,9R, 11aS, 11bR)-3′,6′,10,11b-tetramethylspiro[2,3,4,6,6a,6b,7,8,11,11a-decahydro-1H-benzo[a]fluorene-9,2′-3a,4,5,6,7,7a-hexahydro-3H-furo[3,2-b]pyridine]-3-ol (cyclopamine). In some embodiments, the inhibitor of Smoothened (Smo) and Sonic hedgehog (Shh) is used at a concentration of between about 25 nM to 5 μM, or between about 200 nM to 4 μM, or between about 400 nM to 3 μM, or between about 600 nM to 2 μM, or between about 800 nM to 1 μM, or about 100, 250, 350, 450, 500, 700, 800 nM or about 1.5, 2, 2.5, 3.5 and 4.5 μM.
In some embodiments, The medium comprises a molecule which is an activator of the FGF and MAPK pathway as described above and used at concentrations described herein.
In some embodiments, the protein kinase C activator is selected from the group consisting of a phorbol ester, (1S,3 S,5Z,7R,8E, 11S,12S, 13E,15 S,17R,20R,23R,25S)-25-Acetoxy-1,11,20-trihydroxy-17-[(1R)-1-hydroxyethyl]-5,13-bis(2-methoxy-2-oxoethylidene)-10,10,26,26-tetramethyl-19-oxo-18,27,28,29-tetraoxatetracyclo[21.3.1.13,7.111,15]nonacos-8-en-12-yl (2E,4E)-2,4-octadienoate (Bryostatin I), (1aR,1bS,4aR,7aS,7bS,8R,9R,9aS)-9a-(acetyloxy)-4a,7b-dihydroxy-3-(hydroxymethyl)-1,1,6,8-tetramethyl-5-oxo-1a, 1b,4,4a, 5,7a,7b, 8,9,9a-decahydro-H-cyclopropa[3,4]benzo[1,2-e]azulen-9-yl myristate (TPA), and 5-chloro-N-heptylnaphthalene-1-sulfonamide (SC-10), (2Z)-2-Methyl-2-butenoic acid (1 aR,2S, 5R, 5aS,6S,8aS,9R, 1 0aR)-1a,2,5,5a,6,9,10,10a-octahydro-5,5a-dihydroxy-4-(hydroxymethyl)-1,1,7,9-tetramethyl-11-oxo-1H-2,8a-methanocyclopenta[a]cyclopropa[e]cyclodecen-6-yl ester (PEP 005). In some embodiments, the protein kinase C activator is used at a concentration of between about 100 nM to 10 μM, or between about 200 nM to about 8 μM, or between about 400 nM to about 6 μM, or between about 400 nM to 4 μM, or between about 400 nM to 2 μM, or between about 450 nM to about 1 μM; or about 350, 475, 500, 550, 600, 800, or 900 nM, or about 7, 5, 3, 2.5, 1.5 or 1 μM.
In some embodiments, the (selective) ALK2, ALK3 and ALK6 inhibitor is selected from the group consisting of Noggin (NCBI 9241), 6-[4-(2-piperidin-1-yl ethoxy)phenyl]-3-pyridin-4-ylpyrazolo[1,5-a]pyrimidine (Dorsomorphin), and 4-[6-(4-piperazin-1-ylphenyl)pyrazolo[1,5-a]pyrimidin-3-yl]quinolone (LDN193189). In some embodiments, the (selective) ALK2, ALK3 and ALK6 inhibitor (e.g., Noggin) is used at a concentration of between about 2 ng/ml to 5 μg/ml, or between about 5 ng/ml to 5 μg/ml, or between about 10 ng/ml to 4 μg/ml, or between about 15 ng/ml to 3 μg/ml, or between about 20 ng/ml to 2 μg/ml, or about 5, 18, 20, 25, 28, 30, 50, 60, 70 ng/ml, or about 1, 2.5, 3.5 or 4.5 μg/ml.
In some embodiments the medium further comprises an inhibitor of γ-secretase. In some embodiments, the inhibitor of γ-secretase is selected from the group consisting of is selected from the group consisting of DAPT: tert-butyl (2S)-2-[[(2S)-2-[[2-(3,5-difluorophenyl)acetyl]amino]propanoyl]amino]-2-phenylacetate, Compound E (C-E) (2S)-2-[[2-(3,5-difluorophenyl)acetyl] amino]-N-[(3 S)-1-methyl-2-oxo-5-phenyl-3H-1,4-benzodiazepin-3-yl]propanamide, Dibenzazepine (DBZ): (2S)-2-[[2-(3,5-difluorophenyl)acetyl]amino]-N-[(7S)-5-methyl-6-oxo-7H-benzo[d][1]benzazepin-7-yl]propanamide, Begacestat: 5-chloro-N-[(2S)-4,4,4-trifluoro-1-hydroxy-3-(trifluoromethyl)butan-2-yl]thiophene-2-sulfonamide, and Flurizan: (2R)-2-(3-fluoro-4-phenylphenyl)propanoic acid. In some embodiments, the inhibitor of γ-secretase (e.g., DAPT) is used at a concentration of between about 0.1 μM to 20 μM, or between about 0.5 μM to 15 μM, or between about 0.8 μM to 10 μM, or between about 1 μM to 5 μM, or between about 0.9 μM to 2.5 μM, or about 0.7, 0.8, 0.9, 1, 1.5, 2, 7, 8, 12, 17 or 19 μM.
In some embodiments, the medium further comprises an activator of AKT/PI3K signaling pathway and MAPK signaling pathway, or a molecule which is an repressor of NFκB activity and activator of mitogen-activated protein (MAP) kinase ERK, p38 and JNK, or a compound selected from the group consisting of nicotinamide, nicotinic acid, 5-fluoronicotinamide, isonicotinic acid hydrazide, and nikethamide, as described herein and used at the concentrations described herein.
In some embodiments, the medium for deriving and maintaining a pancreatic spheroid comprises:
In some embodiments, the AMPK signaling activator is selected from the group consisting of thyroid hormone 3 (T3), 5-amino-1-[(2R,3S,5R)-3,4-dihydroxy-5-(hydroxymethyl)oxolan-2-yl]imidazole-4-carboxamide (AICAR), and 3-(diaminomethylidene)-1,1-dimethylguanidine (Metformin). In some embodiments, the AMPK signaling activator (e.g., T3) is used at a concentration of between about 10 nM to 1 M, or between about 200 nM to 0.8 μM, or between about 400 nM to 0.6 M, or between about 600 nM to 0.4 M, or between about 800 nM to 0.2 M, or between about 1 μM to 2 μM, or about 500, 700, 900, 950 nM, or about 1, 1.1, 1.5, 2, 5, 7, 8, 150, 500, 700 or 800 μM.
In some embodiments, the P3 medium further comprises a molecule which is an repressor of NFκBactivity and activator of mitogen-activated protein (MAP) kinase ERK, p38 and JNK; or a compound selected from the group consisting of nicotinamide, nicotinic acid, 5-fluoronicotinamide, isonicotinic acid hydrazide, and nikethamide, as described herein and used at a concentration described herein.
In some embodiments, the early pancreatic endoderm progenitor is characterized by expression of one or more markers selected from: SOX9 (NCBI: 6662), PDX1 (NCBI: 3651), NKX6.1 (NCBI: 4825), and CK19 (3880).
In some embodiments, the late pancreatic endoderm progenitor is characterized by expression of one or more markers selected from: PDX1 (NCBI: 3651), NKX6.1 (NCBI: 4825), NEUROG3 (NCBI: 50674), NKX2.2 (NCBI: 4821), NEUROD1 (NCBI: 4760), and PAX6 (NCBI: 5080).
In some embodiments, the pancreatic endoderm progenitor cell is characterized by expression one or more markers selected from:
In some embodiments, the cell secretes one or more of the following hormones or enzymes: INS(NCBI 3630), GCG (NCBI 2641), SST (NCBI 6750) or PRSS1 (NCBI 5644).
MESP expresses PDX1 which is a key marker of the pancreatic lineage. This highlights the potential of MESP to generate tissues of the pancreatic lineage. Described herein is a 3 step protocol to derived pancreatic spheroids from MESP. The pancreatic spheroid not only expresses PDX1 but also another important pancreatic progenitor marker NKX6.1 (Rezania., et al 2013; Burlison., et al 2008; Nostro et al., 2015) (
In contrast, pancreatic epithelial organoids (PCT/NL2010/000017) derived from the adult pancreas expresses different stem cell markers such as EPCAM and SOX9 (Table 6) and. Different culture conditions are also used to culture the MESP derived pancreatic spheroid compared to the adult pancreatic epithelial organoids (Table 7).
Derivation of Pancreatic Progenitors and Spheroids from MESP
Exemplary methods for producing pancreatic progenitors and spheroids from MESP are described in the Examples and illustrated schematically in
Gene Expression Analysis with Quantitative PCR (qPCR)
Total RNA from the cells was isolated using the TRIzol reagent (thermos scientific) according to manufacturer's protocol. Briefly, lml of trizol was used for not more than 1.5 million cells. Trizol was added to the cells directly after media was removed. The samples were incubated for 15-30 min to completely lyse the cells. 200 μl of the 100% chloroform was added and samples were vigorously mixed and left to stand for 5 min at room temperature. The samples were centrifuge for 15 min at 13,000 RPM in 4° C. and top aqueous layer was retrieve into a new 1.5 ml appendorf tube. Equal volume of 100% Isopropanol was added to the aqueous solution to precipitate the Total DNA and RNA. The samples were left to stand for 10 mins and centrifuge for 10 min at 13,000 RPM in 4° C. The total DNA and RNA pelleted are washed once with 70% ethanol and centrifuge for 5 min at 5,000 RPM at room temperature. The total DNA and RNA is reconstituted with DEPC water. DNA contaminations is removed via DNASE I treatment (Thermo Scientific). The total RNA is clean up using RNA purification kit (PureLink, Invitrogen) according to the manufacturer's protocol. 500 ng of total RNA was input for the reverse transcription process using the SuperScript II reverse transcriptase reagents (Invitrogen) according to the manufacturer's protocol. The cDNA was quantitated using the SYBR FAST qPCR Master Mix (KAPA) reagents and read with the Real-Time PCR System (Applied Biosystem).
For immunofluorescence of 3D suspension cultures, the organoids were washed three times with PBS before fixing with 4% PFA for 30 mins at room temperature. The samples were permeabilized with 0.5% Triton X-100 and blocked with 0.5% Triton X-100+5% BSA respectively for 1 hr. The samples are incubated with the primary antibody diluted in 0.1% Tween-20 containing 5% BSA overnight at 4° C. After 16-24 hr, the samples were washed three times with 0.1% Tween-20 for 15 mins during each wash. The organoids are incubated with secondary antibody diluted in 0.1% Tween-20 containing 5% BSA for 3 hrs at room temperature and subsequently washed three times with 0.1% Tween-20. Hoechst 33342 was added during the last wash. For immunofluorescence of 3D matrigel cultures, the Matrigel containing the cells was mechanically dissociated, transferred to an eppendorff tube and kept on ice. A combination of dispase and low temperature was used to liquefy the Matrigel. After 15 mins, the samples were centrifuged at 1,000 r.p.m for 5 mins and the supernatant was aspirated. The pellet was washed once with cold PBS to remove remaining Matrigel contaminants. The samples were subsequently fixed and stained as described above. All immunofluorescence images of 3D samples were acquired using a confocal microscopy (Olympus FV1000 inverted). ImageJ 1.48k software (Wayne Rasband, NIHR, USA, http://imagej.nih.gov/ij) was used for image processing. Changes in brightness or contrast during processing were applied equally across the entire image.
Derivation of MESP from Human Embryonic Stem Cells (hESC)
H1 human embryonic stem cells were purchased from Wicell. The H1 hESC was used to generate MESP. H1 hESC were culture in 6 well dishes (Falcon) using mTeSR1 media (STEMCELL Technologies). 2 mls of media was provided for each 6 well and media was refreshed daily. hESC were routinely passage every 5-7 days upon confluency. Briefly, 6 well dish was thinly coated with 30× diluted matrigel (200 μl per well) and incubator for 1 hr before use. To passage the cells, the media is aspirated and cells were washed once with 1.5 to 2 ml of 1×PBS (Gibco). After aspirating the 1×PBS, 0.5-1 ml of 1×Dispase (Gibco) was added to each well of the hESC and cells were incubated for 5-7 mins at 37° C. The dispase was removed and cells were washed once with 2 ml of 1×PBS and 1 ml of mTeSR1 media was added to the well. The cells were lifted from the plate with a cell scraper and the hESC colonies were dissociated into cell clumps of 50-100 cells and seeded at a ratio of 1:12 into well pre-coated with matrigel.
To seed hESC for generating MESP (
The differentiation process can be monitored with qPCR of marker genes expression. During differentiation, levels of the pluripotent stem cell markers OCT4 and NANOG would start to decrease in the DE and GUT (
To differentiate GUT into MESP (
The MESP were seeded on Matrigel (BD Biosciences) containing B27 supplement (Invitrogen). MESP were cultured in Advanced Dulbecco's modified Eagle's medium (DMEM)/F12 (Gibco) containing N2 supplement (Gibco), B27 supplement (Gibco), penicillin/streptomycin (Gibco), A83-01 (Stemgent), Dexamethasone (Stemgent), ChIR99021 (Tocris), Valproic Acid (VPA) (Stemgent), human HGF (R&D), human EGF (R&D), Jagged-1 (Anaspec), N6,2′-O-Dibutyryladenosine 3′,5′-cyclic monophosphate sodium salt (dbCAMP) (Sigma-Aldrich), Nicotinamide (Sigma-Aldrich). MESP were passaged every 14-16 days using Dispase and TryLE. Matrigel was mechanically dissociated, transferred to an eppendorff tube and kept on ice to allow a combination of dispase and low temperature to liquefy the Matrigel. After 5 mins, the samples were centrifuged at 1,000 r.p.m for 5 mins and the supernatant was aspirated. Spheroids were then incubated in TryLE at 37° C. for 5 mins before being dissociated into single cells. Cells were centrifuged at 1,000 r.p.m for 5 mins. Cells were washed once with MESP media and then resuspended in MESP media. Cells were seeded at a density of 12,000 cells/cm2. Media was changed every 2 days.
To identify the developmental identity of the MESP along the endoderm lineage, markers targeting different regions of the GUT tube were selected and detected via gene expression analysis with qPCR (
This proliferation capacity of MESP is valuable for the large scale production of cells which is required for regenerative therapy, and in proteomics and genomics studies. To scale up the production of MESP, the cells are seeded in larger vessels of 24 well and 12 well dishes. MESP were seeded at similar density of 12,000 cells/cm2 and the volume of matrigel used was increase proportionally to the volume of the culture chamber. The media was similarly refreshed every 2 days. By increasing the size of the chamber, the number of MESP retrieved also proportionally increased (
To further characterize the unique stem cell state of MESP, the transcriptome is profiled using whole genome microarrays. Briefly, the total RNA from MESP, hESCs, DE and GUT cells were extracted using Trizol reagent, DNASE treated and purified using Purelink RNA kit, using similar approach for gene expression analysis with qPCR. For the microarray, 500 ng DNase-treated total RNA was amplified into biotin labeled cRNA with Illumina Total Prep RNA Amplification Kit (Ambion) according to manufacturer's protocol. Subsequently, 750 ng of cRNA was hybridized, washed and labelled with Cy3-streptavidin onto the array (Illumina HumanHT-12_v4_BeadChips) according to manufacturer's Protocol. Reading of the hybridized chips was done using Illumina HiScan Platform. The data was processed using Genome Studio (Illumina). Samples were subjected to Quantile Normalization. The normalized data was exported into GeneSpring format and to Microsoft Excel for subsequent analysis. Briefly, data was filtered to remove probes which do not have any signal. Generation of the Principal component analysis plots, statistical analysis to generate genes was done with GeneSpring (GeneSpring). Generation of heatmaps and clustering was done using Gene-E (Broard Institute). The cluster analysis shows that the transcriptome of MESP is highly different from hESCs, DE and GUT cells. MESP expresses a unique expression signature as a stem cell state. Many of the early endoderm specific markers such as SOX2, CER1, GATA4, SOX17, CXCR4, FOXA2 and CD34 are not expressed in MESP (
Derivation of MESP from Induced Pluripotent Stem Cells (iPSCs)
Pluripotent stem cells encompass both embryonic stem cells and induced pluripotent stem cells. iPSCs is generated by the nobel winning method by Takahashi and Yamanaka (Takahashi and Yamanaka, 2006) where terminally differentiated somatic cells are converted back into a pluripotent cells state. The iPSC technology has vast application potentials and one of the key breakthroughs includes the modeling of genetic diseases. The disease patient somatic cells such as blood or skin fibroblast can be reverted back to a pluripotent cell state. This disease patient pluripotent stem cell can be used to generate the cell type of interest which harbors the disease phenotype. Thus, this technology potentially allows the modeling of any genetic disease in a dish. It is thus important to show that iPSC can similarly be used to generate MESP for modeling diseases.
iPSCs are generated and characterized as previously described (Chia., et al, nature 2010). Briefly, human MRC5 fibroblast (ATCC) culture in DMEM (Gibco) supplement with 15% fetal bovine serum (Hyclone) were infected with retroviruses harboring the overexpression cassettes for genes OCT4, SOX2, KLF4, CMYC and PRDM14. After 3-5 days infection, the fibroblast was plated on the Mitomycin C inactivated CF-1 feeders. After 24-48 hrs post infection, the cells were culture in DMEM/F12 containing 20% Knockout serum replacement (Gibco), 1 mM L-glutamine, 1% non-essential amino acids, 0.1 mM 2-mercaptoethanol and supplemented with 4-8 ng/ml basic fibroblast growth factor (Invitrogen). The media was refreshed every 2 days. Human iPSC colonies will appear and ready to manually picked 3 weeks post seeding. The pluripotent cell state of the iPSC clones picked was validated by gene expression of pluripotent stem cell markers and ability to form teratomas in the SCID mice (Chia., et al, nature2010).
iPSCs were differentiated to MESP using the same protocol described above for derivation from hESC. The MESP derived from iPSC is morphologically similar to those derived from hESC and expresses similar key MESP markers HNF4A, CDX2, PDX1, CK19 and SOX9 (
Generation of LDLR Knockout MESP from Genome Edited hESCs
A dual expression vector (PX330-2AmCherry) encoding for SpCas9 linked to a mCherry reporter cassette via a T2A peptide, and a single guided RNA (sgRNA), was utilized for the generation of LDLR knockout hESC lines. sgRNAs were designed to target the first protein coding exons for both genes respectively, with additional nucleotide sequences appended to the 5′ and 3′ ends of the oligos intended for BbsI cloning. All sgRNA cloned PX330 vectors were validated via Sanger Sequencing using the following U6 promoter primer sequence (5′-GAGGGCCTATTTCCCATGAT-3′; SEQ ID NO:94), amplify using Stbl3 cells and purified using FavorPrep™ Plasmid DNA Extraction Mini Kit (FAVORGEN Biotech Corp). Nucleofection of hESC was performed using the P3 Primary Cell 4D-Nucleofector® X Kit L (Lonza, # V4XP-3012) following manufacturer's protocol. Briefly, hESCs were grown to 80% confluency in a well of a 6 well dish and harvested as single cells with TrypLE™ (ThermoFisher Scientific). A tube containing a total of 1×106 single cell hESCs was resuspended into 50 μl of the P30 nucleofector solution and mixed with another 50p of the P30 nucleofector solution containing 5 g of plasmid. The final hESC and DNA mixture was transferred into a Nucleocuvette™ and nucleofected with a 4D-Nucleofector™ System using the CM-113 experimental parameter setting. Following nucleofection, hESCs was transferred to a matrigel coated well of a 6 well plate and recovered in mTESR containing 0.5 M Rock Inhibitor Thiazovivin (STEMGENT). After 48 h, mCherry positive hESCs were sorted using FACS and cells were plated as single cells in a 10 cm dish and cultured in mTESR containing Rock Inhibitor for 5-7 days. Upon confluency, single colonies were then picked and expanded individually in mTESR. Each clonal line was later split at a ratio of 1:2, with half of each expanded clonal line retained for maintenance and another half lysed in QuickExtract solution for genomic DNA extraction. gDNA of each clonal line was subsequently used as a PCR template together with specific PCR primers designed to amplify sgRNA targeted regions of approximately 200 bp in size. To identify clones with potential frameshift mutations, PCR products from clones and WT hESC were analysed via gel electrophoresis (3% Agarose; 150V for 5 h) and distinct shifts in band size (arrows) were observed in clones with successful gene targeting (
The LDLR KO hESCs is subjected to similar MESP differentiation protocol (
Generation of Intestinal Organoids from MESP
MESP gene expression profile suggests that this stem cell closely resembles the posterior foregut which has the developmental potential to generate the liver pancreas and intestine. We tested if MESP is able to generated intestinal organoids adopting and modifying differentiation strategies reported by others generating the organoids from hESC (Spence., et al 2011). MESP were cultured in in a media comprised of Advanced Dulbecco's modified Eagle's medium (DMEM)/F12 (Gibco) containing N2 supplement (Gibco), B27 supplement (Gibco), 1× penicillin/streptomycin (Gibco), A83-01 (Stemgent), SB202190 (Tocris), human EGF (R&D), Nicotinamide (Sigma-Aldrich), Noggin (R&D), Wnt3A (R&D), R-spondinl (R&D), N-acetyl cysteine (Sigma) and FGF4 (R&D) for 8 days to induce specification towards the intestinal lineage. After which, spheroids were removed from matri-gel using Dispase and dissociated into single cells using TryLE. Briefly Matrigel was mechanically dissociated, transferred to an eppendorff tube and kept on ice to allow a combination of dispase and low temperature to liquefy the Matrigel. After 5 mins, the samples were centrifuged at 1,000 r.p.m for 5 mins and the supernatant was aspirated. Spheroids were then incubated in TryLE at 37° C. for 5 mins before being dissociated into single cells. Cells were centrifuged at 1,000 r.p.m for 5 mins. Cells were washed once with 12 media which comprises of Advanced Dulbecco's modified Eagle's medium (DMEM)/F12 (Gibco) containing N2 supplement (Gibco), B27 supplement (Gibco), 1× penicillin/streptomycin (Gibco), A83-01 (Stemgent), SB202190 (Tocris), human EGF (R&D), Nicotinamide (Sigma-Aldrich), Noggin (R&D), Wnt3A (R&D), R-spondinl (R&D), N-acetyl cysteine (Sigma) and FGF4 (R&D) Cells were subsequently seeded at a density of 5,000 cells/per well (96 well) and cultured in 12 as suspension culture for 30 days in a 96 well ultra-low attachment plate to derive intestinal organoids.
After 3 weeks of differentiation, coiled-coil structures resembling the small intestine can be observed in dish (
Generation of Liver Organoids from MESPs
The posterior foregut forms the liver organ in the human body. Thus, MESP spheroid has the potential to generate liver organoids. Herein we developed a stepwise induction protocol to the generate human liver organoids from MESPs (
The early hepatic progenitors were subsequently removed from matri-gel using Dispase and dissociated into single cells using TryLE. Briefly, Matrigel was mechanically dissociated, transferred to an eppendorff tube and kept on ice to allow a combination of dispase and low temperature to liquefy the Matrigel. After 5 mins, the samples were centrifuged at 1,000 r.p.m for 5 mins and the supernatant was aspirated. The early hepatic progenitors were then incubated in TryLE at 37° C. for 5 mins before being dissociated into single cells. Cells were centrifuged at 1,000 r.p.m for 5 mins. Cells were washed once with H2 media and then resuspended in H2 media. Cells were seeded at a density of 5,000 cells/per well (96 well) and cultured in Advanced Dulbecco's modified Eagle's medium (DMEM)/F12 (Gibco) containing N2 supplement (Gibco), B27 supplement (Gibco), 1× penicillin/streptomycin (Gibco), A83-01 (Stemgent), ChIR99021 (Tocris), human HGF (R&D), human EGF (R&D), N6,2′-O-Dibutyryladenosine 3′,5′-cyclic monophosphate sodium salt (dbCAMP) (Sigma-Aldrich), (Sigma-Aldrich), BMP7 (R&D) and FGF7/KGF (R&D) as suspension culture for 15 days in a 96 well ultra-low attachment plate to derive late hepatic progenitors (
To derived hepatic organoids, hepatic progenitors were cultured in Clonetics™ HCM™ Hepatocyte Culture Medium (Lonza) containing A83-01, Dexamethasone, Compound-E (EMD Millipore), HGF, BMP7, FGF19, Oncostatin-M (R&D) for at least 3 weeks (
The liver is largely consisting of hepatocyte which is the major metabolic cell type of the organ. Other than the hepatocytes, the other liver parenchyma cell type is the cholangiocytes. The cholangiocytes form the bile ducts in the liver that export the bile secretions from the hepatocytes out of the liver and into the small intestine. To investigate if the liver organoids contain both parenchyma cell type of the liver, the organoids are co-stained with antibodies specific for ALB (specifically expressed in hepatocytes) and cytokeratin 7 (CK7) (specifically expressed in cholangiocytes). From the staining results (
Not only are both parenchyma liver cells found in the liver organoids, the cells are arranged in a specific manner that mimics the in vivo liver tissue (
To determine if the liver organoids are functional, the expression of various metabolic enzymes and transported were detect by qPCR. The liver organoids expresses most of the cytochrome P450 enzymes (CYPs) including CYP3A4, CYP3A7, CYP1A1, CYP2D6, CYP2B6, CYP2C19 and CYP2E1 compared to MESP. In addition, the liver organoids also expresses major UDP-glucuronosyltransferase enzymes UGT1A1, UGT2B15 and UGT2B7 compared to MESP. These enzymes are important for the different phase of detoxification functions of the liver. The CYPs essentially metabolized almost 75% of the drugs in the human body. In the first phase of detoxification in the liver, these enzymes introduce reactive subgroups to the substrates to increase water solubility of the molecules for removal. In the second phase, the UDP-glucuronosyltransferase enzymes conjugate these reactive metabolites from the CYP enzymes with charge groups such as glucuronic acid to increase the mass of this substrate and reducing its reactivity. In the last phase of detoxification, bile transporter such as NTCP and OATP1B3 expressed in the organoids actively transport the detoxified products out of the hepatocytes into the bile canaliculi and towards the bile duct for removal. Cell staining with another important liver transport MRP2 shows that liver organoids expresses the essential transport essential for liver detoxification functions. The expression analysis suggests that the organoids expresses most detoxification enzymes require for all phases of detoxification processes in the liver.
To further validate the enzymatic activity of the CYPs expressed in the organoids, the P450-Glo™ CYP450 Assays (Promega) was used to assay for the activity of various CYPs. Specific assay kits for the each CYP enzyme were used according to manufacturer's protocol. Independent organoids were used for each assay kits specific for detecting CYP3A4, CYP2D6, CYP2B6 and CYP1A2. The total luciferase reading taken from the luminometer is normalized to the total cell number in the organoids. The enzymatic assays showed that the organoids have highly active CYPs enzymatic activity compared HepG2 cell lines commonly employed in the industry for liver studies.
The organoids are also assay for specific liver functions such as albumin secretion. The media from individual organoids are collected after 24 hrs. The amount of albumin in the media was detected using ELISA with a human albumin specific antibody and a spectrophotometer. The exact amount of albumin was determined using a standard control consisting of different concentration of recombinant albumin. The readings from the recombinant albumin of various concentrations generate a standard curve. The standard curve is used to extrapolate the amount of albumin in the media based on its readings in the ELISA. The results shows that liver organoids secretes 30-60 ng/ml/day of albumin compared to media control.
Another major function of the liver is the storage of excess glucose as glycogen in the hepatocytes. Glycogen serves as an important form of energy storage and dysregulation of this process in the liver lead to diseases such as diabetes. The liver organoids were stained with Periodic acid-Schiff (PAS) which detects polysaccharides such as glycogen. The PAS staining shows that the hepatocytes (stained purpled, arrows) in the organoids are capable of the storing glycogen (
In the liver lobule, the hepatocytes and cholangiocytes are connected by a channel known as the bile canaliculi (
To detect the functional bile canaliculi network in the liver organoids, live organoids were treated with CDFDA. Briefly, media was removed and the organoids were first washed three times with PBS containing calcium and magnesium. Next, the organoids were incubated with 5 μg/ml CDFDA (Molecular Probes) and 1 μg/ml Hoechst 33342 for stipulated time at 37° C. Organoids were subsequently washed three times and imaging was performed on a confocal microscopy (Olympus FV1000 inverted). ImageJ 1.48k software (Wayne Rasband, NIHR, USA, http://imagej.nih.gov/ij) was used for image processing. Changes in brightness or contrast during processing were applied equally across the entire image.
Within 30 minutes of treatment with CDFDA, the hepatocytes in the organoids accumulated CDF (
Modeling Disease with Liver Organoids
The successful generation of the genetically modified MESP (example 4) highlighted the potential of modeling diseases of organs that can be generated from MESP. Herein, the LDLR KO MESP was used to generate liver organoids using similar approach with the wild type MESP (example 6). In patient deficient of the LDLR, the liver secretes high levels of the cholesterols in the human body, resulting in hypercholesterolemia. The elevated in levels of cholesterol in the blood stream results in cardiovascular diseases and patients undergo statin treatment to control blood cholesterol levels. To investigate if the LDLR deficient liver organoids mimics the liver organ of a hypercholesterolemia patient deficient in LDLR, we assay for the level of cholesterol secreted by the liver organoids. The media incubated with organoids after 24 hours are collected and the amount of cholesterol in the media is determine using the Amplex® Red Cholesterol Assay Kit (Thermo fisher scientific) according to the manufacturer's protocol. Briefly, media from each organoids is incubated with the reagents provided in the kit and incubated for 30 mins at 37° C. The fluorescence generated is detected and quantify using a fluorescence plate reader. The total amount of cholesterol for lml of the media is tabulated. To test the response of the LDLR deficient organoids to statin treatments, the organoids are incubated with the different concentrations of Pravastatin (Sigma) added into the media. The LDLR deficient liver organoids secreted higher levels of cholesterol compared to the LDLR expressing liver organoids (
To generate organoids in large numbers, the protocol was adapted to generate organoids of comparable size and function in a 96 well dish. Briefly, MESP were cultured in Advanced Dulbecco's modified Eagle's medium (DMEM)/F12 (Gibco) containing N2 supplement (Gibco), B27 supplement (Gibco), 1× penicillin/streptomycin (Gibco), 500 nM A83-01 (Stemgent), 2 μM ChIR99021 (Tocris), 20 ng/ml human HGF (R&D), 50 ng/ml human EGF (R&D), luM Jagged-1 (Anaspec), 300 ng/ml N6,2′-O-Dibutyryladenosine 3′,5′-cyclic monophosphate sodium salt (dbCAMP) (Sigma-Aldrich), 10 mM Nicotinamide (Sigma-Aldrich), 20 ng/ml BMP4, 20 ng/ml BMP7 (R&D) and 25 ng/ml FGF7/KGF (R&D) for 8 days to induce specification towards the hepatic lineage. After which, spheroids were removed from matri-gel, dissociated to single cells using TryPLE and seeded in a 96 well plate in Advanced Dulbecco's modified Eagle's medium (DMEM)/F12 (Gibco) containing N2 supplement (Gibco), B27 supplement (Gibco), 1× penicillin/streptomycin (Gibco), 2.5 mM A83-01 (Stemgent), 2μM ChIR99021 (Tocris), 20 ng/ml human HGF (R&D), 50 ng/ml human EGF (R&D), 300 ng/ml N6,2′-O-Dibutyryladenosine 3′,5′-cyclic monophosphate sodium salt (dbCAMP) (Sigma-Aldrich), 10 mM Nicotinamide (Sigma-Aldrich), 20 ng/ml BMP4, 20 ng/ml BMP7 (R&D) and 25 ng/ml FGF7/KGF (R&D) as suspension culture for 2 weeks. The cells were subsequently cultured in Clonetics™ HCM™ Hepatocyte Culture Medium (Lonza) containing 500 nM A83-01, 30 M Dexamethasone, 500 nM Compound-E (EMD Millipore), 25 ng/ml HGF, 25 ng/ml BMP7, 25 ng/ml FGF19, 20 ng/ml Oncostatin-M (R&D) for another 3-4 weeks to derive hepatic organoids. One organoid was generated for each well of the 96 well format plates (
Generation of Liver Organoids from Adult Liver Stem Cells
The step wise generation of liver progenitors during the derivation of liver organoids from the MESP (example 6) suggests that the method could be applied to other stem cells of hepatic lineage. Correspondingly, the method was successfully adapted for deriving liver organoids from liver stem cells (PCT/SG2016/050270) derived from the adult liver tissue (
The late hepatic progenitors were lastly cultured in Clonetics™ HCM™ Hepatocyte Culture Medium (Lonza) containing A83-01, Dexamethasone, Compound-E (EMD Millipore), HGF, BMP7, FGF19, Oncostatin-M (R&D) for another 3-4 weeks to derive liver organoids. The organoids were co-stained with antibodies specific for ALB and CK7 to check if both parenchyma liver cells (hepatocytes and cholangiocytes) are present in the organoids. The organoids derived from the adult liver stem cells similarly consist of ALB expressing hepatocytes and CK7 expressing cholangiocytes (
Generation of Pancreatic Spheroids from MESP
PDX1 expression in MESP suggests that these spheroid stem cells similar to the posterior foregut have the ability to generate pancreatic tissues. Herein, we employed a step-wise protocol to differentiate the MESP into pancreatic spheroids (
PSC: Pluripotent Stem Cells; ESC: embryonic stem cell; MESP: Multipotent Endodermal Spheroid Progenitors; ECM; Extracellular Matrix; CYP: Cytochrome p450 e.g. CYP3A4: Cytochrome P450, Family 3, Subfamily A, Polypeptide 4; LGR5: Leucine-rich repeat-containing G-protein coupled receptor 5; KRT: Cyto-keratin e.g KRT19: Cyto-keratin 19; AFP: Alpha-Fetoprotein; HNF: Hepatocyte Nuclear Factor e.g. HNF4a: Hepatocyte Nuclear Factor 4 Alpha; IF: Immunofluorescence; E-CAD: E-Cadherin; KI67: Antigen KI-67; SOX: SRY (Sex Determining Region Y)-Box e.g. SOX9: SRY (Sex Determining Region Y)-Box 9; PROM1: Prominin 1; FOXA: Forkhead Box Protein e.g. FOXA2: Forkhead Box Protein A2; ALB: Albumin; PROX1: Prospero Homeobox 1; qPCR: Quantitative polymerase chain reaction; FACS: Fluorescence-activated cell sorting; 2D: 2 dimensional; 3D: 3 dimensional; PAS: Periodic acid Schiff; LDL: Low-density lipoprotein; cAMP: cyclic adenosine monophosphate; BMP: Bone Morphogenetic Protein; HGF: Hepatocyte Growth Factor; FGF: Fibroblast Growth Factor; EGF: Epidermal Growth Factor; TGF-β: Transforming growth factor beta; MAPK; Mitogen-activated protein kinases; extracellular signal-regulated kinases; JNK; c-Jun N-terminal kinases; FGF; Fibroblast Growth Factor; STAT3: Signal transducer and activator of transcription 3; GAB1: GRB2-associated-binding protein 1; AKT/PI3K/mTOR: Protein kinase B/Phosphatidylinositol-4,5-bisphosphate 3-kinase/mechanistic target of rapamycin; NF-κB: nuclear factor kappa-light-chain-enhancer of activated B cells; YAP: Yes-associated protein; IGF; Insulin-like growth factor, IL: Interleukin e.g. IL-6: Interleukin-6, OSM: Oncostatin-M
Biologically pure cultures of the endoderm spheroid progenitor cells described herein were deposited ______, 2016, under terms of the Budapest Treaty with the American Type Culture Collection (ATCC®) (10801 University Boulevard, Manassas, Va. 20110 USA), and given the patent deposit designation number(s) ______, respectively. The microorganism deposit was made under the provisions of the “Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purposes of Patent Procedure.” All restrictions on the availability to the public of these deposited microorganisms will be irrevocably removed upon issuance of a patent based on this application. For the purposes of this disclosure, any isolate having the identifying characteristics of the deposited cells, including subcultures and variants thereof having the identifying characteristics and activity as described herein, are included.
It is understood that the examples and embodiments described herein are for illustrative purposes only and that various modifications or changes in light thereof will be suggested to persons skilled in the art and are to be included within the spirit and purview of this application and scope of the appended claims. All publications, sequence accession numbers, patents, and patent applications cited herein are hereby incorporated by reference in their entirety for all purposes.
| Number | Date | Country | Kind |
|---|---|---|---|
| 10201507675Y | Sep 2015 | SG | national |
This application is a divisional of U.S. patent application Ser. No. 15/760,335, filed Mar. 15, 2018, which is the U.S. National Stage of International Patent Application PCT/SG2016/050448, filed Sep. 15, 2016, which claims priority to Singapore Application No. SG10201507675Y, filed Sep. 15, 2015, which is incorporated by reference herein in its entirety.
| Number | Date | Country | |
|---|---|---|---|
| Parent | 15760335 | Mar 2018 | US |
| Child | 16732948 | US |
