Patent Application
20160256672
The claimed invention relates to therapeutic devices, methods, and applications for patients with acute or chronic liver failure.
According to the U.S. Centers for Disease Control, an estimated 500 million people worldwide are infected with viral hepatitis, and an estimated one million people die annually from related causes. Many other autoimmune and toxic exposures can also lead to end-stage liver disease: 35% of heavy drinkers develop acute alcoholic hepatitis and drugs as common as acetaminophen can lead to acute liver injury. Patients with acute liver failure (ALF) have a mortality of over 80%. Both acute and chronic liver disease can lead to liver failure and require costly transplantation.
An extra-corporeal bioartificial liver (SAL) can extend the life span of ailing patients waiting for a donor organ and also allow the injured liver to regenerate, thus obviating the need for transplantation in some cases. Acute liver failure (ALF) due to viral hepatitis, acetaminophen poisoning, and exacerbation of chronic liver disease may have a fatality rate of over 80%. During liver failure, building up of toxic compounds can lead to brain damage and multiple organ failure. Therefore, liver failure generally requires liver transplantation. However, the limited supply of donors limits the availability of transplantation.
The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee. Exemplary embodiments are illustrated in referenced figures. It is intended that the embodiments and figures disclosed herein are to be considered illustrative rather than restrictive.
Liver function can be substituted by metabolically active hepatocytes to reduce toxic buildup in the blood. The inventors have generated hepatocytes from human iPSCs, which may maintain metabolic function in the hollow fiber based bioartificial device of the present invention. Conventional biologic liver support systems utilize human hepatocytes (cadaveric primary hepatocytes or hepatic cancer cells from HepG2 cell line) or xeno-hepatocytes (pig or dog), as cell source. However these cell sources are a significant limitation of these systems, owing to, among other things, the quality and risk of using either pig hepatocytes or human cancer cells. Animal cells confer a risk of zoonotic disease transmission, making human cells more acceptable. However, HepG2-based human tumor cells pose a theoretic risk for transmission of malignancy through leaky membrane and paracrine factors secreted into the circulation. Moreover, besides human cancer cells, human hepatocytes are scarce.
Alternative approaches to liver transplantation include non-biological (hemodialysis and hemoadsorption) as well as biological liver support systems such as ex vivo liver perfusion and hepatocyte systems (human or pig cells loaded devices). For example, the extracorporeal liver assist device (ELAD), HepatAssist liver support system, molecular adsorbent recirculating system (MARS), and the Prometheus device have been in clinical evaluation. To date, non-biological liver support systems have been shown to be ineffective and, while intuitively more promising, no truly effective biologic device has yet to be developed for routine clinical treatment purpose.
In accordance with various embodiments of the present invention, one potential source of human hepatocytes is induced pluripotent stem cells. Metabolically active human hepatocytes derived from a well-characterized iPSC line are believed to offer a tremendous advantage over current BAL cell sources. For instance, a nearly unlimited supply of hepatocytes could be provided by this method. In some embodiments, either autologous or allogeneic iPSC-hepatocytes (iHeps) can be used. In some embodiments, the inventors have developed a BAL module comprised of iPSC-hepatocytes arrayed on the extracapillary space of hollow fibers that allow the flow of blood or plasma through the intracapillary space, thus mimicking the tissue micro-architecture of blood capillaries exchanging gas, nutrients, and metabolites to and from cells. Using the BAL module, the inventors have developed an example of an iPSC differentiation protocol that yields a homogenous population of functional hepatocytes that maintained plasma protein and glucose secretory functions in a hollow fiber bioreactor device.
In some embodiments, disclosed are differentiation protocols that have three phases with various factors applied in each phase that are summarized by the following chart:
In Phases 1 and 2, the iPSCs are incubated in a two dimensional medium environment. In some embodiments, on day 19 or other suitable days, the iPSCs are harvested for culture in a hollow fiber bioreactor where they are subject to continuous flow of oxygenated media:
The inventors have shown that culturing the cells in a continuous flow hollow fiber bioreactor from day 19-31, in some embodiments, reduces the expression of AFP, the absence of which indicates mature hepatocytes. Thus, the inventors have shown that culturing or maturing of induced hepatocytes in a continuous flow hollow fiber bioreactor may provide superior maturation of hepatocytes. In other embodiments, the inventors have shown that culturing induced pluripotent hepatocytes, or maturing them attached to microcarriers may also yield superior maturation of iPSC hepatocytes.
Below are tables that provide an example of the media formulations for a hepatocyte differentiation protocol:
The technology may be implemented in a bioartificial liver module by seeding the iPSCs into the extracorporeal space of a hollow fiber bioreactor. In some embodiments, blood or plasma could then be pumped through the intracapillary space of the bioreactor to allow the induced hepatocytes to exchange gas, nutrients, and metabolites to and from the cells.
All references cited herein are incorporated by reference in their entirety as though fully set forth. Unless defined otherwise, technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Allen et al., Remington: The Science and Practice of Pharmacy 22nd ed., Pharmaceutical Press (Sep. 15, 2012); Hornyak et al., Introduction to Nanoscience and Nanotechnology, CRC Press (2008); Singleton and Sainsbury, Dictionary of Microbiology and Molecular Biology 3rd ed., revised ed., J. Wiley & Sons (New York, NY 2006); Smith, March's Advanced Organic Chemistry Reactions, Mechanisms and Structure 7th ed., J. Wiley & Sons (New York, N.Y. 2013); Singleton, Dictionary of DNA and Genome Technology 3rd ed., Wiley-Blackwell (Nov. 28, 2012); and Green and Sambrook, Molecular Cloning: A Laboratory Manual 4th ed., Cold Spring Harbor Laboratory Press (Cold Spring Harbor, N.Y. 2012), provide one skilled in the art with a general guide to many of the terms used in the present application. For references on how to prepare antibodies, see Greenfield, Antibodies A Laboratory Manual 2nd ed., Cold Spring Harbor Press (Cold Spring Harbor N.Y., 2013); Köhler and Milstein, Derivation of specific antibody-producing tissue culture and tumor lines by cell fusion, Eur. J. Immunol. 1976 July 6(7):511-9; Queen and Selick, Humanized immunoglobulins, U.S. Pat. No. 5,585,089 (1996 Dec); and Riechmann et al., Reshaping human antibodies for therapy, Nature 1988 Mar. 24, 332(6162):323-7.
One skilled in the art will recognize many methods and materials similar or equivalent to those described herein, which could be used in the practice of the present invention. Indeed, the present invention is in no way limited to the methods described herein. For purposes of the present invention, the following terms are defined below.
“Subject” as used herein includes all animals, including mammals and other animals, including, but not limited to, companion animals, farm animals and zoo animals. The term “animal” can include any living multi-cellular vertebrate organisms, a category that includes, for example, a mammal, a bird, a simian, a dog, a cat, a horse, a cow, a rodent, and the like. Likewise, the term “mammal” includes both human and non-human mammals.
“Therapeutically effective amount” as used herein refers to the quantity of a specified composition, or active agent in the composition, sufficient to achieve a desired effect in a subject being treated. A therapeutically effective amount may vary depending upon a variety of factors, including but not limited to the physiological condition of the subject (including age, sex, disease type and stage, general physical condition, responsiveness to a given dosage, desired clinical effect) and the route of administration. One skilled in the clinical and pharmacological arts will be able to determine a therapeutically effective amount through routine experimentation.
“Treat,” “treating” and “treatment” as used herein refer to both therapeutic treatment and prophylactic or preventative measures, wherein the object is to prevent or slow down (lessen) the targeted condition, disease or disorder (collectively “ailment”) even if the treatment is ultimately unsuccessful. Those in need of treatment may include those already with the ailment as well as those prone to have the ailment or those in whom the ailment is to be prevented.
“Stem cell” as used herein refers to a cell that can continuously produce unaltered daughters and also has the ability to produce daughter cells that have different, more restricted properties. Stem cells include adult and ES cells.
“Progenitor cell” as used herein includes liver cells, as well as cells that have attributes and characteristics of liver cells, such as the expression of markers associated with liver progenitor cells.
“Packaging material” as used herein refers to one or more physical structures used to house the contents of a kit, such as inventive compositions and the like. The packaging material is constructed by well-known methods, preferably to provide a sterile, contaminant-free environment.
“Package” as used herein refers to a suitable solid matrix or material such as glass, plastic, paper, foil, and the like, capable of holding individual kit components.
Thus, for example, a package can be a cryocontainer used to contain suitable quantities 20 of peritoneal stem cells and/or peritoneal cells described herein. The packaging material generally has an external label which indicates the contents and/or purpose of the kit and/or its components.
As used herein, “FGF” means fibroblast growth factor.
“HES2 and “HES3” as used herein refer to cell lines of human embryonic stem cells.
As used herein, “microcarrier” is a support matrix for attachment and growth of anchorage-dependent cells in suspension systems.
As used herein “definitive endoderm (DE)” refers to cells exhibiting the characteristics and morphology including protein or gene expression characteristics of the definitive endoderm.
The present invention is also directed to kits for the induction, propagation and/or isolation of liver progenitor cells. The present invention is also directed toward kits for the transplantation of liver progenitor cells to liver damaged models. Each kit is an assemblage of materials or components. The exact nature of the components configured in each inventive kit depends on its intended purpose. For example, some embodiments are configured for the purpose of inducing and/or propagating hepatocyte precursors. Instructions for use may be included in the kit. “Instructions for use” typically include a tangible expression describing the technique to be employed in using the components of the kit for a desired purpose, such as for induction, propagation, and/or isolation of liver progenitor cells.
Optionally, the kits also contain other useful components, such as those described herein, or buffers (e.g., PBS), growth media, tissue culture plates, multiple well plates, flasks, chamber slides, differentiation media, stem cell media, goat serum, fetal bovine serum, basic fibroblast growth factor, epidermal growth factor, diluents, pharmaceutically acceptable carriers, syringes, catheters, applicators, pipetting or measuring tools, or other useful paraphernalia as will be readily recognized by those of skill in the art.
The materials or components assembled in the kit can be provided to the practitioner stored in any convenient and suitable ways that preserve their operability and utility. For example, the components can be in dissolved, dehydrated, or lyophilized form; they can be provided at room, refrigerated or frozen temperatures. The components are typically contained in suitable packaging material(s).
Cell Types Utilized for Differentiation into Hepatic Function
In some embodiments, disclosed is a biological liver support system that includes hepatocytes. In general, a biological liver support system is comprised of several distinct components: (a) a cell source: human hepatocytes (cadaveric primary hepatocytes or hepatic cancer cells from HepG2 cell line) or xeno-hepatocytes (pig or dog), (b) a bioreactor to house the cells and (c) a perfusion system for blood or plasma [11, 19, 23]. Hollow fiber capillary bioreactors loaded with hepatocytes of pig origin or HepG2 sub clone C3A cells in the extracapillary space (ECS) for detoxification and plasma protein synthesis purposes have been under clinical investigation [24-27]. In these studies, typically the plasma from a liver failure patient is perfused through the intracapillary space (ICS) of bioreactors in order to be cleared of toxic metabolites by ECS hepatocytes via bidirectional mass transport at the membrane (pore size of 0.1 to 0.2 um) in the semi permeable hollow fiber. In clinical trials, the use of extracorporeal BAL systems have resulted in some improvement in patent's neurological score, blood chemistry, and prothrombin time [11, 17, 26, 27].
The limitations of currently available liver support systems are the quality and risk of using either pig hepatocytes or human cancer cells that are incorporated in the liver support systems [23, 28]. Animal cells confer a risk of zoonotic disease transmission, making human cells more acceptable [29]. Unfortunately, a major bottleneck of such a device is the scarce availability of human hepatocytes. HepG2-based human tumor cells pose a theoretic risk for transmission of malignancy through a leaky membrane and paracrine factors secreted into the circulation [23]. A previous study has shown that HepG2 conditioned media can induce transformation of normal cells [30]. Recent evidence suggests oncogenic microRNAs and proteins are secreted by tumor cells via exosomes [31, 32] and could pose serious health risks.
Therefore, the inventors have determined that metabolically active human hepatocytes derived from a well characterized induced pluripotent stem cell (iPSC) line could offer a tremendous advantage over the current BAL cell sources. In some embodiments, either autologous or allogeneic iPSC hepatocytes (iHeps) can be used. Accordingly, the inventors have developed systems and methods for differentiating and maturing hepatocytes from iPSCs as disclosed herein.
Illustrated in
In some embodiments, the differentiation protocol may be roughly broken into 3 phases that can be defined as: (1) phase 1: endoderm induction, (2) phase 2: hepatic specification, and (3) phase 3: hepatic maturation. Following is a description of the specific protocols and factors that may be utilized that roughly fall within each of these three phases. In other embodiments, various phases may overlap, be different lengths, or have factors that span multiple phases, or be broken into additional phases. These three phases are illustrated in the timeline represented in
In some embodiments, certain portions of the differentiation protocol or the entire protocol may be performed in a bioreactor. In these embodiments, the cells of interest are loaded into the bioreactor and media is perfused through the capillaries. In some embodiments, this may be a hollow fiber bioreactor, a continuous flow hollow fiber bioreactor, or other suitable incubators. In some embodiments, a polysulfone hollow fiber Bioreactor cartridge (e.g., as available from Alpha Plan, Germany) with 70-6000 cm2 inner surface area and 0.21 μm pore size may be used for iHep culture. In some embodiments, the bioreactor may contain microporous polyethersufone capillary membranes (e.g., as available from mPES, Membrana, Wuppertal, Germany) or multi-laminate hollow fiber capillaries (e.g., as available from MHF, Mitsubishi, Tokyo, Japan) to enable gas exchange with the cells. In some embodiments, the hollow fibers are arranged in longitudinal fashion with a pack density of 36%-40%, 20%, 50%, or other suitable percentages without waviness inside durable polycarbonate casing of the cartridge. In some embodiments, the fibers may be wavy.
The HFB cartridge may be connected to a reservoir bottle or other container containing growth media, a membrane oxygenator and a pump with peroxide cured silicon tubing or other suitable tubing for incubation. The media from reservoir pumped into the HFB cartridge's vestibule is divided across the hollow fiber mouth for even flow into the ICS of fibers. Before entering into the cartridge, the media is oxygenated (95% O2 and 5% CO2) by an oxygenator module. This closed circuit HFB system was placed in a cell culture incubator at 37° C. In some embodiments, the flow rate of the media in the bioreactor may be 1 ml/minute, 2 ml/minute, 22-30 ml/minute, 40 ml/minute, and may altered depending on the stage of the incubation.
Additionally, in some embodiments, the hepatocytes and/or progenitor cells may be adhered to microcarriers prior to introduction into the extracapillary space of the bioreactor. Microcarriers are a support matrix for attachment and growth of anchorage-dependent cells in suspension systems. They provide high surface area to volume ratios for facilitating a scale-up process of generating hepatocytes. In other embodiments, the hepatocyte cells may be incubated in the bioreactor without microcarriers, for example as free cells or cell clumps (organoids) in the extracapillary space.
In some embodiments, the microcarriers may be collagen coated. In other embodiments, they may be coated with other extra-cellular matrix proteins and small molecules. In some embodiments, the microcarriers may be porous gelatin microcarriers with a diameter of 9-100 um. In some embodiments, gelatin and cytodexill microcarriers may be utilized. In other embodiments, micro carriers may be porous or nonporous, comprised of gelatin, glass, collagen, or cellulose, and have dimensions from 170 μm-6,000 μm. In some embodiments, a pore size of less than 1 μm, 1 μm, 3 μm, 4μm, or 5 μm may be utilized. In some embodiments, the immature hepatocytes may be harvested on day 15, 16, 17, 18, 19, 20, 21, or 22 or other suitable day for culture in a hollow fiber bioreactor. In other embodiments, the hepatocytes may be cultured from day 10 on in a hollow fiber bioreactor. In other embodiments, the iPSCs may be cultured from day 1 onward in the bioreactor.
In general, hepatic cells derived from pluripotent stem cells using current differentiation protocols are immature as demonstrated by presence of alpha fetoprotein (
In some embodiments, a bioartificial liver device may include hollow fibers. A hollow fiber capillary design is utilized for its high surface area and resemblance to natural blood capillary liver microarchitecture for nutrient and gas exchange. This design has been used in other liver support devices such as extracorporeal liver assist device (ELAD), and HepatAssist liver support system [11, 26]. Additional design features such as cartridge having layers of polysulfone membrane sheets where alternate layers filled with cells or blood/plasma flow can be considered. Another design consideration is improving the throughput. In some embodiments, a single cartridge closed circuit system may be utilized. For testing multiple growth conditions, animal experiments, and human clinical studies, a multi-cartridge system can be useful. An integrated multi-cartridge system can allow for parallel production, better quality control and uniformity among bioartificial liver devices.
In some embodiments, the inventors utilize dextran microcarrier beads for growing iHeps in suspension culture (
The BAL devices disclosed herein may be further developed and validated for rescuing disease specific decompensated liver functions. In acute liver failure due to acetaminophen (APAP; paracetamol) poisoning, the liver cannot detoxify ammonia and secrete blood coagulation proteins such as prothrombin resulting in increased intracranial pressure and bleeding [64, 65]. For this purpose, APAP metabolism and lethal concentration, and ammonia detoxification by iHeps needed to be assessed. Moreover, unbiased analysis of metabolites and proteins that are secreted by iHeps can be conducted using a mass spectrometer. The secreted liver proteins and metabolites can be used as biomarkers for defining the therapeutic use window of an iHepBAL in testing the efficacy in an animal and human.
For clinical applications, the iHepBAL device has to reconstitute the function of the damaged liver and improve the health condition of the patient. In vivo preclinical studies in the rat and pig model systems of liver failure will provide insights into safety and efficacy of an iHepBAL device. For preclinical testing, the following acute liver failure animal models, anhepatic rat and acetaminophen toxicity in pig, can be used [66, 67]. Based on plasma proteins, albumin and apoB, secretion data (
In some embodiments, the plasma lines may be divided or separated into multiple parallel lines to be circulated through multiple iHep-BAL devices to improve throughput. In some embodiments, different rates of filtration may be utilized for different amounts of liver support needed. In other embodiments, whole blood may be passed through a bioartificial liver device rather than first separating the plasma from the blood. In other embodiments, hollow fibers may not be used and instead the plasma may be in direct contact with the iHeps. For example, the Academisch Medisch Centrum Bioartificial Liver developed by Chamuleau's group utilizes this configuration. Various other configurations of a bioartificial liver device may be utilized that include iHeps differentiated and cultured accordingly to the methods and in accordance with the devices disclosed herein.
The following examples are provided to better illustrate the claimed invention and are not intended to be interpreted as limiting the scope of the invention. To the extent that specific materials or steps are mentioned, it is merely for purposes of illustration and is not intended to limit the invention. One skilled in the art may develop equivalent means or reactants without the exercise of inventive capacity and without departing from the scope of the invention.
Generally, an example of a multi-step differentiation protocol for generating of hepatocytes is shown in
In this example experiment, the human iPSC line 83iCTRL was obtained from Cedars Sinai Medical Center iPSC core facility [33]. The iPSC 83iCTRL line was established by reprogramming normal human fibroblast using non-integrating expression vector carrying OCT4, SOX2, KLF4, and L-MYC genes. Human embryonic stem cell (hESC) line, WA09 (H9), was obtained from WiCell Research Institute, USA. The iPSCs and hESCs were cultured using serum free chemically defined media, mTeSR1, (STEMCELL Technologies, Canada) with daily media change regimen at 37° C. incubator with 5% CO2. The human liver cancer cell line HepG2 and Huh7.5.1 was maintained on complete Dulbecco's modified Eagle's medium (DMEM) (Fisher Scientific). Complete DMEM was supplemented with 10% fetal bovine serum (FBS), 10 mM Hepes, 10 mM nonessential amino acids, penicillin (100 units/ml), streptomycin (100 mg/ml), and 2 mM Lglutamine (Life Technologies).
For in vitro differentiation, the iPSCs were single cell plated in a 6 well plate, cultured at 37° C. in 5% CO2 and subjected to a 3 week hepatic differentiation protocol. The differentiation steps consisted of three phases, including endoderm induction (day 1-5), hepatic specification (day 6-15) and hepatic maturation (day 16-21). The cytokines were purchased from Peprotech Inc., (Rocky Hill, N.J.) unless otherwise mentioned. The cells were differentiated to endoderm for 5 days using IMDM/F12 or RPMI media (Life Technologies) supplemented with Wnt 3A (40 ng/ml, R and D Systems) and Activin A (100 ng/ml) for the first one day and then treated with Activin A, VEGF (10 ng/ml) and bFGF (10 ng/ml) for an additional 4 days. From day 6 onwards, the media was changed to IMDM/F12 supplemented with BMP4 (50 ng/ml), VEGF (10 ng/ml), EGF (10 ng/ml), TGFα (20 ng/ml), HGF (100 ng/ml), dexamethasone (1×107 M; SigmaAldrich, St. Louis, Mo.) DMSO (1%, SigmaAldrich). From day 12 onwards, BMP4 and TGFα were removed from the cocktail. For hepatocyte maturation, HGF, dexamethasone and oncostatin M (20 ng/ml) were included in the media from day 16 onwards. At day 19 post differentiation, the iPSC hepatocytes or iHeps were harvested for bioreactor culture. To compare the hepatic maturation status between bioreactor cultured iHeps and two dimensional monolayer cultured iHeps, one plate of the cells was continuously differentiated until day 31 in parallel to bioreactor culture. At specific time points, markers for endoderm and hepatic lineage cells were assessed by immunocytochemistry (ICC), flow cytometry and reverse transcription quantitative PCR (RTqPCR).
Preparation of HFB system: The closed circuit bioreactor system comprised a media reservoir, pump, oxygenator, HFB cartridge containing cells, and flow path (tubing and adaptors). A polysulfone hollow fiber Bioreactor cartridge (Alpha Plan, Germany) with 70 cm2 inner surface area and 0.21 μm pore size was used for iHep culture. The hollow fibers are arranged in longitudinal fashion with a pack density of 36%-40% without waviness inside durable polycarbonate casing of the cartridge. The HFB cartridge was connected with a reservoir bottle (FiberCell Systems Inc., USA) containing growth media, a membrane oxygenator (Radnoti, LLC., USA) and a pump (Masterflex L/S Digital Drive pump; ColeParmer, USA) by peroxide cured silicon tubing (ColeParmer, USA). The media from reservoir pumped into the HFB cartridge's vestibule is divided across the hollow fiber mouth for even flow into the ICS of the fibers. Before entering into the cartridge, the media is oxygenated (95% O2 and 5% CO2) by an oxygenator module. This closed circuit HFB system was placed in a cell culture incubator at 37° C.
Preculturing iHeps on microcarrier beads in spinner flask: A total of 9 to 10 million iHeps (day 19 post differentiation) were harvested from the monolayer culture plate, and precultured with laminin coated Cytodex microcarrier beads (Sigma Aldrich) in 150 ml HepatoZymeSFM (Life Technologies) media supplemented with ITS (InsulinTransferrin Selenium, Life Technologies) and EGF (25 ng/ml) in a spinner culture bottle. The spinner bottle culture was performed in a CO2 incubator at 37o C with the stir speed of 40-70 RPM.
Loading iHeps into HFB system: Prior to cell loading, the bioreactor system was primed by perfusion with 500 ml of PBS for 24 hours, then by 500 ml of HepatoZyme-SFM media for 48 hours. For culturing cells, 250 ml of HepatoZyme-SFM media supplemented with 2.5 ml of ITS, EGF (25 ng/ml) was perfused. Before cell loading, the extracapillary space of the bioreactor was coated with the mouse laminin (Mouse Laminin I, Trevigen) according to the manufacture's protocol. After 24 hours of preculturing in a spinner bottle, the cells on the microcarrier beads were harvested by centrifugation, resuspended in 3 ml of HepatoZyme-SFM medium, and loaded into the extracapillary space of the HFB cartridge with a syringe. The media flow rate was set at 2 ml/min. At indicated time points (day 0, 3, 6, 9, and 12) post cell loading, 25 ml of the culture medium was collected from the bioreactor media reservoir bottle, and replaced with same volume of fresh made HepatoZyme-SFM medium containing ITS and EGF. The collected medium was stored at 20° C. for future assays. At day 12 post loading, the cells in the ECS of the bioreactor were also harvested for RNA isolation.
Total RNA was extracted from iHeps cultured in plates or bioreactor using RNeasy Mini Kit (QIAGEN). Human primary hepatocyte RNA from fetal liver was included as a positive control (provided by Samuel W French, UCLA). RNA samples were reverse transcribed using SuperScript III Reverse Transcriptase kit (Life Technologies) with random primers as described by the manufactures. Expression of human hepatic genes was quantified using Platinum SYBR Green qPCR SuperMixUDG with ROX Kit (Invitrogen) by the ViiA7 realtime PCR system (Applied Biosystems). Approximately 100 ng of cDNA was used for each qPCR reaction. The reaction for each marker gene expression was triplicated with the housekeeping human PPIG (cyclophilin G) gene as an internal reference control. The sequences of the primer pairs used for qPCR are given in supplementary Table S1. All cDNAs were amplified under the following conditions: 50° C. for 2 min; 95° C. for 2 min followed by 40 cycles of 95° C. for 15 sec and 60° C. for 1 min.
The iHep culture media samples from both bioreactor and culture plate were collected. The amount of human albumin and apolipoprotein B produced by the iHeps was measured in triplicate by the Human Albumin ELISA kit (Bethyl Laboratories, Inc., Montgomery, Tex.) and the Human Apolipoprotein B ELISA kit (MABTECH, Inc) respectively. Prothrombin was measured by ELISA as per manufacturer's protocol (Molecular Innovations, Inc). Intracellular glycogen was detected by Periodic Acid Schiff (PAS) assay as per manufacturer's protocol (SigmaAldrich). Glucose was measured by a hexokinase enzyme based colorimetric assay.
The differentiating cells were collected at indicated time points, permeabilized using the Cytofix/Cytoperm kit (BD Biosciences) and analyzed using antibodies against SOX17 and CXCR4 (R & D Systems), AFP (Dako, USA) and Albumin (Bethyl Laboratories, Inc) and FOXA2 (Novus Biologicals, Littleton, Colo.). Data was acquired on a BD Fortessa (BD Biosciences) flow cytometer using FACS diva software and analyzed using FlowJo software (TreeStar Inc.).
Cells were fixed with methanol at day 4 post differentiation. Following three PBS washes, the cells were blocked (10% fetal bovine serum, 3% BSA, 0.1% Tritonx 100 in PBS) and incubated with human SOX17 mouse monoclonal primary antibody (BD Biosciences) and FOXA2 rabbit monoclonal primary antibody (Novus Biologicals, Littleton, Colo.) at a dilution of 1:200 for 5 hours to overnight at 4° C. The goat anti-mouse polyclonal antibody (Alexa fluor 594) or goat anti-rabbit polyclonal antibody (Alexa fluor 488) was added as a secondary antibody (Life Technologies, USA) at 1:1000 dilutions and incubated for 1 hour at room temperature. Between antibody changes the cells were washed thrice with PBS. The nuclei were stained with Hoechst dye (Life Technologies, USA).
For the development of an iPSC based bioartificial liver, the different components of the device including metabolically active iPSC hepatocytes and the closed circuit hollow fiber bioreactor system need to be integrated optimally.
The inventors adapted a three stage differentiation program to differentiate iPSCs to mature hepatic lineage cells (
To accurately measure the differentiation efficiency at single cell level, the inventors employed flow cytometry to quantify cells that express endoderm and liver specific markers. The cells were analyzed on day 45 for endoderm specific markers SOX17 and FOXA2 by immunocytochemistry (ICC) (
Generating metabolically active functional hepatocytes from iPSCs is critical for the successful development of an iHep based bioartificial liver device that can provide metabolic and detoxification functions. Therefore, the inventors assessed the activities of cytochrome p450 (CYP), and carbohydrate metabolic pathways in the iHeps. The liver CYP system is crucial for degradation and clearance of endogenous metabolites, hormones and xenobiotics [41-47]. CYP genes are classified as phase I and Phase II enzymes and Phase III transporters. Phase I enzymes function in oxidative reduction reactions and Phase II enzymes act in modifying metabolites by acetylation, sulfation, glucuronidation and glutathione conjugation. Phase III transporters are involved in drug clearance. CYP components have been investigated extensively for their role in metabolism and excretion of pharmaceutical compounds [41-45, 48, 49].
The inventors observed that the basal level expression of many CYP genes, including CYP2D6, CYP2B6, CYP1A2, UGT2B7, UGT2B 15 and SLCo2B 1, were significantly upregulated in iPSC hepatocytes compared to that of undifferentiated iPSCs (
Glycogen, a branched polysaccharide, is stored in liver cells. To test the capacity of the glycogen storage by the iHeps the inventors performed a Periodic Acid Schiff (PAS) assay. The differentiated iPSC hepatocytes (day 20 post-differentiation) were fixed and stained for glycogen. The intensity of glycogen staining was analyzed. Hepatocytes derived from 83iCTR show glycogen staining, along with the positive control Huh7.5.1 hepatoma cell line (
The hollow fiber bioreactor cartridge loaded with functional hepatocytes would serve as a bioartificial liver module. Artificial devices used for compensating the function of failing organs should be capable of replicating essential physiologic functions; thus, the design of the BAL is critical for facilitating the cells to execute respective functions. In liver, plates of hepatocytes are arranged along the sinusoidal capillary spaces where the nutrients, oxygen, metabolites, xenobiotics, hormones, and toxins are exchanged for anabolic and catabolic purposes. Hollow fiber capillary arrayed with iHeps on the outer surface can provide intracapillary flow paths for blood or serum towards metabolic detoxification. The metabolites and waste products from the blood can diffuse through the hollow fiber micropores (size 210 nm) and can be metabolized by cells in the array. The resulting metabolites will be removed by continuous media flow in the hollow fiber bioreactors. With this design, the inventors developed an iHepBAL device.
Large scale production of iPSCs and iPSC hepatocytes is a key step in the development of iHep based BAL. For clinical application, an estimated 5 to 20 billion hepatocytes are required for the treatment of patient with decompensated liver disease. To achieve this, microcarriers are useful for cultivating anchorage dependent cells in suspension which allow large scale and high yield production of iHeps. For a proof of concept study, the inventors used dextran microcarrier beads (6087 Lm in size) for culturing iHeps. The microcarriers were coated with laminin and 10 million iHeps (19 day post-differentiation) were added to the microcarrier in a 250 ml spinner flask (
In order to maintain physiologic functions, the iPSC hepatocytes loaded in the bioartificial liver device have to survive in the extracapillary space microenvironment over a period of time and exchange gas, nutrients, proteins and metabolites. These in vitro parameters of cell viability and functions can be used for assessing the quality and functionality of iHepBAL. The iHeps (1×107 cells) attached to the microcarrier beads were loaded into the extracapillary space of polysulfone hollow fiber bioreactor cartridge (70 cm2) using a syringe. We utilized single cartridge closed circuit set up with a perfusion media volume of 250-500 ml (
Liver specific proteins and metabolites produced by the iHeps were secreted into ICS through 0.21 μm pores on the hollow fibers. This design is similar to in vivo natural liver architecture where the hepatocytes secrete plasma proteins into liver sinusoidal spaces. For functional verification, human plasma proteins albumin, apoB and prothrombin secreted by iHeps into the ICS media were quantified (
In vivo, the generated metabolites and toxic waste products by hepatocytes are continuously removed by the blood flow resulting in maintenance of liver homeostasis. The closed circuit flow system can mimic the in vivo physiological condition compared to that of cells cultured in a two dimensional static condition. Thus, the inventors further characterized the iHeps cultured in the hollow fiber bioreactors. At the end of the experiment on day 12, viable cells (over 80%) were recovered from the bioreactors and used for gene expression analysis. Culturing iPSC hepatocytes in a continuous flow microenvironment improved the maturation state as evidenced by 83 fold reduction of AFP and significant increase in CYP3A4, and Glucose6phosphatase (G6PC) gene expression compared to Day 0 iHeps (
The biological factors and chemicals required for deriving functional hepatocytes from pluripotent stem cells have been studied extensively [34-37, 50-61]. Using the established three phase differentiation protocol, the inventors have generated over 90% homogenous population of hepatic lineage cells (
In general, hepatic cells derived from pluripotent stem cells using current differentiation protocols are immature as demonstrated by presence of alpha fetoprotein (
In some embodiments, a hollow fiber capillary design is utilized for its high surface area and resemblance to natural blood capillary liver microarchitecture for nutrient and gas exchange. This design has been used in other liver support devices such as extracorporeal liver assist device (ELAD), and HepatAssist liver support system [11, 26]. Additional design features such as cartridge having layers of polysulfone membrane sheets where alternate layers filled with cells or blood/plasma flow can be considered. Another design consideration is improving the throughput. In some embodiments, a single cartridge closed circuit system may be utilized. For testing multiple growth conditions, animal experiments, and human clinical studies, a multi-cartridge system can be useful. Integrated multi-cartridge systems can allow for parallel production, better quality control, and uniformity among bioartificial liver devices.
In some embodiments, the inventors utilize dextran microcarrier beads for growing iHeps in suspension culture (
The BAL devices disclosed herein may be further developed and validated for rescuing disease specific decompensated liver functions. In acute liver failure due to acetaminophen (APAP; paracetamol) poisoning, the liver cannot detoxify ammonia and secrete blood coagulation proteins such as prothrombin which results in increased intracranial pressure and bleeding [64, 65]. For this purpose, APAP metabolism and lethal concentration, and ammonia detoxification by iHeps need to be assessed. Moreover, unbiased analysis of metabolites and proteins that are secreted by iHeps can be conducted using a mass spectrometer. The secreted liver proteins and metabolites can be used as biomarkers for defining the therapeutic use window of an iHepBAL in testing the efficacy in an animal and human.
For clinical applications, the iHepBAL device has to reconstitute the function of the damaged liver and improve the health condition of the patient. In vivo preclinical studies in the rat and pig model systems of liver failure will provide insights into safety and efficacy of an iHepBAL device. For preclinical testing, anhepatic rat and acetaminophen toxicity in pig testing can be used [66, 67]. Based on plasma proteins, albumin and apoB, secretion data (
The various methods and techniques described above provide a number of ways to carry out the invention. Of course, it is to be understood that not necessarily all objectives or advantages described may be achieved in accordance with any particular embodiment described herein. Thus, for example, those skilled in the art will recognize that the methods can be performed in a manner that achieves or optimizes one advantage or group of advantages as taught herein without necessarily achieving other objectives or advantages as may be taught or suggested herein. A variety of advantageous and disadvantageous alternatives are mentioned herein. It is to be understood that some preferred embodiments specifically include one, another, or several advantageous features, while others specifically exclude one, another, or several disadvantageous features, while still others specifically mitigate a present disadvantageous feature by inclusion of one, another, or several advantageous features.
Furthermore, the skilled artisan will recognize the applicability of various features from different embodiments. Similarly, the various elements, features and steps discussed above, as well as other known equivalents for each such element, feature or step, can be mixed and matched by one of ordinary skill in this art to perform methods in accordance with principles described herein. Among the various elements, features, and steps some will be specifically included and others specifically excluded in diverse embodiments.
Although the invention has been disclosed in the context of certain embodiments and examples, it will be understood by those skilled in the art that the embodiments of the invention extend beyond the specifically disclosed embodiments to other alternative embodiments and/or uses and modifications and equivalents thereof.
Many variations and alternative elements have been disclosed in embodiments of the present invention. Still further variations and alternate elements will be apparent to one of skill in the art. Among these variations, without limitation, are the methods of deriving insulin-producing cells from pluripotent stem cells, preparing, isolating, or modifying cells used in the described differentiation techniques, derivation of insulin-producing cell lines from the aforementioned techniques, treatment of diseases and/or conditions that relate to the teachings of the invention, techniques and composition and use of solutions used therein, and the particular use of the products created through the teachings of the invention. Various embodiments of the invention can specifically include or exclude any of these variations or elements.
In some embodiments, the numbers expressing quantities of ingredients, properties such as concentration, reaction conditions, and so forth, used to describe and claim certain embodiments of the invention are to be understood as being modified in some instances by the term “about.” Accordingly, in some embodiments, the numerical parameters set forth in the written description and attached claims are approximations that can vary depending upon the desired properties sought to be obtained by a particular embodiment. In some embodiments, the numerical parameters should be construed in light of the number of reported significant digits and by applying ordinary rounding techniques. Notwithstanding that the numerical ranges and parameters setting forth the broad scope of some embodiments of the invention are approximations, the numerical values set forth in the specific examples are reported as precisely as practicable. The numerical values presented in some embodiments of the invention may contain certain errors necessarily resulting from the standard deviation found in their respective testing measurements.
In some embodiments, the terms “a” and “an” and “the” and similar references used in the context of describing a particular embodiment of the invention (especially in the context of certain of the following claims) can be construed to cover both the singular and the plural. The recitation of ranges of values herein is merely intended to serve as a shorthand method of referring individually to each separate value falling within the range. Unless otherwise indicated herein, each individual value is incorporated into the specification as if it were individually recited herein. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g. “such as”) provided with respect to certain embodiments herein is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention otherwise claimed. No language in the specification should be construed as indicating any non-claimed element essential to the practice of the invention.
Groupings of alternative elements or embodiments of the invention disclosed herein are not to be construed as limitations. Each group member can be referred to and claimed individually or in any combination with other members of the group or other elements found herein. One or more members of a group can be included in, or deleted from, a group for reasons of convenience and/or patentability. When any such inclusion or deletion occurs, the specification is herein deemed to contain the group as modified thus fulfilling the written description of all Markush groups used in the appended claims.
Preferred embodiments of this invention are described herein, including the best mode known to the inventor for carrying out the invention. Variations on those preferred embodiments will become apparent to those of ordinary skill in the art upon reading the foregoing description. It is contemplated that skilled artisans can employ such variations as appropriate, and the invention can be practiced otherwise than specifically described herein. Accordingly, many embodiments of this invention include all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. Moreover, any combination of the above-described elements in all possible variations thereof is encompassed by the invention unless otherwise indicated herein or otherwise clearly contradicted by context.
Furthermore, numerous references have been made to patents and printed publications throughout this specification. Each of the above cited references and printed publications are herein individually incorporated by reference in their entirety.
In closing, it is to be understood that the embodiments of the invention disclosed herein are illustrative of the principles of the present invention. Other modifications that can be employed can be within the scope of the invention. Thus, by way of example, but not of limitation, alternative configurations of the present invention can be utilized in accordance with the teachings herein. Accordingly, embodiments of the present invention are not limited to that precisely as shown and described.
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The application claims priority under 35 U.S.C. §119 to U.S. Provisional Application Ser. No. 62/114,425, entitled “Induced Pluripotent Stem Cell-Derived Hepatocyte Based Bioartificial Liver Device,” by Vaithilingaraja Arumugaswami, et al., filed Feb. 10, 2015, the disclosure of which is incorporated in its entirety by this reference.
| Number | Date | Country | |
|---|---|---|---|
| 62114245 | Feb 2015 | US |
