Identification of small molecules for human hepatocyte expansion and iPS differentiation
01 Jun 2013-
TL;DR: A high-throughput screening platform for primary human hepatocytes is developed to identify small molecules in two different classes that can be used to generate renewable sources of functional human liver cells in vitro.
Abstract: Broad Institute of MIT and Harvard (Scientific Planning and Allocation of Resources Committee Grant)
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TL;DR: A microfluidic cell culture device created with microchip manufacturing methods that contains continuously perfused chambers inhabited by living cells arranged to simulate tissue- and organ-level physiology has great potential to advance the study of tissue development, organ physiology and disease etiology.
Abstract: Organ-level physiology is recapitulated in vitro by culturing cells in perfused, microfluidic devices.
2,339 citations
TL;DR: Conditions allowing long-term expansion of adult bile duct-derived bipotent progenitor cells from human liver opens up experimental avenues for disease modeling, toxicology studies, regenerative medicine, and gene therapy.
1,081 citations
Cites background from "Identification of small molecules f..."
...Indeed, a recent study describes a human liver hepatocyte culture system for a period of 1 week with only 10-fold expansion (Shan et al., 2013)....
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TL;DR: Treatment with 15 mM acetaminophen induced a toxic response in the hepatic construct that was similar to published studies on animal and other in vitro models, thus providing a proof-of-concept demonstration of the utility of this liver-on-a-chip platform for toxicity assessment.
Abstract: The inadequacy of animal models in correctly predicting drug and biothreat agent toxicity in humans has resulted in a pressing need for in vitro models that can recreate the in vivo scenario. One of the most important organs in the assessment of drug toxicity is liver. Here, we report the development of a liver-on-a-chip platform for long-term culture of three-dimensional (3D) human HepG2/C3A spheroids for drug toxicity assessment. The bioreactor design allowed for in situ monitoring of the culture environment by enabling direct access to the hepatic construct during the experiment without compromising the platform operation. The engineered bioreactor could be interfaced with a bioprinter to fabricate 3D hepatic constructs of spheroids encapsulated within photocrosslinkable gelatin methacryloyl (GelMA) hydrogel. The engineered hepatic construct remained functional during the 30 days culture period as assessed by monitoring the secretion rates of albumin, alpha-1 antitrypsin, transferrin, and ceruloplasmin, as well as immunostaining for the hepatocyte markers, cytokeratin 18, MRP2 bile canalicular protein and tight junction protein ZO-1. Treatment with 15 mM acetaminophen induced a toxic response in the hepatic construct that was similar to published studies on animal and other in vitro models, thus providing a proof-of-concept demonstration of the utility of this liver-on-a-chip platform for toxicity assessment.
436 citations
TL;DR: In this article, the authors generated HLCs from multiple lineages, using two different protocols, for direct comparison with fresh fetal and adult hepatocytes, and found that HLC maturity was proven by transcript, protein and function to be fetal-like and short of the adult phenotype.
272 citations
TL;DR: It is reported that micropatterned cocultures of primary human hepatocytes with stromal cells (MPCCs) reliably support productive HBV infection, and infection can be enhanced by blocking elements of the hepatocyte innate immune response associated with the induction of IFN-stimulated genes.
Abstract: Hepatitis B virus (HBV) chronically infects 400 million people worldwide and is a leading driver of end-stage liver disease and liver cancer. Research into the biology and treatment of HBV requires an in vitro cell-culture system that supports the infection of human hepatocytes, and accurately recapitulates virus–host interactions. Here, we report that micropatterned cocultures of primary human hepatocytes with stromal cells (MPCCs) reliably support productive HBV infection, and infection can be enhanced by blocking elements of the hepatocyte innate immune response associated with the induction of IFN-stimulated genes. MPCCs maintain prolonged, productive infection and represent a facile platform for studying virus–host interactions and for developing antiviral interventions. Hepatocytes obtained from different human donors vary dramatically in their permissiveness to HBV infection, suggesting that factors—such as divergence in genetic susceptibility to infection—may influence infection in vitro. To establish a complementary, renewable system on an isogenic background in which candidate genetics can be interrogated, we show that inducible pluripotent stem cells differentiated into hepatocyte-like cells (iHeps) support HBV infection that can also be enhanced by blocking interferon-stimulated gene induction. Notably, the emergence of the capacity to support HBV transcriptional activity and initial permissiveness for infection are marked by distinct stages of iHep differentiation, suggesting that infection of iHeps can be used both to study HBV, and conversely to assess the degree of iHep differentiation. Our work demonstrates the utility of these infectious systems for studying HBV biology and the virus’ interactions with host hepatocyte genetics and physiology.
222 citations
Cites background from "Identification of small molecules f..."
...Collectively, these results demonstrate that the MPCC system can serve as a platform for studying the efficacy and mechanism of action of diverse antiviral agents, and has the potential to be expanded to a medium-to-high throughput drug-discovery tool (13, 26)....
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...iPSCs are renewable, can be derived from a single donor, and repeatedly differentiated into iHeps that share features of human hepatocytes (15, 16, 26) (Fig....
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References
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TL;DR: The first free, open-source system designed for flexible, high-throughput cell image analysis, CellProfiler is described, which can address a variety of biological questions quantitatively.
Abstract: Biologists can now prepare and image thousands of samples per day using automation, enabling chemical screens and functional genomics (for example, using RNA interference). Here we describe the first free, open-source system designed for flexible, high-throughput cell image analysis, CellProfiler. CellProfiler can address a variety of biological questions quantitatively, including standard assays (for example, cell count, size, per-cell protein levels) and complex morphological assays (for example, cell/organelle shape or subcellular patterns of DNA or protein staining).
4,578 citations
TL;DR: Because there is no vaccine and no post-exposure prophylaxis for HCV, the focus of primary prevention efforts should be safer blood supply in the developing world, safe injection practices in health care and other settings, and decreasing the number of people who initiate injection drug use.
Abstract: Hepatitis C virus (HCV) is a major cause of liver disease worldwide and a potential cause of substantial morbidity and mortality in the future. The complexity and uncertainty related to the geographic distribution of HCV infection and chronic hepatitis C, determination of its associated risk factors, and evaluation of cofactors that accelerate its progression, underscore the difficulties in global prevention and control of HCV. Because there is no vaccine and no post-exposure prophylaxis for HCV, the focus of primary prevention efforts should be safer blood supply in the developing world, safe injection practices in health care and other settings, and decreasing the number of people who initiate injection drug use.
2,865 citations
"Identification of small molecules f..." refers background in this paper
...FPHs induced functional proliferation of hepatocytes in vitro (FPH1 (1) and FPH2 (2); Fig....
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Journal Article•
TL;DR: This review attempts to integrate the findings of the last three decades and looks toward clues as to the nature of the causes that trigger this fascinating organ and cellular response.
Abstract: Liver regeneration after the loss of hepatic tissue is a fundamental parameter of liver response to injury. Recognized as a phenomenon from mythological times, it is now defined as an orchestrated response induced by specific external stimuli and involving sequential changes in gene expression, growth factor production, and morphologic structure. Many growth factors and cytokines, most notably hepatocyte growth factor, epidermal growth factor, transforming growth factor-α, interleukin-6, tumor necrosis factor-α insulin, and norepinephrine, appear to play important roles in this process. This review attempts to integrate the findings of the last three decades and looks toward clues as to the nature of the causes that trigger this fascinating organ and cellular response.
2,338 citations
01 Jan 2004
Abstract: The laboratory rat (Rattus norvegicus) is an indispensable tool in experimental medicine and drug development, having made inestimable contributions to human health. We report here the genome sequence of the Brown Norway (BN) rat strain. The sequence represents a high-quality ‘draft’ covering over 90% of the genome. The BN rat sequence is the third complete mammalian genome to be deciphered, and three-way comparisons with the human and mouse genomes resolve details of mammalian evolution. This first comprehensive analysis includes genes and proteins and their relation to human disease, repeated sequences, comparative genome-wide studies of mammalian orthologous chromosomal regions and rearrangement breakpoints, reconstruction of ancestral karyotypes and the events leading to existing species, rates of variation, and lineage-specific and lineage-independent evolutionary events such as expansion of gene families, orthology relations and protein evolution.
1,854 citations
TL;DR: It is shown that pancreatic endoderm derived from human embryonic stem (hES) cells efficiently generates glucose-responsive endocrine cells after implantation into mice, and it is demonstrated that implantation of hES cell–derived pancreaticEndoderm protects against streptozotocin-induced hyperglycemia.
Abstract: Development of a cell therapy for diabetes would be greatly aided by a renewable supply of human β-cells. Here we show that pancreatic endoderm derived from human embryonic stem (hES) cells efficiently generates glucose-responsive endocrine cells after implantation into mice. Upon glucose stimulation of the implanted mice, human insulin and C-peptide are detected in sera at levels similar to those of mice transplanted with ~3,000 human islets. Moreover, the insulin-expressing cells generated after engraftment exhibit many properties of functional β-cells, including expression of critical β-cell transcription factors, appropriate processing of proinsulin and the presence of mature endocrine secretory granules. Finally, in a test of therapeutic potential, we demonstrate that implantation of hES cell–derived pancreatic endoderm protects against streptozotocin-induced hyperglycemia. Together, these data provide definitive evidence that hES cells are competent to generate glucose-responsive, insulin-secreting cells. Development of a cellular therapy for the amelioration of diabetes requires a renewable source of human insulin–secreting cells that respond to glucose in a physiologic manner. Currently, cellular replacement is performed either by whole-pancreas transplant or by infusion of isolated primary islets into the portal vein 1 . These procedures, although effective, are not suitable for the general diabetes population, primarily because of the inadequate supply of organs and the necessity of chronic immunosuppression. One approach to overcoming the problem of insufficient supply is to generate islets from proliferative stem cell populations. Currently, the only stem cell population with sufficient proliferative capacity to achieve this goal is human embryonic stem (hES) cells, which proliferate in culture at a rate of >250 population doublings per year 2,3 . Of equal importance, hES cells are capable of efficiently and rapidly differentiating through a series of defined developmental transitions to generate cells of all somatic lineages. This competence has allowed us to produce definitive endoderm cells 4 , foregut, pancreatic and endocrine precursor cells 5 and ultimately insulin-secreting cells 5 . However, in previous studies, functional characterization of hES cells differentiated to endocrine populations showed insulin secretion in response to various secretagogues but not to glucose in vitro 5 or in vivo 6 . Fetal human pancreatic tissues at 14–20 weeks 7–9 or 6–9 weeks 10,11 of age have been shown to develop functionally after implantation in animals. At 6–9 weeks, only a few hormone-expressing endocrine cells, which do not respond to glucose, are present in the fetal human pancreatic anlagen. After implantation, these tissues differentiate
1,849 citations