Adult and iPS-derived non-parenchymal cells regulate liver organoid development through differential modulation of Wnt and TGF-β

Goulart, Ernesto; Caires-Júnior, Luiz C.; Telles-Silva, Kayque A.; Araújo, Bruno Henrique Silva; Kobayashi, Gerson Shigeru; Musso, Camila Manso; Assoni, Amanda F.; Oliveira, Danyllo; Caldini, Élia Garcia; Gerstenhaber, Jonathan A; Raia, Silvano; Lelkes, Peter I.; Zatz, Mayana

doi:10.1186/s13287-019-1367-x

Cited by 41 publications

(39 citation statements)

References 42 publications

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“…Proteomic analysis of four different types of hPSC-LBs (HAEC/dpMSCs; HAEC/hPSC-derived MSCs; hPSC-derived ECs/dpMSCs and entirely hPSC-derived LB) revealed differences in the WNT and TGF-β pathways among the four groups. The high TGF-β expression in HAECs was related to impaired hepatocyte differentiation from hepatoblasts as shown by a decrease in hPSC-LB albumin production, which was probably caused by remodeling of the ECM [73]. In accordance with this, Koui et al showed that hPSC-derived LSECs and hPSC-derived MSCs improved expansion, maintenance and metabolic rate of hPSC-derived hepatocytes compared to that of their primary counterparts (HUVECs and MSCs) in a 2D-coculture [74].…”

Section: Liver Organoids and Vascularizationmentioning

confidence: 84%

“…Other approaches attempting to vascularize LBs have been based on the use of human adipose microvascular endothelial cells (HAMECs) as a source of human ECs [70]. Recently, some studies have succeeded in generating LBs entirely from hPSCs by either a codifferentiation approach [71] or an independent differentiation of hPSCs into ECs, MSCs and hepatoblasts and their subsequent coculture [72][73][74]. Similar to HUVECs and HAMECs, hPSC-derived ECs have been shown to improve the maturation of hepatocytes and the engraftment of hPSC-hepatocytes in in vivo models (Ramanathan et al 2015) [70][71][72]75].…”

Section: Liver Organoids and Vascularizationmentioning

confidence: 99%

“…Despite this, the use of EC derived from hPSCs have been proven to be more suitable for organoid maturation. Two independent studies showed that different sources of ECs can result in different hepatocyte maturation [73,74], with hPSC-derived ECs being the most advantageous source. This was partially explained by the fact that hPSCderived ECs used in these studies were differentiated into liver-specific ECs, LSEC, from hPSCs prior to organoid formation [74].…”

Section: Future Directionsmentioning

confidence: 99%

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The endothelium, a key actor in organ development and hPSC-derived organoid vascularization

et al. 2020

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Over the last 4 decades, cell culture techniques have evolved towards the creation of in vitro multicellular entities that incorporate the three-dimensional complexity of in vivo tissues and organs. As a result, stem cells and adult progenitor cells have been used to derive self-organized 3D cell aggregates that mimic the morphological and functional traits of organs in vitro. These so-called organoids were first generated from primary animal and human tissues, then human pluripotent stem cells (hPSCs) arose as a new tool for organoid generation. Due to their selfrenewal capacity and differentiation potential, hPSCs are an unlimited source of cells used for organoids. Today, hPSC-derived small intestinal, kidney, brain, liver, and pancreas organoids, among others, have been produced and are promising in vitro human models for diverse applications, including fundamental research, drug development and regenerative medicine. However, achieving in vivo-like organ complexity and maturation in vitro remains a challenge. Current hPSC-derived organoids are often limited in size and developmental state, resembling embryonic or fetal organs rather than adult organs. The use of endothelial cells to vascularize hPSC-derived organoids may represent a key to ensuring oxygen and nutrient distribution in large organoids, thus contributing to the maturation of adult-like organoids through paracrine signaling. Here, we review the current state of the art regarding vascularized hPSC-derived organoids (vhPSC-Orgs). We analyze the progress achieved in the generation of organoids derived from the three primary germ layers (endoderm, mesoderm and ectoderm) exemplified by the pancreas, liver, kidneys and brain. Special attention will be given to the role of the endothelium in the organogenesis of the aforementioned organs, the sources of endothelial cells employed in vhPSC-Org protocols and the remaining challenges preventing the creation of ex vivo functional and vascularized organs.

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Section: Liver Organoids and Vascularizationmentioning

confidence: 84%

Section: Liver Organoids and Vascularizationmentioning

confidence: 99%

Section: Future Directionsmentioning

confidence: 99%

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The endothelium, a key actor in organ development and hPSC-derived organoid vascularization

et al. 2020

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“…hiPSCs were differentiated toward definitive endoderm (DE) according to Goulart et al 26 with minor modifications. hiPSC cells were seeded at 2.5 × 104 cells/cm 2 on GELTREX coated plates and cultured for 3 days until reached 40% confluency.…”

Section: Methodsmentioning

confidence: 99%

Long-term single-cell passaging of human iPSC fully supports pluripotency and high-efficient trilineage differentiation capacity

et al. 2020

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Objectives: To establish a straightforward single-cell passaging cultivation method that enables high-quality maintenance of human induced pluripotent stem cells without the appearance of karyotypic abnormalities or loss of pluripotency. Methods: Cells were kept in culture for over 50 passages, following a structured chronogram of passage and monitoring cell growth by population doubling time calculation and cell confluence. Standard procedures for human induced pluripotent stem cells monitoring as embryonic body formation, karyotyping and pluripotency markers expression were evaluated in order to assess the cellular state in long-term culture. Cells that underwent these tests were then subjected to differentiation into keratinocytes, cardiomyocytes and definitive endoderm to evaluate its differentiation capacity. Results: Human induced pluripotent stem cells clones maintained its pluripotent capability as well as chromosomal integrity and were able to generate derivatives from the three germ layers at high passages by embryoid body formation and high-efficient direct differentiation into keratinocytes, cardiomyocytes and definitive endoderm. Conclusions: Our findings support the routine of human induced pluripotent stem cells single-cell passaging as a reliable procedure even after long-term cultivation, providing healthy human induced pluripotent stem cells to be used in drug discovery, toxicity, and disease modeling as well as for therapeutic approaches.

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“…41,52,154 Initial studies focusing on differentiation and maturation of iPSC-hepatocytes in 3D systems yielded promising results for obtaining functional and gene expression profiles that are closer to the activity of primary tissues. [155][156][157] However, in addition to better investigating the mechanistic implications of 3D microenvironments in iPSChepatocyte differentiation, the potential employment of these systems with differentiated cells for drug clearance studies still requires elucidation. Once validated for this context, engineered systems with iPSC-differentiated cells will provide the opportunity of replicating patient-specific properties that relate to the expression of enzymes and transporters.…”

Section: Toxicitymentioning

confidence: 99%

Microengineered systems with iPSC-derived cardiac and hepatic cells to evaluate drug adverse effects

Dame

Ribeiro

2020

Exp Biol Med (Maywood)

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Hepatic and cardiac drug adverse effects are among the leading causes of attrition in drug development programs, in part due to predictive failures of current animal or in vitro models. Hepatocytes and cardiomyocytes differentiated from human induced pluripotent stem cells (iPSCs) hold promise for predicting clinical drug effects, given their human-specific properties and their ability to harbor genetically determined characteristics that underlie inter-individual variations in drug response. Currently, the fetal-like properties and heterogeneity of hepatocytes and cardiomyocytes differentiated from iPSCs make them physiologically different from their counterparts isolated from primary tissues and limit their use for predicting clinical drug effects. To address this hurdle, there have been ongoing advances in differentiation and maturation protocols to improve the quality and use of iPSC-differentiated lineages. Among these are in vitro hepatic and cardiac cellular microsystems that can further enhance the physiology of cultured cells, can be used to better predict drug adverse effects, and investigate drug metabolism, pharmacokinetics, and pharmacodynamics to facilitate successful drug development. In this article, we discuss how cellular microsystems can establish microenvironments for these applications and propose how they could be used for potentially controlling the differentiation of hepatocytes or cardiomyocytes. The physiological relevance of cells is enhanced in cellular microsystems by simulating properties of tissue microenvironments, such as structural dimensionality, media flow, microfluidic control of media composition, and co-cultures with interacting cell types. Recent studies demonstrated that these properties also affect iPSC differentiations and we further elaborate on how they could control differentiation efficiency in microengineered devices. In summary, we describe recent advances in the field of cellular microsystems that can control the differentiation and maturation of hepatocytes and cardiomyocytes for drug evaluation. We also propose how future research with iPSCs within engineered microenvironments could enable their differentiation for scalable evaluations of drug effects. Impact statement Cardiac and hepatic adverse drug effects are among the leading causes of attrition in preclinical and clinical drug development programs as well as marketing withdrawals. The insufficiency of animal testing models has led to considerable interest in the employment of cardiac and hepatic models using human-induced pluripotent stem cells (iPSCs) for drug toxicity testing. However, current batches of iPSC-derived cardiomyocytes and hepatocytes are variable and not matured as adult primary tissues, which limit their prediction of drug effects. This article discusses how the use of microfluidics can create microenvironments to better control differentiation protocols and increase the physiological relevance of iPSC-derived cardiomyocytes and hepatocytes. Development and standardization of technologies will enable evaluation of the potential value of cellular microsystems to improve the in vitro models used in drug development programs. Future steps in this field include controlled connections of organ systems to better recreate clinical metabolism and pharmacokinetics.

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Adult and iPS-derived non-parenchymal cells regulate liver organoid development through differential modulation of Wnt and TGF-β

Cited by 41 publications

References 42 publications

The endothelium, a key actor in organ development and hPSC-derived organoid vascularization

The endothelium, a key actor in organ development and hPSC-derived organoid vascularization

Long-term single-cell passaging of human iPSC fully supports pluripotency and high-efficient trilineage differentiation capacity

Microengineered systems with iPSC-derived cardiac and hepatic cells to evaluate drug adverse effects

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