The promotion of hepatic maturation of human pluripotent stem cells in 3D co-culture using type I collagen and Swiss 3T3 cell sheets

Laboratory of Biochemistry and Molecular Biology, Graduate School of Pharmaceutical Sciences, Osaka University, Osaka 565-0871, Japan.
Biomaterials (Impact Factor: 8.56). 03/2012; 33(18):4526-34. DOI: 10.1016/j.biomaterials.2012.03.011
Source: PubMed


Hepatocyte-like cells differentiated from human embryonic stem cells (hESCs) or human induced pluripotent stem cells (hiPSCs) are known to be a useful cell source for drug screening. We recently developed an efficient hepatic differentiation method from hESCs and hiPSCs by sequential transduction of FOXA2 and HNF1α. It is known that the combination of three-dimensional (3D) culture and co-culture, namely 3D co-culture, can maintain the functions of primary hepatocytes. However, hepatic maturation of hESC- or hiPSC-derived hepatocyte-like cells (hEHs or hiPHs, respectively) by 3D co-culture systems has not been examined. Therefore, we utilized a cell sheet engineering technology to promote hepatic maturation. The gene expression levels of hepatocyte-related markers (such as cytochrome P450 enzymes and conjugating enzymes) and the amount of albumin secretion in the hEHs or hiPHs, which were 3D co-cultured with the Swiss 3T3 cell sheet, were significantly up-regulated in comparison with those in the hEHs or hiPHs cultured in a monolayer. Furthermore, we found that type I collagen synthesized in Swiss 3T3 cells plays an important role in hepatic maturation. The hEHs or hiPHs that were 3D co-cultured with the Swiss 3T3 cell sheet would be powerful tools for medical applications, such as drug screening.

1 Follower
18 Reads
  • Source
    • "), hydroxyethylmethacrylate and ethoxyethylmethacrylate copolymers (Marekova et al., 2013) 3T3-J2 fibroblasts (Cho et al., 2008), bone marrow mesenchymal stem cells (Marekova et al., 2013), human adipose-derived stem cells (No da et al., 2012) ESC Carry problems of ethics and immunorejection Inner mass cells or primordial germ cells 3D spheroid culture system, rotating bioreactor, hollow fiber (Subramanian et al., 2014), biodegradable polymer scaffold (Wang et al., 2012), type I collagen and Swiss 3T3 cell sheets (Nagamoto et al., 2012) STO feeder cells, MLSgt20 cells, HSC (Ishii et al., 2010), HepG2 cells (Lee et al., 2009), xeno-free extracellular matrix (Farzaneh et al., 2014) iPSC Create chimeras by germ line transmission and tetraploid complementation Skin and nucleated blood cells and other terminally differentiated cells Hollow fiber/organoid (Amimoto et al., 2011), micro-cavitary hydrogel (MCG) system (Lau et al., 2013), type I collagen and Swiss 3T3 cell sheets (Nagamoto et al., 2012), multicomponent hydrogel fibers (Du et al., 2014) Bone marrow mesenchymal stem cells (Mobarra et al., 2014), endothelial cells (Du et al,. 2014), liver nonparenchymal cell line TWNT-1 (Javed et al., 2014) HPC Lack of sources Liver Biomatrix scaffolds (Wang et al., 2011), 3D collagen gel matrix, fibroblast feeder layer culture system (Lazaro et al., 1998) "
    [Show abstract] [Hide abstract]
    ABSTRACT: Various liver diseases result in terminal hepatic failure, and liver transplantation, cell transplantation and artificial liver support systems are emerging as effective therapies for severe hepatic disease. However, all of these treatments are limited by organ or cell resources, so developing a sufficient number of functional hepatocytes for liver regeneration is a priority. Liver regeneration is a complex process regulated by growth factors (GFs), cytokines, transcription factors (TFs), hormones, oxidative stress products, metabolic networks, and microRNA. It is well-known that the function of isolated primary hepatocytes is hard to maintain; when cultured in vitro, these cells readily undergo dedifferentiation, causing them to lose hepatocyte function. For this reason, most studies focus on inducing stem cells, such as embryonic stem cells (ESCs), induced pluripotent stem cells (iPSCs), hepatic progenitor cells (HPCs), and mesenchymal stem cells (MSCs), to differentiate into hepatocyte-like cells (HLCs) in vitro. In this review, we mainly focus on the nature of the liver regeneration process and discuss how to maintain and enhance in vitro hepatic function of isolated primary hepatocytes or stem cell-derived HLCs for liver regeneration. In this way, hepatocytes or HLCs may be applied for clinical use for the treatment of terminal liver diseases and may prolong the survival time of patients in the near future.
    Protein & Cell 06/2015; 6(8). DOI:10.1007/s13238-015-0180-2 · 3.25 Impact Factor
  • Source
    • "Nonetheless, we have designed iMPCCs to be modular in that interactions between different donors of iPSC- HHs and donor-matched stromal cells can be studied without significant changes to iPSC-HH homotypic interactions on the micropatterned ECM domains. Limited in situ cell observation by conventional microscopy and nutrient transport limitations makes high-throughput screening in 3D iPSC-HH cultures (Nagamoto et al., 2012; Ogawa et al., 2013; Takayama et al., 2013; Takebe et al., 2013) non-trivial. "
    [Show abstract] [Hide abstract]
    ABSTRACT: Primary human hepatocytes (PHHs) are a limited resource for drug screening, their quality for in vitro use can vary considerably across different lots, and a lack of available donor diversity restricts our understanding of how human genetics affect drug-induced liver injury (DILI). Induced pluripotent stem cell-derived human hepatocyte-like cells (iPSC-HHs) could provide a complementary tool to PHHs for high-throughput drug screening, and ultimately enable personalized medicine. Here, we hypothesized that previously developed iPSC-HH-based micropatterned co-cultures (iMPCCs) with murine embryonic fibroblasts could be amenable to long-term drug toxicity assessment. iMPCCs, created in industry-standard 96-well plates, were treated for 6 days with a set of 47 drugs, and multiple functional endpoints (albumin, urea, ATP) were evaluated in dosed cultures against vehicle-only controls to enable binary toxicity decisions. We found that iMPCCs correctly classified 24 of 37 hepatotoxic drugs (65% sensitivity), while all 10 non-toxic drugs tested were classified as such in iMPCCs (100% specificity). On the other hand, conventional confluent cultures of iPSC-HHs failed to detect several liver toxins that were picked up in iMPCCs. Results for DILI detection in iMPCCs were remarkably similar to published data in PHH-MPCCs (65% versus 70% sensitivity) that were dosed with the same drugs. Furthermore, iMPCCs detected the relative hepatotoxicity of structural drug analogs and recapitulated known mechanisms of acetaminophen toxicity in vitro. In conclusion, iMPCCs could provide a robust tool to screen for DILI potential of large compound libraries in early stages of drug development using an abundant supply of commercially available iPSC-HHs. © The Author 2015. Published by Oxford University Press on behalf of the Society of Toxicology. All rights reserved. For permissions, please email:
    Toxicological Sciences 02/2015; DOI:10.1093/toxsci/kfv048 · 3.85 Impact Factor
  • Source
    • "This cell source has most of the advantageous properties of the ESCs [136], while being patient-specific and less controversial [137]–[139]. It has been shown that these cells can be differentiated to many cell types including hepatocytes [140] and cardiomyocytes [141]. Also, there are few studies that use iPSCs for tissue engineering purposes in the literature. "
    [Show abstract] [Hide abstract]
    ABSTRACT: The field of tissue engineering has been growing in the recent years as more products have made it to the market and as new uses for the engineered tissues have emerged, motivating many researchers to engage in this multidisciplinary field of research. Engineered tissues are now not only considered as end products for regenerative medicine, but also have emerged as enabling technologies for other fields of research ranging from drug discovery to biorobotics. This widespread use necessitates a variety of methodologies for production of tissue engineered constructs. In this review, these methods together with their non-clinical applications will be described. First, we will focus on novel materials used in tissue engineering scaffolds; such as recombinant proteins and synthetic, self assembling polypeptides. The recent advances in the modular tissue engineering area will be discussed. Then scaffold-free production methods, based on either cell sheets or cell aggregates will be described. Cell sources used in tissue engineering and new methods that provide improved control over cell behavior such as pathway engineering and biomimetic microenvironments for directing cell differentiation will be discussed. Finally, we will summarize the emerging uses of engineered constructs such as model tissues for drug discovery, cancer research and biorobotics applications.
    12/2012; 6. DOI:10.1109/RBME.2012.2233468
Show more