A Novel Human Placental Barrier Model Based on Trophoblast Stem Cells Derived from Human Induced Pluripotent Stem Cells.

The placenta acts as an interface between the fetus and the expecting mother. Various drugs and environmental pollutants can pass through the human placental barrier and may harm the developing fetus. Currently available in vitro placental barrier models are often inadequate, because they are lacking the functional trophoblast cells. Therefore, we developed and characterized a new human placental model using trophoblast stem cells (TSCs) derived from human induced pluripotent stem cells. Umbilical vein endothelial cells, fibroblast, and TSCs were cocultured using micromesh cell culture technique. These cells formed a tight three-layered structure. This coculture model induced progressive fusion of TSCs and formed a syncytialized epithelium that resembles the in vivo syncytiotrophoblast. Our model allowed the cultured trophoblasts to form microvilli and to reconstitute expression and physiological localization of membrane transport proteins, such as transporter for ATP-binding cassette subfamily B member 1, ATP-binding cassette subfamily C member 3, and glucose transporter-1. Drug permeability assays were performed using five compounds. The results from the permeability assays were comparable to the ones obtained with ex vivo placental models. In conclusion, we developed a novel coculture model mimicking human placenta that provides a useful tool for the studies on transfer of substances between the mother and fetus. Impact statement Compared with the currently available in vitro placental barrier models, a novel three-dimensional coculture placental barrier model presented in this study morphologically and functionally modeled the true placental barrier. The use of human trophoblast stem cells from human induced pluripotent stem cells substantially improved the current model. The use of micromesh sheet as a bioscaffold facilitated the formation of a good multilayer structure, which is closer to the physical appearance of the placenta observed in human.

A comparative study of key physiological stem cell parameters between three human trophoblast cell lines.

Human trophoblast stem cells (TSCs) play a key role in the placenta. These cells are proliferative, undifferentiated, and can differentiate into mature trophoblast cell types. However, primary human TSCs are difficult to obtain. In our previous study, we established TSCs from human induced pluripotent stem cells (TShiPSC). Here, we aimed to characterize the identity of these TShiPSC cells by comparing them with BeWo choriocarcinoma cells and primary TSCs (CT cells). Compared with BeWo cells, CT and TShiPSC cells showed high secretion of human chorionic gonadotrophin (hCG) and syncytiotrophoblast differentiation ability. Global gene microarray analysis results showed that CT and TShiPSC cells, unlike BeWo cells, could be classified in the same group. Compared with BeWo cells, CT and TShiPSC cells showed high expression levels of TSC-specific genes and low expression of cancer adhesion and invasion genes. Analysis of placental barrier integrity showed that TShiPSC cells could form a good barrier. Prospective studies using TShiPSC cells hold great promise for elucidating the pathogenesis of infertility due to trophoblast defects.

Three-Dimensional Cell Sheet Construction Method with a Polyester Micromesh Sheet.

Cell sheet engineering has become important in a variety of fields, including regenerative medicine and transplantation. Our research group previously developed micromesh cultures that enable cells to form a cell sheet on a microstructured mesh sheet. Here, we present a more usable micromesh culture and devices that make it possible, aiming for widespread use. The devices are mainly constituted of a polyester micromesh sheet and three-dimensional (3D)-printed simple frames that fix the mesh sheet on it. Cells such as fibroblast Tig-1-20 cells, hepatoma HepG2 cells, or mesenchymal stem cells (MSC) were easily seeded on the polyester mesh sheet in the device and cultured for 16 days, which was followed by the formation of a 100-400-µm-thick cell sheet. The cell sheet was very robust, easy to handle, and could be readily removed from the device for subsequent analysis. Optical coherence tomography revealed the structure of the cell sheet as having the mesh sheet layer in the center of the cell sheet. Confocal microscopy demonstrated that Tig-1-20 cells in the cell sheet were aligned according to the shape of the mesh apertures, indicating that cell orientation can be controlled with this micromesh culture. As for another application, the device was used to construct a multilayered cell sheet that consists of three different types of cells. Furthermore, for mass production, the device frames were made using polyoxymethylene (POM) instead of 3D printing materials. Using the POM devices, a large MSC sheet for 10 cm dishes was successfully produced 7 days after cell seeding. This micromesh culture may become one of the useful cell sheet construction methods in future for medical and research fields. Impact statement Currently, cell sheets are constructed, for example, on a temperature-responsive polymer-coated dish or a porous membrane. These cell sheets are widely used but are not completely suitable in terms of robustness, ease of handling, cost, ease of microscopic cell observation, or nutrient supply. We previously reported that the micromesh culture can provide a three-dimensional (3D) cell sheet that has advantages for cell observation and nutrient supply. In this study, the micromesh culture was enhanced with a polyester micromesh sheet and a series of devices of polyoxymethylene, helping us to produce a robust, cost-effective, easily layered, and easy-to-use 3D cell sheet.

Compact fluidic system for functional assessment of pancreatic islets.

Transplantation of pancreatic islets is becoming a promising therapy for people with type I diabetes. In this study, we present a compact fluidic system that enables assessment of islet functionality ex vivo for efficient islet transplantation. The fluidic system includes a micromesh sheet-embedded chip. Islets can be loaded easily on the micromesh sheet and observed clearly by microscopy. Islets on the mesh sheet mainly remained in place during perfusion and did not get damaged by hydraulic pressure because of high porosity of the micromesh sheet. The fluidic system was assembled with a sample fraction chip of polydimethylsiloxane. The chip includes a channel and columns, both having surfaces that were super-hydrophilized so that solutions could flow smoothly within the chip by gravity. Using mouse pancreatic islets, a dynamic glucose-stimulated insulin secretion test was performed to examine the performance of the fluidic system. The system successfully analyzed levels and patterns of insulin secretion upon exposure of the islets to low- and high-glucose solutions in turns, thus demonstrating its capacity to assess islet functions more easily and cost-effectively.

Establishment of human trophoblast stem cells from human induced pluripotent stem cell-derived cystic cells under micromesh culture.

BACKGROUND: Trophoblasts as a specific cell lineage are crucial for the correct function of the placenta. Human trophoblast stem cells (hTSCs) are a proliferative population that can differentiate into syncytiotrophoblasts and extravillous cytotrophoblasts. Many studies have reported that chemical supplements induce the differentiation of trophoblasts from human induced pluripotent stem cells (hiPSCs). However, there have been no reports of the establishment of proliferative hTSCs from hiPSCs. Our previous report showed that culturing hiPSCs on micromesh as a bioscaffold induced cystic cells with trophoblast properties. Here, we aimed to establish hTSCs from hiPSCs. METHODS: We used the micromesh culture technique to induce hiPSC differentiation into trophoblast cysts. We then reseeded and purified cystic cells. RESULTS: The cells derived from the reseeded cysts were highly proliferative. Low expression levels of pluripotency genes and high expression levels of TSC-specific genes were detected in proliferative cells. The cells could be passaged, and further directional differentiation into syncytiotrophoblast- and extravillous cytotrophoblast-like cells was confirmed by marker expression and hormone secretion. CONCLUSIONS: We established hiPSC-derived hTSCs, which may be applicable for studying the functions of trophoblasts and the placenta. Our experimental system may provide useful tools for understanding the pathogenesis of infertility owing to trophoblast defects in the future.

Nuclear transplantation between allogeneic cells through topological reconnection of plasma membrane in a microfluidic system.

Previous studies have demonstrated that somatic cells fused with pluripotent stem cells can be reprogrammed on the basis of reprogramming factors acquired from the latter. However, fusion-reprogrammed cells are deemed unsuitable for therapeutic applications mainly because conventional fusion techniques often yield tetraploid fusants that contain exogenous genes acquired from the fusion partners. Here, we present a novel cell-cell topological reconnection technique and demonstrate its application to nuclear transplantation between a somatic cell and a stem cell without nuclei mixing. As a proof of concept, a microfluidic fusion chip embodied with a microslit (4 µm in width) to prevent nuclei mixing was developed and used to perform one-to-one electrofusion of a target somatic cell (Jurkat cell) with an induced pluripotent stem (iPS) cell. To extract its cytoplasm, the target cell was first topologically connected to a sacrificial iPS cell by electrofusion via a microslit, followed by shear flow removal of the latter to obtain a cytoplasm-depleted nucleus of the target cell. Then, to replace the lost cytoplasm, topological reconnection to a second iPS cell was performed similarly by electrofusion, followed by shear flow separation of the target cell to enable it acquire most of the iPS cytoplasm, but without nuclei mixing. Microscopic observation of target cells harvested and cultured post hoc in a microwell confirmed that they manifested cell division. Taken together, these results demonstrate the potential application of the cell-cell topological reconnection technique to somatic cell nuclear transplantation for the generation of autologous pluripotent stem cells.

A Three-dimensional Cell Culture Method with a Micromesh Sheet and Its Application to Hepatic Cells.

In vitro 3D cultures of hepatocytes are increasingly being used to assess human hepatic metabolism and toxicity in drug development. Here, we developed an in vitro 3D cell culture method with a microstructured mesh sheet and applied it to culturing human hepatoma HepG2 cells. The micromesh sheet is constituted of fine mesh strands and apertures that are each much larger than a single cell in size. Proliferating on a micromesh sheet, HepG2 cells spread out in a planar manner and then formed a multilayered cell sheet, so that cell-cell adhesion was dominant over cell-substrate adhesion as being different from 2D cultures. In micromesh cultures, the increase rate in thickness of the cell mass was visually slower than that in spheroid cultures, enabling us to clearly observe inside cells of the cell population by microscopy. Micromesh-cultured HepG2 cells showed higher viability compared with spheroid-cultured cells. The multilayered HepG2 cell sheet increased expression of hepatic marker genes and induced cell polarization with bile canalicular membranes. Furthermore, a combination of micromesh cultures with medium perfusion further induced expression of hepatic marker genes in HepG2 cells; especially CYP1A1 and CYP1A2 mRNA increased 86-fold and 43-fold compared with 2D controls, respectively, which were much higher than those in spheroid cultures. Thus, this simple and versatile micromesh culture method holds some advantages over traditional spheroid cultures and is expected to be instrumental in culturing more differentiated hepatic cells such as HepaRG cells and primary hepatocytes for future preclinical testing.

Development of trophoblast cystic structures from human induced pluripotent stem cells in limited-area cell culture.

We developed a novel engineering technique to induce differentiation of human induced pluripotent stem cells (hiPSCs) into organoids mimicking the trophectoderm (TE). Here, hiPSCs were cultured on a limited area of 2-4 mm in diameter. After 15-20 days, spherical cysts appeared on the surface of the limited area. Secretion of human chorionic gonadotrophin (hCG) began to increase after ∼ 20 days and remained dramatically elevated over the next 20 days. Limited-area-cultured cysts exhibited expression of hCG, which was a result of epithelial differentiation. Low expression levels of pluripotent genes and high expression levels of trophoblast lineage-specific genes were detected in the cells of spherical cysts. Multinucleated syncytia trophoblast was observed in the reseeded cystic cells. We observed hiPSC-derived cysts that morphologically resembled trophectoderm in vivo. The limited-area cell culture induced a three-dimensional (3D) trophectoderm organoid, which has potential for use in the study of human trophoblast differentiation and placental morphogenesis.

Self-organization of human iPS cells into trophectoderm mimicking cysts induced by adhesion restriction using microstructured mesh scaffolds.

Cellular dynamics leading to the formation of the trophectoderm in humans remain poorly understood owing to limited accessibility to human embryos for research into early human embryogenesis. Compared to animal models, organoids formed by self-organization of stem cells in vitro may provide better insights into differentiation and complex morphogenetic processes occurring during early human embryogenesis. Here we demonstrate that modulating the cell culture microenvironment alone can trigger self-organization of human induced pluripotent stem cells (hiPSCs) to yield trophectoderm-mimicking cysts without chemical induction. To modulate the adhesion microenvironment, we used the mesh culture technique recently developed by our group, which involves culturing hiPSCs on suspended micro-structured meshes with limited surface area for cell adhesion. We show that this adhesion-restriction strategy can trigger a two-stage self-organization of hiPSCs; first into stem cell sheets, which express pluripotency signatures until around day 8-10, then into spherical cysts following differentiation and self-organization of the sheet-forming cells. Detailed morphological analysis using immunofluorescence microscopy with both confocal and two-photon microscopes revealed the anatomy of the cysts as consisting of a squamous epithelial wall richly expressing E-cadherin and CDX2. We also confirmed that the cysts exhibit a polarized morphology with basal protrusions, which show migratory behavior when anchored. Together, our results point to the formation of cysts which morphologically resemble the trophectoderm at the late-stage blastocyst. Thus, the mesh culture microenvironment can initiate self-organization of hiPSCs into trophectoderm-mimicking cysts as organoids with potential application in the study of early embryogenesis and also in drug development.

Cell Adhesion Minimization by a Novel Mesh Culture Method Mechanically Directs Trophoblast Differentiation and Self-Assembly Organization of Human Pluripotent Stem Cells.

Mechanical methods for inducing differentiation and directing lineage specification will be instrumental in the application of pluripotent stem cells. Here, we demonstrate that minimization of cell-substrate adhesion can initiate and direct the differentiation of human pluripotent stem cells (hiPSCs) into cyst-forming trophoblast lineage cells (TLCs) without stimulation with cytokines or small molecules. To precisely control cell-substrate adhesion area, we developed a novel culture method where cells are cultured on microstructured mesh sheets suspended in a culture medium such that cells on mesh are completely out of contact with the culture dish. We used microfabricated mesh sheets that consisted of open meshes (100∼200 µm in pitch) with narrow mesh strands (3-5 µm in width) to provide support for initial cell attachment and growth. We demonstrate that minimization of cell adhesion area achieved by this culture method can trigger a sequence of morphogenetic transformations that begin with individual hiPSCs attached on the mesh strands proliferating to form cell sheets by self-assembly organization and ultimately differentiating after 10-15 days of mesh culture to generate spherical cysts that secreted human chorionic gonadotropin (hCG) hormone and expressed caudal-related homeobox 2 factor (CDX2), a specific marker of trophoblast lineage. Thus, this study demonstrates a simple and direct mechanical approach to induce trophoblast differentiation and generate cysts for application in the study of early human embryogenesis and drug development and screening.

Single DNA molecule denaturation using laser-induced heating.

This paper proposes targeted in situ denaturation through laser-induced heating to partially amplify relevant sequences from a long DNA strand. It uses 5 kb of DNA as a sample, labeling both strands with quantum dots, with one strand immobilized on a solid surface. We irradiated a targeted DNA sequence with a focused infrared laser to elevate its temperature, monitoring the process by microscope. The denaturation was detected in real time by separating quantum dots on each strand. Results showed that complete separation of the strands occurred within a few seconds of laser irradiation, which raised the temperature to approximately 90 °C.

Dielectrophoretic cell trapping and parallel one-to-one fusion based on field constriction created by a micro-orifice array.

Micro-orifice based cell fusion assures high-yield fusion without compromising the cell viability. This paper examines feasibility of a dielectrophoresis (DEP) assisted cell trapping method for parallel fusion with a micro-orifice array. The goal is to create viable fusants for studying postfusion cell behavior. We fabricated a microfluidic chip that contained a chamber and partition. The partition divided the chamber into two compartments and it had a number of embedded micro-orifices. The voltage applied to the electrodes located at each compartment generated an electric field distribution concentrating in micro-orifices. Cells introduced into each compartment moved toward the micro-orifice array by manipulation of hydrostatic pressure. DEP assisted trapping was used to keep the cells in micro-orifice and to establish cell to cell contact through orifice. By applying a pulse, cell fusion was initiated to form a neck between cells. The neck passing through the orifice resulted in immobilization of the fused cell pair at micro-orifice. After washing away the unfused cells, the chip was loaded to a microscope with stage top incubator for time lapse imaging of the selected fusants. The viable fusants were successfully generated by fusion of mouse fibroblast cells (L929). Time lapse observation of the fusants showed that fused cell pairs escaping from micro-orifice became one tetraploid cell. The generated tetraploid cells divided into three daughter cells. The fusants generated with a smaller micro-orifice (diameter approximately 2 mum) were kept immobilized at micro-orifice until cell division phase. After observation of two synchronized cell divisions, the fusant divided into four daughter cells. We conclude that the presented method of cell pairing and fusion is suitable for high-yield generation of viable fusants and furthermore, subsequent study of postfusion phenomena.