Matrix Culture Research Articles

Constructing Cross-Linked Nanofibrous Scaffold via Dual-Enzyme-Instructed Hierarchical Assembly.

To explore the potential of step-by-step assembly in the fabrication of biological materials, we designed and synthesized two peptide-based molecules for enzyme-instructed hierarchical assembly. Upon the treatment of alkaline phosphatase, one molecule undergoes enzyme-instructed self-assembly forming uniformed nanofibers. The other one that can self-assemble into vesicles undergoes enzyme-induced transformation of self-assembly converting vesicles into irregular aggregates upon the treatment of carboxylesterase. Coadministration of two enzymes to a mixture of these two molecules in a stage-by-stage fashion leads to a physically knotted nanofibrous scaffold that is applicable as a nanostructured matrix for cell culture.

Self‐Healing, High Adherent, and Antioxidative LbL Multilayered Film for Enhanced Cell Adhesion

AbstractCell adhesion acts as a complex process that is hardly promoted through surfaces with a single performance. Controlling the cell's adhesion on the interfaces between the synthetic materials and the biological environments remains a real challenge in the biomedical fields. Herein, based on RGD peptide modified carboxymethyl chitosan (CCS‐R) and dopamine modified oxidized alginate (OALG‐D), a layer‐by‐layer (LbL) self‐assemble multilayered (CCS‐R/OALG‐D)15 film is developed. The multifunctional (CCS‐R/OALG‐D)15 film with a self‐healing, highly adherent, biocompatible, and antioxidative biomatrix property for the cell adhesion is fabricated with the Schiff‐base bond, electrovalent bond, and hydrogen bond. The LbL multilayered film exhibits the significant cell adhesion ability for the normal human dermal fibroblast cells and the coral Acropopa formosa cells. Furthermore, it holds high self‐healing efficiency (88.8%), excellent adhesion strength (adhesive stress: 61.1 MPa), and outstanding antioxidation (free radical scavenging rate: 86.8%). Furthermore, it is demonstrated that the LbL multifunctional film, as a culture matrix, has prolonged the survival of the coral Acropopa formosa. Therefore, the incorporation of a self‐healing, high adhesion, superior cell adhesion, and antioxidation property into a single LbL self‐assembly film provides a promising platform for regenerative medicine.

Fabrication of Bioactive Inverted Colloidal Crystal Scaffolds Using Expanded Polystyrene Beads.

Inverted colloidal crystal (ICC) hydrogel scaffolds have emerged as a new class of three-dimensional cell culture matrix that represents a unique opportunity to reproduce lymphoid tissue microenvironments. ICC geometry promotes the formation of stromal cell networks and their interaction with hematopoietic cells, a core cellular process in lymphoid tissues. When subdermally implanted, ICC hydrogel scaffolds direct unique foreign body responses to form a vascularized stromal tissue with prolonged attraction of hematopoietic cells, which together resemble lymphoid tissue microenvironments. While conceptually simple, fabrication of ICC hydrogel scaffold requires multiple steps and laborious handling of delicate materials. Here, we introduce a facile route for ICC hydrogel scaffold fabrication using expanded polystyrene (EPS) beads. EPS beads shrink and fuse in a tunable manner under pressurized thermal conditions, which serves as colloidal crystal templates for ICC scaffold fabrication. Inclusion of collagen in the precursor solution greatly simplified preparation of bioactive hydrogel scaffolds. The resultant EPS-templated bioactive ICC hydrogel scaffolds demonstrate characteristic features required for lymphoid tissue modeling in both in vitro and in vivo settings. We envision that the presented method will facilitate widespread implementation of ICC hydrogel scaffolds for lymphoid tissue engineering and other emerging applications. Impact statement Inverted colloidal crystal (ICC) hydrogel scaffolds have emerged as a new class of three-dimensional cell culture matrix that represents a unique opportunity for lymphoid tissue modeling and other emerging novel bioengineering applications. While conceptually simple, fabrication of the ICC hydrogel scaffold requires multiple steps and laborious handling of delicate materials with highly toxic chemicals. The presented method for ICC hydrogel scaffold fabrication using expanded polystyrene (EPS) beads is simple, cost-effective, and involves less toxic chemicals than conventional methods, while retaining comparable biological significance. We envision that EPS bead-based hydrogel scaffold fabrication will greatly facilitate the widespread implementation of ICC hydrogel scaffolds and their practical applications.

Optimization system of mesenchymal stem cells for tissue engineering chondrogenic differentiation

Articular cartilage defect is a common joint damage, however, due to the particularity of cartilage tissue, its repair ability is limited. Thus, articular cartilage damage often changes to more severe osteoarthritis which brings huge pain and economic burden to patients. The development of cartilage tissue engineering brings good news to these patients, but also faces great challenges. Because of the characteristics of mesenchymal stem cells(MSCs) including autologous source, amplification and cartilage differentiation potential, MSCs are often used as the seed cells of cartilage tissue engineering and got widely attention. In the commonly used treatment strategy of articular cartilage differentiated from mesenchymal cells, in vitro pre culture and differentiation showed good therapeutic effects.The mesenchymal stem cells collected from the patient were concentrated, cultured and induced differentiation in vitro, a certain number of differentiated chondrocytes were obtained, and then the cells and culture matrix were transplanted to the patient together. However, the tissue engineering cartilage obtained from mesenchymal stem cells cannot fully meet the requirements of clinical treatment. Moreover, due to the differences of disease type, degree and individuality, there are various optimized factors for the treatment of MSCs derived tissue-engineered cartilage transplantation. For the same type of disease treatment, the optimization system still has no unified recognized standard. In order to obtain better adapt to the human body and meet the clinical requirements of articular cartilage, this review focus onoptimized factors of MSCs in the treatment of orthopedic diseases, summarize and analysis the research status, and discuss the induced factorsfrom three aspects: environmental factors, scaffold selection and seed cells.It provides an idea for using mesenchymal stem cells to obtain better tissue-engineered cartilage and to establish a better optimization system.

Secretome Analysis Performed During in vitro Cardiac Differentiation: Discovering the Cardiac Microenvironment.

Human pluripotent stem cells are an important tool for the study of developmental processes, such as cardiomyogenic differentiation. Despite the advances made in this field, the molecular and cellular signals involved in the commitment of embryonic stem cells to the cardiac phenotype are still under investigation. Therefore, this study focuses on identifying the extracellular signals involved in in vitro cardiac differentiation of human embryonic stem cells. Using a three-dimensional cardiomyogenic differentiation protocol, the conditioned medium and the extracellular matrix (ECM) of embryoid body cultures were collected and characterized at four specific time points. Mass spectrometry (MS) and antibody array analysis of the secretome identified a number of secreted proteins related to signaling pathways, such as Wnt and TGFβ, as well as many ECM proteins. When comparing the proteins identified at selected time points, our data pointed out protein interactions and biological process related to cardiac differentiation. Interestingly, the great changes in secretome profile occurred during the cardiac progenitor specification. The secretome results were also compared with our previous RNAseq data, indicating that the secreted proteins undergo some level of gene regulation. During cardiac commitment it was observed an increase in complexity of the ECM, and some proteins as IGFBP7, FN1, HSPG2, as well as other members of the basal lamina could be highlighted. Thus, these findings contribute valuable information about essential microenvironmental signals working on cardiomyogenic differentiation that may be used in future strategies for cardiac differentiation, cardiomyocyte maturation, and in advances for future acellular therapies.

Principal component analysis of MALDI-TOF MS of whole-cell foodborne pathogenic bacteria

The rapid and accurate identification of foodborne pathogenic bacteria is of great importance for human health. Matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS) can be used to rapidly and sensitively identify microorganisms but is limited by the expensive protein databases available. In this study, we established a whole-cell method for the identification of foodborne pathogenic bacteria, using MALDI-TOF MS and principal component analysis (PCA), which did not use protein extractions or expensive protein databases. Thirty strains comprising six common foodborne pathogenic bacteria, namely, Shigella flexneri, Escherichia coli, Staphylococcus aureus, Salmonella enteritidis, Pseudomonas aeruginosa, and Listeria monocytogenes were analyzed using MALDI-TOF MS. The culture time, matrix, and spotting method were optimized based on peak intensity and deviation. A PCA was performed to analyze the mass spectrometry results of six samples and proved capable of identifying significant changes in those samples. It was found that directly applying MALDI-TOF MS analysis to whole-cell bacteria, without protein extraction, exhibited rich peak contents and a high level of reproducibility. MALDI-TOF MS combined with PCA is a promising method of rapidly identifying pathogens in food products.

Simple Time-lapse Imaging for Quantifying the Hydrostatic Production of Oxygenic Photogranules.

Oxygenic photogranules (OPGs) are dense, three-dimensional aggregates containing a syntrophic, light-driven microbial community. Their temporal and spatial development interests microbial ecologists working at the bioprocess engineering interface, as this knowledge can be used to optimize biotechnological applications, such as wastewater treatment and biomass valorization. The method presented here enables the high-throughput quantification of photogranulation. OPGs are produced from a loose sludge-like microbial matrix in hydrostatic batch cultures exposed to light. This matrix transforms into a consolidated, roughly spherical aggregate over time. Photogranulation is quantified by time-lapse imaging coupled to automated image analysis. This allows studying the development of many OPGs simultaneously and in a fully automated way to systematically test what factors drive photogranulation. The protocol can also be used to quantify other types of (a)biotic aggregation.

Reticulated PVA Foams: Preparation, Characterization and in vitro Evaluation for Potential 3D Microbiological Culture

The development of three-dimensional matrices for microbial cultures is a promising strategy by mimic the natural environments of cells and promoting adhesion, growth and cellular proliferation. In the current study, polyvinyl alcohol (PVA) foams were produced by the combination of gas foaming and freeze-drying for applied as microorganism 3D matrix. The spectra FTIR indicated consume of hydroxyl groups that evidences the crosslinking reaction and promoted increases in thermal stability. Morphological analysis by SEM revealed an interconnected porous structure in the PVA foams. Besides, the crosslinking reaction did not affect pore size and morphology. Meanwhile, cross-linked PVA foam (G-PVAFL) exhibited an increase of compressive modulus and compressive strength. The swelling ratio reached 85% and degradation kinetics of G-PVAFL showed a suitable profile to support microorganism in the biofilm formation. Escherichia coli (Gram-negative bacteria), Staphylococcus aureus (Gram-positive bacteria) and Candida albicans (yeast) were efficiently immobilized in G-PVAFL. In addition, cells morphology were preserved for all species investigated. Besides, Sugar Fermentation Test with Escherichia coli immobilized indicates the preservation of proliferative and metabolic activity. These results indicate that G-PVAFL is suitable as a matrix for 3D microbiological culture.

Role of Extracellular Matrix in Three-dimensional Muscle Cell Culture

Role of Extracellular Matrix in Three-dimensional Muscle Cell Culture

Isolation and ex vivo Expansion of Human Limbal Epithelial Progenitor Cells.

Limbal stem cell transplantation has been used successfully to treat patients with limbal stem cell deficiency all over the world. However, long term clinical results often proved less satisfactory due to the low quality of the graft or inadequate properties of transplanted cells. To enhance the ex vivo expansion of human limbal epithelial stem or progenitor cells (LEPC) by preserving stem cell phenotype and to improve subsequent transplantation efficiency, cell-matrix interactions ex vivo should mimic the condition in vivo. The laminin isoforms preferentially expressed in the limbal niche can be used as a culture matrix for epithelial tissue engineering. We recently published the expansion of LEPC on various laminin isoforms and observed that laminin alpha 5-derived matrices support the efficient expansion of LEPC compared to tissue culture plates and other laminin isoforms by preserving stem/progenitor cell phenotype. Here, we describe an optimized protocol for the isolation of LEPC from cadaveric corneal limbal tissue by collagenase digestion and efficient expansion of LEPC using recombinant human laminin-511 E8 fragment (LN-511E8) as culture substrate.

The Regulatory Functionality of Exosomes Derived from hUMSCs in 3D Culture for Alzheimer's Disease Therapy.

Reducing amyloid-β (Aβ) accumulation could be a potential therapeutic approach for Alzheimer's disease (AD). Particular functional biomolecules in exosomes vested by the microenvironment in which the original cells resided can be transferred to recipient cells to improve pathological conditions. However, there are few reports addressing whether exosomes derived from cells cultured on scaffolds with varying dimension can reduce Aβ deposition or ameliorate cognitive decline for AD therapy. Herein, both 3D graphene scaffold and 2D graphene film are used as the matrix for human umbilical cord mesenchymal stem cell culture, from which the supernatants are obtained to isolate exosomes. The levels of 195 kinds of miRNAs and proteins, including neprilysin, insulin-degrading enzyme and heat shock protein 70, in 3D-cultured exosomes (3D-Exo) are dramatically different from those obtained from 2D culture. Hence, 3D-Exo could up-regulate the expression of α-secretase and down-regulate the β-secretase to reduce Aβ production in both AD pathology cells and transgenic mice, through their special cargo. With rescuing Aβ pathology, 3D-Exo exerts enhanced therapeutic effects on ameliorating the memory and cognitive deficits in AD mice. These findings provide a novel clinical application for scaffold materials and functional exosomes derived from stem cells.

Developing a self-organized tubulogenesis model of human renal proximal tubular epithelial cells in vitro.

Three-dimensional tissue culture models which recapitulate the phenotype and function of human renal tissue have attracted significant interest as valuable tools for studying kidney development, disease pathophysiology, and nephrotoxicity. Here, a layer-by-layered three-dimensional (3D) co-culture technique was employed to bioengineer an improved human proximal tubule tissue model through incorporating human renal proximal tubule epithelial cells (RPTECs) with two types of interstitial cells on the layered extracellular matrix-like culture matrix. The resulting cultures were characterized by their growth profile, metabolic and proliferative activity, morphological characteristics as well as their functional gene expression. Our results found that the cultures were able to enable the self-organization of RPTECs and promote the tubule-like structure formation in vitro. A well-defined lumen structure and polarized expression of some key protein markers including actin, P-gp, Na+ -K+ -ATPase, and SGLT2 were also observed in the 3D co-cultures. Moreover, compared to the 3D monocultures, the tubule-like structures formed within the 3D co-cultures displayed more significant polarity and enhanced functional gene expression. This suggested the important role played by the renal stromal cells in supporting the tubulogenesis and differentiation of RPTECs. Thus, the 3D co-culture model reported here would benefit bioengineering approaches toward more physiologically relevant proximal tubule tissue in vitro, providing more robust tool not only for better understanding kidney development and pathophysiology but also for drug screening for nephrotoxicity.

Gut Microbiota Imbalance is Related to Sporadic Colorectal Neoplasms. A Pilot Study

(1) Background: Colorectal cancer (CRC) development is sustained by multiple factors including the gut microbiota, as suggested by a growing body of evidence. Most CRCs have a sporadic (non-hereditary) onset and develop from sporadic colorectal adenomas/polyp (SCA/P). In the present study, we investigated the characteristic of anaerobic microorganisms in stool samples obtained from 20 patients with SCA/P and 20 subjects without evidence of proliferative lesions at colonoscopy (Controls). (2) Material and Methods: We designed this clinical trial using adaptive randomization by minimization. Selective culture media and Matrix Assisted Laser Desorption Ionization Time of Flight (MALDI-TOF) mass spectrometry techniques were used to identify the components of microbiota. The data obtained revealed a different variability of gut microbiota in stool samples of controls and SCA/P subjects. (3) Results: The most interesting difference was observed for Bacteroides species, which represent the 50% of all bacterial species identified in the stool samples: two species, Bacteroides stercoris and Parabacteroides distasonis, were found only in the feces from control group, whereas Bacteroides fragilis and Prevotella melaningenica species were presents only in SCA/P patients. Among Gram+ bacteria also, specific species were found in the two groups of feces: Clostridium clostridioforme, Propionibacterium avidum and Pediococcus pentasaceus were identified only in controls, while Eubacterium limosum, Clostridium innocuum and Corybebacterium xerosus were identified in SCA/P stool samples only. (4) Conclusions: Our findings suggest that, compared to control stool samples, a different intestinal microbiota is present in SCA/P stool samples, that may create a micro-environment predisposing for the development of proliferative phenomena. As a consequence, gut microbiota manipulation could be a future target for personalized treatments.

Synergistic Adhesiveness of Fibronectin with PHSRN Peptide in Gelatin Mixture Promotes the Therapeutic Potential of Human ES-Derived MSCs.

Mesenchymal stem cells (MSCs) are promising candidates for cell therapy owing to their therapeutic effect in various diseases. In general, MSCs grow efficiently in serum-containing culture media, indicating an essential role of adhesion in their mesenchymal lineage-specific propagation. Nevertheless, the use of non-human supplements in culture (xeno-free issue) in addition to the lack of control over unknown factors in the serum hampers the clinical transition of MSCs. In this study, embryonic stem cell derived mesenchymal stem cells (ES-MSCs) were used owing to their scalable production, and they expressed a series of MSC markers same as adipose-derived MSCs. The affinity of the culture matrix was increased by combining fibronectin coating with its adjuvant peptide, gelatin, or both (FNGP) on tissue culture polystyrene to compare the regenerative, therapeutic activities of ES-MSCs with a cell binding plate as a commercial control. The FNGP culture plate promoted pivotal therapeutic functions of ES-MSCs as evidenced by their increased stemness as well as anti-inflammatory and proangiogenic effects in vitro. Indeed, after culturing on the FNGP plates, ES-MSCs efficiently rescued the necrotic damages in mouse ischemic hindlimb model. This study suggests a potential solution by promoting the surface affinity of culture plates using a mixture of human fibronectin and its adjuvant PHSRN peptide in gelatin. The FNGP plate is expected to serve as an effective alternative for serum-free MSC expansion for bench to clinical transition.

In vitro endothelial cell migration from limbal edge-modified Quarter-DMEK grafts

Endothelial cell migration plays a crucial role in achieving corneal clearance after corneal transplantation when using smaller-sized endothelial grafts to increase the donor pool. In this study we investigated how different strategies of Quarter-Descemet Membrane Endothelial Keratoplasty (Quarter-DMEK) limbal graft edge modification influence peripheral endothelial cell migration in an in vitro culture environment. For this study, 15 Quarter-DMEK grafts, prepared from 7 corneas deemed ineligible for transplantation but with intact and viable endothelial cells, were embedded in a cooled biocompatible, thermoresponsive matrix for culture. The limbal edge of ten Quarter-DMEK grafts were modified, either by using a small diameter punch or by peripheral radial cuts. All Quarter-DMEK grafts showed substantial collective endothelial cell migration from the radial cut graft edges, as observed by light microscopy at standardized time intervals. Grafts were retrieved from the polymer matrix after the two-week culture for immunohistochemistry analyses of the newly formed cell monolayers; this demonstrated the presence of tightly packed and viable cells that showed higher migratory ability at the leading edge. Peripheral endothelial cell migration, however, was not triggered by increasing cell exposure to free space through surgical modifications of the far periphery. Our data suggest that alterations in the far peripheral area of Quarter-DMEK grafts were insufficient to triggering cell migration from the limbal graft edge. This may be due to transient-amplifying cells that reside in the far periphery and which lack cytokinetic directional cues. Understanding the migration capacity of the peripheral endothelium could unlock cells’ therapeutic potential which are, at present, routinely discarded from transplantation. Encouraging peripheral cell migration may also improve clinical outcomes from Quarter-DMEK, but a more effective solution is required prior to clinical implementation of modified grafts.

Silk-CNT Mediated Fibroblast Stimulation toward Chronic Wound Repair.

BackgroundDiabetic patients suffer from chronic wounds partly due to altered function of fibroblasts. Fibroblasts of diabetic patients synthesize collagen I (COLI) at a much higher level than collagen III (COLIII), resulting in delayed tissue granulation and, consequently, a delay in the overall wound healing process.MethodsWe aimed to revive the matrix protein productivity of diabetic fibroblasts by employing aligned, electrically conductive and biocompatible spider silk-CNT fibers as a cell culture matrix to mediate the electrical stimulation of fibroblasts to induce cell polarization and activation.ResultsA 5.2 and 42.7 fold increase in COLI and COLIII production was induced in diabetic fibroblasts. The stimulated cells synthesized a substantially high level of COLIII to reduce the abnormally high COLI/COLIII ratio, and the matrix metalloproteinases expression was markedly suppressed. The protein expression profile was consistent with favorable wound healing. The modulation effect was also demonstrated in normal fibroblasts of healthy individuals, suggesting that the developed method can be utilized generally for connective tissue repair. Silkworm silk-CNT fibers corroborated similar effects on restoring the function of diabetic fibroblasts.ConclusionsThe approach of using an engineered biopolymer matrix to remedy dysfunctional fibroblasts of patients offers the opportunity of developing personalized cell therapy for noninvasive treatments and inspires the design of multi-functional biometrics for effective tissue regeneration.

Microbiological Control of Cellular Products: The Relevance of the Cellular Matrix, Incubation Temperature, and Atmosphere for the Detection Performance of Automated Culture Systems

Background: The microbiological control of cellular products sometimes causes significant procedural issues for quality control laboratories. According to the European Pharmacopoeia (EP), the microbiological control of cellular products requires a 7- to 14-day incubation period at two different incubation temperatures using aerobic and anaerobic growth media. However, the suitability of these test conditions for efficient quality control can be influenced by many conditions, such as the expected microbial spectrum of contamination or the texture and composition of the cellular product. Because of interference, direct inoculation and membrane filtration as reference methods of pharmacopoeia are largely unsuitable for the microbiological control of cellular products; therefore, alternative and, above all, automated methods are the focus of interest. Objective: The aim of our study was to evaluate the method suitability and possible effects of cell matrix, incubation temperature, and oxygen pressure on the detection performance of automated culture systems. Methods: The BacT/ALERT® 3D^TM Dual T system (bioMérieux, Nürtingen, Germany) was used to evaluate the factors influencing automated microbiological control of cellular products. The tests were performed using microbial strains recommended by the EP for microbiological method suitability testing and additional relevant possible contaminants of human-derived stem-cell products under varying culture and cell matrix conditions. Results: All contaminants were detected by the system in the required period of 2–5 days. Low incubation temperatures (22°C) had overall negative effects on the detection kinetics of each type of microbial contamination. The adverse effects of the accompanying cell matrix on the detection properties of the system could be compensated in our study by incubation at 32°C in both the aerobic and the anaerobic culture conditions. Conclusion: Automated culture techniques represent a sufficient approach for the microbiological control of cellular products. The negative effects of the cell matrix and microbial contamination on the detection performance can be compensated by the application of variable culture conditions in the automated culture system.

Mastery of Cutimed Sorbact dressings

Cutimed Sorbact is a unique wound dressing that uses no antibiotics but has antibacterial effects. It is a hydrophobic dressing that acts by irreversibly binding to the hydrophobic extracellular surfaces of bacteria and fungi, thereby deactivating them. The active component in the dressing is a fatty acid derivative, dialkylcarbamoylchloride. The dressing has been used in many clinical scenarios, including on infected wounds, in prevention of infection on wounds and on central blood line puncture sites, as skin substitute in burn wounds, on donor sites and as a cell culture matrix. The optimal wound environment for the dressing is a relatively dry environment to enhance the hydrophobic mechanism of action, and secondary dressings should be selected accordingly. The dressing should be removed when saturated by fluids, including blood, and dressing-to-wound-bed contact is essential for good outcomes. It is not to be used on the upper eyelids, because it will inhibit eyelid closure. The dressing can be used as a first-line choice in wound-dressing antibacterial stewardship. Once the use of the dressing has been mastered in the different scenarios, it becomes an integral part of daily wound care.

Bio-engineering of fetal cartilage for in utero spina bifida repair.

During in utero surgical spina bifida repair, a multi-layer closure is used to cover the defect. These soft tissues, however, might be not sufficient to protect the spinal cord during the future life. Our goal is to develop a more rigid protective tissue construct consisting of bioengineered cartilage and skin. Ovine fetal chondrocytes were tested for their in vitro chondrogenic potential in three-dimensional cultures. Scaffolds based on natural biopolymers (collagen I, fibrin glue) were loaded with varying amounts of fetal chondrocytes and assessed for their ability to support cartilage formation in vitro. The bioengineered constructs were analyzed using cartilage-specific histology stainings and compared to native fetal cartilage. Fetal chondrocytes actively produced cartilage extracellular matrix in three-dimensional cultures, even at high passages. Among all bioengineered scaffolds, only the collagen I-based hydrogels loaded with high densities of fetal chondrocytes showed cartilage-like structure in vitro but also extensive shrinking. Fetal chondrocytes represent a good cell source for cartilage bioengineering. Collagen I scaffolds support cartilage formation in vitro, but the construct shrinking constitutes a major limitation. Future steps include the identification of suitable bioprintable materials which maintain their shape and size, as well as the analysis of the interphase between bioengineered cartilage and skin.

Pharmaceutical Research on Liver Diseases Using iPS Cell and Genome Editing Technologies

The liver is a major organ responsible for maintaining the body's homeostasis and xenobiotic metabolism. Liver transplantation is essential for the alleviation of many severe liver diseases. However, there are many patients who cannot receive liver transplants because of donor shortage. Therefore development of effective therapeutic drugs that can replace the need for liver transplantation is desired. To this end, model cells that faithfully reproduce hepatic functions are essential. It is expected that human induced pluripotent stem cell (iPS)-derived hepatocyte-like cells, which faithfully reproduce hepatic functions, would be a valuable tool for drug discovery. Hepatic differentiation from human iPS cells has been performed using growth factors, but the hepatic differentiation efficiency was quite low and liver functions of human iPS cell-derived hepatocyte-like cells were lower than those of primary human hepatocytes. Therefore we tried to improve the hepatic differentiation technology using gene transfer, genome editing, three-dimensional culture, and extracellular matrix technologies. As a result, the purity of human iPS cell-derived hepatocyte-like cells was improved into 90% or more, and the liver functions of human iPS cell-derived hepatocyte-like cells were improved to a level comparable to primary human hepatocytes. In this article, we introduce the research results we have acquired over the last decade.