Learning Towards Maturation of Defined Feeder-free Pluripotency Culture Systems: Lessons from Conventional Feeder-based Systems.

Pluripotent stem cells (PSCs) are widely recognized as one of the most promising types of stem cells for applications in regenerative medicine, tissue engineering, disease modeling, and drug screening. This is due to their unique ability to differentiate into cells from all three germ layers and their capacity for indefinite self-renewal. Initially, PSCs were cultured using animal feeder cells, but these systems presented several limitations, particularly in terms of Good Manufacturing Practices (GMP) regulations. As a result, feeder-free systems were introduced as a safer alternative. However, the precise mechanisms by which feeder cells support pluripotency are not fully understood. More importantly, it has been observed that some aspects of the need for feeder cells like the optimal density and cell type can vary depending on conditions such as the developmental stage of the PSCs, phases of the culture protocol, the method used in culture for induction of pluripotency, and intrinsic variability of PSCs. Thus, gaining a better understanding of the divergent roles and necessity of feeder cells in various conditions would lead to the development of condition-specific defined feeder-free systems that resolve the failure of current feeder-free systems in some conditions. Therefore, this review aims to explore considerable feeder-related issues that can lead to the development of condition-specific feeder-free systems.

Isolation and propagation of bovine blood-derived macrophages using a mixed culture with bovine endothelial B46 cells.

Macrophages are innate immune cells with multiple functions such as phagocytosis, cytokine production, and antigen presentation. Since macrophages play critical roles in some bacterial infectious diseases in cattle, including tuberculosis, paratuberculosis, and brucellosis, the in vitro culturing of bovine macrophages is useful for evaluating host-pathogen interactions at the cellular and molecular levels. We have previously reported the establishment of two immortalized bovine liver sinusoidal cell lines, endothelial B46 cells and myofibroblast-like A26 cells (Cell Biology International, 40, 1372-1379, 2016). In this study, we investigated the use of these cell lines as feeder cells that support the proliferation of bovine blood-derived macrophages (BBMs). Notably, the B46 cell line efficiently acts as feeder cells for the propagation of BBMs. Compared with primary cultured vascular endothelial cells, the infinite proliferation ability of B46 cells is more beneficial for preparing confluent feeder layers. In conclusion, this study provides a simple and efficient protocol for the isolation and propagation of BBMs using a primary mixed culture of bovine whole blood with B46 feeder cells. Isolated BBMs are expected to be useful for developing in vitro models for studying the interactions between bovine pathogens and host immune cells.

Generation of canine induced pluripotent stem cells under feeder-free conditions using Sendai virus vector encoding six canine reprogramming factors.

Although it is in its early stages, canine induced pluripotent stem cells (ciPSCs) hold great potential for innovative translational research in regenerative medicine, developmental biology, drug screening, and disease modeling. However, almost all ciPSCs were generated from fibroblasts, and available canine cell sources for reprogramming are still limited. Furthermore, no report is available to generate ciPSCs under feeder-free conditions because of their low reprogramming efficiency. Here, we reanalyzed canine pluripotency-associated genes and designed canine LIN28A, NANOG, OCT3/4, SOX2, KLF4, and C-MYC encoding Sendai virus vector, called 159cf. and 162cf. We demonstrated that not only canine fibroblasts but also canine urine-derived cells, which can be isolated using a noninvasive and straightforward method, were successfully reprogrammed with or without feeder cells. ciPSCs existed in undifferentiated states, differentiating into the three germ layers in vitro and in vivo. We successfully generated ciPSCs under feeder-free conditions, which can promote studies in veterinary and consequently human regenerative medicines.

Purification of Mouse Embryonic Fibroblasts (MEFs).

Mouse embryonic fibroblasts (MEFs) are primary fibroblasts purified from mouse embryos at a defined time post-fertilization. MEFs have versatile applications, including use as feeder cell layers or sources of untransformed primary cells for a variety of biological assays. MEFs are most commonly isolated between embryonic day (E)12.5 and E13.5 but can be isolated from embryos as early as E8.5 and as late as E15.5. The individual embryos are harvested by carefully removing uterine tissue, yolk sac, and placenta. The embryos are euthanized, and non-mesenchymal tissues, such as the fetal liver and heart, are removed before tissue homogenization. The remaining fetal tissue is homogenized by mechanical mincing using a sterile blade, followed by enzymatic digestion and resuspension. During tissue dissociation, the duration of trypsin-EDTA/DNase digestion and enzyme concentration are critical parameters to produce high-quality MEFs with the highest rates of cell viability and proliferation potential. MEFs can be cryopreserved at passage (P) 0 if >80% confluent, passaged for further expansion before freezing down, or directly utilized for downstream applications, i.e., preparation as feeder cell layers. Primary MEFs possess a limited proliferation capacity of ∼20 cell divisions, beyond which the percentage of senescent cells rapidly increases; thus, cultures should only be expanded/passaged to a maximum of P5. Critical for cell viability during cryopreservation and thawing of MEFs is the slow decrease in temperature when freezing, the rapid increase when thawing, the use of a cryoprotective agent, and an optimal cell density. While it is critical to generate high-quality MEFs to standardize and optimize preparation procedures and utilize fresh reagents, some variability in proliferation capacity and cell viability between MEF preparations remains. Thus, MEF preparation, culture, and cryopreservation procedures are continuously being optimized. © 2023 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol: Purification, passaging, and expansion of MEFs Supporting Protocol: Cryopreservation and thawing of MEFs.

Development of Reproducible and Scalable Culture Conditions for In Vitro Maintenance of Pig Embryonic Stem Cells Using the Sandoz Inbred Swiss Mouse Thioguanine-Resistant Ouabain-Resistant Cell Line as a Feeder Layer.

Feeder cells play a crucial role in maintaining the pluripotency of embryonic stem cells (ESCs) by secreting various extrinsic regulators, such as extracellular matrix (ECM) proteins and growth factors. Although primary mouse embryonic fibroblasts (MEFs) are the most widely used feeder cell type for the culture of ESCs, they have inevitable disadvantages such as batch-to-batch variation and labor-intensive isolation processes. Here, we revealed that the Sandoz inbred Swiss Mouse (SIM) thioguanine-resistant ouabain-resistant (STO) cell line, an immortalized cell line established from mouse SIM embryonic fibroblasts, can be used as a feeder layer for in vitro culture of authentic pig ESCs instead of primary MEFs. First, the expression of genes encoding ECM proteins and growth factors was analyzed to compare their secretory functions as feeder cells. Quantitative real-time polymerase chain reaction (qPCR) showed that the gene expression of these pluripotency-associated factors was downregulated in STO cells compared to primary MEFs of similar density. Therefore, subsequent optimization of the culture conditions was attempted using higher STO cell densities. Notably, pig ESCs cultured on STO cell density of 3 × (187,500 cells/cm2) exhibited the most similar pluripotent state to pig ESCs cultured on primary MEF density of 1 × (62,500 cells/cm2), as determined by alkaline phosphatase staining, qPCR, and immunocytochemistry. In addition, pig ESCs cultured on STO cell density of 3 × formed complex teratoma containing multiple types of tissues derived from all three germ layers. Our culture conditions using optimal STO cell density can be applied to fields requiring reproducible and scalable production of pig ESCs, such as preclinical research and cellular agriculture.

Development of Genetically Engineered Feeder Cells for Natural Killer Cell Expansion.

BACKGROUND/AIM: To obtain sufficient numbers of high-quality natural killer (NK) cells, we developed feeder cells using synthetic biology techniques. MATERIALS AND METHODS: K562 cells were engineered to express membrane bound interleukin-2 (mbIL2) or interleukin-13 (mbIL13). RESULTS: The incubation of human primary NK cells isolated from peripheral blood mononuclear cells (PBMCs) with these feeder cells significantly increased the number of activated NK cells compared to K562 parental cells. Fluorescence-activated cell sorting (FACS) analysis demonstrated that NKG2D activating receptors were abundant on the surface of NK cells expanded by K562-mbIL2 or mbIL13 cells. NK cells expanded on K562-mbIL2 or mbIL13 lysed cancer cells more effectively than those cultured with normal K562 cells. Using NK cells incubated with our feeder cells, we developed anti-CD19 chimeric antigen receptor (CAR)-NK cells. They showed robust cytotoxic effect against CD19 positive cancer cell line. CONCLUSION: Our newly developed feeder cells could provide useful tools for NK cell therapy.

Rapid, Cost-Efficient, Enzyme-Free Passaging of Human Pluripotent Stem Cells on Feeder Cells by Ethylenediaminetetraacetic Acid-Mediated Dis-Adhesion.

Human pluripotent stem cells (human embryonic stem cells, hESCs, and human induced pluripotent stem cells, hiPSCs) were originally cultured on different types of feeder cells for maintenance in an undifferentiated state in long-term culture. This approach has been supplanted to a large extent by feeder-free culture protocols, but these involve more costly reagents and can promote a transition to a primed state, which restricts the cells' differentiation capacity. In both feeder and feeder-free conditions, the harvesting of hESC or hiPSC colonies for passaging is a necessary procedure for expanding the cultures. To provide an easy and high-yield procedure for passaging hESCs/hiPSCs cultured on feeder cells, we have established a harvesting method using dis-adhesion elicited by the calcium chelator ethylenediaminetetraacetic acid (EDTA). We have assessed the yield and quality of the resultant passaged cells by comparing this approach to the original mechanical harvesting approach, in which colonies are isolated with a scalpel under a microscope (mechanical harvesting was chosen as a comparator to avoid the reagent variability associated with enzymatic harvesting). In one set of experiments, two different hESC lines were maintained on a feeder cell layer of human foreskin fibroblasts. Each line was subjected to multiple passages using EDTA-based or mechanical harvesting and assessed for colony size and morphology, cell density, stemness marker expression, differentiation to the three germ layers in embryoid bodies, and genomic aberrations. In another set of experiments, we used EDTA-based harvesting on two different hiPSC lines and obtained similar results. EDTA-induced dis-adhesion saved time and gave a higher yield of colonies of a more favorable size and more uniform morphology compared to mechanical harvesting. It was also faster than enzymatic harvesting and not prone to enzyme batch variability. The EDTA-induced dis-adhesion method also facilitates the transfer of hESC/hiPSC lines from feeder cell-based culture to feeder-free conditions if desired for downstream use and analysis.

Comparison of the different anti-CD16 antibody clones in the activation and expansion of peripheral blood NK cells.

Natural killer (NK) cells are promising tool for cancer treatment. Methods have been developed for large-scale NK cell expansion, including feeder cell-based methods or methods involving stimulation with NK cell activating signals, such as anti-CD16 antibodies. Different clones of anti-CD16 antibodies are available; however, a comprehensive comparison of their differential effects on inducing NK cell activation and expansion has not been conducted among these various clones under the same experimental conditions. Herein, we found that the NK cell expansion rate differed depending on the various anti-CD16 antibodies (CB16, 3G8, B73.1, and MEM-154) coated on microbeads when stimulated with genetically engineered feeder cells, K562­membrane-bound IL­18, and mbIL­21 (K562­mbIL­18/-21). Only the CB16 clone combination caused enhanced NK cell expansion over K562­mbIL­18/-21 stimulation alone with similar NK cell functionality. Treatment with the CB16 clone once on the initial day of NK cell expansion was sufficient to maximize the combination effect. Overall, we developed a more enhanced NK expansion system by merging a feeder to effectively stimulate CD16 with the CB16 clone.

The Influence of Feeder Cell-Derived Extracellular Matrix Density on Thymic Epithelial Cell Culture.

The thymus is responsible for the selection and development of T cells, having an essential role in the establishment of adaptive immunity. Thymic epithelial cells (TECs) are key players in T cell development interacting with thymocytes in the thymic 3D environment. Feeder-layer cells have been frequently used as platforms for the successful establishment of TEC cultures. Nevertheless, the role of the feeder cell-derived extracellular matrix (ECM) on TEC cultures was not previously reported. Therefore, this work aimed at assessing the effect of the ECM produced by feeder cells cultured at two different densities on the establishment of TEC culture. Due to the high surface area and porosity, electrospun fibrous meshes were used to support ECM deposition. The feeder cell-derived ECM was efficiently recovered after decellularization, maintaining the composition of major proteins. All the decellularized matrices were permeable and showed an increase in surface mechanical properties after decellularization. TEC cultures confirmed that the ECM density impacts cellular performance, with higher densities showing a decreased cellular activity. Our findings provide evidence that feeder cell-derived ECM is a suitable substrate for TEC culture and can potentially be applied in thymus bioengineering.

Development of substrates for the culture of human pluripotent stem cells.

Although human pluripotent stem cell (hPSC) lines were initially established in culture using feeder cells, the development of culture media and substrates is essential for safe, stable, high-quality, and efficient production of large numbers of cells. Many researchers are now culturing hPSCs in chemically defined media and on culture substrates without feeder cells. In this review, we first discuss the problems with Matrigel, which has long been used as a culture substrate. Then, we summarize the development of extracellular matrix proteins for hPSCs, which are now the mainstream alternative, and synthetic substrates that are expected to be the future mainstream alternative. We also highlight three-dimensional culture for suitable mass production of hPSCs.

The adaptation of bovine embryonic stem cells to the changes of feeder layers.

Although the feeder-free culture system has been established, the microenvironment provided by the feeder cells still possesses a unique advantage in maintaining the long-term stability and the rapid proliferation of pluripotent stem cells (PSCs). The aim of this study is to discover the adaptive ability of PSCs upon changes of feeder layers. In this study, the morphology, pluripotent marker expression, differentiation ability of bovine embryonic stem cells (bESCs) cultured on low-density, or methanol fixed mouse embryonic fibroblasts were examined by immunofluorescent staining, Western blotting, real-time reverse transcription polymerase chain reaction, and RNA-seq. The results showed that the changes of feeder layers did not induce the rapid differentiation of bESCs, while resulting in the differentiation initiation and alteration of pluripotent state of bESCs. More importantly, the expression of endogenous growth factors and extracellular matrix were increased, and the expression of cell adhesion molecules was altered, which indicated that bESCs may compensate some functions of the feeder layers upon its changes. This study shows the PSCs have the self-adaptive ability responded to the feeder layer alteration.

The use of human-derived feeder layers for the cultivation of transplantable human epidermal cell sheet to repair second degree burn wounds.

BACKGROUND AND OBJECTIVES: Human epidermal cell sheet (human-ECS) is a feasible treatment option for wound injury. Traditionally, researchers often use murine 3T3 fibroblast cells as feeder layer to support human epidermal cell sheet grafts, thus increase risk to deliver animal-borne infection. To overcome the potential risks involved with xenotransplantation, we develop human foreskin fibroblast cell as feeder layer culture system and investigate the effects of human-ECS on second-degree burn wound healing in mini-pig in order to develop more effective and safer therapies to enhance wound healing in human. MATERIALS AND METHODS: Human epidermal keratinocytes and fibroblasts were isolated from foreskin tissue and were co-cultured to manufacture human-ECS. The cell morphology was monitored with phase-contrast microscopy, the stem cell markers were assessed by flow cytometry, and by colony-forming efficiency (CFE) assay. The structure of human-ECS was observed by hematoxylin and eosin staining. Expression of cytokines in human-ECS was confirmed by enzyme-linked immunosorbent assay. Second-degree burn wounds were created on the dorsal of miniature pig to evaluate the effect of oil gauze, oil gauze combined with commercial epidermal growth factor (EGF) cream, and oil gauze combined with human-ECS. Wound healing rate, histological examination, and Masson staining were measured to observe the wound repair efficacy. Real-time PCR and Western blot were utilized to detect the expression level of EGF and interleukin 6 (IL-6). RESULTS: Stratified human-ECS with 6-7 layers of epidermal cells was successfully cultivated with human-derived feeder cells, in which epidermal cell highly expressed CD49f and CFE was 3% ± 0.45%. Application of human-ECS induced a higher wound healing rate than commerical EGF cream and oil gauze control. The expression of EGF in human-ECS group was higher than those in the other groups; however, the expression of IL-6 was significantly decreased at day 14 by human-ECS treatment group. CONCLUSIONS: Human-derived feeder cells are suitable for cultivation of human-ECS, avoiding pathogen transmission. Human-ECS could enhance second-degree burn wound healing, and its promoting effect involved secreting a variety of cytokines to regulate tissue reparative process.

Optimization of bovine embryonic fibroblast feeder layer prepared by Mitomycin C.

Feeder cells play important roles in In-vitro culture of stem cells. However, the preparation protocol of feeder cells produced by bovine embryonic fibroblast cells (bEFs) is still lack. In this study, the preparation of bEF-feeder by Mitomycin C was optimized with different concentrations and treatment time. The cell viability of bEFs was detected by CCK8 and 5-Ethynyl-2'-deoxyuridine. The growth of bESCs in each bEFs-feeder group was assessed by alkaline phosphatase staining and CCK8. Quantitative real time PCR was used to detect the mRNA expression of pluripotency-related genes of bESCs. Results showed that the proliferation of bEFs was significantly repressed while bEFs were treated with 14 ug/mL or 16 ug/mL Mitomycin C for 3 h, and the cell viability within 2-4 days after treatment was consistent with the 1st day. The numbers of bESCs clones in bEF-feeder treated with 14 µg/mL Mitomycin C for 3 h or 16 µg/mL Mitomycin C for 3 h were significantly higher than that in bEF-feeder treated with 8 µg/mL Mitomycin C for 8 h or bEFs treated with 6 µg/mL Mitomycin C for 9 h. The mRNA expression of pluripotency-related genes in bESCs cultured by bEF-feeder were higher than the MEF-feeder, the clone morphology of bESCs cultured in bEF-feeder was rounder and sharper than the MEF-feeder. In conclusion, the bEF-feeder prepared with 14 µg/mL Mitomycin C for 3 h or 16 µg/mL Mitomycin C for 3 h could effectively maintains the growth of bESCs, and bEF-feeder is more suitable for bESCs culture than the MEF-feeder.

The progress in techniques for culturing human limbal epithelial stem cells.

In vitro culture of human limbal epithelial stem cells (hLESCs) is crucial to cell therapy in the treatment of limbal stem cell deficiency, a potentially vision-threatening disease that is characterized by persistent corneal epithelial defects and corneal epithelium conjunctivalization. Traditionally, hLESCs are cultivated based on either limbal tissue explants or single-cell suspensions in culture media containing xenogenous components, such as fetal bovine serum and murine 3T3 feeder cells. Plastic culture dishes and human amniotic membranes are classical growth substrates used in conventional hLESC culture systems. The past few decades have witnessed considerable progress and innovations in hLESC culture techniques to ensure a higher level of biosafety and lower immunogenicity for further cell treatment, including complete removal of xenogenous components from culture media, the application of human-derived feeder cells, and the development of novel scaffolds. Three-dimensional artificial niches and three-dimensional culture techniques have also been established to simulate the real microenvironment of limbal crypts for better cell outgrowth and proliferation. All these progresses ensure that in vitro cultured hLESCs are more adaptable to translational stem cell therapy for limbal stem cell deficiency.

Feeder cells treated with ethanol can be used to maintain self-renewal and pluripotency of human pluripotent stem cells.

Feeder cells play an important role in the culture of human pluripotent stem cells (hPSCs) in vitro. Previously, we used methanol as a fixative to prepare feeder cells for the cultivation of pluripotent stem cells (PSCs), and this method could maintain the self-renewal and pluripotency of PSCs. However, methanol is toxic, and so here we examined whether ethanol could be used to prepare feeder cells as a fixative for hPSC culturing. Primed, naïve, and extended human embryonic stem cells and induced pluripotent stem cells can maintain self-renewal and undifferentiated potential on feeder cells treated with ethanol for an extended period. RNA sequencing analysis showed that the expression of collagen-related genes in hPSCs cultured on feeder cells treated with ethanol was significantly lower as compared with hPSCs cultured on feeder cells treated with mitomycin C. Therefore, we speculate that the signaling pathway mediated by collagen-related genes may, at least in part, contribute to the maintenance of self-renewal and pluripotency of PSCs induced by feeder cells treated with chemicals.

In Situ Hydrogelation of Cellular Monolayers Enables Conformal Biomembrane Functionalization for Xeno-Free Feeder Substrate Engineering.

The intricate functionalities of cellular membranes have inspired strategies for deriving and anchoring cell-surface components onto solid substrates for biological studies, biosensor applications, and tissue engineering. However, introducing conformal and right-side-out cell membrane coverage onto planar substrates requires cumbersome protocols susceptible to significant device-to-device variability. Here, a facile approach for biomembrane functionalization of planar substrates is demonstrated by subjecting confluent cellular monolayer to intracellular hydrogel polymerization. The resulting cell-gel hybrid, herein termed GELL (gelated cell), exhibits extraordinary stability and retains the structural integrity, membrane fluidity, membrane protein mobility, and topology of living cells. In assessing the utility of GELL layers as a tissue engineering feeder substrate for stem cell maintenance, GELL feeder prepared from primary mouse embryonic fibroblasts not only preserves the stemness of murine stem cells but also exhibits advantages over live feeder cells owing to the GELL's inanimate, non-metabolizing nature. The preparation of a xeno-free feeder substrate devoid of non-human components is further shown with HeLa cells, and the resulting  HeLa GELL feeder effectively sustains the growth and stemness of both murine and human induced pluripotent stem cells. The study highlights a novel bio-functionalization strategy that introduces new opportunities for tissue engineering and other biomedical applications.

Exogenous pyruvate and recombinant human basic fibroblast growth factor maintain pluripotency and enhance global metabolic activity of bovine embryonic stem cells grown on low-density feeder layers.

A suitable microenvironment or niche is essential for self-renewal and pluripotency of stem cells cultured in vitro, including bovine embryonic stem cells (bESCs). Feeder cells participate in the construction of stem cell niche by secreting growth factors and extracellular matrix proteins. In this study, metabolomics and transcriptomics analyses were used to investigate the effects of low-density feeder cells on bESCs. The results showed that bESCs co-cultured with low-density feeder cells experienced a decrease in pluripotent gene expression, cell differentiation, and a reduction of central carbon metabolic activity. When cell-permeable pyruvate (Pyr) and recombinant human basic fibroblast growth factor (rhbFGF) were added to the culture system, the pluripotency of bESCs on low-density feeder layers was rescued, and acetyl-coenzyme A (AcCoA) synthesis and fatty acid de novo synthesis increased. In addition, rhbFGF enhances the effects of Pyr and activates the overall metabolic level of bESCs grown on low-density feeder layers. This study explored the rescue effects of exogenous Pyr and rhbFGF on bESCs cultured on low-density feeder layers, which will provide a reference for improvement of the PSC culture system through the supplementation of energy metabolites and growth factors.

Ex vivo expansion of natural killer cells for hematological cancer immunotherapy: a systematic review and meta-analysis.

The present systematic review aimed to investigate natural killer (NK) cell ex vivo expansion protocols within the scope of clinical trials targeting hematological cancer and to conduct a meta-analysis to assess the effect of NK cell infusion on survival. Research articles of clinical studies in which cell products produced by ex vivo expansion, consisting of a certain amount of NK cells and infused to patients with hematological cancer, were included in the systematic review. We conducted a proportion analysis with random effects for product purity and viability values. Studies having control groups were included in the survival meta-analysis. Among 11.028 identified records, 21 were included in the systematic review. We observed statistically significant heterogeneity for viability (I2 = 97.83%, p < 0.001) and purity values (I2 = 99.95%, p < 0.001), which was attributed to the diversity among isolation and expansion protocols. In addition, the survival meta-analysis findings suggested that NK cell therapy favors disease-free survival (DFS) of patients with myeloid malignancies but limited to only two clinical studies (odds ratio = 3.40 (confidence interval:1.27-9.10), p = 0.01). While included protocols yielded cell products with acceptable viability, the utility of immunomagnetic methods; feeder cells such as K562 expressing membrane-bound IL15 and 4-1BBL or expressing membrane-bound IL21 and 4-1BBL might be preferable to achieve better purity. In conclusion, NK cell therapy has a potential to improve DFS of patients with myeloid malignancies.

Imaging Analysis of Neurotrophic Effects by CCN2 Protein in Neuronal Precursor Cells Derived from Human-Induced Pluripotent Stem Cells.

Human-induced pluripotent stem cells (hiPSCs) are useful tools to examine human neuronal maturation processes. In this chapter, we describe the maturation of human neuronal precursor cells derived from hiPSCs by cellular communication network family member 2, also known as connective tissue growth factor. We describe the (1) preparation of feeder cells for undifferentiated culture of hiPSCs, (2) undifferentiated culture of hiPSCs, (3) induction of neuronal precursor cells from hiPSCs, (4) maturation of neuronal precursor cells from hiPSCs, (5) immunofluorescent staining of neuronal cells from hiPSCs, and (6) immunofluorescence analysis.

Feeder cell-dependent primary culture of single blastula-derived embryonic cell lines from marine medaka (Oryzias dancena).

Fish embryonic stem cells (ESCs) are derived from blastomeres that have been cultured from blastula embryos. The most widely used method for derivation of fish ESCs is the culture of blastomeres that have been isolated from approximately 10 blastula embryos under feeder-free conditions. However, this method leads to intercellular genetic heterogeneity among the cultured cells, which is a major obstacle to the development of stable ESC culture conditions. In this study, to establish ESC lines with intercellular genetic homogeneity at the early stage of culture, we attempted to derive embryonic cell lines from single blastula-derived blastomeres of marine medaka (Oryzias dancena) in a feeder cell culture system. Using basic fibroblast growth factor-expressing feeder cells during primary culture, we successfully established 22 single blastula-derived embryonic cell lines that could be subcultured more than 20 times. In contrast, we were unable to efficiently derive cell lines using wild-type feeder cells and under feeder-free conditions. The established cell lines exhibited ESC-like cell characteristics in terms of alkaline phosphatase activity, pluripotency-related gene expression, and embryoid body formation. The results of this study will contribute to the development of methods for derivation of fish ESCs.