Single Cell Culture System Research Articles

Forming Single-Cell-Derived Colon Cancer Organoid Arrays on a Microfluidic Chip for High Throughput Tumor Heterogeneity Analysis.

Single-cell-derived tumor organoids (STOs) possess a distinct genetic background, making them valuable tools for demonstrating tumor heterogeneity. In order to fulfill the high throughput demands of STO assays, we have developed a microfluidic chip containing 30 000 microwells, which is dedicated to a single cell culture approach for selective expansion and differential induction of cancer stem cells. The microwells are coated with a hydrophilic copolymer to eliminate cell adhesion, and the cell culture is supported by poly(ethylene glycol) (PEG) to establish a nonadhesive culture environment. By utilizing an input cell density of 7 × 103·mL-1, it is possible to construct a 4000 single cell culture system through stochastic cell occupation. We demonstrate that the addition of 15% PEG10000 in the cell culture medium effectively prevents cell loss while facilitating tumor stem cell expansion. As were demonstrated by HCT116, HT29, and SW480 colon cancer cells, the microfluidic approach achieved a STO formation rate of ∼20%, resulting in over 800 STOs generated from a single culture. Comprehensive analysis through histomorphology, immunohistochemistry, drug response evaluation, assessment of cell invasion, and biomarker detection reveals the heterogeneity among individual STOs. Specifically, the smaller STOs exhibited higher invasion and drug resistance capabilities compared with the larger ones. The developed microfluidic approach effectively facilitates STO formation and offers promising prospects for investigating tumor heterogeneity, as well as conducting personalized therapy-focused drug screening.

Synoviocytes assist in modulating the effect of Ross River virus infection in micromass-cultured primary human chondrocytes.

Introduction. Ross River virus (RRV) is a mosquito-borne virus prevalent in Australia and the islands of the South Pacific, where it causes an arthritogenic illness with a hallmark feature of severe joint pain. The joint space is a unique microenvironment that contains cartilage and synovial fluid. Chondrocytes and synoviocytes are crucial components of the joint space and are known targets of RRV infection.Hypothesis/Gap statement. Understanding the relationship between synoviocytes and chondrocytes during RRV infection will provide further insights into RRV-induced joint pathology.Methodology. To better understand the unique dynamics of these cells during RRV infection, we used primary chondrocytes cultured in physiologically relevant micromasses. We then directly infected micromass chondrocytes or infected primary fibroblast-like synoviocytes (FLS), co-cultured with micromass chondrocytes. Micromass cultures and supernatants were collected and analysed for viral load with a PCR array of target genes known to play a role in arthritis.Results. We show that RRV through direct or secondary infection in micromass chondrocytes modulates the expression of cellular factors that likely contribute to joint inflammation and disease pathology, as well as symptoms such as pain. More importantly, while we show that RRV can infect micromass-cultured chondrocytes via FLS infection, FLS themselves affect the regulation of cellular genes known to contribute to arthritis.Conclusion. Single-cell culture systems lack the complexity of in vivo systems, and understanding the interaction between cell populations is crucial for deciphering disease pathology, including for the development of effective therapeutic strategies.

Effect of gut microbiome-derived metabolites and extracellular vesicles on hepatocyte functions in a gut-liver axis chip

Metabolism, is a complex process involving the gut and the liver tissue, is difficult to be reproduced in vitro with conventional single cell culture systems. To tackle this challenge, we developed a gut-liver-axis chip consisting of the gut epithelial cell chamber and three-dimensional (3D) uniform-sized liver spheroid chamber. Two cell culture chamber compartments were separated with a porous membrane to prevent microorganisms from passing through the chamber. When the hepG2 spheroids cultured with microbiota-derived metabolites, we observed the changes in the physiological function of hepG2 spheroids, showing that the albumin and urea secretion activity of liver spheroids was significantly enhanced. Additionally, the functional validation of hepG2 spheroids treated with microbiota-derived exosome was evaluated that the treatment of the microbiota-derived exosome significantly enhanced albumin and urea in hepG2 spheroids in a gut-liver axis chip. Therefore, this gut-liver axis chip could be a potentially powerful co-culture platform to study the interaction of microbiota and host cells.Graphical

Generation of bispecific antibodies by structure-guided redesign of IgG constant regions.

Bispecific antibodies (BsAbs) form an exciting class of bio-therapeutics owing to their multispecificity. Although numerous formats have been developed, generation of hetero-tetrameric IgG1-like BsAbs having acceptable safety and pharmacokinetics profiles from a single cell culture system remains challenging due to the heterogeneous pairing between the four chains. Herein, we employed a structure-guided approach to engineer mutations in the constant domain interfaces (CH1-CL and CH3-CH3) of heavy and κ light chains to prevent heavy-light mispairing in the antigen binding fragment (Fab) region and heavy-heavy homodimerization in the Fc region. Transient co-transfection of mammalian cells with heavy and light chains of pre-existing antibodies carrying the engineered constant domains generates BsAbs with percentage purity ranging from 78% to 85%. The engineered BsAbs demonstrate simultaneous binding of both antigens, while retaining the thermal stability, Fc-mediated effector properties and FcRn binding properties of the parental antibodies. Importantly, since the variable domains were not modified, the mutations may enable BsAb formation from antibodies belonging to different germline origins and isotypes. The rationally designed mutations reported in this work could serve as a starting point for generating optimized solutions required for large scale production.

Hypoxia promotes a perinatal-like progenitor state in the adult murine epicardium

The epicardium is a reservoir of progenitors that give rise to coronary vasculature and stroma during development and mediates cardiac vascular repair. However, its role as a source of progenitors in the adult mammalian heart remains unclear due to lack of clear lineage markers and single-cell culture systems to elucidate epicardial progeny cell fate. We found that in vivo exposure of mice to physiological hypoxia induced adult epicardial cells to re-enter the cell cycle and to express a subset of developmental genes. Multiplex single cell transcriptional profiling revealed a lineage relationship between epicardial cells and smooth muscle, stromal cells, as well as cells with an endothelial-like fate. We found that physiological hypoxia promoted a perinatal-like progenitor state in the adult murine epicardium. In vitro clonal analyses of purified epicardial cells showed that cell growth and subsequent differentiation is dependent upon hypoxia, and that resident epicardial cells retain progenitor identity in the adult mammalian heart with self-renewal and multilineage differentiation potential. These results point to a source of progenitor cells in the adult heart that can be stimulated in vivo and provide an in vitro model for further studies.

Advancing animal health and disease research in the lab with three-dimensional cell culture systems.

The development of three-dimensional cell culture systems representative of tissues from animals of veterinary interest is accelerating research that seeks to address specific questions tied to animal health. In terms of their relevance and complexity, these in vitro models can be seen as a midpoint between the more reductionist single-cell culture systems and complex live animals. Organoids in particular represent a significant development due to their organised multicellular structure that more closely represents in vivo tissues than any other cell culture technology previously developed. In this review, we provide an overview of the different three-dimensional cell culture systems available to veterinary researchers and give examples of their application in contexts relating to animal health.

Development of a refined ex vivo model of peritoneal adhesion formation, and a role for connexin 43 in their development.

Despite many advances across the surgical sciences, post-surgical peritoneal adhesions still pose a considerable risk in modern-day procedures and are highly undesirable. We have developed a novel mouse peritoneal strip ex vivo adhesion model which may serve to bridge the gap between single cell culture systems and in vivo animal drug testing for the assessment of potential anti-adhesion agents, and study of causality of the process. We investigated the optimal conditions for adhesion formation with mouse peritoneal tissue strips by modifying an existing ex vivo rat model of peritoneal adhesions. We assessed the impact of the following conditions on the formation of adhesions: contact pressure, abrasions, and the presence of clotted blood. Macroscopic adhesions were detected in all mouse peritoneal strips exposed to specific conditions, namely abrasions and clotted blood, where peritoneal surfaces were kept in contact with pressure using cotton gauze in a tissue cassette. Adhesions were confirmed microscopically. Interestingly, connexin 43, a gap junction protein, was found to be upregulated at sites of adhesions. Key features of this model were the use of padding the abraded tissue with gauze and the use of a standardised volume of clotted blood. Using this model, peritoneal strips cultured with clotted blood between abraded surfaces were found to reproducibly develop adhesion bands at 72h. Our goal is to develop a model that can be used in genetically modified mice in order to dissect out the role of particular genes in adhesion formation and to test drugs to prevent adhesion formation.

A Single-Cell Culture System for Dissecting Microenvironmental Signaling in Development and Disease of Cartilage Tissue.

Cartilage tissue is comprised of extracellular matrix and chondrocytes, a cell type with very low cellular turnover in adults, providing limited capacity for regeneration. However, in development a significant number of chondrocytes actively proliferate and remodel the surrounding matrix. Uncoupling the microenvironmental influences that determine the balance between clonogenic potential and terminal differentiation of these cells is essential for the development of novel approaches for cartilage regeneration. Unfortunately, most of the existing methods are not applicable for the analysis of functional properties of chondrocytes at a single cell resolution. Here we demonstrate that a novel 3D culture method provides a long-term and permissive in vitro niche that selects for highly clonogenic, colony-forming chondrocytes which maintain cartilage-specific matrix production, thus recapitulating the in vivo niche. As a proof of concept, clonogenicity of Sox9IRES–EGFP mouse chondrocytes is almost exclusively found in the highest GFP+ fraction known to be enriched for chondrocyte progenitor cells. Although clonogenic chondrocytes are very rare in adult cartilage, we have optimized this system to support large, single cell-derived chondrogenic organoids with complex zonal architecture and robust chondrogenic phenotype from adult pig and human articular chondrocytes. Moreover, we have demonstrated that growth trajectory and matrix biosynthesis in these organoids respond to a pro-inflammatory environment. This culture method offers a robust, defined and controllable system that can be further used to interrogate the effects of various microenvironmental signals on chondrocytes, providing a high throughput platform to assess genetic and environmental factors in development and disease.

Efficient Neural Differentiation using Single-Cell Culture of Human Embryonic Stem Cells.

In vitro differentiation of human embryonic stem cells (hESCs) has transformed the ability to study human development on both biological and molecular levels and provided cells for use in regenerative applications. Standard approaches for hESC culture using colony type culture to maintain undifferentiated hESCs and embryoid body (EB) and rosette formation for differentiation into different germ layers are inefficient and time-consuming. Presented here is a single-cell culture method using hESCs instead of a colony-type culture. This method allows maintenance of the characteristic features of undifferentiated hESCs, including expression of hESC markers at levels comparable to colony type hESCs. In addition, the protocol presents an efficient method for neural progenitor cell (NPC) generation from single-cell type hESCs that produces NPCs within 1 week. These cells highly express several NPC marker genes and can differentiate into various neural cell types, including dopaminergic neurons and astrocytes. This single-cell culture system for hESCs will be useful in investigating the molecular mechanisms of these processes, studies of certain diseases, and drug discovery screens.

Relationship between Early Peak Temperature and Neutropenia Duration in Acute Leukemia Patients after Chemotherapy and Its Mechanism

To investigate the relationship between early peak body temperature and neutropenia duration and its potential mechanism. A total of 111 patients with CR1 phase acute leukemia (AL) with neutropenia infection were enrolled in this study. The relationship between early peak body temperature and neutropenia duration was analyzed retrospectively, and the IL-6 serum level in patients with different peak of body temperature was detected, and the single cell culture system in vitro was established, the incorparation rate of EdU in vivo was detected, and the effect of IL-6 on mouse hematopoietic stem cells /progenitor cells was analyzed. Out of 111 patients with nentropenia, the body temperature <38 °C and the neutropenia duration 9.5±3.69 d were observed in 44 patients, while the body temperature >38 °C and neutropenia duration 7.33±4.20 d were observed in 69 patients, the differences between 2 groups was statistically signficant (P<0.05). The EdU test showed that the number of EdU+ hematopoietic stem cells and progenitor cells increased. The IL-6 level was different in patients with different peaks of initial bady temperature (P<0.05). The results of amimal experiment showed that the IL-6 could promote the proliferation of hematopoietic stem cells/ progenitor cells in vitro and in vivo. For patients with neutropenic infection when initial body temperature peak is <38 °C, the probability of neutropenia duration prolonging after chamotherapy increases, which may relate with promotive effect of pro-inflammatory cytokins on prliferation of hematopoietic stem cells/progenitor cells.

Alcohol-tolerant mutants of cyanobacterium Synechococcus elongatus PCC 7942 obtained by single-cell mutant screening system.

Enhancement of alcohol tolerance in microorganisms is an important strategy for improving bioalcohol productivity. Although cyanobacteria can be used as a promising biocatalyst to produce various alcohols directly from CO2 , low productivity, and low tolerance against alcohols are the main issues to be resolved. Nevertheless, to date, a mutant with increasing alcohol tolerance has rarely been reported. In this study, we attempted to select isopropanol (IPA)-tolerant mutants of Synechococcus elongatus PCC 7942 using UV-C-induced random mutagenesis, followed by enrichment of the tolerant candidates in medium containing 10 g/L IPA and screening of the cells with a high growth rate in the single cell culture system in liquid medium containing 10 g/L IPA. We successfully acquired the most tolerant strain, SY1043, which maintains the ability to grow in medium containing 30 g/L IPA. The photosynthetic oxygen-evolving activities of SY1043 were almost same in cells after 72 h incubation under light with or without 10 g/L IPA, while the activity of the wild-type was remarkably decreased after the incubation with IPA. SY1043 also showed higher tolerance to ethanol, 1-butanol, isobutanol, and 1-pentanol than the wild type. These results suggest that SY1043 would be a promising candidate to improve alcohol production using cyanobacteria. Biotechnol. Bioeng. 2017;114: 1771-1778. © 2017 Wiley Periodicals, Inc.

Dnd Is a Critical Specifier of Primordial Germ Cells in the Medaka Fish.

SummaryPrimordial germ cell (PGC) specification occurs early in development. PGC specifiers have been identified in Drosophila, mouse, and human but remained elusive in most animals. Here we identify the RNA-binding protein Dnd as a critical PGC specifier in the medaka fish (Oryzias latipes). Dnd depletion specifically abolished PGCs, and its overexpression boosted PGCs. We established a single-cell culture procedure enabling lineage tracing in vitro. We show that individual blastomeres from cleavage embryos at the 32- and 64-cell stages are capable of PGC production in culture. Importantly, Dnd overexpression increases PGCs via increasing PGC precursors. Strikingly, dnd RNA forms prominent particles that segregate asymmetrically. Dnd concentrates in germ plasm and stabilizes germ plasm RNA. Therefore, Dnd is a critical specifier of fish PGCs and utilizes particle partition as a previously unidentified mechanism for asymmetric segregation. These findings offer insights into PGC specification and manipulation in medaka as a lower vertebrate model.

A single cell culture system using lectin-conjugated magnetite nanoparticles and magnetic force to screen mutant cyanobacteria.

Cyanobacteria can be utilized as a potential biocatalyst for the production of biofuels and biochemicals directly from CO2. Useful mutants of cyanobacteria, which can grow rapidly or are resistant to specific metabolic products, are essential to improve the productivity of biofuels. In this study, we developed a single cell culture system to effectively screen mutant cyanobacteria using magnetite nanoparticles and magnetic force. Lens culinaris Agglutinin (LCA) was selected as a lectin, which binds to the surface of Synechococcus elongatus PCC7942 cells and the LCA-conjugated magnetite cationic liposomes (MCLs) were developed for magnetic labeling of PCC7942 cells. The MCL-labeled PCC7942 cells were magnetically patterned at a single cell level by using 6,400 iron pillars of the pin-holder device. The device enabled 1,600 single cells to be arrayed in one square centimeter. We cultured the patterned cells in liquid medium and achieved higher colony-forming ratio (78.4%) than that obtained using conventional solid culture method (4.8%). Single cells with different properties could be distinguished in the single cell culture system depending on their growth. Furthermore, we could selectively pick up the target cells and subsequently perform efficient isolation culture. The ratio of successful isolation culture using the developed method was 13 times higher than that of the conventional methods. Thus, the developed system would serve as a powerful tool for screening mutant cyanobacteria.

Innovations in studying in vivo cell behavior and pharmacology in complex tissues – microvascular endothelial cells in the spotlight

Many studies on the molecular control underlying normal cell behavior and cellular responses to disease stimuli and pharmacological intervention are conducted in single-cell culture systems, while the read-out of cellular engagement in disease and responsiveness to drugs in vivo is often based on overall tissue responses. As the majority of drugs under development aim to specifically interact with molecular targets in subsets of cells in complex tissues, this approach poses a major experimental discrepancy that prevents successful development of new therapeutics. In this review, we address the shortcomings of the use of artificial (single) cell systems and of whole tissue analyses in creating a better understanding of cell engagement in disease and of the true effects of drugs. We focus on microvascular endothelial cells that actively engage in a wide range of physiological and pathological processes. We propose a new strategy in which in vivo molecular control of cells is studied directly in the diseased endothelium instead of at a (far) distance from the site where drugs have to act, thereby accounting for tissue-controlled cell responses. The strategy uses laser microdissection-based enrichment of microvascular endothelium which, when combined with transcriptome and (phospho)proteome analyses, provides a factual view on their status in their complex microenvironment. Combining this with miniaturized sample handling using microfluidic devices enables handling the minute sample input that results from this strategy. The multidisciplinary approach proposed will enable compartmentalized analysis of cell behavior and drug effects in complex tissue to become widely implemented in daily biomedical research and drug development practice.

Abstract 5357: Evaluation of colony formation and growth of malignant glioma cells in soft agar using advanced colony counter system

Abstract [Introduction] Malignant gliomas are the most common primary tumors of the central nervous system. The prognosis of patients with malignant gliomas is poor in spite of current intensive therapy because of inevitable regrowth or recurrence. Since glioma stem-like cells (GSCs) are thought to play critical role in initiation, regrowth and recurrence, effective therapy against GSCs is sought for. Efficient anticancer therapies should suppress not only the growth of tumor mass but also colony formation initiated by single cancer stem cell. Therefore, it is essential to analyze the effect of anticancer therapies on both tumor growth and colony formation. Regarding tumor growth, multicellular tumor spheroid (MCTS) model is thought to be more plausible method than 2-D culture because MCTSs resemble intervascular regions of solid tumors, that is, MCTSs reproduce 3-D structure-specific features such as intense paracrine, cell-cell adhesion related signaling, direct intercellular transport of materials, gradient of oxygen and nutrients, and lower drug penetration into the spheroids. Since GSCs form MCTSs in the standard stem cell culture media, viability and growth of GSCs have been analyzed mainly in this culture condition. However, regarding analysis of clonogenicity, colony formation assay using GSCs has been a challenge because GSCs aggregate in the stem cell culture media and evaluation of the accurate colony number requires single cell culture system that might need large number of wells/plates, especially for combination treatment, and might not be always appropriate for high-throughput screening. Therefore, a better approach is necessary. GelCountTM colony counter was developed to analyze both number and volume of colonies. In this study, we used soft agar and GelCountTM to analyze colony formation and growth of malignant glioma cells including GSCs. [Materials and Methods] Human glioma stem-like cells GSC11 and GSC20, or human malignant glioma cell line U87-MG were cultured in agarose gel and treated with erlotinib, sorafenib, imatinib or a polynuclear platinum compound BBR3610. The colony number and the calculated total colony volume (total biomass) were analyzed using GelCountTM colony counter system (Oxford Optronix Inc., UK). [Results] Glioma cells formed 3-D colonies in agarose, and the colony number and the total biomass increased according to the Gompertz function that is known to correlate with tumor growth in vivo. Each drug suppressed increase of colony number and total biomass in comparable concentration to that in 2-D culture. [Conclusions] GelCountTM allowed accurate, rapid monitoring of colony number and size at many time points without harm to the cultures. This system can be high throughput screening method using GSCs and also bridge the gap between 2-D culture assays and in vivo tumor formation assays. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5357. doi:1538-7445.AM2012-5357

The Time Course of Long-Distance Signaling in Radiation-Induced Bystander EffectIn VivoinArabidopsis thalianaDemonstrated Using Root Micro-Grafting

The radiation-induced bystander effect has been demonstrated in whole organisms as well as in multicellular tissues in vitro and single-cell culture systems in vitro. However, the time course of bystander signaling, especially in whole organisms, is not clear. Long-distance bystander/abscopal effects in vivo in plants have been demonstrated by our group. Plant grafting is a useful experimental tool for studying the root-shoot signaling of plants. In the present study, we developed a root micro-grafting technique with young seedlings of Arabidopsis thaliana in which the bystander signaling communication of root-to-shoot could easily be stopped or started at specific times after root irradiation. Using this methodology, we demonstrated the time course of long-distance signaling in radiation-induced bystander effects at the level of the organism using the expression level of the AtRAD54 gene as a biological end point. Briefly, an 8-h accumulation of damage signals in bystander parts after irradiation was essential for eliciting a bystander response. The protraction of signal accumulation was not related to the transmission speed of signaling molecules in plants and did not result from the delayed initiation of bystander signals in targeted root cells. It was suggested that the bystander effect might be induced jointly by multiple bystander signals initiated at different stages after irradiation. Moreover, reactive oxygen species (ROS) were shown to be implicated in the response process of bystander cells to radiation damage signals rather than in the generation of bystander signals in targeted cells.

Tracing blastomere fate choices of early embryos in single cell culture

AbstractBlastomeres of early vertebrate embryos undergo numerous fate choices for division, motility, pluripotency maintenance and restriction culminating in various cell lineages. Tracing blastomere fate choices at the single cell level in vitro has not been possible because of the inability to isolate and cultivate early blastomeres as single cells. Here we report the establishment of single cell culture system in the fish medaka, enabling the isolation and cultivation of individual blastomeres from 16- to 64-cell embryos for fate tracing at the single cell level in vitro. Interestingly, these blastomeres immediately upon isolation exhibit motility, lose synchronous divisions and even stop dividing in &amp;8805;50% cases, suggesting that the widely accepted nucleocytoplasmic ratio controlling synchronous divisions in entire embryos does not operate on individual blastomeres. We even observed abortive division, endomitosis and cell fusion. Strikingly, ~5% of blastomeres in single cell culture generated extraembryonic yolk syncytial cells, embryonic stem cells and neural crest-derived pigment cells with timings mimicking their appearance in embryos. We revealed the maternal inheritance of key lineage regulators and their differential expression in cleavage embryos. Therefore, medaka blastomeres possess the accessibility for single cell culture, previously unidentified heterogeneity in motility, division, gene expression and intrinsic ability to generate major extraembryonic and embryonic lineages without positioning cues. Our data demonstrate the fidelity and potential of the single cell culture system for tracking blastomere fate decisions under defined conditions in vitro.

In vitro lung slices: a powerful approach for assessment of lung pathophysiology

This review summarizes the existing literature on the use of in vitro lung slices to study pulmonary physiology, pharmacology, pathogenesis and toxicity. Since in vitro lung slices maintain cell–cell and cell–matrix relationships in a highly controllable and accessible setting, they offer many advantages over both in vivo and single-cell culture systems. With the advent of high-production slicers, lung slices can be rapidly and reproducibly generated, including from animals treated in vivo. Slices can then be treated in vitro and analyzed using high-throughput technology. Therefore, the lung-slice system offers broad, current and unrealized potential for the detection of toxicity and the delineation of pathophysiologic and therapeutic mechanisms.

CD61/ Integrin β3 Ligation Contributes to the Thrombopoietin-Mediated Niche Function of Mouse Hematopoietic Stem Cells.

Abstract 383Hematopoietic stem cells (HSCs) reside in a specialized microenvironment, bone marrow (BM) ‘niche' for maintaining self-renewal and multi-lineage differentiation capacities through the life. We recently reported that murine HSCs (CD34- c-Kit+ Sca-1+Lin-: CD34-KSL) but not CD34+KSL hematopoietic progenitors highly express CD61 (integrin β3), correlating well with long-term repopulating activity (LTRA) of HSCs (Umemoto et al., J. Immunol, 2006). However, the roles of CD61 on HSC regulation and underlying molecular mechanisms in the BM niche are to date unknown. We herein demonstrate a novel role of CD61 using CD61-deficient (CD61-/-) and mutant CD61 mice that exhibit 747 tyrosine residue substituted with alanine in the cytoplasmic tail of β3 integrin subunit (CD61Y747A KI) whereby the integrin inside-out and outside-in signaling are impaired. We found that the CD61-deficiency influences the sensitivity to 5-fluorouracil (5-FU) administration (0.25 mg/g i.v. one shot), increasing the mortality of CD61-/- mice (CD61-/- mice died within 14 days, n=20, vs. WT mice survive over 90 days, n=20, P<0.0001). HSCs per se appeared to be responsible for these results because CD61-/- CD34-KSL cells but not CD61-/- CD34+KSL cells showed accelerated cell-cycle progression compared to those of WT, suggesting that CD61 might contribute to maintenance of quiescent state of HSCs within the BM. It is known that thrombopoietin (TPO) acts on not only potent hematopoietic differentiation from HSCs but also for maintaining quiescent state of HSCs. Accordingly, we examined the relationship between TPO effect and integrin signaling through CD61 in HSCs. The in vitro single cell culture system showed the decreased cell proliferation and survival in CD61-/- CD34-KSL cells that were cultured in the presence of TPO (50 ng/mL), compared to the WT CD34-KSL cells (P<0.01). In contrast, CD61-deficency did not influence the phenotype of cultured CD34-KSL cells in the presence of stem cell factor (SCF, 50 ng/ml). Interestingly, these results were mirrored by CD61Y747A KI CD34-KSL cells and their littermate control CD34-KSL cells. Furthermore, when 40 CD34-KSL cells after 5 days culture in the presence of TPO and they were then applied into long-term competitive repopulation assay, CD61Y747A KI CD34-KSL cells showed less LTRA than that of WT (P<0.01). These results indicated that tyrosine at 747 of the β3 integrin subunit was required for the TPO-dependent expansion/maintenance of HSC in vitro. Whereas, to initiate integrin outside-in signaling in the presence of TPO, CD34-KSL cells were cultured on a well of 96-well plates pre-coated with various CD61 ligands (either vitronectin, 5 μg/ml or osteopontin, 5 μg/mL), resulting in the enhancement of their LTRA, compared to without these ligands. Since these ligands could interact with integrin receptors besides CD61, we utilized the antibody to CD61, 2C9.G2 that is capable of phosphorylating CD61 to mimic the actions of outside-in signaling. Using CD34-KSL cells were cultured with 2C9.G2, we further confirmed that CD61 signaling allowed CD34-KSL cells to maintain undivided state in vitro and increase LTRA in vivo, which were dependent upon the presence of TPO but neither SCF nor other growth factors including insulin growth factor-2 and fibroblast growth factor-1. More interestingly, both stimulation with CD61 ligation and TPO influenced ex vivo expansion of HSCs when started at 40 CD34-KSL cells in a 96-well culture well. Collectively, our results indicate that CD61/β3 integrin signaling plays important roles in TPO-mediated expansion/maintenance of HSC within the BM niche, which are possibly mediated through the tyrosine phosphorylation at 747 tyrosine residue of the β3 integrin cytoplasmic domain. Disclosures:No relevant conflicts of interest to declare.

Enrichment and Clonal Culture of Progenitor Cells During Mouse Postnatal Liver Development in Mice

Stem and progenitor cells exist in normal postnatal livers. However, it has not been possible to clonally isolate or analyze postnatal liver stem/progenitor-like cells (PLSCs) derived from noninjured livers because of a lack of specific surface markers. This study aimed to establish a primary culture system for clone-sorted PLSCs. To investigate proliferation and differentiation of PLSCs, subpopulations of nonparenchymal cells derived from noninjured livers were purified and cultured using a single-cell culture system. Cells were grown in fetal liver cell-derived conditioned medium in the presence of the Rho-associated kinase (ROCK) inhibitor Y-27632. We identified CD13 and CD133 as markers expressed on the PLSC-containing population in noninjured livers and established an efficient single-cell culture system to clonally analyze PLSCs. Culture of PLSCs is difficult, even using conditioned medium, but the addition of Y-27632 increased PLSC cell proliferation. The proportion of progenitor cells among nonparenchymal cells decreased during postnatal liver development; however, a PLSC population was still preserved in 3-month-old mice. Long-term cultivated cells derived from clone-sorted cells in normal livers were established and were called normal-liver-derived stem-like cells (NLS cells). NLS cells could differentiate into hepatocyte-like and cholangiocyte-like cells under appropriate culture conditions and underwent self-renewal-like activity in serial reclone-sorted culture. CD13 and CD133 were expressed on progenitor cells derived from fetal and postnatal liver, whereas CD49f (integrin alpha6 subunit) was strongly expressed only on PLSCs. These results demonstrate the presence of progenitor cells in the CD13(+)CD49f(+)CD133(+) subpopulation of nonhematopoietic cells derived from noninjured postnatal livers.