ABSTRACT
High-throughput 3D spheroid formation from human induced pluripotent stem cells (hiPSCs) can be easily performed using the unique microfabric vessels EZSPHERE, resulting in effective and large scale generation of differentiated cells such as cardiomyocytes or neurons. Such hiPSC-derived cardiomyocytes (hiPSC-CMs) or neurons are very useful in the fields of regenerative medicine or cell-based drug safety tests. Previous studies indicated that 3D spheroids arising from hiPSCs are effectively differentiated into high quality hiPSC-CMs by controlling Wnt signals through utilization of the microfabric vessels EZSPHERE. Here, we describe a simple and highly efficient protocol for generating a large number of uniformly sized hiPSC spheroids and inducing them for cardiac differentiation using the EZSPHERE. This method comprises the collection and dissociation of the spheroids from cardiac differentiation medium, in the middle stage of the whole cardiac differentiation process, and re-seeding the obtained single cells into the EZSPHERE to re-aggregate them into uniform hiPSC-CM spheroids with controlled size. This re-aggregation process promotes non-canonical Wnt signal-related cardiac development and improves the purity and maturity of the hiPSC-CMs generated. Graphic abstract: Overview of cardiac differentiation from iPSCs by spheroid formation and reaggregation using EZSPHERE.
ABSTRACT
The tumorigenicity and toxicity of induced pluripotent stem cells (iPSCs) and their derivatives are major safety concerns in their clinical application. Recently, we developed granulocyte-macrophage colony-stimulating factor (GM-CSF)-producing proliferating myeloid cells (GM-pMCs) from mouse iPSCs as a source of unlimited antigen-presenting cells for use in cancer immunotherapy. As GM-pMCs are generated by introducing c-Myc and Csf2 into iPSC-derived MCs and are dependent on self-produced GM-CSF for proliferation, methods to control their proliferation after administration should be introduced to improve safety. In this study, we compared the efficacy of two promising suicide gene systems, herpes simplex virus-thymidine kinase (HSV-TK)/ganciclovir (GCV) and inducible caspase-9 (iCasp9)/AP1903, for safeguarding GM-pMCs in cancer immunotherapy. The expression of HSV-TK or iCasp9 did not impair the fundamental properties of GM-pMCs. Both of these suicide gene-expressing cells selectively underwent apoptosis after treatment with the corresponding apoptosis-inducing drug, and they were promptly eliminated in vivo. iCasp9/AP1903 induced apoptosis more efficiently than HSV-TK/GCV. Furthermore, high concentrations of GCV were toxic to cells not expressing HSV-TK, whereas AP1903 was bioinert. These results suggest that iCasp9/AP1903 is superior to HSV-TK/GCV in terms of both safety and efficacy when controlling the fate of GM-pMCs after priming antitumor immunity.
ABSTRACT
Interaction of cancer cells with cancer-associated fibroblasts (CAFs) plays critical roles in tumor progression. Recently we proposed a new tumor invasion mechanism in which invasive cancer cells individually migrate on elongate protrusions of CAFs (CAF fibers) in 3-D collagen matrix. In this mechanism, cancer cells interact with fibronectin fibrils assembled on CAFs mainly through integrin-α5ß1. Here we tested whether this mechanism is applicable to the collective invasion of cancer cells, using two E-cadherin-expressing adenocarcinoma cell lines, DLD-1 (colon) and MCF-7 (breast). When hybrid spheroids of DLD-1 cells with CAFs were embedded into collagen gel, DLD-1 cells collectively but very slowly migrated through the collagen matrix in contact with CAFs. Epidermal growth factor and tumor necrosis factor-α promoted the collective invasion, possibly by reducing the E-cadherin junction, as did the transforming growth factor-ß inhibitor SB431542 by stimulating the outgrowth of CAFs. Transforming growth factor-ß itself inhibited the cancer cell invasion. Efficient collective invasion of DLD-1 cells required large CAF fibers or their assembly as stable adhesion substrates. Experiments with function-blocking Abs and siRNAs confirmed that DLD-1 cells adhered to fibronectin fibrils on CAFs mainly through integrin-α5ß1. Anti-E-cadherin Ab promoted the single cell invasion of DLD-1 cells by dissociating the E-cadherin junction. Although the binding affinity of MCF-7 cells to CAFs was lower than DLD-1, they also collectively invaded the collagen matrix in a similar fashion to DLD-1 cells. Our results suggest that the direct interaction with CAFs, as well as environmental cytokines, contributes to the collective invasion of cancers.
Subject(s)
Cancer-Associated Fibroblasts/pathology , Collagen/metabolism , Fibroblasts/pathology , Fibronectins/metabolism , Integrin alpha5beta1/metabolism , Neoplasm Invasiveness/pathology , A549 Cells , Adenocarcinoma/pathology , Amides/pharmacology , Benzamides/pharmacology , Cancer-Associated Fibroblasts/metabolism , Cell Adhesion , Cell Line, Tumor , Cell Movement , Chromones/pharmacology , Colonic Neoplasms/metabolism , Colonic Neoplasms/pathology , Connective Tissue/pathology , Dioxoles/pharmacology , Epidermal Growth Factor/metabolism , Fibroblasts/metabolism , Humans , Immunohistochemistry/methods , MCF-7 Cells , Morpholines/pharmacology , Neoplasm Invasiveness/physiopathology , Pyridines/pharmacology , Spheroids, Cellular/pathology , Transforming Growth Factor beta/antagonists & inhibitors , Tumor Necrosis Factor-alpha/metabolismABSTRACT
Cardiomyocytes differentiated from human induced pluripotent stem cells (hiPSCs) have great potential for regenerative medicine and drug discovery. In this study, we developed a novel protocol to more reproducibly and efficiently induce cardiomyocytes. A large quantity of uniformly sized spheroids were generated from hiPSCs using microfabricated vessels and induced into cardiac differentiation. In the middle of the cardiac differentiation process, spheroids were then dissociated into single cells and reaggregated into smaller spheroids using the microfabricated vessels. This reaggregation process raised WNT5A and WNT11 expression levels and improved the quality of cardiomyocyte population compared to that in a control group in which dissociation and reaggregation were not performed.
ABSTRACT
Immunotherapy using dendritic cells (DCs) is a promising treatment modality for cancer. However, the limited number of functional DCs from peripheral blood has been linked to the unsatisfactory clinical efficacies of current DC-based cancer immunotherapies. We previously generated proliferating antigen-presenting cells (APCs) by genetically engineering myeloid cells derived from induced pluripotent stem cells (iPSC-pMCs), which offer infinite functional APCs for broad applications in cancer therapy. Herein, we aimed to further enhance the antitumor effect of these cells by genetic modification. GM-CSF gene transfer did not affect the morphology, or surface phenotype of the original iPSC-pMCs, however, it did impart good viability to iPSC-pMCs. The resultant cells induced GM-CSF-dependent CD8+ T cell homeostatic proliferation, thereby enhancing antigen-specific T cell priming in vitro. Administration of the tumor antigen-loaded GM-CSF-producing iPSC-pMCs (GM-pMCs) efficiently stimulated antigen-specific T cells and promoted effector cell infiltration of the tumor tissues, leading to an augmented antitumor effect. To address the potential tumorigenicity of iPSC-derived products, irradiation was applied and found to restrict the proliferation of GM-pMCs, while retaining their T cell-stimulatory capacity. Furthermore, the irradiated cells exerted an antitumor effect equivalent to that of bone marrow-derived DCs obtained from immunocompetent mice. Additionally, combination with immune checkpoint inhibitors increased the infiltration of CD8+ or NK1.1+ effector cells and decreased CD11b+/Gr-1+ cells without causing adverse effects. Hence, although GM-pMCs have certain characteristics that differ from endogenous DCs, our findings suggest the applicability of these cells for broad clinical use and will provide an unlimited source of APCs with uniform quality.
Subject(s)
Dendritic Cells , Granulocyte-Macrophage Colony-Stimulating Factor , Animals , Antigens, Neoplasm/genetics , Granulocyte-Macrophage Colony-Stimulating Factor/genetics , Lymphocyte Activation , Mice , T-Lymphocytes, CytotoxicABSTRACT
Various scalable three-dimensional culture systems for regenerative medicine using human induced pluripotent stem cells (hiPSCs) have been developed to date. However, stable production of hiPSCs with homogeneous qualities still remains a challenge. Here, we describe a novel and simple embryoid body (EB) formation system using unique microfabricated culture vessels. Furthermore, this culture system is useful for high throughput EB formation and rapid generation of differentiated cells such as neural stem cells (NSCs) from hiPSCs. The period of NSC differentiation was significantly shortened under high EB density culture conditions. Simultaneous mass production of a pure population of NSCs was possible within 4 days. These results indicate that the novel culture system might not only become a unique tool to obtain new insights into developmental biology based on human stem cells, but also provide an important tractable platform for efficient and stable production of NSCs for clinical applications.
Subject(s)
Blood Vessels/pathology , Embryoid Bodies/pathology , Neural Stem Cells/physiology , Pluripotent Stem Cells/physiology , Regenerative Medicine/methods , Cell Differentiation , Cells, Cultured , Humans , Microtechnology , Organ Culture TechniquesSubject(s)
Induced Pluripotent Stem Cells , Regenerative Medicine , Cell Culture Techniques , HumansABSTRACT
Schizosaccharomyces pombe carboxypeptidase Y (CPY) is synthesized as a zymogen and transported into the vacuole where maturation and activation occurs. The 110-kDa S. pombe CPY precursor is processed twice and finally converted to a mature form consisting of polypeptides of approximately 19 and 32 kDa linked by a single disulfide bond. In Saccharomyces cerevisiae, maturation of CPY occurs mostly through the activity of vacuolar aspartyl protease Pep4p, whereas a Pep4p homolog has not been found in the S. pombe genome database. Based on analysis of protease-deficient mutants, we found that S. pombe CPY was not able to be processed or activated in isp6Δpsp3Δ double disruptants. Both Isp6p and Psp3p are subtilase-type serine proteases with related sequences. Moreover, alkaline phosphatase of S. pombe was found to be localized at the vacuolar membrane and was also unprocessed in isp6Δpsp3Δ double disruptants. Vacuolar localization of GFP-fused Isp6p and Psp3p was determined by fluorescence microscopy. These results suggest that the two serine proteases Isp6p and Psp3p are functional in the vacuole and are involved in proteolytic processing of vacuolar proteins.
Subject(s)
Alkaline Phosphatase/metabolism , Cathepsin A/metabolism , Enzyme Precursors/metabolism , Protein Processing, Post-Translational , Schizosaccharomyces pombe Proteins/metabolism , Schizosaccharomyces/enzymology , Alkaline Phosphatase/chemistry , Alkaline Phosphatase/genetics , Amino Acid Sequence , Cathepsin A/chemistry , Cathepsin A/genetics , Enzyme Precursors/chemistry , Enzyme Precursors/genetics , Molecular Sequence Data , Protein Transport , Schizosaccharomyces/chemistry , Schizosaccharomyces/genetics , Schizosaccharomyces pombe Proteins/chemistry , Schizosaccharomyces pombe Proteins/genetics , Sequence Alignment , Serine Proteases/genetics , Serine Proteases/metabolism , Vacuoles/enzymology , Vacuoles/genetics , Vacuoles/metabolismABSTRACT
Yeasts combine the ease of genetic manipulation and fermentation of a microorganism with the capability to secrete and modify foreign proteins according to a general eukaryotic scheme. Their rapid growth, microbiological safety, and high-density fermentation in simplified medium have a high impact particularly in the large-scale industrial production of foreign proteins, where secretory expression is important for simplifying the downstream protein purification process. However, secretory expression of heterologous proteins in yeast is often subject to several bottlenecks that limit yield. Thus, many studies on yeast secretion systems have focused on the engineering of the fermentation process, vector systems, and host strains. Recently, strain engineering by genetic modification has been the most useful and effective method for overcoming the drawbacks in yeast secretion pathways. Such an approach is now being promoted strongly by current post-genomic technology and system biology tools. However, engineering of the yeast secretion system is complicated by the involvement of many cross-reacting factors. Tight interdependence of each of these factors makes genetic modification difficult. This indicates the necessity of developing a novel systematic modification strategy for genetic engineering of the yeast secretion system. This mini-review focuses on recent strategies and their advantages for systematic engineering of yeast strains for effective protein secretion.
Subject(s)
Genetic Engineering , Metabolic Networks and Pathways/genetics , Saccharomyces cerevisiae Proteins/genetics , Saccharomyces cerevisiae Proteins/metabolism , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae/metabolism , Humans , Recombinant Proteins/metabolismABSTRACT
Previously, we achieved approximately 30-fold enhanced secretion of the protease-sensitive model protein human growth hormone (hGH) by multiple gene deletion of seven obstructive proteases in the fission yeast Schizosaccharomyces pombe. However, intracellular retention of secretory hGH was found in the resultant multiprotease-deficient strains. As a solution, genetic modification of the intracellular trafficking pathway that is related to intracellular retention of hGH was attempted on a protease octuple deletant strain. Vacuolar accumulation of the intracellularly retained hGH was identified by secretory expression of hGH fused with EGFP, and three vacuolar protein sorting (vps)-deficient strains, vps10Delta, vps22Delta, and vps34Delta, were determined on account of their hGH secretion efficiency. The mutant vps10Delta was found to be effective for hGH secretion, which suggested a role for vps10 in the vacuolar accumulation of the intracellularly retained hGH. Finally, vps10 deletion was performed on the protease octuple deletant strain, which led to an approximately 2-fold increase in hGH secretion. This indicated the possible application of secretory-pathway modification and multiple protease deletion for improving heterologous protein secretion from the fission yeast S. pombe.
Subject(s)
Human Growth Hormone/metabolism , Peptide Hydrolases/deficiency , Recombinant Proteins/metabolism , Schizosaccharomyces pombe Proteins/genetics , Schizosaccharomyces/genetics , Schizosaccharomyces/metabolism , Vacuoles/metabolism , Gene Deletion , Human Growth Hormone/genetics , Humans , Peptide Hydrolases/genetics , Protein Transport , Recombinant Proteins/geneticsABSTRACT
The fission yeast Schizosaccharomyces pombe is a particularly useful model for studying the function and regulation of genes from higher eukaryotes. The genome of Sc. pombe has been sequenced, and DNA microarray, proteome and transcriptome analyses have been carried out. Among the well-characterized yeast species, Sc. pombe is considered an attractive host for the production of heterologous proteins. Expression vectors for high-level expression in Sc. pombe have been developed and many foreign proteins have been successfully expressed. However, further improvements in the protein-expressing host systems are still required for the production of heterologous proteins involved in post-translational modification, metabolism and intracellular trafficking. This minireview focuses on recent advances in heterologous protein production by use of engineered fission-yeast strains.
Subject(s)
Recombinant Proteins/biosynthesis , Recombinant Proteins/genetics , Schizosaccharomyces/genetics , Genetic Engineering , Genetic Vectors/genetics , Oligonucleotide Array Sequence Analysis , Polymerase Chain Reaction , Schizosaccharomyces/metabolismABSTRACT
We report a gap-filling sequence between SPBPB21E7.09 (in contig c1348) and SPBPB10D8.01 (in contig pB10D8) on the left arm of chromosome 2 in the fission yeast, Schizosaccharomyces pombe. The sequence was determined from a BAC clone overlapping SPBPB21E7.01c (eno102) (in contig c1348) and SPBC1683.07 (mal1) (in contig pB10D8). The gap-filling sequence is 17,881 bp in length and contains five putative open reading frames, which were systematically named as SPBC460.01c, SPBC460.02c, SPBC460.03, SPBC460.04c and SPBC460.05. Their deduced amino acid sequences respectively include protein motifs corresponding to amino acid permease, glutathione S-transferase C-terminal domain, taurine catabolism dioxygenase TauD TfdA family and major facilitator superfamily, whereas their functions are unknown.
Subject(s)
Chromosomes, Fungal , Schizosaccharomyces/genetics , Amino Acid Motifs , Amino Acid Sequence , Base Sequence , DNA, Fungal/chemistry , DNA, Fungal/genetics , Gene Library , Molecular Sequence Data , Open Reading Frames , Polymerase Chain Reaction , Sequence AlignmentABSTRACT
The creation of protease-deficient mutants to avoid product degradation is one of the current strategies employed to improve productivity and secretion efficiency of heterologous protein expression. We previously constructed a set of single protease-deficient mutants of the fission yeast Schizosaccharomyces pombe by respective disruption of 52 protease genes, and we succeeded in confirming useful disruptants (Idiris et al., Yeast 23:83-99, 2006). In the present study, we attempted multiple deletions of 13 protease genes, single deletions of which were previously confirmed as being beneficial for reducing extracellular product degradation. Using PCR-based gene replacement, a series of multiple deletion strains was constructed by multiple disruption of a maximum of seven protease genes. Effects of the resultant multiple deletion strains on heterologous expression were then measured by practical expression of a proteolytically sensitive model protein, the human growth hormone (hGH). Time profiles of hGH secretion from each resultant mutant demonstrated significantly enhanced hGH productivity with processing of the multiple protease deletions. The data clearly indicated that disruption of multiple protease genes in the fission yeast is an effective method for controlling proteolytic degradation of heterologous proteins particularly susceptible to proteases.
Subject(s)
Biotechnology/methods , Peptide Hydrolases/chemistry , Schizosaccharomyces pombe Proteins/physiology , Schizosaccharomyces/enzymology , Schizosaccharomyces/genetics , Cell Proliferation , Gene Deletion , Genetic Vectors , Genome, Fungal , Human Growth Hormone/genetics , Humans , Models, Genetic , Mutation , Peptide Hydrolases/metabolism , Polymerase Chain Reaction , Recombinant Proteins/chemistry , Schizosaccharomyces pombe Proteins/chemistryABSTRACT
One of the major problems hindering effective production and purification of heterologous proteins from the fission yeast Schizosaccharomyces pombe is proteolytic degradation of the recombinant gene products by host-specific proteases. As an initial solution to this problem, we constructed a protease-deficient disruptant set by respective disruption of 52 Sz. pombe protease genes. Functional screening of the resultant set was performed by observing secretory production of a proteolytically sensitive model protein, human growth hormone (hGH). The results indicated that some of the resultant disruptants were effective in reducing hGH degradation, as observed during the hGH expression procedure and mainly as a result of unknown serine- and/or cysteine-type proteases in the culture medium. These findings also demonstrated that construction of a protease-deficient strain set is not only useful for practical application in protein production, but also for functional screening, specification and modification of proteases in Sz. pombe, where further investigations of proteolytic processes and improvement through multiple gene manipulations are required.
Subject(s)
Human Growth Hormone/biosynthesis , Peptide Hydrolases/deficiency , Recombinant Proteins/biosynthesis , Schizosaccharomyces pombe Proteins/genetics , Schizosaccharomyces/genetics , Biotechnology/methods , Blotting, Southern , DNA, Fungal/chemistry , DNA, Fungal/genetics , Human Growth Hormone/genetics , Human Growth Hormone/metabolism , Mutagenesis, Insertional , Peptide Hydrolases/genetics , Peptide Hydrolases/metabolism , Polymerase Chain Reaction , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Schizosaccharomyces/enzymology , Schizosaccharomyces/metabolism , Schizosaccharomyces pombe Proteins/metabolismABSTRACT
To determine the intermolecular force on protein-protein interaction (PPI) by atomic force microscopy (AFM), a photograft-polymer spacer for protein molecules on both surfaces of the substrate and AFM probe tip was developed, and its effectiveness was assessed in a PPI model of a pair of human serum albumin (HSA) and its monoclonal antibody (anti-HSA). A carboxylated photoiniferter, N-(dithiocarboxy)sarcosine, was derivatized on both surfaces of the glass substrate and AFM probe tip, and subsequently water-soluble nonionic vinyl monomers, N,N-dimethylacrylamide (DMAAm), were graft-polymerized on them upon ultraviolet light irradiation. DMAAm-photograft-polymerized spacers with carboxyl groups at the growing chain end but with different chain lengths on both surfaces were prepared. The proteins were covalently bound to the carboxyl terminus of the photograft-polymer chain using a water-soluble condensation agent. The effects of the graft-spacer length on the profile of the force-distance curves and on the unbinding characteristics (unbinding force and unbinding distance) were examined in comparison with those in the case of the commercially available poly(ethylene glycol) (PEG) spacer. The frequency of the nonspecific adhesion force profile was markedly decreased with the use of the photograft spacers. Among the force curves detected, a high frequency of single-peak curves indicating the unbinding process of a single pair of proteins and a very low frequency of multiple-peak profiles were observed for the photograft spacers, regardless of the graft chain length, whereas a high frequency of no-force peaks was noted. These observations were in marked contrast with those for the PEG spacer. The force peak values determined ranged from 88 to 94 pN, irrespective of the type of spacer, while the standard deviation of force distribution observed for the photograft spacer was lower than that for the PEG spacer, indicating that the photograft spacers provide a higher accuracy of force determination.
Subject(s)
Antibodies/chemistry , Antigen-Antibody Reactions , Antigens/chemistry , Microscopy, Atomic Force/methods , Adhesiveness , Antibodies, Monoclonal/chemistry , Biocompatible Materials/chemistry , Glass , Humans , Light , Polyethylene Glycols/chemistry , Polymers/chemistry , Proteins/chemistry , Sarcosine/analogs & derivatives , Sarcosine/chemistry , Serum Albumin/chemistry , Stress, Mechanical , Surface Properties , Thiocarbamates/chemistry , Tissue AdhesionsABSTRACT
The "most primitive" living vertebrate the hagfish has a dimeric proteinase inhibitor, a protein homologous to human alpha2-macroglobulin, in its plasma at high concentration. Although the hagfish proteinase inhibitor has been isolated and its function and quaternary structure studied, its primary structure, subunit composition and fragmentation process remain unclear. In this study, hagfish proteinase inhibitor cDNA was cloned, sequenced and cDNA-deduced amino acid sequence was analyzed. A large fraction of homosubunits in the dimeric structure of the protein has undergone a cleavage at a specific arginyl residue (Arg833) while the rest retained their chain integrity without being processed. Thus random combinations of processed and nonprocessed subunits in the dimeric structure of this protein result in different molecular conformers and generate a complicated multiband pattern in SDS-PAGE. It was further demonstrated by proteolytic analysis that the hagfish inhibitor has no susceptible arginyl residues within its bait region and thus incapable of trapping arginine specific proteinases. This implies that the specific subunit cleavage at Arg833 was caused by an unknown arginine specific proteinase which escaped from the entrapment by the hagfish inhibitor.