Improved Production of Induced Pluripotent Stem Cells Using Dot Pattern Culture Plates.

The rate of cell proliferation is a crucial factor in cell production under good manufacturing practice (GMP) control. In this study, we identified a culture system for induced pluripotent cells (iPSCs) that supports cell proliferation and viability and maintains the cells in an undifferentiated state even at 8 days after seeding. This system involves the use of dot pattern culture plates that have been coated with a chemically defined scaffold which has high biocompatibility. Under cell starvation conditions, where medium exchange was not performed for 7 days or where the amount of medium exchange was reduced to half or a quarter, iPSC viability and lack of differentiation were maintained. The rate of cell viability in this culture system was greater than generally obtained by standard culture methods. The cells in this compartmentalized culture system could be induced to differentiate in a controlled and consistent manner: differentiation of endoderm occurred in a controlled and consistent manner: endoderm, mesoderm, and ectoderm could be consistently induced to differentiate in the cultures. In conclusion, we have developed a culture system that supports high viability in iPSCs and allows their controlled differentiation. This system has the potential for use in GMP-based production of iPSCs for clinical purposes.

Efficient production of recombinant proteins in suspension CHO cells culture using the Tol2 transposon system coupled with cycloheximide resistance selection.

DNA recombination techniques in mammalian cells has been applied to the production of therapeutic proteins for several decades. To be used for commercial production, established cell lines should stably express target proteins with high productivity and acceptable quality for human use. In the conventional transfection method, the screening process is laborious and time-consuming since superior cell lines had to be selected from an enormous number of transfected cell pools and clonal cell lines with a wide variety of transgene insertion locations. In this study, we demonstrated that the combination of a Tol2 transposon system and cell selection by cycloheximide resistance is an efficient method to express therapeutic proteins, such as human antibody in suspension culture of Chinese hamster ovary cells. The resulting stable cell lines showed constant productivity and cell growth over a long enough cultivation periods for recombinant protein production. We anticipate that this approach will prove widely applicable to protein production in research and development of pharmaceutical products.

A clinical-grade HLA haplobank of human induced pluripotent stem cells matching approximately 40% of the Japanese population.

BACKGROUND: Human induced pluripotent stem cells (iPSCs) are expected to be useful for regenerative medicine for many diseases. Many researchers have focused on and enabled the generation of differentiated cells or tissue-like structures, including organoids, which help to ameliorate target diseases. To promote such cell therapies, we established a clinically applicable iPSC haplobank matching as many people as possible in Japan. METHODS: Through cooperation with several organizations, we recruited donors whose human leukocyte antigens (HLAs) involved in immunorejection were homozygous. The peripheral or umbilical cord blood collected from the donors was used for iPSC production by electroporation of episomal vectors. These iPSC lines were then subjected to testing, including genome analyses and sterility, to maximize safety. FINDINGS: We constructed a clinical-grade haplobank of 27 iPSC lines from 7 donors according to good manufacturing practice regulations. However, reasons to avoid using iPSC lines include the presence of residual episomal vectors or genetic mutations in cancer-related genes. CONCLUSIONS: This haplobank provides HLA-matched iPSC lines for approximately 40% of the Japanese population. Since the haplobank's release in 2015, these iPSC lines have been used in more than 10 clinical trials. The establishment of this haplobank is an important step toward the clinical application of iPSCs in cell therapies. FUNDING: This study was supported by a research center network for the realization of regenerative medicine of the Japan Agency for Medical Research and Development (AMED) under grant number JP20bm0104001h0108.

Adhesion Characteristics of Human Pancreatic Islets, Duct Epithelial Cells, and Acinar Cells to a Polymer Scaffold.

We reported in 2018 that among several extracellular matrices, fibronectin, type I collagen, type IV collagen, laminin I, fibrinogen, and bovine serum albumin, fibronectin is particularly useful for adhesion of porcine pancreatic tissue. Subsequently, we developed a technology that enables the chemical coating of the constituent motifs of fibronectin onto cell culture dishes. In this experiment, we used islets (purity ≥ 90%), duct epithelial cells (purity ≥ 60%), and acinar cells (purity ≥ 99%) isolated from human pancreas according to the Edmonton protocol published in 2000 and achieved adhesion to the constituent motifs of fibronectin. A solution including cGMP Prodo Islet Media was used as the assay solution. In islets, adhesion was enhanced with the constitutive motifs of fibronectin compared with uncoated islets. In the functional evaluation of islets, insulin mRNA expression and insulin secretion were enhanced by the constitutive motif of fibronectin compared with non-coated islets. The stimulation index was comparable between non-coated islets and fibronectin motifs. In duct epithelial cells, adhesion was mildly promoted by the fibronectin component compared with non-coated component, while in acinar cells, adhesion was inhibited by the fibronectin component compared with the non-coated component. These data suggest that the constitutive motifs of fibronectin are useful for the adhesion of islets and duct epithelial cells.

Induced hepatic stem cells are suitable for human hepatocyte production.

Human hepatocytes were transfected with Sendai virus vectors (SeV) expressing OCT3/4, SOX2, KLF4, and C-MYC to produce hepatocyte-derived induced pluripotent stem cells (iPSCs). The messenger RNA (mRNA) expression of undifferentiated markers (passage 19-21) and hepatocyte-specific markers (HSMs) (passage 0-20) in 48 established hepatocyte-derived iPSC-like colonies was examined. Among the 48 clones, 10 clones continuously expressed HSM mRNA (HNF1ß and HNF4α) in passage 0-20. The colonies which expressed HSMs (iTS-L cells: induced tissue-specific stem cells from liver) showed a different tendency in microarray and methylation analyses to fibroblast-derived iPSCs (strain: 201B7). iTS-L cells were less likely to form teratomas in mice than iPSCs (He). The iTS-L cells were differentiated into hepatocyte-like cells more efficiently than iPSCs (He) or iPSCs (201B7). These data suggest that SeV expressing OCT3/4, SOX2, KLF4, and C-MYC induce the generation of iPSCs and iTS-L cells.

Semi-3D cultures using Laminin 221 as a coating material for human induced pluripotent stem cells.

It was previously believed that human induced pluripotent stem cells (hiPSCs) did not show adhesion to the coating material Laminin 221, which is known to have specific affinity for cardiomyocytes. In this study, we report that human mononuclear cell-derived hiPSCs, established with Sendai virus vector, form peninsular-like colonies rather than embryonic stem cell-like colonies; these peninsular-like colonies can be passaged more than 10 times after establishment. Additionally, initialization-deficient cells with residual Sendai virus vector adhered to the coating material Laminin 511 but not to Laminin 221. Therefore, the expression of undifferentiated markers tended to be higher in hiPSCs established on Laminin 221 than on Laminin 511. On Laminin 221, hiPSCs15M66 showed a semi-floating colony morphology. The expression of various markers of cell polarity was significantly lower in hiPSCs cultured on Laminin 221 than in hiPSCs cultured on Laminin 511. Furthermore, 201B7 and 15M66 hiPSCs showed 3D cardiomyocyte differentiation on Laminin 221. Thus, the coating material Laminin 221 provides semi-floating culture conditions for the establishment, culture and induced differentiation of hiPSCs.

Generation of hypoimmunogenic induced pluripotent stem cells by CRISPR-Cas9 system and detailed evaluation for clinical application.

In order to expand the promise of regenerative medicine using allogeneic induced pluripotent stem cells (iPSCs), precise and efficient genome editing of human leukocyte antigen (HLA) genes would be advantageous to minimize the immune rejection caused by mismatches of HLA type. However, clinical-grade genome editing of multiple HLA genes in human iPSC lines remains unexplored. Here, we optimized the protocol for good manufacturing practice (GMP)-compatible CRISPR-Cas9 genome editing to deplete the three gene locus (HLA-A, HLA-B, and CIITA genes) simultaneously in HLA homozygous iPSCs. The use of HLA homozygous iPSCs has one main advantage over heterozygous iPSCs for inducing biallelic knockout by a single gRNA. RNA-seq and flow cytometry analyses confirmed the successful depletion of HLAs, and lineage-specific differentiation into cardiomyocytes was verified. We also confirmed that the pluripotency of genome-edited iPSCs was successfully maintained by the three germ layers of differentiation. Moreover, whole-genome sequencing, karyotyping, and optical genome mapping analyses revealed no evident genomic abnormalities detected in some clones, whereas unexpected copy number losses, chromosomal translocations, and complex genomic rearrangements were observed in other clones. Our results indicate the importance of multidimensional analyses to ensure the safety and quality of the genome-edited cells. The manufacturing and assessment pipelines presented here will be the basis for clinical-grade genome editing of iPSCs.

Laminin-511 Activates the Human Induced Pluripotent Stem Cell Survival via α6ß1 Integrin-Fyn-RhoA-ROCK Signaling.

In human induced pluripotent stem cells (hiPSCs), laminin-511/α6ß1 integrin interacts with E-cadherin, an intercellular adhesion molecule, to induce the activation of the phosphatidylinositol 3-kinase (PI3K)-dependent signaling pathway. The interaction between laminin-511/α6ß1 integrin and E-cadherin, an intercellular adhesion molecule, results in protection against apoptosis through the proto-oncogene tyrosine-protein kinase Fyn(Fyn)-RhoA-ROCK signaling pathway and the Ras homolog gene family member A (RhoA)/Rho kinase (ROCK) signaling pathway (the major pathway for cell death). In this article, the impact of laminin-511 on hiPSC on α6ß1 integrin-Fyn-RhoA-ROCK signaling is discussed and explored along with validation experiments. PIK3CA mRNA (mean [standard deviation {SD}]: iMatrix-511, 1.00 [0.61]; collagen+MFGE8, 0.023 [0.02]; **P < 0.01; n = 6) and PIK3R1 mRNA (mean [SD]: iMatrix-511, 1.00 [0.79]; collagen+MFGE8, 0.040 [0.06]; *P < 0.05; n = 6) were upregulated by iMatrix-511 resulting from an increased expression of Integrin α6 mRNA (mean [SD]: iMatrix-511, 1.00 [0.42]; collagen+MFGE8, 0.23 [0.05]; **P < 0.01; n = 6). The iMatrix-511 increased the expression of p120-Catenin mRNA (mean [SD]: iMatrix-511, 1.00 [0.71]; collagen+MFGE8, 0.025 [0.03]; **P < 0.01; n = 6) and RAC1 mRNA (mean [SD]: iMatrix-511, 1.00 [0.28]; collagen+MFGE8, 0.39 [0.15]; **P < 0.01; n = 6) by increasing the expression of E-cadherin mRNA (mean [SD]: iMatrix-511, 1.00 [0.38]; collagen+MFGE8, 0.16 [0.11]; **P < 0.01; n = 6). As a result, iMatrix-511 increased the expression of P190 RhoGAP (GTPase-activating proteins) mRNA, such as ARHGAP1 mRNA (mean [SD]: iMatrix-511, 1.00 [0.57]; collagen+MFGE8, 0.032 [0.03]; **P < 0.01; n = 6), ARHGAP4 mRNA (mean [SD]: iMatrix-511, 1.00 [0.56]; collagen+MFGE8, 0.039 [0.049]; **P < 0.01; n = 6), and ARHGAP5 mRNA (mean [SD]: iMatrix-511, 1.00 [0.39]; collagen+MFGE8, 0.063 [0.043]; **P < 0.01; n = 6). Western blotting showed that phospho-Rac1 remained in the cytoplasm and phospho-Fyn showed nuclear transition in iPSCs cultured on iMatrix-511. Proteome analysis showed that PI3K signaling was enhanced and cytoskeletal actin was activated in iPSCs cultured on iMatrix-511. In conclusion, laminin-511 strongly activated the cell survival by promoting α6ß1 integrin-Fyn-RhoA-ROCK signaling in hiPSCs.

A chemically-defined plastic scaffold for the xeno-free production of human pluripotent stem cells.

Clinical use of human pluripotent stem cells (hPSCs) is hampered by the technical limitations of their expansion. Here, we developed a chemically synthetic culture substrate for human pluripotent stem cell attachment and maintenance. The substrate comprises a hydrophobic polyvinyl butyral-based polymer (PVB) and a short peptide that enables easy and uniform coating of various types of cell culture ware. The coated ware exhibited thermotolerance, underwater stability and could be stored at room temperature. The substrate supported hPSC expansion in combination with most commercial culture media with an efficiency similar to that of commercial substrates. It supported not only the long-term expansion of examined iPS and ES cell lines with normal karyotypes during their undifferentiated state but also directed differentiation of three germ layers. This substrate resolves major concerns associated with currently used recombinant protein substrates and could be applied in large-scale automated manufacturing; it is suitable for affordable and stable production of clinical-grade hPSCs and hPSC-derived products.

Protocol for the generation of human induced hepatic stem cells using Sendai virus vectors.

Our recent study demonstrated the generation of induced tissue-specific stem/progenitor (iTS/iTP) cells by the transient overexpression of reprogramming factors combined with tissue-specific selection. Here, we present a protocol to reprogram human hepatocytes to generate human induced tissue-specific liver stem (iTS-L) cells. Human hepatocytes are transfected with Sendai virus vectors (SeV) expressing OCT3/4, SOX2, KLF4, and c-MYC. iTS-L cells continuously express mRNA of hepatocyte-specific markers (HNF1ß and HNF4α) and do not form teratomas. For complete details on the use and execution of this protocol, please refer to Nakashima et al. (2022).1.

A rapid method for simultaneous evaluation of free light chain content and aggregate content in culture media of Chinese hamster ovary cells expressing monoclonal antibodies for cell line screening.

The goal of developing a monoclonal antibody (mAb) production process is high productivity and high quality. Because the productivity and quality of mAbs depend on cell line properties, the selection of cell lines suitable for large-scale production is an important stage in process development for mAb production. The light chain (LC) is important for antibody folding and assembly in the endoplasmic reticulum; cell lines that secrete a large amount of LCs in the medium secrete high-quality antibodies with high productivity. LC contents in culture media have been estimated by western blotting, reverse-phase high-performance liquid chromatography, and enzyme-linked immunosorbent assay. However, these analyses require fine tuning of experimental conditions for each antibody analyzed. Here we report a rapid and simple high-sensitivity size-exclusion chromatography (HS-SEC) method to evaluate the contents of low-molecular weight species (LMWS, mainly consisting of LC monomers and dimers) and high-molecular weight species (HMWS, aggregates) in the media for cell line screening. Because LMWS and HMWS are important indicators of productivity and quality, respectively, for cell line screening, HS-SEC will be useful in the first step of cell line selection needed for large-scale production.

Approaches to Quality Risk Management When Using Single-Use Systems in the Manufacture of Biologics.

Biologics manufacturing technology has made great progress in the last decade. One of the most promising new technologies is the single-use system, which has improved the efficiency of biologics manufacturing processes. To ensure safety of biologics when employing such single-use systems in the manufacturing process, various issues need to be considered including possible extractables/leachables and particles arising from the components used in single-use systems. Japanese pharmaceutical manufacturers, together with single-use suppliers, members of the academia and regulatory authorities have discussed the risks of using single-use systems and established control strategies for the quality assurance of biologics. In this study, we describe approaches for quality risk management when employing single-use systems in the manufacturing of biologics. We consider the potential impact of impurities related to single-use components on drug safety and the potential impact of the single-use system on other critical quality attributes as well as the stable supply of biologics. We also suggest a risk-mitigating strategy combining multiple control methods which includes the selection of appropriate single-use components, their inspections upon receipt and before releasing for use and qualification of single-use systems. Communication between suppliers of single-use systems and the users, as well as change controls in the facilities both of suppliers and users, are also important in risk-mitigating strategies. Implementing these control strategies can mitigate the risks attributed to the use of single-use systems. This study will be useful in promoting the development of biologics as well as in ensuring their safety, quality and stable supply.

Titer of trastuzumab produced by a Chinese hamster ovary cell line is associated with tricarboxylic acid cycle activity rather than lactate metabolism.

Achieving high productivity and quality is the final goal of therapeutic antibody development, but the productivity and quality of antibodies are known to be substantially dependent on the nature of the cell lines expressing the antibodies. We characterized two contrasting cell lines that produce trastuzumab, namely cell line A with a high titer and a low aggregate content and cell line B with a low titer and a high aggregate content to identify the causes of the differences. We observed the following differences: cell growth (A > B), proportion of defucosylated oligosaccharides on antibodies (A < B), and proportion of covalent antibody aggregates (A > B). Our results suggest that the high monoclonal antibody (mAb) titers in cell line A is associated with the high proliferation and is not caused by the lactate metabolism shift (switching from lactate production to net lactate consumption). Rather, these differences can be accounted for by the following: levels of tricarboxylic acid cycle intermediates (A > B), ammonium ion levels (A ≤ B), and oxidative stress (A > B).

Comparison of antibody molecules produced from two cell lines with contrasting productivities and aggregate contents.

Cell culture processes that produce therapeutic antibodies with high productivity (titer) and low aggregate content reduce the risk of adverse effects and expense to patients. To elucidate the mechanism of aggregate formation, we compared trastuzumab samples produced from two contrasting cell lines: cell line A, which exhibits high titer and low aggregate content, and cell line B, which exhibits low titer and high aggregate content. Cell line B produced significantly fewer (approximately 1/3) antibodies compared with cell line A and contained higher (approximately 3-fold) percentages of aggregates. The aggregates of antibodies found in the protein A-purified samples of cell line B were associated mostly with noncovalent interactions. Cell line B exhibited a low content of monomers/dimers of light chains in the medium and within cells. Because light chains are essential for the correct folding of heavy chains and secretion of mature antibodies, the characteristics of cell line B may be attributed to low levels of light chain production. In addition, protein A-purified antibodies from cell line B (but not those from cell line A) contained fragments that are expected to expose the hydrophobic CH3 domain, which may serve as nuclei for aggregation.

Efficient folding/assembly in Chinese hamster ovary cells is critical for high quality (low aggregate content) of secreted trastuzumab as well as for high production: stepwise multivariate regression analyses.

When developing cell culture processes for therapeutic antibodies, the low content of aggregated proteins is the most critical because administering aggregated antibody molecules might result in adverse effects such as immunogenicity. To characterize cells with high productivity and quality, we determined factors that are closely related to antibody titer, which is a productivity indicator, and the area percentage of high molecular weight species in cultivated media, which is equivalent to aggregate content and is used as a quality indicator. We examined the factors influencing antibody titer and aggregate content using various data from 28 cell lines throughout their culture periods from growth to death phases. Our study using correlation analysis revealed that statistically significant correlations between factors and indicators changes with sampling points, hence we thought that various factors would influence each indicator simultaneously. To understand the relationship between these factors and titer/aggregates contents, we performed stepwise multiple linear regression analyses and deduced a multiple linear model for each indicator. The titer was found to positively associate with specific growth rate and specific production rate and negatively with intracellular heavy chain content. The aggregate content was found to positively associate with protein disulfide isomerase mRNA level and negatively with light chain secreted into culture media, specific production rate, intracellular light chain content, and specific growth rate. Our observations suggest that correct and efficient assembling and/or folding of an antibody molecule in an endoplasmic reticulum are important for high titer and low aggregates contents.

Fully human monoclonal antibody directed to proteolytic cleavage site in severe acute respiratory syndrome (SARS) coronavirus S protein neutralizes the virus in a rhesus macaque SARS model.

BACKGROUND: There is still no effective method to prevent or treat severe acute respiratory syndrome (SARS), which is caused by SARS coronavirus (CoV). In the present study, we evaluated the efficacy of a fully human monoclonal antibody capable of neutralizing SARS-CoV in vitro in a Rhesus macaque model of SARS. METHODS: The antibody 5H10 was obtained by vaccination of KM mice bearing human immunoglobulin genes with Escherichia coli-producing recombinant peptide containing the dominant epitope of the viral spike protein found in convalescent serum samples from patients with SARS. RESULTS: 5H10, which recognized the same epitope that is also a cleavage site critical for the entry of SARS-CoV into host cells, inhibited propagation of the virus and pathological changes found in Rhesus macaques infected with the virus through the nasal route. In addition, we analyzed the mode of action of 5H10, and the results suggested that 5H10 inhibited fusion between the virus envelope and host cell membrane. 5H10 has potential for use in prevention and treatment of SARS if it reemerges. CONCLUSIONS: This study represents a platform to produce fully human antibodies against emerging infectious diseases in a timely and safe manner.

Regulation of DNA fragmentation: the role of caspases and phosphorylation.

DNA fragmentation is a hallmark of apoptosis that is induced by apoptotic stimuli in various cell types. Apoptotic signal pathways, which eventually cause DNA fragmentation, are largely mediated by the family of cysteinyl aspartate-specific protease caspases. Caspases mediate apoptotic signal transduction by cleavage of apoptosis-implicated proteins and the caspases themselves. In the process of caspase activation, reversible protein phosphorylation plays an important role. The activation of various proteins is regulated by phosphorylation and dephosphorylation, both upstream and downstream of caspase activation. Many kinases/phosphatases are involved in the control of cell survival and death, including the mitogen-activated protein kinase signal transduction pathways. Reversible protein phosphorylation is involved in the widespread regulation of cellular signal transduction and apoptotic processes. Therefore, phosphatase/kinase inhibitors are commonly used as apoptosis inducers/inhibitors. Whether protein phosphorylation induces apoptosis depends on many factors, such as the type of phosphorylated protein, the degree of activation and the influence of other proteins. Phosphorylation signaling pathways are intricately interrelated; it was previously shown that either induction or inhibition of phosphorylation causes cell death. Determination of the relationship between protein and phosphorylation helps to reveal how apoptosis is regulated. Here we discuss DNA fragmentation and protein phosphorylation, focusing on caspase and serine/threonine protein phosphatase activation.

Okadaic acid induces DNA fragmentation via caspase-3-dependent and caspase-3-independent pathways in Chinese hamster ovary (CHO)-K1 cells.

DNA fragmentation is a hallmark of apoptosis that occurs in a variety of cell types; however, it remains unclear whether caspase-3 is required for its induction. To investigate this, we produced caspase-3 knockout Chinese hamster ovary (CHO)-K1 cells and examined the effects of gene knockout and treatment with caspase-3 inhibitors. Okadaic acid (OA) is a potent inhibitor of the serine/threonine protein phosphatases (PPs) PP1 and PP2A, which induce apoptotic cellular reactions. Treatment of caspase-3(-/-) cells with OA induced DNA fragmentation, indicating that caspase-3 is not an essential requirement. However, in the presence of benzyloxycarbonyl-Asp-Glu-Val-Asp (OMe) fluoromethylketone (z-DEVD-fmk), DNA fragmentation occurred in CHO-K1 cells but not in caspase-3(-/-) cells, suggesting that caspase-3 is involved in OA-induced DNA fragmentation that does not utilize DEVDase activity. In the absence of caspase-3, DEVDase activity may play an important role. In addition, OA-induced DNA fragmentation was reduced but not blocked in CHO-K1 cells, suggesting that caspase-3 is involved in caspase-independent OA-induced DNA fragmentation. Furthermore, OA-induced cleavage of caspase-3 and DNA fragmentation were blocked by pretreatment with the wide-ranging serine protease inhibitor 4-(2-aminoethyl)-benzenesulfonyl fluoride hydrochloride. These results suggest that serine proteases regulate DNA fragmentation upstream of caspase-3.

pH Condition in temperature shift cultivation enhances cell longevity and specific hMab productivity in CHO culture.

Controlling cell proliferation during cell culturing is an effective way to improve the production yield in mammalian cell culture. We examined the effect of temperature shifts (TS) under pH control conditions in Chinese hamster ovary cells. When we shifted the culture temperature from 37 degrees C to 31 degrees C before a stationary phase at pH 6.8 (TS/pH 6.8), cell viability remained high, and the final human monoclonal antibody (hMab) concentration increased to 2.3 times that in the culture remaining at 37 degrees C. However, there were no significant effects on the cell viability or production yield with the same TS at pH 7.0 (TS/pH 7.0). The average specific hMab productivity and mRNA level of TS/pH 7.0 were the same as that of TS/pH 6.8. The control of cell growth by the TS or the addition of rapamycin was effective in the maintenance of cell viability, but there was no significant increase of the average specific hMab productivity in the culture where cell proliferation was controlled with rapamycin. The hMab mRNA concentration decreased to 55%-65% at a 37 degrees C culture with the addition of actinomycin D. In contrast, actinomycin D did not affect the mRNA level in the TS culture. This result suggested that the increase in the mRNA level in the TS condition was caused by an increase in mRNA stability. In this study, we show that TS can produce two unrelated effects: a prolongation of cell longevity and an improvement in mRNA stability.