Background aims: With the continuous development and advancement of human pluripotent stem cell (PSC)derived cell therapies, an ever-increasing number of clinical indications can benefit from their application. Due to the capacity for PSCs to form teratomas, safety testing is required to ensure the absence of residual PSCs in a cell product. To mitigate these limitations, in vitro analytical methods can be utilized as quality control after the production of a PSC-derived cell product. Sensitivity of these analytic methods is critical in accurately quantifying residual PSC in the final cell product. In this study, we compared the sensitivity of three in vitro assays: qPCR, ddPCR and RT-LAMP. Methods: The spike-in samples were produced from three independent experiments, each spiked with different PSC lines (PSC1, NH50191, and WA09 referred to as H9) into a background of primary fibroblasts (Hs68). These samples were then subjected to qPCR, ddPCR and RT-LAMP to determine their detection limit in measuring a commonly used PSC marker, LIN28A. Results: The results indicated that the three analytic methods all exhibited consistent results across different cell-line spiked samples, with ddPCR demonstrating the highest sensitivity of the three methods. The LIN28A ddPCR assay could confidently detect 10 residual PSCs in a million fibroblasts. Discussion: In our hand, ddPCR LIN28A assay demonstrated the highest sensitivity for detection of residual PSCs compared to the other two assays. Correlating such in vitro safety results with corresponding in vivo studies demonstrating the tumorigenicity profile of PSC-derived cell therapy could accelerate the safe clinical translation of cell therapy. (c) 2024 International Society for Cell & Gene Therapy. Published by Elsevier Inc. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/)

The reliable and cost-efficient manufacturing of monoclonal antibodies (mAbs) is essential to fulfil their ever-growing demand. Cell death in bioreactors reduces productivity and product quality, and is largely attributed to apoptosis. In perfusion bioreactors, this leads to the necessity of a bleed stream, which negatively affects the overall process economy. To combat this limitation, death-resistant Chinese hamster ovary cell lines were developed by simultaneously knocking out the apoptosis effector proteins Bak1, Bax, and Bok with CRISPR technology. These cell lines were cultured in fed-batch and perfusion bioreactors and compared to an unmodified control cell line. In fed-batch, the death-resistant cell lines showed higher cell densities and longer culture durations, lasting nearly a month under standard culture conditions. In perfusion, the death-resistant cell lines showed slower drops in viability and displayed an arrest in cell division after which cell size increased instead. Pertinently, the death-resistant cell lines demonstrated the ability to be cultured for several weeks without bleed, and achieved similar volumetric productivities at lower cell densities than that of the control cell line. Perfusion culture reduced fragmentation of the mAb produced, and the death-resistant cell lines showed increased glycosylation in the light chain in both bioreactor modes. These data demonstrate that rationally engineered death-resistant cell lines are ideal for mAb production in perfusion culture, negating the need to bleed the bioreactor whilst maintaining product quantity and quality.

Chinese hamster ovary (CHO) cells are the primary platform for the production of biopharmaceuticals. To increase yields, many CHO cell lines have been genetically engineered to resist cell death. However, the kinetics that governs cell fate in bioreactors are confounded by many variables associated with batch processes. Here, we used CRISPR-Cas9 to create combinatorial knockouts of the three known BCL-2 family effector proteins: Bak1, Bax, and Bok. To assess the response to apoptotic stimuli, cell lines were cultured in the presence of four cytotoxic compounds with different mechanisms of action. A population-based model was developed to describe the behavior of the resulting viable cell dynamics as a function of genotype and treatment. Our results validated the synergistic antiapoptotic nature of Bak1 and Bax, while the deletion of Bok had no significant impact. Importantly, the uniform application of apoptotic stresses permitted direct observation and quantification of a delay in the onset of cell death through Bayesian inference of meaningful model parameters. In addition to the classical death rate, a delay function was found to be essential in the accurate modeling of the cell death response. These findings represent an important bridge between cell line engineering strategies and biological modeling in a bioprocess context.

Background Exciting pre-clinical data have confirmed that human pluripotent stem cell derived cardiomyocytes (PSC-CMs) can remuscularise the injured or diseased heart, with several clinical trials now in planning or recruitment stages worldwide. However, ventricular arrhythmias are a predictable complication following engraftment of intramyocardially injected PSC-CMs. Therefore, there is an urgent unmet need to gain mechanistic insights and treatment strategies to control or prevent these engraftment arrhythmias (EAs). Methods We used a porcine model of myocardial infarction and PSC-CM transplantation to investigate efficacy of pharmacologic and catheter based anti-arrhythmic strategies in mitigating EAs. Furthermore, cell doses were robustly phenotyped using single cell ribonucleic acid sequencing and high parameter flow cytometry to identify cellular characteristics predictive of arrhythmogenesis. Results Combination therapy with amiodarone and ivabradine significantly reduced EA rate and burden following PSC-CM transplantation. Catheter ablation was also a feasible and effective treatment strategy which could be considered in the case of pharmacologically refractory arrhythmias. In addition, we show that EAs are mechanistically linked to cellular heterogeneity in the input PSC-CM and resultant graft. Specifically, we identify atrial and pacemaker-like cardiomyocytes as culprit arrhythmogenic subpopulations. We further describe two unique surface marker signatures, SIRPA + /CD90 - /CD200 + and SIRPA + /CD90 - /CD200 - , which identify arrhythmogenic and non-arrhythmogenic cardiomyocytes respectively. Conclusion Our data deepens mechanistic understanding of EAs and suggests that modifications to current PSC-CM production and/or selection protocols could ameliorate this problem. We further show that current clinical pharmacologic and interventional anti-arrhythmic strategies can control and potentially abolish these arrhythmias, an important safety consideration given several impending clinical trials.

Chinese hamster ovary (CHO) cells are widely used in biopharmaceutical production. Improvements to cell lines and bioprocesses are constantly being explored. One of the major limitations of CHO cell culture is that the cells undergo apoptosis, leading to rapid cell death, which impedes reaching high recombinant protein titres. While several genetic engineering strategies have been successfully employed to reduce apoptosis, there is still room to further enhance CHO cell lines performance. 'Omics analysis is a powerful tool to better understand different phenotypes and for the identification of gene targets for engineering. Here, we present a comprehensive review of previous CHO 'omics studies that revealed changes in the expression of apoptosis-related genes. We highlight targets for genetic engineering that have reduced, or have the potential to reduce, apoptosis or to increase cell proliferation in CHO cells, with the final aim of increasing productivity.

Background The monoclonal antibody m102.4 is a potent, fully human antibody that neutralises Hendra and Nipah viruses in vitro and in vivo. We aimed to investigate the safety, tolerability, pharmacokinetics, and immunogenicity of m102.4 in healthy adults. Methods In this double-blind, placebo-controlled, single-centre, dose-escalation, phase 1 trial of m102.4, we randomly assigned healthy adults aged 18-50 years with a body-mass index of 18.0-35.0 kg/m(2) to one of five cohorts. A sentinel pair for each cohort was randomly assigned to either m102.4 or placebo. The remaining participants in each cohort were randomly assigned (5:1) to receive m102.4 or placebo. Cohorts 1-4 received a single intravenous infusion of m102.4 at doses of 1 mg/kg (cohort 1), 3 mg/kg (cohort 2), 10 mg/kg (cohort 3), and 20 mg/kg (cohort 4), and were monitored for 113 days. Cohort 5 received two infusions of 20 mg/kg 72 h apart and were monitored for 123 days. The primary outcomes were safety and tolerability. Secondary outcomes were pharmacokinetics and immunogenicity. Analyses were completed according to protocol. The study was registered on the Australian New Zealand Clinical Trials Registry, ACTRN12615000395538. Findings Between March 27, 2015, and June 16, 2016, 40 (52%) of 77 healthy screened adults were enrolled in the study. Eight participants were assigned to each cohort (six received m102.4 and two received placebo). 86 treatment-emergent adverse events were reported, with similar rates between placebo and treatment groups. The most common treatment-related event was headache (12 [40%] of 30 participants in the combined m102.4 group, and three [30%] of ten participants in the pooled placebo group). No deaths or severe adverse events leading to study discontinuation occurred. Pharmacoldnetics based on those receiving m102.4 (n=30) were linear, with a median half-life of 663.3 h (range 474.3-735.1) for cohort 1, 466.3 h (382.8-522.3) for cohort 2, 397.0 h (333. 9-491- 8) for cohort 3, and 466 . 7 h (351.0-889.6) for cohort 4. The elimination kinetics of those receiving repeated dosing (cohort 5) were similar to those of single-dose recipients (median elimination half-time 472.0 [385.6-592.0]). Anti-m102.4 antibodies were not detected at any time-point during the study. Interpretation Single and repeated dosing of m102.4 were well tolerated and safe, displayed linear pharmacokinetics, and showed no evidence of an immunogenic response. This study will inform future dosing regimens for m102.4 to achieve prolonged exposure for systemic efficacy to prevent and treat henipavirus infections. Copyright (C) 2020 Elsevier Ltd. All rights reserved.

Chinese hamster ovary (CHO) cells are the predominant host cell line for the production of biopharmaceuticals, a growing industry currently worth more than $188 billion USD in global sales. CHO cells undergo programmed cell death (apoptosis) following different stresses encountered in cell culture, such as substrate limitation, accumulation of toxic by-products, and mechanical shear, hindering production. Genetic engineering strategies to reduce apoptosis in CHO cells have been investigated with mixed results. In this review, a contemporary understanding of the real complexity of apoptotic mechanisms and signaling pathways is described; followed by an overview of antiapoptotic cell line engineering strategies tested so far in CHO cells.

Undifferentiated human embryonic stem cells have a distinct morphology (hESC). Changes in cell morphology during culture can be indicative of differentiation. hESC, maintained in diverse medias, demonstrated alterations in morphological parameters and subsequent alterations in underlying transcript expression and lineage differentiation. Analysis of morphological parameters showed distinct and significant differences between the undefined, less defined and Xeno-free medias while still maintaining pluripotency markers. This suggested that the less defined media may be creating dynamic instability in the cytoskeleton, with the cytoskeleton becoming more stabilised in the Xeno-free media as demonstrated by smaller and rounder cells. Examination of early lineage markers during undirected differentiation using d5 embryoid bodies demonstrated increased mesodermal lineage preference as compared to endodermal or ectoderm in cells originally cultured in Xeno-free media. Undefined media showed preference for mesoderm and ectoderm lineages, while less defined media (BSA present) demonstrated no preference. These data reveal that culture media may produce fundamental changes in cell morphology which are reflected in early lineage differentiation choice.

Human pluripotent stem cells (hPSCs) are viewed as promising candidates for applications in regenerative medicine and therapy due to their proliferative and pluripotent properties. However, obtaining clinically significant numbers of hPSCs remains a limiting factor and impedes their use in therapeutic applications. Conventionally, hPSCs are cultured on two-dimensional surfaces coated with a suitable substrate, such as Matrigel™. This method, however, requires a large surface area to generate sufficient cell numbers to meet clinical needs and is therefore impractical as a manufacturing platform for cell expansion. In addition, the use of enzymes for cell detachment and small molecule inhibitors to increase plating efficiency may impact future cell behavior when used for routine subculturing. In this study, we describe a protocol to generate and maintain hPSC aggregates in a three-dimensional suspension culture by utilizing thermoresponsive nanobridges. The property of the polymer used in the nanobridges enables passaging and expansion through a temperature change in combination with mechanically applied shear to dissociate aggregates; thus, we eliminate the need of enzymes or small molecules for cell dissociation and viability, respectively. Utilizing this platform, maintenance of human embryonic stem cells for three continuous passages demonstrated high expression levels in key pluripotent markers.

Abstract Androgen-deprivation therapy is the standard treatment for prostate cancer. Despite the initial response, a fraction of cases manifest progression to castration-resistant prostate cancer. PCa recurrence is possibly due to androgen-independent cancer stem cells that reinitiate tumor growth. The properties of cancer stem cells versus highly proliferative progenitor cells remain to be further characterized at the cellular and gene expression level. To address the molecular basis of CSC switch from androgen dependency to independence, we have employed patient-derived xenograft models (LAPC-9 and BM-18) that have different androgen sensitivity properties. We have performed microarray and proteomic analysis of BM-18 tumor tissues prior to and following castration as well as androgen replacement. To characterize the cancer stem cells in these models we have isolated different subpopulations by FACS cytometry, based on combination of selected markers (CD44 and ALDH activity measured by ALDELFUOR assay) aiming to assess the transcriptomic profile of these different populations. To assess response of these CD44+/-/ALDH+/- cells to androgen targeting compounds we have established an androgen-sensitive xenograft model (PNPCa) and tested it by ex vivo tissue culture and organoid viability assays. Proteomic and microarray analysis of bulk BM-18 tumors indicates enrichment of stem cell markers upon castration: CD44, NKX3.1 and ALDH1 isoforms. Readministration of testosterone induces decrease of these markers. Castration induces a rapid tumor volume decrease in the BM-18 as opposed to LAPC-9, reflecting different androgen-dependent cell states. CD44+/-/ALDHhigh/low subpopulations were isolated by flow cytometry and for transcriptomic analysis. Castration in the BM18 model induces an increase in the subpopulations of CD44+/ALDHlow and CD44+/ALDHhigh, while ALDHsingle and ALDHhigh total populations are decreased. Both CD44 expression and ALDH activity is increased in the LAPC-9 model upon castration. Treatment of PNPCa tumor parts ex vivo with enzalutamide inhibited expression of androgen receptor and keratin 8 expression. CD44+ organoids derived from the same tissue did not show altered viability after enzalutamide treatment. Different androgen-independent cancer stem cell populations may be distinguished by ALDH activity status and CD44. Androgen-independent cells in the BM-18 androgen-dependent model are reflected by enrichment of CD44 expression. The least abundant subpopulation CD44+/ALDHhigh is present in the LAPC-9 and expanded upon castration, while in the BM-18 model it is exclusively present after castration, reflecting different androgen sensitivity. Ongoing analysis may elucidate the molecular mechanisms controlling cancer stem cell fates, androgen sensitivity and drug resistance. Citation Format: Sofia Karkampouna, Marta De Menna, Markus Germann, Joel Grosjean, Peter Clark Gray, George Niklaus Thalmann, Marianna Kruithof-de Julio. Characterization of cancer stem cell subpopulations from patient-derived xenografts of advanced prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3053.

The development of next-generation sequencing technologies has opened new opportunities to better characterize complex eukaryotic cells. Chinese hamster ovary (CHO) cells play a primary role in therapeutic protein production, with currently five of the top ten blockbuster drugs produced in CHO. However, engineering superior CHO cells with improved production features has had limited success to date and cell lines are still developed through the generation and screening of large strain pools. Here, we applied RNA sequencing to contrast a high and a low monoclonal antibody producing cell line. Rigorous experimental design achieved high reproducibility between biological replicates, remarkably reducing variation to less than 10%. More than 14 000 gene-transcripts are identified and surprisingly 58% are classified as differentially expressed, including 2900 with a fold change higher than 1.5. The largest differences are found for gene-transcripts belonging to regulation of apoptosis, cell death, and protein intracellular transport GO term classifications, which are found to be significantly enriched in the high producing cell line. RNA sequencing is also performed on subclones, where down-regulation of genes encoding secreted glycoproteins is found to be the most significant change. The large number of significant differences even between subclones challenges the notion of identifying and manipulating a few key genes to generate high production CHO cell lines.

You have accessJournal of UrologyStem Cell Research: Stem Cell Research II1 Apr 2018MP81-16 MOLECULAR CHARACTERIZATION OF CANCER STEM CELLS FROM PROSTATE CANCER XENOGRAFTS Sofia Karkampouna, Maria De Menna, Markus Germann, Joel Grosjean, Peter Clark Gray, George N. Thalmann, and Marianna Kruithof-de Julio Sofia KarkampounaSofia Karkampouna More articles by this author , Maria De MennaMaria De Menna More articles by this author , Markus GermannMarkus Germann More articles by this author , Joel GrosjeanJoel Grosjean More articles by this author , Peter Clark GrayPeter Clark Gray More articles by this author , George N. ThalmannGeorge N. Thalmann More articles by this author , and Marianna Kruithof-de JulioMarianna Kruithof-de Julio More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2018.02.2724AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Prostate cancer recurrence after androgen deprivation therapy is attributed to the existence of androgen-independent cancer stem cells, which differentiate into androgen-dependent cancer cells that reinitiate tumor growth. Phenotypic properties of cancer stem cell subpopulations versus highly proliferative progenitor cells remain to be further characterized in terms of androgen receptor signaling, quiescence/proliferation, tumorigenicity and therapy-resistance. METHODS To address the molecular basis of CSC switch from Androgen-dependency to independency we employed two patient-derived xenograft models (LAPC-9, BM-18) that have different androgen sensitivity properties. We performed microarray and proteomic analysis of tumor tissues prior to/ following castration and after androgen replacement. Subpopulations are isolated based on combination of selected markers to assess their transcriptomic changes, in vitro and in vivo self-renewal. RESULTS Castration induces rapid tumor volume decrease of BM-18 and stabilization of tumor burden in the LAPC-9, reflecting different androgen dependent cell states. Proteomic analysis of bulk BM-18 tumors (intact, 14 days post castration, 50 days post Androgen replacement), indicates an enrichment of stem cell markers upon castration; CD44, NKX3.1 and ALDH1 isoforms. Microarray analysis has confirmed upregulation of CD44 and ALDH1A1 at castrated state, versus downregulation upon androgen replacement at early time points (24 hours). We isolated CD44+/-ALDHhigh/low subpopulations by flow cytometry for transcriptome analysis. Castration in the BM-18 model induces an increase in the subpopulations of CD44+/ALDHlow (from 0.44% to 2.3%) and CD44+/ALDHhigh (from 0.06% to 0.38%). However, the same subpopulations are not increased in the LAPC-9 model upon castration, while only the CD44-/ALDHhigh subset is enriched (from 3.5% to 7.1%). Organoids derived from bulk BM-18 and LAPC-9 tumors are reestablishing tumor formation upon intraosseous inoculation. CONCLUSIONS Different androgen-independent cancer stem cell subpopulations may be distinguished by the ALDH activity status in combination with CD44. The androgen-independent cells in the BM-18 androgen-dependent model are reflected by enrichment of CD44 expression, as well as a rare double positive population. In the androgen-independent LAPC-9 model, CD44 expression is decreased potentially reflecting androgen-dependent CD44+ cells, and ALDH activity increased in CD44- cells. © 2018FiguresReferencesRelatedDetails Volume 199Issue 4SApril 2018Page: e1102-e1103 Advertisement Copyright & Permissions© 2018MetricsAuthor Information Sofia Karkampouna More articles by this author Maria De Menna More articles by this author Markus Germann More articles by this author Joel Grosjean More articles by this author Peter Clark Gray More articles by this author George N. Thalmann More articles by this author Marianna Kruithof-de Julio More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

ABSTRACTFor decades, Chinese hamster ovary (CHO) cells have been the preferred host for therapeutic monoclonal antibody (mAb) production; however, increasing mAb titer by rational engineering remains a challenge. Our previous proteomic analysis in CHO cells suggested that a higher content of glutathione (GSH) might be related to higher productivity. GSH is an important antioxidant, cell detoxifier, and is required to ensure the formation of native disulfide bonds in proteins. To investigate the involvement of GSH in mAb production, we generated stable CHO cell lines overexpressing genes involved in the first step of GSH synthesis; namely the glutamate‐cysteine ligase catalytic subunit (Gclc) and the glutamate‐cysteine ligase modifier subunit (Gclm). The two genes were reconstructed from our RNA‐Seq de novo assembly and then were functionally annotated. Once the sequences of the genes were confirmed using proteogenomics, a transiently expressed mAb was introduced into cell lines overexpressing either Gclc or Gclm. The new cell lines were compared for mAb production to the parental cell line and changes at the proteome level were measured using SWATH. As per our previous proteomics observations, overexpressing Gclm improved productivity, titer, and the frequency of high producer clones by 70%. In contrast, overexpressing Gclc, which produced a higher amount of GSH, did not increase mAb production. We show that GSH cannot be linked to higher productivity and that Gclm may be controlling other cellular processes involved in mAb production yet to be elucidated. Biotechnol. Bioeng. 2017;114: 1825–1836. © 2017 Wiley Periodicals, Inc.

CRIPTO (CR-1, TDGF1) is a cell surface/secreted oncoprotein actively involved in development and cancer. Here, we report that high expression of CRIPTO correlates with poor survival in stratified risk groups of prostate cancer (PCa) patients. CRIPTO and its signaling partner glucose-regulated protein 78 (GRP78) are highly expressed in PCa metastases and display higher levels in the metastatic ALDH(high) sub-population of PC-3M-Pro4Luc2 PCa cells compared with non-metastatic ALDH(low). Coculture of the osteotropic PC-3M-Pro4Luc2 PCa cells with differentiated primary human osteoblasts induced CRIPTO and GRP78 expression in cancer cells and increases the size of the ALDH(high) sub-population. Additionally, CRIPTO or GRP78 knockdown decreases proliferation, migration, clonogenicity and the size of the metastasis-initiating ALDH(high) sub-population. CRIPTO knockdown reduces the invasion of PC-3M-Pro4Luc2 cells in zebrafish and inhibits bone metastasis in a preclinical mouse model. These results highlight a functional role for CRIPTO and GRP78 in PCa metastasis and suggest that targeting CRIPTO/GRP78 signaling may have significant therapeutic potential.

The tumor microenvironment (TME) has been recognized as an integral component of malignancies in breast and prostate tissues, contributing in confounding ways to tumor progression, metastasis, therapy resistance, and disease recurrence. Major components of the TME are immune cells, fibroblasts, pericytes, endothelial cells, mesenchymal stroma/stem cells (MSCs), and extracellular matrix (ECM) components. Herein, we discuss the molecular and cellular heterogeneity within the TME and how this presents unique challenges and opportunities for treating breast and prostate cancers.

Biotechnology and BioengineeringVolume 114, Issue 8 p. 1633-1637 Issue Information - TOCFree Access Biotechnology and Bioengineering: Volume 114, Number 8, August 2017 First published: 26 June 2017 https://doi.org/10.1002/bit.26150AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinkedInRedditWechat Volume114, Issue8August 2017Pages 1633-1637 RelatedInformation