Culture Lines Research Articles

Low Molecular Weight Peptides Derived from Iranian Scorpion (Odontobuthus bidentatus) Venom Induces Apoptosis in the Hepatocellular Carcinoma Cell Line (HepG2) in 3D Cell Culture

Low Molecular Weight Peptides Derived from Iranian Scorpion (Odontobuthus bidentatus) Venom Induces Apoptosis in the Hepatocellular Carcinoma Cell Line (HepG2) in 3D Cell Culture

STRAIN SELECTION AND CULTIVATION OF FOOT-AND-MOUTH DISEASE VIRUSES FOR THE DEVELOPMENT OF A TRIVALENT INACTIVATED VACCINE

Foot-and-mouth disease is a well-known transboundary animal disease and has been recognised as a priority disease by the Steering Committee of the Global Framework for the Progressive Control of Transboundary Animal Diseases in Europe. The territory of the Republic of Kazakhstan is generally free of foot-and-mouth disease, however, sporadic cases of foot-and-mouth disease occur in various regions of the country as a result of importation from unfavorable countries. Epizootic safety in the Republic of Kazakhstan is maintained by vaccinating animals in the border areas of the southern and eastern regions. Inactivated vaccines against serotypes A, O, Asia produced by the All-Russian Research Institute of Animal Health (Russia) are used for vaccination. Continuous seromonitoring is carried out to maintain the level of post-vaccination antibodies at a high level, ensuring the immunity of animals to foot-and-mouth disease. However, despite all the measures taken, sporadic outbreaks of foot-and-mouth disease cause damage to livestock farming in the Republic of Kazakhstan. The effectiveness of preventive measures using vaccination depends on the strain used in the vaccine. The closer the strain used in the vaccine is genetically to the viruses circulating in that area, the more effective the preventive measures will be and the research conducted to create the most immunogenic domestic anti-foot-and-mouth disease vaccines have scientific and practical significance. This article presents the results of the selection of foot-and-mouth disease virus strains previously circulating in the territory of the Republic of Kazakhstan and studies their cultural and biological properties. Studies of the above-mentioned strains on adaptation to a transplantable cell culture line were conducted, the stability of cultural properties was studied over 10 generations and the stability was confirmed by the PCR method. Also, based on the studies, it was found that the maximum accumulation of the virus in cell culture occurs at a multiplicity of infection of 0.01-0.1 TCID50/cell. The reproductive activity of the studied isolates was within 6.5-7.25 lg TCID50/cm3 when cultivated for 16 hours.

Multiplexed, image-based pooled screens in primary cells and tissues with PerturbView.

Optical pooled screening (OPS) is a scalable method for linking image-based phenotypes with cellular perturbations. However, it has thus far been restricted to relatively low-plex phenotypic readouts in cancer cell lines in culture due to limitations associated with in situ sequencing of perturbation barcodes. Here, we develop PerturbView, an OPS technology that leverages in vitro transcription to amplify barcodes before in situ sequencing, enabling screens with highly multiplexed phenotypic readouts across diverse systems, including primary cells and tissues. We demonstrate PerturbView in induced pluripotent stem cell-derived neurons, primary immune cells and tumor tissue sections from animal models. In a screen of immune signaling pathways in primary bone marrow-derived macrophages, PerturbView uncovered both known and novel regulators of NF-κB signaling. Furthermore, we combine PerturbView with spatial transcriptomics in tissue sections from a mouse xenograft model, paving the way to in situ screens with rich optical and transcriptomic phenotypes. PerturbView broadens the scope of OPS to a wide range of models and applications.

Physical modelling study on wave damping induced by an idealized floating kelp farm

A physical modelling study was carried out to investigate random wave damping promoted by an idealized floating kelp farm. The experimental conditions spanned intermediate water depths and both linear and nonlinear water waves. Unlike previous studies of wave damping promoted by vegetation, the floating kelp farm was placed close to the water surface with a ratio between vegetation height and water depth close to 0.25. The wave transmission coefficient induced by the floating kelp farm ranged between 0.56 and 0.96. This coefficient decreased for longer floating kelp farms and it was a function of the ratio between kelp farm length and incident wavelength and of the relative wave depth. Spectral analysis showed that wave damping was not frequency-dependent for wave frequencies close to the peak frequency. The wave transmission coefficients of a floating kelp farm with about 100 culture lines and with an extension of approximately 200 m were similar to those of submerged detached breakwaters with a relative crest freeboard smaller than -0.4. Furthermore, the bulk drag coefficient of near-surface idealized floating kelp farms can be modelled as a function of the Keulegan-Carpenter number. This study highlights the potential viability of nature-based solutions such as floating kelp farms for coastal protection.

Y-box binding protein 1/cyclin A1 axis specifically promotes cell cycle progression at G2/M phase in ovarian cancer

Y-box binding protein 1 (YBX1) promotes oncogenic transformation and tumor growth. YBX1 plays a role in regulation of cell cycle promotion via upregulation of cell cycle-related genes. In ovarian cancer, YBX1 also promotes tumor growth, but the mechanisms of YBX1 in cell growth and cell cycle in ovarian cancer remain not to be fully understood. Here, we investigated whether YBX1-dependent cancer cell proliferation was specifically associated with expression of cell cycle related genes in ovarian cancer. Protein and mRNA expression levels of YBX1 and cell cycle-related genes in ovarian cancer cell lines and tissues were determined by western blot analysis, immunohistochemical analysis and reverse transcription-quantitative PCR. Cell cycle analysis was performed by flow cytometry. Luciferase assay and Chromatin immunoprecipitation assay were used to investigate a transcriptional function of YBX1. YBX1 silencing induced marked growth suppression in 4 cell lines (group A), moderate suppression in 5 cell lines (group B), and no suppression in 3 cell lines (group C) among 12 ovarian cancer cell lines in culture. The YBX1 silencing induced cell cycle arrest at G2/M phase and suppressed expression of cyclin A1 gene in group A and B cell lines, but not in group C cell lines. Cyclin A1 silencing specifically suppressed cell proliferation in group A cell lines and partially in group B cell lines, but not at all in group C cell lines. YBX1 mRNA levels were significantly correlated with cyclin A1 mRNA levels in patients with high-grade serous carcinoma. Augmented YBX1 expression plays a key role in tumor growth promotion in ovarian cancer in its close association with cyclin A1.

Selenium and Triple Negative Breast Cancer

Background. The deadliest, most dangerous subtype of breast cancer is triple-negative, which lacks treatment targets and accounts for 30% of all breast cancer-related deaths worldwide. TNBC is characterized by the expression of no estrogen, proges- terone, or human epidermal growth factor 2 receptors. This suggests that new treatment modalities with fewer adverse effects are required. Objective. The aim of the present study was to investigate the therapeutic potential of selenium compounds as an adjuvant therapy for Triple Negative Breast Cancer (TNBC), either on their own or in conjunction with nutritional supplements and chemotherapy medications. Methods. Using the keywords “selenium” and “triple negative breast cancer”, a thorough search was conducted in the PubMed database, yielding 23 articles. The following factors were taken into consideration for inclusion: studies using TNBC cell culture lines or in vivo tumors/specimens; full-text articles from the PubMed database; studies published in the English language; experiments with statistically significant results; and selenium used alone or in combination with other antioxidants or chemotherapy. This led to the evaluation of 13 articles in this review. Results. The results show that selenium therapy increased the anti-cancer drug’s effects and produced tumor cytotoxicity, while reducing the cellular features of the cancer (hyperproliferation, growth, and metastasis). Discussion. This study evaluated the various selenium compounds tested, the cell lines and model organisms used, the assays performed, and the cellular pathways affected. Conclusion. Examining the possible benefits of selenium in TNBC treatment highlights the need for more studies to confirm selenium compounds as viable co-therapeutic agents.

Reversible chemoresistance of pancreatic cancer grown as spheroids

Better in vitro models are needed to identify active drugs to treat pancreatic adenocarcinoma (PAC) patients. We used 3D hanging drop cultures to produce spheroids from five PAC cell lines and tested nine FDA-approved drugs in clinical use. All PAC cell lines in 2D culture were sensitive to three drugs (gemcitabine, docetaxel and nab-paclitaxel), however most PAC (4/5) 3D spheroids acquired profound chemoresistance even at 10 µM. In contrast, spheroids retained sensitivity to the investigational drug triptolide, which induced apoptosis. The acquired chemoresistance was also transiently retained when cells were placed back into 2D culture and six genes potentially associated with chemoresistance were identified by microarray and confirmed using quantitative RT-PCR. We demonstrate the additive effect of gemcitabine and erlotinib, from the 12 different combinations of nine drugs tested. This comprehensive study shows spheroids as a useful multicellular model of PAC for drug screening and elucidating the mechanism of chemoresistance.

Chemical Modification of Pactamycin Leads to New Compounds with Retained Antimicrobial Activity and Reduced Toxicity.

Pactamycin (PCT), an antibiotic produced by Streptomyces pactum, is a five-membered ring aminocyclitol that is active against a variety of Gram-positive and Gram-negative microorganisms, as well as several animal tumor lines in culture and in vivo. Pactamycin targets the small ribosomal subunit and inhibits protein synthesis in bacteria, archaea, and eukaryotes, but due to its toxicity is used only as a tool for biochemical research. Prompted by the successful and well-established procedure for the derivatization of antibiotics, we modified pactamycin by tethering basic amino acids to the free primary amino group of the aminocyclitol ring. Specifically, lysine, ornithine, and histidine were conjugated via an amide bond, and the antimicrobial activity of the derivatives was evaluated both in vivo and in vitro. According to our results, their antimicrobial activity was maintained at almost equal levels, while their toxicity was reduced compared to the parent molecule. These findings suggest that the new pactamycin derivatives can be considered as promising pharmacophores for the development of new antimicrobials that are able to combat the dangerously increasing resistance of pathogens to antibiotics.

Comparison of two cultivation methods for domesticating wild red algal eucheumatoids for use in the seaweed industry

AbstractEucheumatoid cultivation is an important alternative livelihood for small-scale farmers in coastal areas in east Malaysia. Over the last 50 years, the same clones of the red eucheumatoid Kappaphycus alvarezii and Kappaphycus striatus have been vegetatively propagated for cultivation resulting in low genetic diversity. This has made the cultivars more vulnerable to pests and diseases, which has been further exacerbated by climate change. There is an urgent need, therefore, to develop new cultivars that are resilient to pests and diseases, as well as environmental change. In this study, wild eucheumatoids were collected from four regions in Semporna, East Malaysia: Kerindingan, Sibuhun, Sebangkat and Omadal. These eucheumatoids were then grown at a commercial seaweed farm in Silungun, Semporna using two methods: i) tie-tie and ii) basket net for 60 days and their performance was evaluated based on growth and survival rates. A higher mean specific growth rate (SGR) was recorded by wild-collected Kappaphycus spp. (2.15 ± 1.90% day−1) and Eucheuma denticulatum (2.83 ± 3.27% day−1) when tie-tie was used. However, survival of the wild-collected eucheumatoids was better with the basket net, which led to a reduction in grazing and prevented detachment from culture lines. Of 212 original individuals, 72 survived throughout the study, despite the prevalence of pests and ice-ice disease. Of these, 46.15% Kappaphycus spp. and 50.75% E. denticulatum survived when cultivated using the basket net and 17.20% Kappaphycus spp. and 30.77% E. denticulatum survived when cultivated using tie-tie. The mean SGR and survival rate were significantly correlated with temperature, salinity, pH, current velocity and rainfall (p < 0.05). The surviving wild-collected Kappaphycus spp. and E. denticulatum have the potential to be developed into new cultivars for possible commercial use.

Digital battleground: An examination of anti-refugee discourse on Twitter against Ukrainians displaced by Russia’s invasion of Ukraine

Russia’s war of aggression in Ukraine has triggered Europe’s largest refugee crisis since World War II. In this case study, we investigate the prevalence and types of anti-refugee discourse about Ukrainian refugees on Twitter. Previous studies primarily focused on public discourse and attitudes toward racialized refugees and immigrants; the Ukrainian refugee crisis is unique in that it is one of the few instances of a recent refugee crisis involving people who do not come from mostly racialized communities. Using Communalytic, a computational social science tool for studying public discourse on social media, we automatically collected and identified toxic posts mentioning Ukrainian refugees during the first year of Russia’s full-scale invasion of Ukraine. We focused on posts containing toxic language, as this is where we are most likely to find examples of anti-refugee sentiments. Based on a manual analysis of 2,045 toxic posts referencing Ukrainian refugees, the most prevalent ones were politically motivated and included partisan content (33 percent), followed by posts containing expressions countering anti-refugee narratives (20 percent). These findings highlight the escalating politicization and polarization of discussions about Ukrainian refugees both online and offline. Furthermore, 53 percent of the sample aligned with pro-Kremlin narratives against Ukraine. By exploiting anti-refugee sentiments and leveraging existing political and cultural fault lines in the West, pro-Kremlin messages on Twitter contribute to diminishing support for Ukrainian refugees, minimizing the severity of the war, and undermining international support for Ukraine.

Comparative phylogenetic analysis of Siberian isolates of bovine pestiviruses

The results of comparative phylogenetic analysis of 52 isolates of bovine pestiviruses of three species circulating among highly productive dairy cattle in Siberia, as well as those present in the samples of commercial fetal serum, transfected cell culture lines and live vaccines on four genes: Npro, Erns, E1 and E2 are presented. The obtained data were compared with the results of the 5'UTR gene sequencing obtained earlier. The results confirmed the circulation in Siberia of eleven subtypes of BVDV-1 (a,b,c,d,f,g,i,j, k,p,r), three subtypes of BVDV-2 (a,b,c) and one BVDV -3(a). The genetic profiles of the isolates for the 5'UTR, Npro, Erns and E1 genes matched completely. The exceptions were three BVDV-1a isolates (R/FBS/96, N/MDBK/08 and SA/FBS/08) detected in infected cell cultures that clustered in a clade formed by BVDV-1j reference sequences. The remaining isolates previously shown to have 100% sequence identity of the 5'UTR had nucleotide sequence similarities of the Npro, Erns, E1, and E2 genes ranging from 96-99%, which also implies that they are closely related. The results of the BVDV-2 isolates sequencing showed a complete correspondence with the genome sequences identified in the previous studies. Comparative sequencing also confirmed the circulation of H Italian-Brazilian pestivirus isolates (BVBD-3a) in Siberia, but all the isolates were divided into two subclades. The first included the isolates isolated from fetal sera and cell cultures, while the second included those isolated from the vaccines and internal organs of diseased animals during outbreaks in farms. The genetic diversity of bovine pestiviruses, their origin and variability are discussed.

Leveraging chorionic villus biopsies for the derivation of patient-specific trophoblast stem cells.

Human trophoblast stem (TS) cells are an informative in vitro model for the generation and testing of biologically meaningful hypotheses. The goal of this project was to derive patient-specific TS cell lines from clinically available chorionic villus sampling biopsies. Cell outgrowths were captured from human chorionic villus tissue specimens cultured in modified human TS cell medium. Cell colonies emerged early during the culture and cell lines were established and passaged for several generations. Karyotypes of the newly established chorionic villus-derived trophoblast stem (TS CV ) cell lines were determined and compared to initial genetic diagnoses from freshly isolated chorionic villi. Phenotypes of TSCV cells in the stem state and following differentiation were compared to cytotrophoblast-derived TS (TS CT ) cells. TSCV and TSCT cells uniformly exhibited similarities in the stem state and following differentiation into syncytiotrophoblast and extravillous trophoblast cells. Chorionic villus tissue specimens provide a valuable source for TS cell derivation. They expand the genetic diversity of available TS cells and are associated with defined clinical outcomes. TSCV cell lines provide a new set of experimental tools for investigating trophoblast cell lineage development.

The Antinomies of Latvian Literary Realism

This article traces the social and literary contexts of Latvian literary realism. During its rise in the nineteenth century, Latvian realism was seen as developing along the general lines of European culture that helped to foster aesthetic diversity and innovative potential. These processes are the focus of the first part of the article. The subsequent reshaping and distortion of the concept of realism during the period of Soviet occupation are then scrutinized in some detail. The investigation follows in the footsteps of the current rise of interest in realism, considering its importance in a broad spectrum of contexts, and claims that realism is an innovative aesthetic practice of great historical and contemporary relevance. However, it has also frequently been misunderstood or consciously misinterpreted by representatives of other aesthetic trends as well as misused by political regimes. The article points toward the necessity of restoring a sound interpretation of realist aspirations and their social and aesthetic contributions.

Receptor tyrosine kinase inhibition leads to regression of acral melanoma by targeting the tumor microenvironment.

Acral melanoma (AM) is an aggressive melanoma variant that arises from palmar, plantar, and nail unit melanocytes. Compared to non-acral cutaneous melanoma (CM), AM is biologically distinct, has an equal incidence across genetic ancestries, typically presents in advanced stage disease, is less responsive to therapy, and has an overall worse prognosis. Independent analysis of published genomic and transcriptomic sequencing identified that receptor tyrosine kinase (RTK) ligands and adapter proteins are frequently amplified, translocated, and/or overexpressed in AM. To target these unique genetic changes, a zebrafish acral melanoma model was exposed to a panel of narrow and broad spectrum multi-RTK inhibitors, revealing that dual FGFR/VEGFR inhibitors decrease acral-analogous melanocyte proliferation and migration. The potent pan-FGFR/VEGFR inhibitor, Lenvatinib, uniformly induces tumor regression in AM patient-derived xenograft (PDX) tumors but only slows tumor growth in CM models. Unlike other multi-RTK inhibitors, Lenvatinib is not directly cytotoxic to dissociated AM PDX tumor cells and instead disrupts tumor architecture and vascular networks. Considering the great difficulty in establishing AM cell culture lines, these findings suggest that AM may be more sensitive to microenvironment perturbations than CM. In conclusion, dual FGFR/VEGFR inhibition may be a viable therapeutic strategy that targets the unique biology of AM.

UM171 suppresses breast cancer progression by inducing KLF2

PurposeBreast cancer is the most frequent cancer in women with significant death rate. Morbidity is associated with drug resistance and metastasis. Development of novel drugs is unmet need. The aim of this study is to show potent anti-neoplastic activity of the UM171 compound on breast cancer cells and its mechanism of action.MethodsThe inhibitory effect of UM171 on several breast cancer (BC) cell lines was examined using MTT and colony-forming assays. Cell cycle and apoptosis assays were utilized to determine the effect of UM171 on BC cell proliferation and survival. Wound healing scratch and transwell migration assays were used to examine the migration of BC cell lines in culture. Xenograft of mouse model with 4T1 cells was used to determine inhibitory effect of UM171 in vivo. Q-RT-PCR and western blotting were used to determine the expression level of genes effected by UM171. Lentivirus-mediated shRNAs were used to knockdown the expression of KLF2 in BC cells.ResultsUM171 was previously identified as a potent agonist of human hematopoietic stem cell renewal and inhibitor of leukemia. In this study, UM171 was shown to inhibit the growth of multiple breast cancer cell lines in culture. UM171-mediated growth inhibition was associated with the induction of apoptosis, G2/M cell cycle arrest, lower colony-forming capacity, and reduced motility. In a xenotransplantation model of mouse triple-negative breast cancer 4T1 cells injected into syngeneic BALB/c mice, UM171 strongly inhibited tumor growth at a level comparable to control paclitaxel. UM171 increased the expression of the three PIM genes (PIM1-3) in breast cancer cells. Moreover, UM171 strongly induced the expression of the tumor suppressor gene KLF2 and cell cycle inhibitor P21CIP1. Accordingly, knockdown of KLF2 using lentivirus-mediated shRNA significantly attenuated the growth suppressor activity of UM171. As PIM1-3 act as oncogenes and are involved in breast cancer progression, induction of these kinases likely impedes the inhibitory effect of KLF2 induction by UM171. Accordingly, combination of UM171 with a PAN-PIM inhibitor LGH447 significantly reduced tumor growth in culture.ConclusionThese results suggested that UM171 inhibited breast cancer progression in part through activation of KLF2 and P21. Combination of UM171 with a PAN-PIM inhibitor offer a novel therapy for aggressive forms of breast cancer.

COMPARATIVE ANALYSIS OF CULTURE-BOUND WORDS IN “A THOUSAND SPLENDID SUNS” AND ITS TRANSLATIONS INTO RUSSIAN AND UZBEK LANGUAGES

This article explores how the translation of Khaled Hosseini’s “A Thousand Splendid Suns” into Russian and Uzbek reflects the influence of language and culture on the story's reception. It focuses on the translation of culture-bound words and stylistic devices, analyzing the methods used to retain the novel's emotional impact and cultural depth across different languages. By examining specific examples of translated text, the study highlightsthe techniques translators use to make the narrative relatable and understandable for diverse audiences. The analysis demonstrates how adaptations in translation can affect readers' understanding and enjoyment of the story across cultural lines.

A genetically inducible endothelial niche enables vascularization of human kidney organoids with multilineage maturation and emergence of renin expressing cells

Vascularization plays a critical role in organ maturation and cell type development. Drug discovery, organ mimicry, and ultimately transplantation hinge on achieving robust vascularization of in vitro engineered organs. Here, focusing on human kidney organoids, we overcame this hurdle by combining a human induced pluripotent stem cell (iPSC) line containing an inducible ETS translocation variant 2 (ETV2) (a transcription factor playing a role in endothelial cell development) that directs endothelial differentiation in vitro, with a non-transgenic iPSC line in suspension organoid culture. The resulting human kidney organoids show extensive endothelialization with a cellular identity most closely related to human kidney endothelia. Endothelialized kidney organoids also show increased maturation of nephron structures, an associated fenestrated endothelium with de novo formation of glomerular and venous subtypes, and the emergence of drug-responsive renin expressing cells. The creation of an engineered vascular niche capable of improving kidney organoid maturation and cell type complexity is a significant step forward in the path to clinical translation. Thus, incorporation of an engineered endothelial niche into a previously published kidney organoid protocol allowed the orthogonal differentiation of endothelial and parenchymal cell types, demonstrating the potential for applicability to other basic and translational organoid studies.

Alimentary Images as Metaphor of Education

AbstractThe aim of this paper is to explore how the history of images and conceptual metaphors resulting from them that we use in educational reflections are formed regardless of if they are problematized in practical life. Insight into history shows how these images are shaped not only by our own experiences and by the context of our lives, but also by the history of such images, which are unconsciously inscribed in our metaphorical speech through so called “residues of meaning”. The paper clarifies this, using the examples of alimentary images, that are a transition from nutrix (wet nurse) to nutritor (teacher). The text offers selected examples of consideration of alimentary images. These are among the most primitive and therefore the deepest images of human experience. This history is an example of a cultural line that goes from ancient educational imagination to the more recent forms of such images, even if always with different accents.

Participatory Evaluation of Thirty (30) New Rice (Oryza Sativa L.) Lines Developed from Anther Culture under Senegal River Valley Growing Conditions

Rice is one of the most widely used staple foods in the world. However, its production is still very limited compared to the exponentially growing demand. Senegal, with an average consumption of 90 kg/habitant, is the third largest importer of rice in Africa. Aware of the impact of imports on the population, Senegal has set itself the objective of achieving food self-sufficiency by 2022 with an average yield of 5 tonnes/ha. ISRA, through its research, contributes to the achievement of this objective by developing adapted technologies. It is within this framework that a participatory evaluation of 30 new anther culture lines was conducted at the ISRA research station in Fanaye during the hot dry season of 2020. Surveys were conducted among producers through guided tours, while observations and measurements were made to collect agro-morphological parameters and characteristics preferred by producers. The agronomic results analysed and combined with the surveys made it possible to select the 10 best performing varieties. Among these 10 best performing lines, 6 were chosen by the producers. They are KF18134, KF18040, KF18068, KF18103, KF18124, KF18048, KF18005, KF18017, KF18001, KF18024. Crop yield is the first characteristic chosen by the growers. It was followed by short cycle characteristics, panicle load, low bird damage and medium height.

Pre-clinical evaluation of clinically relevant iPS cell derived neuroepithelial stem cells as an off-the-shelf cell therapy for spinal cord injury.

Preclinical transplantations using human neuroepithelial stem (NES) cells in spinal cord injury models have exhibited promising results and demonstrated cell integration and functional improvement in transplanted animals. Previous studies have relied on the generation of research grade cell lines in continuous culture. Using fresh cells presents logistic hurdles for clinical transition regarding time and resources for maintaining high quality standards. In this study, we generated a good manufacturing practice (GMP) compliant human iPS cell line in GMP clean rooms alongside a research grade iPS cell line which was produced using standardized protocols with GMP compliant chemicals. These two iPS cell lines were differentiated into human NES cells, from which six batches of cell therapy doses were produced. The doses were cryopreserved, thawed on demand and grafted in a rat spinal cord injury model. Our findings demonstrate that NES cells can be directly grafted post-thaw with high cell viability, maintaining their cell identity and differentiation capacity. This opens the possibility of manufacturing off-the-shelf cell therapy products. Moreover, our manufacturing process yields stable cell doses with minimal batch-to-batch variability, characterized by consistent expression of identity markers as well as similar viability of cells across the two iPS cell lines. These cryopreserved cell doses exhibit sustained viability, functionality, and quality for at least 2 years. Our results provide proof of concept that cryopreserved NES cells present a viable alternative to transplanting freshly cultured cells in future cell therapies and exemplify a platform from which cell formulation can be optimized and facilitate the transition to clinical trials.