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1.
Cytotherapy ; 25(1): 59-67, 2023 01.
Article in English | MEDLINE | ID: mdl-36319564

ABSTRACT

BACKGROUND: Engineered tissues and cell therapies based on human induced pluripotent stem cells (iPSCs) represent a promising approach for novel medicines. However, iPSC-derived cells and tissues may contain residual undifferentiated iPSCs that could lead to teratoma formation after implantation into patients. As a consequence, highly sensitive and specific methods for detecting residual undifferentiated iPSCs are indispensable for safety evaluations of iPSC-based therapies. The present study provides an approach for identifying potential marker genes for iPSC impurities in iPSC-derived cells using RNA sequencing data from iPSCs and various differentiated cell types. METHODS: Identifying iPSC marker genes for each cell type individually provided a larger and more specific set of potential marker genes than considering all cell types in the analysis. Thus, the authors focused on identifying markers for iPSC impurities in iPSC-derived cardiomyocytes (iCMs) and validated the selected genes by reverse transcription quantitative polymerase chain reaction. The sensitivity of the candidate genes was determined by spiking different amounts of iPSCs into iCMs and their performance was compared with the previously suggested marker lin-28 homolog A (LIN28A). RESULTS: Embryonic stem cell-related gene (ESRG), long intergenic non-protein coding RNA 678 (LINC00678), CaM kinase-like vesicle-associated (CAMKV), indoleamine 2,3-dioxygenase 1 (IDO1), chondromodulin (CNMD), LINE1-type transposase domain containing 1 (L1DT1), LIN28A, lymphocyte-specific protein tyrosine kinase (LCK), vertebrae development-associated (VRTN) and zinc finger and SCAN domain containing 10 (ZSCAN10) detected contaminant iPSCs among iCMs with a limit of detection that ranged from 0.001% to 0.1% depending on the gene and iCM batch used. CONCLUSIONS: Using the example of iCMs, the authors provide a strategy for identifying a set of highly specific and sensitive markers that can be used for quality assessment of iPSC-derived products.


Subject(s)
Induced Pluripotent Stem Cells , Humans , Induced Pluripotent Stem Cells/metabolism , Cell Differentiation/genetics , Embryonic Stem Cells
2.
Front Immunol ; 10: 1340, 2019.
Article in English | MEDLINE | ID: mdl-31281311

ABSTRACT

Ofatumumab is the first, fully human, anti-CD20 monoclonal antibody in Phase 3 development for multiple sclerosis (MS). The study focused on changes in lymphocyte subsets in blood and lymphoid tissues and on potential novel biomarkers as a result of anti-CD20 antibody action in Cynomolgus monkeys treated with human equivalent doses of subcutaneous (s.c.) ofatumumab on Days 0, 7, and 14. Axillary lymph nodes (LNs) and blood samples were collected at various time points until Day 90. Lymphocyte subsets were quantified by flow cytometry, while morphological and immune cell changes were assessed by imaging mass cytometry (IMC), immunohistochemistry (IHC), in situ hybridization (ISH), and transcriptome analyses using single-cell methodology. Ofatumumab treatment resulted in a potent and rapid reduction of B cells along with a simultaneous drop in CD20+ T cell counts. At Day 21, IHC revealed B-cell depletion in the perifollicular and interfollicular area of axillary LNs, while only the core of the germinal center was depleted of CD20+CD21+ cells. By Day 62, the perifollicular and interfollicular areas were abundantly infiltrated by CD21+ B cells and this distribution returned to the baseline cytoarchitecture by Day 90. By IMC CD20+CD3+CD8+ cells could be identified at the margin of the follicles, with a similar pattern of distribution at Day 21 and 90. Single-cell transcriptomics analysis showed that ofatumumab induced reversible changes in t-distributed stochastic neighbor embedding (t-SNE) defined B-cell subsets that may serve as biomarkers for drug action. In summary, low dose s.c. ofatumumab potently depletes both B cells and CD20+ T cells but apparently spares marginal zone (MZ) B cells in the spleen and LN. These findings add to our molecular and tissue-architectural understanding of ofatumumab treatment effects on B-cell subsets.


Subject(s)
Antibodies, Monoclonal, Humanized/pharmacology , B-Lymphocytes , Genomics , Lymph Nodes , Lymphocyte Depletion , Mass Spectrometry , Single-Cell Analysis , Animals , B-Lymphocytes/cytology , B-Lymphocytes/immunology , Gene Expression Profiling , In Situ Hybridization , Lymph Nodes/cytology , Lymph Nodes/immunology , Macaca fascicularis
3.
Biophys Rev ; 1(4): 177, 2009 Dec.
Article in English | MEDLINE | ID: mdl-28510029

ABSTRACT

Electropulsation is one of the nonviral methods successfully used to deliver genes into living cells in vitro and in vivo. This approach shows promise in the field of gene and cellular therapies. The present review focuses on the processes supporting gene electrotransfer in vitro. In the first part, we will report the events occurring before, during, and after pulse application in the specific field of plasmid DNA electrotransfer at the cell level. A critical discussion of the present theoretical considerations about membrane electropermeabilization and the transient structures involved in the plasmid uptake follows in a second part.

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