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Genome-wide analysis of single nucleotide variants allows for robust and accurate assessment of clonal derivation in cell lines used to produce biologics.

ABSTRACT: A clonally derived (or "monoclonal") cell line is a cell population derived from a single progenitor cell. Clonally derived cell lines are required for many biotechnological applications. For instance, recombinant mammalian cells used to produce therapeutic proteins are expected by regulatory authorities to be clonally derived. Assurance of clonal derivation (or "clonality") is usually obtained from the characterization of the procedure used for cell cloning, for instance by assessing the success rate of single-cell sorting but not by assessing the cell line itself. We have developed a method to assess clonal derivation directly from the genetic makeup of cells. The genomic test of clonality is based on whole-genome sequencing and statistical analysis of single nucleotide variants. This approach quantifies the clonal fractions present in nonclonal samples and it provides a measure of the probability that a cell line is derived from a single cell. Upon experimental validation of the test, we show that it is highly accurate and that it can robustly detect minor clonal fractions of as little as 1% of the cell population. Moreover, we find that it is applicable to various cell line development protocols. This approach can simplify development protocols and shorten timelines while ensuring clonal derivation with high confidence.

SUBMITTER: Kuhn A

PROVIDER: S-EPMC7818110 | biostudies-literature | 2020 Aug

REPOSITORIES: biostudies-literature

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Genome-wide analysis of single nucleotide variants allows for robust and accurate assessment of clonal derivation in cell lines used to produce biologics.

Kuhn Alexandre A Le Fourn Valérie V Fisch Igor I Mermod Nicolas N

Biotechnology and bioengineering 20200824 12

A clonally derived (or "monoclonal") cell line is a cell population derived from a single progenitor cell. Clonally derived cell lines are required for many biotechnological applications. For instance, recombinant mammalian cells used to produce therapeutic proteins are expected by regulatory authorities to be clonally derived. Assurance of clonal derivation (or "clonality") is usually obtained from the characterization of the procedure used for cell cloning, for instance by assessing the succes ...[more]

PMID: 32779734

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Project description:BACKGROUND:The role of brown fat in non-shivering thermogenesis and the discovery of brown fat depots in adult humans has made it the subject of intense research interest. A renewable source of brown adipocyte (BA) progenitors would be highly valuable for research and therapy. Directed differentiation of human pluripotent stem (hPS) cells to white or brown adipocytes is limited by lack of cell purity and scalability. Here we describe an alternative approach involving the identification of clonal self-renewing human embryonic progenitor (hEP) cell lines following partial hPS cell differentiation and selection of scalable clones. METHODS:We screened a diverse panel of hPS cell-derived clonal hEP cell lines for adipocyte markers following growth in adipocyte differentiation medium. The transcriptome of the human hES-derived clonal embryonic progenitor cell lines E3, C4ELS5.1, NP88, and NP110 representing three class of definitive adipocyte progenitors were compared to the relatively non-adipogenic line E85 and adult-derived BAT and SAT-derived cells using gene expression microarrays, RT-qPCR, metabolic analysis and immunocytochemistry. Differentiation conditions were optimized for maximal UCP1 expression. RESULTS:Many of the differentiated hEP cell lines expressed the adipocyte marker, FAPB4, but only a small subset expressed definitive adipocyte markers including brown adipocyte marker, UCP1. Class I cells (i.e., E3) expressed CITED1, ADIPOQ, and C19orf80 but little to no UCP1. Class II (i.e., C4ELS5.1) expressed CITED1 and UCP1 but little ADIPOQ and LIPASIN. Class III (i.e., NP88, NP110) expressed CITED1, ADIPOQ, C19orf80, and UCP1 in a similar manner as fetal BAT-derived (fBAT) cells. Differentiated NP88 and NP110 lines were closest to fBAT cells morphologically in adiponectin and uncoupling protein expression. But they were more metabolically active than fBAT cells, had higher levels of 3-hydroxybutyrate, and lacked expression of fetal/adult marker, COX7A1. The hEP BA progenitor lines were scalable to 17 passages without loss of differentiation capacity and could be readily rederived. CONCLUSIONS:Taken together, these data demonstrate that self-renewing adipocyte progenitor cells can be derived from hES cells and that they are functionally like BAT cells but with unique properties that might be advantageous for basic research and for development of cell-based treatments for metabolic diseases.

Project description:Background: Most human breast cancer cell lines currently in use were developed and are cultured under ambient (21%) oxygen conditions. While this is convenient in practical terms, higher ambient oxygen could increase oxygen radical production, potentially modulating signaling pathways. We have derived and grown a series of four human breast cancer cell lines under 5% oxygen, and have compared their properties to those of established breast cancer lines growing under ambient oxygen. Methods: Cell lines were characterized in terms of appearance, cellular DNA content, mutation spectrum, hormone receptor status, pathway utilization and drug sensitivity. Results: Three of the four lines (NZBR1, NZBR2, and NZBR4) were triple negative (ER-, PR-, HER2-), with NZBR1 also over-expressing EGFR. NZBR3 was HER2+ and ER+ and also over-expressed EGFR. Cell lines grown in 5% oxygen showed increased expression of the hypoxia-inducible factor 1 (HIF-1) target gene carbonic anhydrase 9 (CA9) and decreased levels of ROS. As determined by protein phosphorylation, NZBR1 showed low AKT pathway utilization while NZBR2 and NZBR4 showed low p70S6K and rpS6 pathway utilization. The lines were characterized for sensitivity to 7-hydroxytamoxifen, doxorubicin, paclitaxel, the PI3K inhibitor BEZ235 and the HER inhibitors lapatinib, afatinib, dacomitinib, and ARRY-380. In some cases they were compared to established breast cancer lines. Of particular note was the high sensitivity of NZBR3 to HER inhibitors. The spectrum of mutations in the NZBR lines was generally similar to that found in commonly used breast cancer cell lines but TP53 mutations were absent and mutations in EVI2B, LRP1B, and PMS2, which have not been reported in other breast cancer lines, were detected. The results suggest that the properties of cell lines developed under low oxygen conditions (5% O2) are similar to those of commonly used breast cancer cell lines. Although reduced ROS production and increased HIF-1 activity under 5% oxygen can potentially influence experimental outcomes, no difference in sensitivity to estrogen or doxorubicin was observed between cell lines cultured in 5 vs. 21% oxygen.

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Genome-wide analysis of single nucleotide variants allows for robust and accurate assessment of clonal derivation in cell lines used to produce biologics.

Publications

Genome-wide analysis of single nucleotide variants allows for robust and accurate assessment of clonal derivation in cell lines used to produce biologics.

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