QOLP-35. PEDIATRIC POST-MORTEM TISSUE DONATION DURING A PANDEMIC: A MODEL OF COLLABORATION

BACKGROUND Obtaining post-mortem tissue from pediatric oncology patients is not only critical to research, but studies show that participating in the process can also help grieving families heal. Since 2019, the national Gift from a Child program — a multi-institutional effort to increase the rate of rapid autopsies for pediatric CNS tumor patients — has made significant progress. Collecting high-quality post-mortem tissue has advanced research through cell line generation and genomic analyses. Unfortunately, some autopsy programs temporarily shut down during the COVID-19 pandemic. METHODS We retrospectively reviewed autopsies of four patients treated at Memorial Sloan Kettering (MSK) who underwent limited brain post-mortem examination at Weill Cornell Medicine College (WCMC) from June 2020 to June 2021. We collected patient demographics; DNR status; time of death and procedure; restrictions due to the COVID-19 pandemic; and results of the tissue analysis. Each case presented unique challenges and the timing of securing parental consent varied. RESULTS Three of four specimens were processed within 12 hours of the time of death. Two Spanish-speaking families required interpreters services to obtain consent. In all cases tumor aliquots were flash frozen for further study. All specimens contained viable tumor and cell line generation was successful in one case. All families/caregivers expressed gratitude for the opportunity to participate and for the handling of the procedures. DISCUSSION Despite the sensitive nature of these cases, clinicians should offer the option of a rapid autopsy to caregivers of pediatric patients based on the scientific need and the positive effect it has on grieving families. This paper outlines the logistical efforts required for these donations to take place and provides a framework for providers to offer rapid autopsy as an option for families through this program.

Isolation in Natural Host Cell Lines of Wolbachia Strains wPip from the Mosquito Culex pipiens and wPap from the Sand Fly Phlebotomus papatasi

Endosymbiotic intracellular bacteria of the genus Wolbachia are harboured by many species of invertebrates. They display a wide range of developmental, metabolic and nutritional interactions with their hosts and may impact the transmission of arboviruses and protozoan parasites. Wolbachia have occasionally been isolated during insect cell line generation. Here, we report the isolation of two strains of Wolbachia, wPip and wPap, during cell line generation from their respective hosts, the mosquito Culex pipiens and the sand fly Phlebotomus papatasi. wPip was pathogenic for both new C. pipiens cell lines, CPE/LULS50 and CLP/LULS56, requiring tetracycline treatment to rescue the lines. In contrast, wPap was tolerated by the P. papatasi cell line PPL/LULS49, although tetracycline treatment was applied to generate a Wolbachia-free subline. Both Wolbachia strains were infective for a panel of heterologous insect and tick cell lines, including two novel lines generated from the sand fly Lutzomyia longipalpis, LLE/LULS45 and LLL/LULS52. In all cases, wPip was more pathogenic for the host cells than wPap. These newly isolated Wolbachia strains, and the novel mosquito and sand fly cell lines reported here, will add to the resources available for research on host–endosymbiont relationships, as well as on C. pipiens, P. papatasi, L. longipalpis and the pathogens that they transmit.

Differential expression of miRNAs and functional role of mir-200a in high and low productivity CHO cells expressing an Fc fusion protein

Objectives We used miRNA and proteomic profiling to understand intracellular pathways that contribute to high and low specific productivity (Qp) phenotypes in CHO clonally derived cell lines (CDCLs) from the same cell line generation project. Results Differentially expressed (DE) miRNAs were identified which are predicted to target several proteins associated with protein folding. MiR-200a was found to have a number of predicted targets associated with the unfolded protein response (UPR) which were shown to have decreased expression in high Qp CDCLs and have no detected change at the mRNA level. MiR-200a overexpression in a CHO CDCL was found to increase recombinant protein titer by 1.2 fold and Qp by 1.8 fold. Conclusion These results may suggest a role for miR-200a in post-transcriptional regulation of the UPR, presenting miR-200a as a potential target for engineering industrially attractive CHO cell phenotypes.

Cell-to-cell expression dispersion of B-cell surface proteins is linked to genetic variants in humans

Variability in gene expression across a population of homogeneous cells is known to influence various biological processes. In model organisms, natural genetic variants were found that modify expression dispersion (variability at a fixed mean) but very few studies have detected such effects in humans. Here, we analyzed single-cell expression of four proteins (CD23, CD55, CD63 and CD86) across cell lines derived from individuals of the Yoruba population. Using data from over 30 million cells, we found substantial inter-individual variation of dispersion. We demonstrate, via de novo cell line generation and subcloning experiments, that this variation exceeds the variation associated with cellular immortalization. We detected a genetic association between the expression dispersion of CD63 and the rs971 SNP. Our results show that human DNA variants can have inherently-probabilistic effects on gene expression. Such subtle genetic effects may participate to phenotypic variation and disease outcome.

Cell-to-cell expression dispersion of B-cell surface proteins displays genetic variation among humans

ABSTRACTVariability in gene expression across a population of homogeneous cells is known to influence various biological processes. In model organisms, natural genetic variants were found that modify expression dispersion (variability at a fixed mean) but whether such effects exist in humans has not been fully demonstrated. Here, we analyzed single-cell expression of four proteins (CD23, CD55, CD63 and CD86) across cell lines derived from individuals of the Yoruba population. Using data from over 30 million cells, we found substantial inter-individual variation of dispersion. We demonstrate, via de novo cell line generation and subcloning experiments, that this variation exceeds the variation associated with cellular immortalization. By association mapping, we linked the expression dispersion of CD63 to the rs971 SNP. Our results show that human DNA variants can have inherently-probabilistic effects on gene expression. Such subtle genetic effects may participate to phenotypic variation and disease predisposition.