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Omics analyses of 3T3-J2 feeder cells to identify culture conditions that support the long-term culture of human pancreatic progenitor cells

ABSTRACT: Multipotent pancreatic progenitor cells have been successfully cultured, expanded and differentiated into cells of the adult human pancreas. The long-term culture of these progenitors (termed cPP) relies on support from mitotically inactivated 3T3-J2 mouse embryonic fibroblasts. The 3T3-J2-derived signals that support cPP expansion and maintain the fine balance between self-renewal and lineage commitment in this co-culture system remain unknown. To unmask these signals, we exploited a striking disparity in morphology and the capacity to self-renew of cPP cultured on 3T3-J2 cells from different sources. We deployed a comparative approach to identify (1) genes that define expandable cPP through transcriptome analysis and (2) key factors secreted by 3T3-J2 using selective enrichment of newly synthesised proteins and mass spectrometry analysis. Using cPP from three genetic backgrounds cultured on either supportive or non-supportive 3T3-J2, these analyses revealed 100 transcription factor genes that are significantly down-regulated in cPP cultured on non-supportive 3T3-J2. Analysis of differentially expressed 3T3-J2 proteins identified 53 whose expression is higher in supportive 3T3-J2. These results provide significant insight into the requirements not only for optimal cPP culture and expansion but also for progenitor cell types in other organ systems that rely on 3T3-J2 co-culture, most notably keratinocytes for clinical applications.

ORGANISM(S): Mus Musculus (mouse)

SUBMITTER: Norris Ray Dunn

PROVIDER: PXD041758 | JPOST Repository | Wed Apr 24 00:00:00 BST 2024

REPOSITORIES: jPOST

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Project description:Developmental checkpoints in stem/progenitor cells are critical to the determination, commitment and differentiation into distinct lineages. Cancer cells often retain expression of lineage-specific checkpoint proteins, but their potential impact in cancer remains elusive. T lymphocytes mature in the thymus following a highly orchestrated developmental process that entails the successive rearrangements and expression of T-cell receptor (TCR) genes. Low affinity recognition of self-peptide/MHC complexes (self-pMHC) presented by thymic epithelial cells by the TCR of CD4+CD8+ (DP) cortical thymocytes transduces positive selection signals that ultimately shape the developing T cell repertoire. DP thymocytes not receiving these signals die by lack of stimulation whereas those that recognize self-pMHC with high affinity undergo TCR-mediated apoptosis and negative selection. In T-cell acute lymphoblastic leukaemia (T-ALL), leukaemic transformation of maturating thymocytes results from the acquisition of multiple genetic and epigenetic alterations in oncogenes and tumour suppressor genes, that disrupt the normal regulatory circuits and drive clonal expansion of differentiation-arrested lymphoblasts. We show here that TCR triggering by negatively-selecting self-pMHC prevented T-ALL development and leukaemia maintenance in mice. Induction of TCR signalling by high affinity self-pMHC or treatment with monoclonal antibodies to the CD3 signalling chain (anti-CD3) caused massive leukaemic cell death and a gene expression program resembling that of thymocyte negative selection. Importantly, anti-CD3 treatment hampered leukaemogenesis in mice transplanted with either mouse or patient-derived T-ALLs. These data provide a rationale for targeted therapy based on anti-CD3 treatment of T-ALL patients and demonstrate that endogenous developmental checkpoint proteins are amenable to therapeutic intervention in cancer cells. Gene expression data from four culture conditions was performed for the following cells: ALL-SIL-TCRÎ±/Î²-GFP co-cultured on OP9-DL1 for 48 h, ALL-SIL-TCRÎ±/Î²-GFP without co-culture, ALL-SIL cells transduced with TLX shRNA (sh-TLX), and ALL-SIL transduced with sh-control vectors. All conditions are performed in two replicates.

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Omics analyses of 3T3-J2 feeder cells to identify culture conditions that support the long-term culture of human pancreatic progenitor cells

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