Biology subjects

Kufrin, V.

Publications and source records attributed to Kufrin, V..

3 recordsLinked to original sources

bioRxiv2024

Generation and application of novel hES cell reporter lines for the differentiation and maturation of hPS cell-derived islet-like clusters

The significant advances in the differentiation of human pluripotent stem (hPS) cells into pancreatic endocrine cells, including functional {beta}-cells, have been based on a detailed understanding of the underlying developmental mechanisms. However, the final differentiation steps, leading from endocrine progenitors to mono-hormonal and mature pancreatic endocrine cells, remain to be fully understood and this is reflected in the remaining shortcomings of the hPS cell-derived islet cells (SC-islet cells), which include a lack of {beta}-cell maturation and variability among different cell lines. Additional signals and modifications of the final differentiation steps will have to be assessed in a combinatorial manner to address the remaining issues and appropriate reporter lines would be useful in this undertaking. Here we report the generation and functional validation of human pluripotent stem cell reporter lines that can monitor the generation of INS+ and GCG+ cells and their resolution into mono-hormonal cells (INS eGFP, INS eGFP/ GCG mCHERRY) as well as {beta}-cell maturation (INS eGFP/ MAFA mCHERRY) and function (INS GCaMP6). The reporter hPS cell lines maintained strong and widespread expression of pluripotency markers and differentiated efficiently into definitive endoderm and pancreatic progenitor (PP) cells. PP cells from all lines differentiated efficiently into islet cell clusters that robustly expressed the corresponding reporters and contained glucose-responsive, insulin-producing cells. To demonstrate the applicability of these hES cell reporter lines in a high-content live imaging approach for the identification of optimal differentiation conditions, we adapted our differentiation procedure to generate SC-islet clusters in microwells. This allowed the live confocal imaging of multiple SC-islets for a single condition and, using this approach, we found that the use of the N21 supplement in the last stage of the differentiation increased the number of monohormonal {beta}-cells without affecting the number of -cells in the SC-islets. The hPS cell reporter lines and the high-content live imaging approach described here will enable the efficient assessment of multiple conditions for the optimal differentiation and maturation of SC-islets.

developmental biology↗

bioRxiv2023

The histone modifier KAT2A presents a selective target in a subset of well-differentiated microsatellite-stable colorectal cancers

BackgroundLysine acetyltransferase 2A (KAT2A) plays a pivotal role in epigenetic gene regulation across various types of cancer. In colorectal cancer (CRC), upregulation of KAT2A is associated with a more aggressive phenotype. Our study aims to elucidate the molecular underpinnings of KAT2A dependency in CRC and assess the consequences of KAT2A depletion. MethodsWe conducted a comprehensive analysis by integrating CRISPR-Cas9 screening data with genomics, transcriptomics, and global acetylation patterns in CRC cell lines to pinpoint molecular markers indicative of KAT2A dependency. Additionally, we characterized the phenotypic effect of a CRISPR-Cas9-mediated KAT2A knockout and chemical inhibition of KAT2A in CRC cell lines and patient- derived 3D spheroid cultures. ResultsOur findings reveal that KAT2A dependency is closely associated with a lower mutational burden and increased differentiation grade in CRC cell lines, independent of the KAT2A expression levels. KAT2A dependent CRC cell lines display enriched H3K27ac marks at gene loci linked to enterocytic differentiation. Loss of KAT2A leads to decreased cell growth and viability, downregulation of proliferation- and stem cell-associated genes, and induction of differentiation markers. ConclusionA specific subset of CRCs with a more differentiated phenotype relies on KAT2A. For these CRC cases, KAT2A might represent a promising novel therapeutic target.

cancer biology↗

bioRxiv2023

Regulation of multiple signaling pathways promotes the consistent expansion of human pancreatic progenitors in defined conditions

The unlimited expansion of human progenitor cells in vitro could unlock many prospects for regenerative medicine. However, it remains an important challenge as it requires the decoupling of the mechanisms supporting progenitor self-renewal and expansion from those mechanisms promoting their differentiation. This study focuses on the expansion of human pluripotent stem (hPS) cell derived pancreatic progenitors (PP) to advance novel therapies for diabetes. We obtained mechanistic insights into PP expansion requirements and, through a hypothesis-driven iterative approach, identified conditions for the robust and unlimited expansion of hPS cell derived PP cells under GMP-compliant conditions. We show that the combined stimulation of specific mitogenic pathways, suppression of retinoic acid signaling and inhibition of selected branches of the TGF{beta} and Wnt signaling pathways are necessary for the effective decoupling of PP proliferation from differentiation. This enabled the reproducible, 2000-fold, over ten passages and 40-45 days, expansion of PDX1+/SOX9+/NKX6-1+ PP cells. Transcriptome analyses confirmed the stabilisation of PP identity and the effective suppression of differentiation. Using these conditions, PDX1+/SOX9+/NKX6-1+ PP cells, derived from different, both XY and XX, hPS cells lines, were enriched to nearly 90% homogeneity and expanded with very similar kinetics and efficiency. Furthermore, non-expanded and expanded PP cells, from different hPS cell lines, were differentiated in microwells into homogeneous islet-like clusters (SC-islets) with very similar efficiency. These clusters contained abundant {beta}-cells of comparable functionality as assessed by glucose-stimulated insulin secretion assays. These findings established the signaling requirements to decouple PP proliferation from differentiation and allowed the consistent expansion of hPS cell derived PP cells. They will enable the establishment of large banks of GMP-derived PP cells derived from diverse hPS cell lines. This approach will streamline SC-islet production for further development of the differentiation process, diabetes research, personalized medicine and cell therapies.

developmental biology↗