The embryo instructs the allocation of cell states to spatially regulate functions. In the blastocyst, patterning of trophoblast (TR) cells ensures successful implantation and placental development... Show moreThe embryo instructs the allocation of cell states to spatially regulate functions. In the blastocyst, patterning of trophoblast (TR) cells ensures successful implantation and placental development. Here, we defined an optimal set of molecules secreted by the epiblast (inducers) that captures in vitro stable, highly self-renewing mouse trophectoderm stem cells (TESCs) resembling the blastocyst stage. When exposed to suboptimal inducers, these stem cells fluctuate to form interconvertible subpopulations with reduced self-renewal and facilitated dif-ferentiation, resembling peri-implantation cells, known as TR stem cells (TSCs). TESCs have enhanced capacity to form blastoids that implant more efficientlyin utero due to inducers maintaining not only local TR proliferation and self-renewal, but also WNT6/7B secretion that stimulates uterine decidualization. Overall, the epiblast main-tains sustained growth and decidualization potential of abutting TR cells, while, as known, distancing imposed by the blastocyst cavity differentiates TR cells for uterus adhesion, thus patterning the essential functions of implantation. Show less
Kester, L.; Seinstra, D.; Rossum, A.G.J. van; Vennin, C.; Hoogstraat, M.; Velden, D. van der; ... ; Rheenen, J. van 2022
Purpose: Extensive work in preclinical models has shown that microenvironmental cells influence many aspects of cancer cell behavior, including metastatic potential and their sensitivity to... Show morePurpose: Extensive work in preclinical models has shown that microenvironmental cells influence many aspects of cancer cell behavior, including metastatic potential and their sensitivity to therapeutics. In the human setting, this behavior is mainly correlated with the presence of immune cells. Here, in addition to T cells, B cells, macrophages, and mast cells, we identified the relevance of nonimmune cell types for breast cancer survival and therapy benefit, including fibroblasts, myoepithelial cells, muscle cells, endothelial cells, and seven distinct epithelial cell types.Experimental Design: Using single-cell sequencing data, we generated reference profiles for all these cell types. We used these reference profiles in deconvolution algorithms to optimally detangle the cellular composition of more than 3,500 primary breast tumors of patients that were enrolled in the SCAN-B and MATADOR clinical trials, and for which bulk mRNA sequencing data were available.Results: This large data set enables us to identify and subsequently validate the cellular composition of miaoenvironments that distinguish differential survival and treatment benefit for different treatment regimens in patients with primary breast cancer. In addition to immune cells, we have identified that survival and therapy benefit are characterized by various contributions of distinct epithelial cell types.Conclusions: From our study, we conclude that differential survival and therapy benefit of patients with breast cancer are characterized by distinct microenvironments that include specific populations of immune and epithelial cells. Show less
Groen, N.; Leenders, F.; Mahfouz, A.; Munoz-Garcia, A.; Muraro, M.J.; Graaf, N. de; ... ; Carlotti, F. 2021
The maintenance of pancreatic islet architecture is crucial for proper beta-cell function. We previously reported that disruption of human islet integrity could result in altered beta-cell identity... Show moreThe maintenance of pancreatic islet architecture is crucial for proper beta-cell function. We previously reported that disruption of human islet integrity could result in altered beta-cell identity. Here we combine beta-cell lineage tracing and single-cell transcriptomics to investigate the mechanisms underlying this process in primary human islet cells. Using drug-induced ER stress and cytoskeleton modification models, we demonstrate that altering the islet structure triggers an unfolding protein response that causes the downregulation of beta-cell maturity genes. Collectively, our findings illustrate the close relationship between endoplasmic reticulum homeostasis and beta-cell phenotype, and strengthen the concept of altered beta-cell identity as a mechanism underlying the loss of functional beta-cell mass. Show less
Groen, N.; Leenders, F.; Mahfouz, A.; Munoz-Garcia, A.; Muraro, M.J.; Graaf, N. de; ... ; Carlotti, F. 2021
The maintenance of pancreatic islet architecture is crucial for proper β-cell function. We previously reported that disruption of human islet integrity could result in altered β-cell identity. Here... Show moreThe maintenance of pancreatic islet architecture is crucial for proper β-cell function. We previously reported that disruption of human islet integrity could result in altered β-cell identity. Here we combine β-cell lineage tracing and single-cell transcriptomics to investigate the mechanisms underlying this process in primary human islet cells. Using drug-induced ER stress and cytoskeleton modification models, we demonstrate that altering the islet structure triggers an unfolding protein response that causes the downregulation of β-cell maturity genes. Collectively, our findings illustrate the close relationship between endoplasmic reticulum homeostasis and β-cell phenotype, and strengthen the concept of altered β-cell identity as a mechanism underlying the loss of functional β-cell mass. Show less
Sen, M.Y.; Mooijman, D.; Chialastri, A.; Boisset, J.C.; Popovic, M.; Heindryckx, B.; ... ; Oudenaarden, A. van 2021
Wnt dependency and Lgr5 expression define multiple mammalian epithelial stem cell types. Under defined growth factor conditions, such adult stem cells (ASCs) grow as 3D organoids that recapitulate... Show moreWnt dependency and Lgr5 expression define multiple mammalian epithelial stem cell types. Under defined growth factor conditions, such adult stem cells (ASCs) grow as 3D organoids that recapitulate essential features of the pertinent epithelium. Here, we establish long-term expanding venom gland organoids from several snake species. The newly assembled transcriptome of the Cape coral snake reveals that organoids express high levels of toxin transcripts. Single-cell RNA sequencing of both organoids and primary tissue identifies distinct venom-expressing cell types as well as proliferative cells expressing homologs of known mammalian stem cell markers. A hard-wired regional heterogeneity in the expression of individual venom components is maintained in organoid cultures. Harvested venom peptides reflect crude venom composition and display biological activity. This study extends organoid technology to reptilian tissues and describes an experimentally tractable model system representing the snake venom gland. Show less
Baron, C.S.; Barve, A.; Muraro, M.J.; Linden, R. van der; Dharmadhikari, G.; Lyubimova, A.; ... ; Oudenaarden, A. van 2019
Much of current molecular and cell biology research relies on the ability to purify cell types by fluorescence-activated cell sorting (FACS). FACS typically relies on the ability to label cell... Show moreMuch of current molecular and cell biology research relies on the ability to purify cell types by fluorescence-activated cell sorting (FACS). FACS typically relies on the ability to label cell types of interest with antibodies or fluorescent transgenic constructs. However, antibody availability is often limited, and genetic manipulation is labor intensive or impossible in the case of primary human tissue. To date, no systematic method exists to enrich for cell types without a priori knowledge of cell-type markers. Here, we propose GateID, a computational method that combines single-cell transcriptomics with FACS index sorting to purify cell types of choice using only native cellular properties such as cell size, granularity, and mitochondrial content. We validate GateID by purifying various cell types from zebrafish kidney marrow and the human pancreas to high purity without resorting to specific antibodies or transgenes. Show less
Gurp, L. van; Muraro, M.J.; Dielen, T.; Seneby, L.; Dharmadhikari, G.; Gradwohl, G.; ... ; Koning, E.J.P. de 2019
During pancreatic development, endocrine cells appear from the pancreatic epithelium when Neurog3-positive cells delaminate and differentiate into alpha-, beta-,gamma- and delta-cells. The... Show moreDuring pancreatic development, endocrine cells appear from the pancreatic epithelium when Neurog3-positive cells delaminate and differentiate into alpha-, beta-,gamma- and delta-cells. The mechanisms involved in this process are still incompletely understood. We characterized the temporal, lineage-specific developmental programs during pancreatic development by sequencing the transcriptome of thousands of individual pancreatic cells from E12.5 to E18.5 in mice, and identified all known cell types that are present in the embryonic pancreas, but focused specifically on alpha- and beta-cell differentiation by enrichment of a MIP-GFP reporter. We characterized transcriptomic heterogeneity in the tip domain based on proliferation, and characterized two endocrine precursor clusters marked by expression of Neurog3 and Fev. Pseudotime analysis revealed specific branches for developing alpha- and beta-cells, which allowed identification of specific gene regulation patterns. These include some known and many previously unreported genes that appear to define pancreatic cell fate transitions. This resource allows dynamic profiling of embryonic pancreas development at single cell resolution and reveals novel gene signatures during pancreatic differentiation into alpha- and beta-cells. Show less
Kopper, O.; Witte, C.J. de; Lohmussaar, K.; Valle-Inclan, J.E.; Hami, N.; Kester, L.; ... ; Clevers, H. 2019
Ovarian cancer (OC) is a heterogeneous disease usually diagnosed at a late stage. Experimental in vitro models that faithfully capture the hallmarks and tumor heterogeneity of OC are limited and... Show moreOvarian cancer (OC) is a heterogeneous disease usually diagnosed at a late stage. Experimental in vitro models that faithfully capture the hallmarks and tumor heterogeneity of OC are limited and hard to establish. We present a protocol that enables efficient derivation and long-term expansion of OC organoids. Utilizing this protocol, we have established 56 organoid lines from 32 patients, representing all main subtypes of OC. OC organoids recapitulate histological and genomic features of the pertinent lesion from which they were derived, illustrating intra-and interpatient heterogeneity, and can be genetically modified. We show that OC organoids can be used for drug-screening assays and capture different tumor subtype responses to the gold standard platinum-based chemotherapy, including acquisition of chemoresistance in recurrent disease. Finally, OC organoids can be xenografted, enabling in vivo drug-sensitivity assays. Taken together, this demonstrates their potential application for research and personalized medicine. Show less
