biostudies-arrayexpressKarolina ZielinskaMus musculushttps://www.ebi.ac.uk/biostudies/studies/E-MTAB-15617The fetal liver (FL) plays a fundamental role in the ontogeny of the hematopoietic system, by transiently providing a fertile microenvironment for the maturation, proliferation and expansion of fetal hematopoietic progenitors, as well as definitive hematopoietic stem cells (HSCs). Nonetheless, the cellular make up and identity of hematopoietic stem and progenitor cell niches in the FL remain underexplored. Here we employed bulk mRNA-seq to investigate the spatiotemporal dynamics and functional relevance of putative niche cells and HSCs in the FL microenvironment. We find that at peak stages of FL hematopoiesis, pro-hematopoietic cytokines are promiscuously expressed by endothelial, mesenchymal cells and hepatoblasts, which form multicellular consortia that collectively provide unrestricted access to supportive factors throughout the entire tissue. Nevertheless, during peak hematopoietic stages, hepatoblasts are most abundant, express highest levels of growth factors and regulate fetal erythropoiesis and HSC expansion through production of Kit ligand. This highly conducive FL microenvironment is transient and gets rapidly remodeled through hepatoblast differentiation, leading to the downregulation of hematopoietic factors and the contraction of supportive niches, which temporarily coincide with the exit of HSCs towards emergent BM tissues.biostudies-arrayexpressSample Collection - Female mice (6-12 weeks) were raised in groups of five and 72 hours before mating exposed to bedding material from a male cage. In the evening (after 5 p.m.) one female each was transferred to a cage containing a single housed male. The consecutive morning (before 9 a.m.) the breeding pair was separated, and females analyzed for the presence of a vaginal plug. That timepoint was designated as E0.5. Females were only mated for one night and 12 days later examined for a possible pregnancy. Embryos were extracted from the previously euthanized pregnant dams through a small abdominal incision and further dissected under a stereo microscope (Leica). Fetal livers were dissected using forceps and separated into single lobes using a surgical scalpel under a stereo microscope the lobes were fixed in 2 % paraformaldehyde (diluted in PBS) (6 h, 4 °C), washed twice in ice-cold PBS for five minutes and subsequently dehydrated in 30 % sucrose in PBS (24 – 48 h, 4 °C). The liver lobes were placed into base molds (15×15×5 mm) and completely covered in OCT medium, snap-frozen using liquid nitrogen and stored at -80 °C until use. Dissected FLs were placed into 5 mL digestion medium (DMEM GlutaMAX, 10 mM HEPES, 10 % fetal bovine serum (FBS) in a 6-well cell culture dish, disintegrated by gentle pulling motions using two micro-forceps, collagenase (Type 2, 0.04 g/mL) and Deoxyribonuclease I (0.2 mg/mL) added and thoroughly resuspended using a 1 mL pipette. The cell suspension was immediately incubated for 45 minutes at 37 °C with gentle sample agitation. Following incubation, 5 mL of ice-cold calcium- and magnesium-free phosphate-buffered saline (PBS) containing 10 % FBS were added, the suspension filtered through a 70 μm cell strainer, centrifuged at 500 g (5 minutes, 4 °C), the supernatant discarded, and the pellet resuspended into a single cell solution in PBS. Single cell suspensions were incubated with TruStain fcXTM (5 minutes, 4 °C) and subsequently immunostained using pre-conjugated antibodies (30 minutes, 4° C). All employed antibodies are listed in Table 1, including commercial sources. Next, the cells were washed twice in ice-cold PBS and resuspended in PBS containing DAPI (0.5 mg/mL), analyzed on a LSR II Fortessa (BD Biosciences). Post-acquisition data analysis was performed using FlowJo 10 software. Immunostained single cell solutions were prepared following the flow cytometry protocol explained above.Sequencing - Sequencing was performed on the Illumina HiSeq 4000 (single end 125 bp) using the TruSeq SBS Kit v4-HS (Illumina, Inc, California, USA).Nucleic Acid Extraction - Phenotypically defined stromal cell populations were separated using a FACS Aria (BD Biosciences) and collected directly into RNase-free microfuge tubes containing 400 μL RLT lysis buffer (QIAGEN) supplemented with 4 μL β-mercaptoethanol and frozen down on dry ice for storage at - 80 °C. Between 6’000 and maximal 50’000 cells were collected depending on the abundance of each cell type. RNA was extracted from thawed cell lysates using the RNeasy Plus Micro Kit (QIAGEN) following the manufacturer’s instructions and genomic DNA depleted using the supplied gDNA eliminator columns. RNA quality was judged by RIN values measured on a Bioanalyzer 2100 (Agilent, Waldbronn, Germany). Every analyzed sample displayed a RIN value above 9Library Construction - Library preparation, cluster generation, and sequencing were performed at the functional genomics center Zurich (FGCZ, Zurich, Switzerland). The libraries were prepared using the SMART-Seq2 protocol (Picelli et al., 2014).OrganizationMINSEQE ScoreAssays and DataProcessed DataMAGE-TAB FilesData Transformation - Raw counts were converted for exploratory analysis to counts per million (CPM). For differential expression analysis EdgeR was used with its internal normalization method TMM (Trimmed Mean of M-values).Sequence Alignment - Quality control and processing of RNA sequencing data was achieved using the SUSHI platform provided by the FGCZ, Zurich 71. The following integrated apps and settings were used: FastQC, FastQ Screen, STAR alignment to GRCm38.p5, featureCounts (Liao et al., 2014) and EdgeR for differential expression analysis adding a background expression of 10 reads.MetabolomicsUnknownTranscriptomicsGenomicsProteomicsIllumina HiSeq 4000RNA-seq of coding RNAMus musculusTissue-scale mapping of hematopoietic supportive niches reveals a critical role of hepatoblasts in the regulation of fetal liver hematopoiesis and stem cell maintenanceAnjali Vijaykumar, Patrick M. Helbling, Flavian Thelen, Serena Fazio, YeVin Mun, Ana Luisa Pereira, Karolina A. Zielińska, Paul Büschl, Kathrin Loosli, Stephan Isringhausen, Bjoern Menze, Takashi Nagasawa, Thomas Zerkatje, Ingo Roeder, Alvaro Gomariz, Markus G. Manz, Tomomasa Yokomizo, César Nombela-ArrietaKarolina ZielinskaCésar Nombela-ArrietafalseRNA-seq of mouse fetal liver endothelial cells, hepatoblasts and mesenchymal stromal cells at embryonic time points E13.5 and E17.5The fetal liver (FL) plays a fundamental role in the ontogeny of the hematopoietic system, by transiently providing a fertile microenvironment for the maturation, proliferation and expansion of fetal hematopoietic progenitors, as well as definitive hematopoietic stem cells (HSCs). Nonetheless, the cellular make up and identity of hematopoietic stem and progenitor cell niches in the FL remain underexplored. Here we employed bulk mRNA-seq to investigate the spatiotemporal dynamics and functional relevance of putative niche cells and HSCs in the FL microenvironment. We find that at peak stages of FL hematopoiesis, pro-hematopoietic cytokines are promiscuously expressed by endothelial, mesenchymal cells and hepatoblasts, which form multicellular consortia that collectively provide unrestricted access to supportive factors throughout the entire tissue. Nevertheless, during peak hematopoietic stages, hepatoblasts are most abundant, express highest levels of growth factors and regulate fetal erythropoiesis and HSC expansion through production of Kit ligand. This highly conducive FL microenvironment is transient and gets rapidly remodeled through hepatoblast differentiation, leading to the downregulation of hematopoietic factors and the contraction of supportive niches, which temporarily coincide with the exit of HSCs towards emergent BM tissues.2025-10-30T00:00:00Z2026-05-27T18:08:44.815Z2025-09-22T13:52:15.53ZE-MTAB-15617ERP180446EFO_0002944EFO_0004170EFO_0004917EFO_0005518EFO_0003816EFO_0003738EFO_0004184