Capture of authentic embryonic stem cells from rat blastocysts.

Embryonic stem (ES) cells have been available from inbred mice since 1981 but have not been validated for other rodents. Failure to establish ES cells from a range of mammals challenges the identity of cultivated stem cells and our understanding of the pluripotent state. Here we investigated derivation of ES cells from the rat. We applied molecularly defined conditions designed to shield the ground state of authentic pluripotency from inductive differentiation stimuli. Undifferentiated cell lines developed that exhibited diagnostic features of ES cells including colonization of multiple tissues in viable chimeras. Definitive ES cell status was established by transmission of the cell line genome to offspring. Derivation of germline-competent ES cells from the rat paves the way to targeted genetic manipulation in this valuable biomedical model species. Rat ES cells will also provide a refined test-bed for functional evaluation of pluripotent stem cell-derived tissue repair and regeneration.

Thymic epithelial cell fate and potency in early organogenesis assessed by single cell transcriptional and functional analysis.

During development, cortical (c) and medullary (m) thymic epithelial cells (TEC) arise from the third pharyngeal pouch endoderm. Current models suggest that within the thymic primordium most TEC exist in a bipotent/common thymic epithelial progenitor cell (TEPC) state able to generate both cTEC and mTEC, at least until embryonic day 12.5 (E12.5) in the mouse. This view, however, is challenged by recent transcriptomics and genetic evidence. We therefore set out to investigate the fate and potency of TEC in the early thymus. Here using single cell (sc) RNAseq we identify a candidate mTEC progenitor population at E12.5, consistent with recent reports. Via lineage-tracing we demonstrate this population as mTEC fate-restricted, validating our bioinformatics prediction. Using potency analyses we also establish that most E11.5 and E12.5 progenitor TEC are cTEC-fated. Finally we show that overnight culture causes most if not all E12.5 cTEC-fated TEPC to acquire functional bipotency, and provide a likely molecular mechanism for this changed differentiation potential. Collectively, our data overturn the widely held view that a common TEPC predominates in the E12.5 thymus, showing instead that sublineage-primed progenitors are present from the earliest stages of thymus organogenesis but that these early fetal TEPC exhibit cell-fate plasticity in response to extrinsic factors. Our data provide a significant advance in the understanding of fetal thymic epithelial development and thus have implications for thymus-related clinical research, in particular research focussed on generating TEC from pluripotent stem cells.

Restoration of Runx1 expression in the Tie2 cell compartment rescues definitive hematopoietic stem cells and extends life of Runx1 knockout animals until birth.

Mice deficient in the runt homology domain transcription factor Runx1/AML1 fail to generate functional clonogenic hematopoietic cells and die in utero by embryonic day 12.5. We previously generated Runx1 reversible knockout mice, in which the Runx1 locus can be restored by Cre-mediated recombination. We show here that selective restoration of the Runx1 locus in the Tie2 cell compartment rescues clonogenic hematopoietic progenitors in early Runx1-null embryos and rescues lymphoid and myeloid lineages during fetal development. Furthermore, fetal liver cells isolated from reactivated Runx1 embryos are capable of long-term multilineage lymphomyeloid reconstitution of adult irradiated recipients, demonstrating the rescue of definitive hematopoietic stem cells. However, this rescue of the definitive hematopoietic hierarchy is not sufficient to rescue the viability of animals beyond birth, pointing to an essential role for Runx1 in other vital developmental processes.

The differentiation program of embryonic definitive hematopoietic stem cells is largely alpha4 integrin independent.

Previous analyses of the roles of alpha4 integrins in hematopoiesis by other groups have led to conflicting evidence. alpha4 integrin mutant cells developing in [alpha4 integrin(-/-): wt] chimeric mice are not capable of completing lymphomyeloid differentiation, whereas conditional inactivation of alpha4 integrin in adult mice has only subtle effects. We show here that circumventing the fetal stage of hematopoietic stem cell (HSC) development by transplantation of embryonic alpha4 integrin(-/-) cells into the adult microenvironment results in robust and stable long-term generation of alpha4 integrin(-/-) lymphoid and myeloid cells, although colonization of Peyer patches and the peritoneal cavity is significantly impaired. We argue here that collectively, our data and the data from other groups suggest a specific requirement for alpha4 integrin during the fetal/neonatal stages of HSC development that is essential for normal execution of the lymphomyeloid differentiation program.

Multifunctional reversible knockout/reporter system enabling fully functional reconstitution of the AML1/Runx1 locus and rescue of hematopoiesis.

Mice deficient in the runt homology domain transcription factor Runx1 die of severe anemia in utero by embryonic day (E)12.5. A reactivatable Runx1 knockout embryonic stem cell (ESC) and mouse systems were generated by the targeted insertion of a loxP-flanked multipartite gene stop/trap cassette designed to simultaneously ablate the expression of Runx1 and report on the activity of its promoters. The cassette's in-frame LacZ reporter enabled activities of the proximal and the distal promoters to be differentially monitored. Although Runx1-null ESCs were capable of primitive erythroid differentiation in vitro, their capacity to generate granulocyte/macrophage or mixed myelo-erythroid embryoid bodies was lost. Cre-mediated reactivation restored Runx1 structural integrity and rescued the hematopoietic differentiation potential of ESCs. Mice with the reactivated allele survived, showed no hematopoietic deficit, and expressed all major splice isoforms of Runx1 appropriately. This multipurpose mouse model will be useful for the analysis of the critical Runx1-dependent check-point(s) in hematopoietic development.

Progressive divergence of definitive haematopoietic stem cells from the endothelial compartment does not depend on contact with the foetal liver.

The yolk sac and the para-aortic splanchnopleura/aorta-genital ridges-mesonephros (P-Sp/AGM) region are the main sites of haematopoietic activity in the mouse embryo at the pre-liver stage of development. By day 11.5 of gestation, the AGM region is capable of autonomous initiation and expansion of definitive haematopoietic stem cells (HSCs). By day 12.5, HSC activity in the AGM region is reduced whilst a second wave of HSCs begins to emerge in the yolk sac. We show here that HSCs emerging in both locations are marked by co-expression of the endothelial-specific marker VE-cadherin and the pan-leukocyte antigen CD45. Phenotypic characterisation using CD31, TIE2, FLK1, Ac-LDL receptors, and CD34 markers demonstrated significant similarities between this VE-cadherin+CD45+ ;double-positive' population and endothelial cells suggesting a common origin for these cells. The double-positive fraction also expressed the stem cell markers Kit, Sca1 and AA4.1. Long-term transplantation experiments demonstrated that the double-positive population, which constituted less than 0.05% of the day 11.5 AGM region and the day 12.5 yolk sac, is highly enriched for HSCs. In vitro assays showed that this population is also enriched for myeloid progenitors. During foetal liver colonization, circulating HSCs remained within the VE-cadherin+ cell fraction, although their phenotypic similarity with endothelial cells became less prominent. Upon liver colonisation the majority of HSCs downregulated VE-cadherin, expression of which was completely lost in the adult bone marrow. Partial loss of VE-cadherin expression in HSCs can be observed extra hepatically in the advanced AGM region by E12.5. Similarly, the CD34+KIT+ population in the placenta, recently identified as a reservoir of HSCs, partly lose VE-cadherin expression by E12.5. By culturing isolated E11.5 AGM region and E12.5 yolk sac we show that the developmental switch from a ;primary' VE-cadherin+CD45+ to a more ;advanced' VE-cadherin-CD45+ phenotype does not require contact of HSCs with the liver and is probably a function of developmental time.