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Summary Stem cell-based therapy for type 1 diabetes would benefit from implementation of a cell purification step at the pancreatic endoderm stage. This would increase the safety of the final cell product, allow the establishment of an intermediate-stage stem cell bank, and provide a means for upscaling β cell manufacturing. Comparative gene expression analysis revealed glycoprotein 2 ( GP2 ) as a specific cell surface marker for isolating pancreatic endoderm cells (PECs) from differentiated hESCs and human fetal pancreas. Isolated GP2 + PECs efficiently differentiated into glucose responsive insulin-producing cells in vitro. We found that in vitro PEC proliferation declines due to enhanced expression of the cyclin-dependent kinase (CDK) inhibitors CDKN1A and CDKN2A . However, we identified a time window when reducing CDKN1A or CDKN2A expression increased proliferation and yield of GP2 + PECs. Altogether, our results contribute tools and concepts toward the isolation and use of PECs as a source for the safe production of hPSC-derived β cells. [ABSTRACT FROM AUTHOR]

The generation of T cells depends on the migration of hematopoietic progenitor cells to the thymus throughout life. The identity of the thymus-settling progenitor cells has been a matter of considerable debate. Here we found that thymopoiesis was initiated by a first wave of T cell lineage-restricted progenitor cells with limited capacity for population expansion but accelerated differentiation into mature T cells. They gave rise to αβ and γδ T cells that constituted Vγ3+ dendritic epithelial T cells. Thymopoiesis was subsequently maintained by less-differentiated progenitor cells that retained the potential to develop into B cells and myeloid cells. In that second wave, which started before birth, progenitor cells had high proliferative capacity but delayed differentiation capacity and no longer gave rise to embryonic γδ T cells. Our work reconciles conflicting hypotheses on the nature of thymus-settling progenitor cells. [ABSTRACT FROM AUTHOR]

Lineage commitment is regulated during hematopoiesis, with stepwise loss of differentiation potential ultimately resulting in lineage commitment. In this study we describe a novel population of B/NK bipotent precursors among common lymphoid progenitors in the fetal liver and the bone marrow. The absence of T cell precursor potential, both in vivo and in vitro, is due to low Notchl expression and secondary to inhibition of E2A activity by members of the inhibitor of DNA binding (Id) protein family. Our results demonstrate a new, Id protein-dependent, molecular mechanism of Notchl repression, operative in both fetal and adult common lymphoid pro-genitors, where T cell potential is selectively inhibited without affecting either the B or NK programs. This study identifies Id pro-teins as negative regulators of T cell specification, before B and NK commitment, and provides important insights into the transcriptional networks orchestrating hematopoiesis. [ABSTRACT FROM AUTHOR]

Characterizing thymic settling progenitors is important to understand the pre-thymic stages of T cell development, essential to devise strategies for T cell replacement in lymphopenic patients. We studied thymic settling progenitors from murine embryonic day 13 and 18 thymi by two complementary in vitro and in vivo techniques, both based on the "hanging drop" method. This method allowed colonizing irradiated fetal thymic lobes with E13 and/or E18 thymic progenitors distinguished by CD45 allotypic markers and thus following their progeny. Colonization with mixed populations allows analyzing cell autonomous differences in biologic properties of the progenitors while colonization with either population removes possible competitive selective pressures. The colonized thymic lobes can also be grafted in immunodeficient male recipient mice allowing the analysis of the mature T cell progeny in vivo, such as population dynamics of the peripheral immune system and colonization of different tissues and organs. Fetal thymic organ cultures revealed that E13 progenitors developed rapidly into all mature CD3(+) cells and gave rise to the canonical γδ T cell subset, known as dendritic epithelial T cells. In comparison, E18 progenitors have a delayed differentiation and were unable to generate dendritic epithelial T cells. The monitoring of peripheral blood of thymus-grafted CD3(-/-) mice further showed that E18 thymic settling progenitors generate, with time, larger numbers of mature T cells than their E13 counterparts, a feature that could not be appreciated in the short term fetal thymic organ cultures.