Genetic Tagging During Human Mesoderm Differentiation Reveals Tripotent Lateral Plate Mesodermal Progenitors

Abstract

Although clonal studies of lineage potential have been extensively applied to organ specific stem and progenitor cells, much less is known about the clonal origins of lineages formed from the germ layers in early embryogenesis. We applied lentiviral tagging followed by vector integration site analysis (VISA) with high-throughput sequencing to investigate the ontogeny of the hematopoietic, endothelial and mesenchymal lineages as they emerge from human embryonic mesoderm. In contrast to studies that have used VISA to track differentiation of self-renewing stem cell clones that amplify significantly over time, we focused on a population of progenitor clones with limited self-renewal capability. Our analyses uncovered the critical influence of sampling on the interpretation of lentiviral tag sharing, particularly among complex populations with minimal clonal duplication. By applying a quantitative framework to estimate the degree of undersampling we revealed the existence of tripotent mesodermal progenitors derived from pluripotent stem cells, and the subsequent bifurcation of their differentiation into bipotent endothelial/hematopoietic or endothelial/mesenchymal progenitors.

Mapping molecular landmarks of human skeletal ontogeny and pluripotent stem cell-derived articular chondrocytes

“…Fetal cartilage was dissected from 17-week old fetal tissues, enzymatically digested and re-aggregated for up to 48 in vitro. PSC-derived chondrogenic aggregates were generated as described above from a stable line generated in the Evseenko lab expressing GFP 64 . Primary and PSC-derived cell aggregates were then implanted sub-cutaneously into 6, 2 month old, athymic male rats (Harlan labs); no randomization nor blinding was used.…”

The mesenchymoangioblast, mesodermal precursor for mesenchymal and endothelial cells

“…These progenitors are capable to generate all mesodermal lineages, including blood, endothelial cells, bone, cartilage, and adipocytes. Lentiviral tagging experiments suggest that CD326 − CD56 + KDR dim population can be specified into bipotential mesenchymal/endothelial and hematopoietic/endothelial progenitors [67]. Thus, CD326 − CD56 + KDR dim cells described by Evseenko et al [66] likely represent a more immature mesodermal population which emerges before MB and HB specification occurs.…”

Cytoreductive conditioning intensity predicts clonal diversity in ADA-SCID retroviral gene therapy patients

“…More details are available in another study we conducted using this method, as well as in the original Chao2 manuscript. 25,34 Study approval…”