From stem cells to T cells, in vitro applications and in vivo implications

Abstract

The thymus provides the necessary environmental cues for the differentiation and generation of T-cells. The thymus is continuously colonized by a rare subset of bone-marrow derived progenitors, which in humans are characterized as CD34+CD45RA+CD7+ cells. Further characterization and directed expansion of this important progenitor subset would represent an important step in developing T-cell and thymus regenerative approaches. Here we show the induced generation of human progenitor T-cells (pro-T) with a thymus-colonizing phenotype from multiple sources of stem cells cocultured on OP9-DL1 stromal cells. To determine whether pro-T cells generated in vitro possess an intrinsic ability to home, engraft and reconstitute a thymus in vivo, sorted CD34+ CD7++ pro-T cells were injected into immunodeficient mouse strains, which can support human multi-lineage differentiation from CD34+ hematopoietic stem cells (HSCs). Our findings showed that T-lineage progenitors generated in vitro exhibit key properties of being able to home to, settle, and effectively reconstitute the thymus of immunodeficient mice. Additionally, in vitro-generated pro-T cells, when transferred together with purified HSCs, were able to dramatically enhance the thymus reconstituting ability of HSC-derived progenitors in vivo. Additionally, we used human induced pluripotent stem cells generated from dermal fibroblasts of patients with Rag1 mutations, which result in severe combined immunodeficiency (SCID) or Omenn syndrome, and differentiated these into T-lineage cells in vitro in order to assess the alterations in T cell development and V(D)J recombination. Taken together, the generation of humanized mice reconstituted with in vitro derived progenitor T-subsets offers a new means of therapeutic evaluation and the potential to rapidly restore the T-cell compartment for the treatment of immunodeficiencies.