Characterization in vitro and engraftment potential in vivo of human progenitor T cells generated from hematopoietic stem cells

Abstract

T-cell development follows a defined set of stage-specific differentiation steps. However, molecular and cellular events occurring at early stages of human T-cell development remain to be fully elucidated. To address this, human umbilical cord blood (UCB) hematopoietic stem cells (HSCs) were induced to differentiate to the T lineage in OP9-DL1 cocultures. A developmental program involving a sequential and temporally discrete expression of key differentiation markers was revealed. Quantitative clonal analyses demonstrated that CD34 ؉ CD38 ؊ and CD34 ؉ CD38 lo subsets of UCB contain a similarly high Tlineage progenitor frequency, whereas the frequency in CD34 ؉ CD38 ؉/hi cells was 5-fold lower. Delta-like/Notch-induced signals increased the T-cell progenitor frequency of CD34 ؉ CD38 ؊/lo cells differentiated on OP9-DL1, and 2 distinct progenitor subsets, CD34 ؉ CD45RA ؉ CD7 ؉؉ CD5 ؊ CD1a ؊ (proT1) and CD34 ؉ CD45RA ؉ CD7 ؉؉ CD5 ؉ CD1a ؊ (proT2), were identified and their thymus engrafting capacity was examined, with proT2 cells showing a 3-fold enhanced reconstituting capacity compared with the proT1 subset. Furthermore, in vitrogenerated CD34 ؉ CD7 ؉؉ progenitors effectively engrafted the thymus of immunodeficient mice, which was enhanced by the addition of an IL-7/IL-7 antibody complex. Taken IntroductionT cells develop within the thymus from bone marrow-derived hematopoietic progenitors, and follow a series of stage-specific differentiation events, which are broadly characterized by the developmentally coordinated expression of CD4 and CD8. 1 The initial stages of human T-cell development include precursors that express the stem cell marker CD34, 2,3 which is also present on hematopoietic stem cells (HSCs) and on multipotent or lineage-specified progenitor cells. Within the known hierarchy of T-cell development, the earliest precursor subset is further defined by the lack of CD3, CD4, CD8, and CD1a expression. 4 Although immature stages of T-cell development are typically delineated as CD34 ϩ CD1a Ϫ and CD34 ϩ CD1a ϩ cells, these populations remain heterogeneous. Of note, CD7 expression is considered to be one of the earliest cell surface markers known to appear during T lymphopoiesis. 2,5 Importantly, the transition from CD34 ϩ CD7 ϩ CD1a Ϫ to CD34 ϩ CD7 ϩ CD1a ϩ by early thymocytes is associated with T-cell commitment, as a small percentage (ϳ 10%) of these cells show rearrangement at the T-cell receptor ␤-chain (TCR␤) locus. 6 In addition, CD34 ϩ CD7 ϩ CD1a ϩ cells appear to be T-lineage restricted, as these cells show low precursor activity toward non-T-cell lineages. 7 Current understanding of the aforementioned early stages has been obtained from analyses of human fetal or adult thymocyte subsets, and by analyzing T-cell development in vitro using xenogeneic engraftment of mouse fetal thymus organ cultures (FTOCs). 8,9 Although these systems have provided important insight into T-cell development, the capacity to evaluate specific progenitor populations has remained difficult to assess given the requirem...

Concise Review: Hematopoietic Stem Cell Transplantation: Targeting the Thymus

“…Nevertheless, it appears that this population of CLPs is skewed toward a B lymphocyte fate, whereas cord blood progenitors with a CD34þCD7þCD45RAþ phenotype express T/NK cell lineage-affiliated genes and show enhanced T lineage differentiation potential [44]. Furthermore, recent studies have demonstrated that following ex vivo Notch ligand stimulation of CD34þ progenitors, it is the differentiated CD34þCD7þ CD45RAþ CLP subset that displays augmented in vivo thymus colonization and a more rapid T-cell differentiation than conventional CD34þ progenitors [45][46][47]. Thus, the precursors that settle the human thymus under physiological conditions are likely to share this CD34þCD7þCD45RAþ phenotype.…”

Identification and Functional Characterization of Ion Channels in CD34+ Hematopoietic Stem Cells from Human Peripheral Blood

“…During steady-state hematopoiesis, CD34 + HSCs circulate at low numbers in the peripheral blood, suggesting continuous migration of these cells between the bone marrow and organs (Kronenwett et al, 2000). Despite the wellrecognized molecular phenotype of CD34 + HSCs, the regulatory mechanisms for proliferation and the molecular signals mediating mobilization, migration, and differentiation are only partially understood (Awong et al, 2009;Han et al, 2003;Nielsen and MaNagny, 2009;Steidl et al, 2002). Previous studies suggest that HSCs also are able to differentiate into nonhematopoietic cell types such as hepatocytes and cardiomyocytes (Lagasse et al, 2000;Orlic et al, 2001).…”

Natural killer cell-triggered vascular transformation: maternal care before birth?

“…administration of freshly isolated cells versus cells expanded in culture (24 h in StemSpan SFEM medium with CC100 Cytokine Cocktail); 84,85 . tail vein versus intrafemoral cell injection; 86,87 . with or without human IL-15/IL-15Ra complex treatment at 6 and 7 weeks of age and at day 6.5 after mating.…”