Development of CD4+CD25+FoxP3+ regulatory T cells from cord blood hematopoietic progenitor cells.

Adult stem cells are capable of generating all of the cells of the hematopoietic system, and this process is orchestrated in part by the interactions between these cells and the stroma. T cell progenitors emerge from the stem cell compartment and migrate to the thymus, where their terminal differentiation and maturation occur, and it is during this phase that selection shapes the immune repertoire. Notch ligands, including Delta-like 1 (DL1), play a critical role in this lymphoid differentiation. To mimic this in vitro, stroma-expressing DL1 have been used to generate CD4(+)CD8(+) double-positive and single-positive T cells from hematopoietic stem/progenitor cells. This system provides a robust tool to investigate thymopoiesis; however, its capacity to generate regulatory T cells (Tregs) has yet to be reported. Natural Tregs (nTregs) develop in the thymus and help maintain immune homeostasis and have potential clinical use as a cell therapy for modulation of autoimmune disease or for transplant tolerization. Here, we describe for the first time the development of a population of CD4(+)CD25(+) CD127(lo)FoxP3(+) cells that emerge in coculture of cord blood (CB) CD34(+) progenitors on OP9-DL1 stroma. These hematopoietic progenitor-derived CD4(+)CD25(+) Tregs have comparable suppressor function with CB nTregs in vitro. The addition of IL-2 to the coculture enhanced the expansion and survival of this population significantly. This manipulable culture system, therefore, generates functional Tregs and provides a system to elucidate the mechanism of Treg development.

Potential for clinical ex vivo expansion of cord blood haemopoietic stem cells using non-haemopoietic factor supplements.

The establishment of culture systems that promote haemopoietic stem cell (HSC) self-renewal and expansion ex vivo will increase the clinical potential of umbilical cord blood (CB) HSC transplantation. Studies defining key signalling pathways that regulate development and expansion of HSC in vivo have greatly facilitated development of protocols for expanding HSC in ex vivo culture. Recently a number of soluble factors with novel stem cell expansion activity have been identified as part of pathways associated with mesodermal induction, or as factors produced by supportive stroma. These have been reported to support, to varying degrees, HSC self-renewal under in vitro conditions. Here we review the activities of these new factors and consider their future potential as components in ex vivo expansion culture for CB HSC. Finally we discuss the challenges associated with applying these factors to clinically relevant culture systems.

Bone morphogenetic protein 4 contributes to the maintenance of primitive cord blood hematopoietic progenitors in an ex vivo stroma-noncontact co-culture system.

Establishment of conditions supporting hematopoietic stem cell (HSC) maintenance and expansion ex vivo is critical for wider clinical application of cord blood (CB) transplantation. AFT024 is a murine fetal liver cell line that expands primitive hematopoietic cells via a process that is not understood. Here we show that bone morphogenic protein 4 (BMP4) is produced by AFT024 and contributes significantly to the maintenance of co-cultured CB-derived primitive cells. Significant amounts of BMP4 mRNA are produced by the supportive AFT024 stromal cell line, and secreted BMP4 protein accumulates in AFT024 conditioned medium. Blockade of BMP4 activity in this coculture model using neutralizing BMP4 monoclonal antibody reduced expansion of primitive CB cells on the basis of phenotypic (CD34(+)CD38(-)) and functional criteria [long-term culture initiating cells (LTC-IC)] and significantly reduced the capacity of the cultured CB stem cells to support repopulation in the nonobese diabetic-severe combined immunodeficiency (NOD-SCID) xenograft model. Therefore, BMP4 is a key growth factor for maintenance of HSC and contributes to the unique properties of the AFT024 stromal noncontact culture.