Hematopoietic Progenitors with Slow Divisional Kinetics Give Rise to T Cell Precursors in the Thymus of the SCID Mouse Transplantation Model and Represent the Subset with Primitive Function.

Introduction. We have shown in the past that slow divisional kinetics of hematopoietic stem cells (HSC) are correlated with prolonged self renewal and increased frequency of long term culture initiating cells (LTC-IC) and myeloid-lymphoid initiating cells (ML-IC) in vitro. In the present study we have demonstrated that only the slow dividing fraction (SDF) of human CD34+ HSC, but not the fast dividing fraction (FDF), is able to engraft in the thymus and lymph nodes of NOD/SCID mice.

Methods. Human CD34+ HSC were isolated from umbilical cord blood, stained with the membrane fluorescence marker PKH26, cultivated in cytokine rich media for five days and then sorted for their PKH26 fluorescence (dim versus bright). Sorted cells were injected into the tail vein of sublethally irradiated NOD/SCID mice (2×10e5 cells / animal). Mice were sacrificed at defined time points from week 2 to week 18 after transplantation.

Results. Flow cytometric analysis of the samples collected from the marrow, spleen, thymus, and lymph nodes of the NOD/SCID mice indicated that both SDF and FDF were able to engraft in the bone marrow of the mice, establishing a hematopoietic system, i.e. erythroid and myeloid progenitor cells were recovered from the bone marrow of SDF and FDF reconstituted mice. The most remarkable finding was that only progeny cells of SDF were found in the mouse lymph nodes and thymus where, beginning approximately at week 15, they differentiated into T cells. CD4+CD8+ double positive T progenitor cells were recovered in the thymus and CD4+ as well as CD8+ T cells were present in the spleen and the lymph nodes. However, at least until 18 weeks after reconstitution, very few B cells were present in the NOD/SCID spleen, whereas monocytes reached close to normal values. We are in the process of evaluating the functionality of the human T cells that matured in the NOD/SCID mouse environment. Global gene expression profiles of the two subpopulations (SDF and FDF) were analyzed using a ‘human transcriptome cDNA microarray’. We found that several molecular markers for stem cells were highly expressed in the SDF as compared to FDF: CD133 (Prominin), MDR1 (multiple drug resistance gene 1), clqr1 (complement component 1 receptor 1), Hoxa9, Cdx1 and Hesx1 (which encode homeodomain proteins).

Conclusion. Our data indicated that only SDF was enriched for primitive HSC, which in turn were able to engraft in the thymus and lymph nodes in the SCID mouse transplantation model. These cells were able to give rise to T cell precursors and mature T cells. This observation has provided unequivocal evidence that the SDF is associated with primitive HSC function, and is consistent with the results of differential gene expression analysis in SDF versus FDF. The significance and nature of the cell-cell contacts in the microenvironment of the SCID mouse model is currently being examined.