Identification of human T‐lymphoid progenitor cells in CD34⁺CD38^low and CD34⁺CD38⁺ subsets of human cord blood and bone marrow cells using NOD‐SCID fetal thymus organ cultures

Abstract

In contrast to myeloid and B‐lymphoid differentiation, which take place in the marrow environment, development of T cells requires the presence of thymic stromal cells. We demonstrate in this study that human CD34+, CD34+CD38+ and CD34+CD38low cells from both cord blood and adult bone marrow reproducibly develop into CD4+CD8+T cells when introduced into NOD‐SCID embryonic thymuses and further cultured in organotypic cultures. Such human/mouse FTOC (fetal thymic organ culture) thus represents a reproducible and sensitive system to assess the T‐cell potential of human primitive progenitor cells. The frequency of T‐cell progenitors among cord‐blood‐derived CD34+ cells was estimated to be 1/500. Furthermore, the differentiation steps classically observed in human thymus were reproduced in NOD‐SCID FTOC initiated with cord blood and human marrow CD34+ cells: immature human CD4lowCD8−sCD3−TCRαβ–CD5+CD1a+T cells were mixed with CD4+CD8+ cells and more mature CD4+CD8−TCRαβ+cells. However, in FTOC initiated with bone marrow T progenitors, <10% double‐positive cells were observed, whereas this proportion increased to 50% when cord blood CD34+ cells were used, and most CD4+ cells were immature T cells. These differences may be explained by a lower frequency of T‐cell progenitors in adult samples, but may also suggest differences in the thymic signals required by bone marrow versus cord blood T progenitors. Finally, since cytokine‐stimulated CD34+CD38low cells retained their ability to generate T cells, these FTOC assays will be of value to monitor, when combined with other biological assays, the influence of different expansion protocols on the potential of human stem cells.

In vitro identification of human pro-B cells that give rise to macrophages, natural killer cells, and T cells

“…Analysis of human cells collected from these thymic lobes showed that a high number of CD4 high human cells were recovered in lobes seeded with unfractionated cells (not shown) or CD79a ϩ -enriched CDw127 ϩ cells. A few (5%) CD4 high cells were double-positive (DP) CD4 ϩ CD8 ϩ cells ( Figure 5), which, as shown previously, 33 indicates T-cell differentiation; we cannot exclude, however, the contribution of non-T cells to the CD4 ϩ fraction. The low number of human cells collected from the thymic lobes precluded any extensive phenotyping and/or molecular analysis of T-cell receptor (TCR) rearrangement.…”

“…Differentiation was evaluated in fetal thymic organ culture (FTOC) exactly as described previously in detail, 33 using E14 embryonic thymic lobes from NOD-SCID mice. Each lobe was incubated with either 10 000 unfractionated day 14 cultured cells, CDw127 ϩ CD79a ϩ CD19 Ϫ or CDw127 ϩ CD19 ϩ cells.…”

Functional Human T Lymphocyte Development from Cord Blood CD34+ Cells in Nonobese Diabetic/Shi-scid, IL-2 Receptor γ Null Mice

“…FTOC using murine thymus have enabled the identification of T cell progenitors, such as human CD34 ϩ cells (8,9). NOD/SCID mice transplanted with CD34 ϩ cells have been known to reconstitute these T cells in some cases (13).…”

“…An alternative approach, however, is also necessary, because this method requires human fetal tissue and is technically difficult. FTOC using murine thymus has been useful for the identification of T cell progenitors among human CD34 ϩ cells (8). However, the resultant T cells exhibit an immature phenotype, and their functional status has not been fully studied (9).…”

Efficiency of transgenic T cell generation from gene-marked cultured human CD34+ cord blood cells is determined by their maturity and the cytokines present in the culture medium

“…3 For limiting dilution experiments, harvested cells were co-incubated with NOD-SCID lobes and irradiated CD34 + cells as described before. 29 At the end of the culture, lobes were mechanically disrupted with a small tissue grinder to obtain a single cell suspension for evaluation with flow cytometry.…”