Abstract
Hematopoietic stem cells are responsible for the generation of the entire blood system through life. This characteristic relies on their ability to self renew and on their multi-potentiality. Thus quantification of the number of hematopoietic stem cells in a given cell population requires to show both properties in the studied cell populations. Although xenografts models that support human hematopoietic stem cells have been described, such in vivo experimental systems remain restrictive for high throughput screening purposes for example. In this work we developed a conditional tetracycline inducible system controlling the expression of the human NOTCH ligand Delta-like 1 in the murine stromal MS5 cells. We cultured hematopoietic immature cells enriched in progenitor/stem cells in contact with MS5 cells that conditionally express Delta-like 1, in conditions designed to generate multipotential lineage differentiation. We show that upon induction or repression of DL1 expression during co-culture, human immature CD34+CD38−/low(CD45RA−CD90+) cells can express their B, T, NK, granulo/monocytic and erythroid potentials in a single well, and at the single cell level. We also document the interference of low NOTCH activation with human B and myelo/erythroid lymphoid differentiation. This system represents a novel tool to precisely quantify human hematopoietic immature cells with both lymphoid and myeloid potentials.
Highlights
The hematopoietic system originates from the proliferation and differentiation of a rare population of cells named the hematopoietic stem cell (HSC)
DL1 expression was induced within a few hours of doxycyclin treatment and remained high for 2–3 days without doxycyclin re-feeding of cultures; compatible with the fact that doxycyclin is still present during this period (Figure 1C, 1F)
In accordance with these data, we show that constitutive DL1 expression allow T cell development from immature cord blood (CB) cells up to the double positive (DP) CD4+CD8+ stage, a proportion of which expressed surface CD3
Summary
The hematopoietic system originates from the proliferation and differentiation of a rare population of cells named the hematopoietic stem cell (HSC). The most conventional way to study these primitive cells is to serially transplant a given cell population into irradiated compatible mouse recipients [2] Essential, this assay remains restrictive and costly. We have previously shown that multi-potential development from single human primitive cells from cord blood (CB) was possible in vitro Such approach requires to initiate cultures on a mouse stromal feeder, for instance MS5 cells, and later to split proliferating clones into two independent cultures, MS5 co-cultures [8] and Fetal-thymic-organ-culture (FTOC) [9], to circumvent different human exclusive lineages [10]. Another strategy proposed to mix two stromal feeders that express or not the NOTCH ligand DL4 [11] or to culture cells in presence of low levels of inhibitors of NOTCH, such as gamma secretase inhibitors (GSI) [12], in order to drive multiple differentiations from cultures of single defined human cells
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