Laure Coulombel

The human embryo, but not its yolk sac, generates lympho-myeloid stem cells: Mapping multipotent hematopoietic cell fate in intraembryonic mesoderm

We have traced emerging hematopoietic cells along human early ontogeny by culturing embryonic tissue rudiments in the presence of stromal cells that promote myeloid and B cell differentiation, and by assaying T cell potential in the NOD-SCID mouse thymus. Hematogenous potential was present inside the embryo as early as day 19 of development in the absence of detectable CD34+ hematopoietic cells, and spanned both lymphoid and myeloid lineages from day 24 in the splanchnopleural mesoderm and derived aorta where CD34+ progenitors appear at day 27. By contrast, hematopoietic cells arising in the third week yolk sac, as well as their progeny at later stages, were restricted to myelopoiesis and therefore are unlikely to contribute to definitive hematopoiesis in man.

Expression and Function of Integrins on Hematopoietic Progenitor Cells

The growth and differentiation of hematopoietic stem cells are highly dependent on regulatory molecules produced by stromal cells of the marrow environment. Evidence has accumulated over the past years which shows that adhesive receptors on hematopoietic cells and their ligands on stromal cells and extracellular matrix play a crucial role in these interactions. Integrins of the beta 1 family, mostly VLA-4 and VLA-5, are the best characterized and have been identified on committed progenitor cells of the hematopoietic hierarchy as well as on more primitive stem cells defined by their long-term repopulating capacity assayed in vitro as well as in vivo. Functional assays demonstrate that most progenitor cells efficiently bind to ECM components through beta 1 integrins and lineage- and maturation stage-specific differences have been described. Evidence exists on the direct control of late erythroid differentiation by VLA-4, but whether or not the triggering of beta 1 integrins is critically required for hematopoietic stem cell functioning at more immature steps is unclear. Many other integrin and non-integrin receptors involved in adhesive interactions are expressed on hematopoietic progenitor cells and tightly regulated during differentiation but their function is still controversial. Our main purpose in this review is to describe recent advances in the knowledge of integrin expression on hematopoietic progenitor cells in both mouse and man. The emerging importance of the synergy between integrins and cytokine signalling pathways in the regulation of hematopoietic differentiation will also be discussed.

NK cells differentiated from bone marrow, cord blood and peripheral blood stem cells exhibit similar phenotype and functions

In the present study, we investigated the differentiation of human NK cells from bone marrow, cord blood and mobilized peripheral blood purified CD34+ stem cells using a potent culture system. Elutriated CD34+ stem cells were grown for several weeks in medium supplemented with stem cell factor (SCF) and IL‐15 in the presence or absence of a murine stromal cell line (MS‐5). Our data indicate that IL‐15 induced the proliferation and maturation of highly positive CD56+ NK cells in both types of culture, although murine stromal cells slightly increased the proliferation of NK cells. NK cells differentiated in the presence of MS‐5 were mostly CD56+u2009CD7− and a small subset expressed CD16. These in vitro differentiated CD56+ NK cells displayed cytolytic activity against the HLA class Iu2009− target K562. The CD56+u2009CD16+ subset also lysed NK‐resistant Daudi cells. Neither of these NK subsets were shown to express Fas ligand. Total CD56+ cells expressed high amounts of transforming growth factor‐β and granulocyte‐macrophage colony‐stimulating factor, but no IFN‐γ. Investigation of NK receptor expression showed that most CD56+ cells expressed membrane CD94 and NKG2‐A mRNA. PCR analysis revealed that p58 was also expressed in these cells. The role of CD94 in NK cell‐mediated cytotoxicity was assessed on human HLA‐B7‐transfected murine L cells. While a low cytotoxic activity towards HLA‐B7 cells was observed, the HLA‐DR4 control cells were killed with high efficiency. These studies demonstrate that cytolytic and cytokine‐producing NK cells may be derived from adult and fetal precursors by IL‐15 and that these cells express a CD94 receptor which may influence their lytic potential.

In vitro and in vivo evidence for the long-term multilineage (myeloid, B, NK, and T) reconstitution capacity of ex vivo expanded human CD34+ cord blood cells

The aim of the present report is to describe clinically relevant culture conditions that support the expansion of primitive hematopoietic progenitors/stem cells, with maintenance of their hematopoietic potential as assessed by in vitro assays and the NOD-SCID in vivo repopulating capacity.CD34(+) cord blood (CB) cells were cultured in serum-free medium containing stem cell factor, Flt3 ligand, megakaryocyte growth and development factor, and granulocyte colony-stimulating factor. After 14 days, the primitive functions of expanded and nonexpanded cells were determined in vitro using clonogenic cell (colony-forming cells, long-term culture initiating cell [LTC-IC], and extended [E]-LTC-IC) and lymphopoiesis assays (NK, B, and T) and in vivo by evaluating long-term engraftment of the bone marrow of NOD-SCID mice. The proliferative potential of these cells also was assessed by determining their telomere length and telomerase activity. Levels of expansion were up to 1,613-fold for total cells, 278-fold for colony-forming unit granulocyte-macrophage, 47-fold for LTC-IC, and 21-fold for E-LTC-IC. Lymphoid B-, NK, and T-progenitors could be detected. When the expanded populations were transplanted into NOD-SCID mice, they were able to generate myeloid progenitors and lymphoid cells for 5 months. These primitive progenitors engrafted the NOD-SCID bone marrow, which contained LTC-IC at the same frequency as that of control transplanted mice, with conservation of their clonogenic capacity. Moreover, human CD34(+)CDl9(-) cells sorted from the engrafted marrow were able to generate CD19(+) B-cells, CD56(+)CD3(-) NK cells, and CD4(+)CD8(+)alphabetaTCR(+) T-cells in specific cultures. Our expansion protocol also maintained the telomere length in CD34(+) cells, due to an 8.8-fold increase in telomerase activity over 2 weeks of culture. These experiments provide strong evidence that expanded CD34(+) CB cells retain their ability to support long-term hematopoiesis, as shown by their engraftment in the NOD-SCID model, and to undergo multilineage differentiation along all myeloid and the B-, NK, and T-lymphoid pathways. The expansion protocol described here appears to maintain the hematopoietic potential of CD34(+) CB cells, which suggests its relevance for clinical applications.

Identification of human T-lymphoid progenitor cells in CD34+ CD38low and CD34+ CD38+ subsets of human cord blood and bone marrow cells using NOD-SCID fetal thymus organ cultures.

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.

Rat liver biliary epithelial cells support long‐term production of haemopoietic progenitors from human CD34+ cells

In this study we report the supportive activity of rat liver epithelial cells (RLEC) on human haemopoiesis in the absence of exogeneously supplied growth factors. RLEC is a rat cell line derived from primitive biliary cells with epithelial characteristics which induce the long‐term differentiation of hepatocytes through cell–cell contacts. We have established the ability of these cells to sustain long‐term survival and multilineage differentiation of human haemopoietic progenitors from unfractionated bone marrow and growth‐factor mobilized peripheral blood cells, and from human CD34+ and CD34+ CD38− haemopoietic cells, with a higher efficiency than the murine MS‐5 stromal cell line: the numbers of committed progenitors recovered from RLEC cocultures after 8 weeks were 3‐fold higher than fromMS‐5 cocultures, with an unusually high BFU‐E production. Furthermore, using diffusible insert cultures, we demonstrated that, despite the lack of strong adhesive interaction between haemopoietic cells and RLEC, physical proximity was absolutely required for optimum stimulation of LTC‐IC by RLEC. Taken together, these results show that biliary epithelial cells support human haemopoiesis and cause speculation that common mechanisms might be used by RLEC to regulate both the hepatocyte and the haemopoietic progenitors differentiation.

L’hémangioblaste, précurseur commun des cellules endothéliales et hématopoïétiques

On a dabord designe SOllS Ie terme d« hemangioblastes » les amas de cellules mesoderrniques qui, dans Ie sac vitellin de lembryon, donnent naissance aux premiers vaisseaux sanguins contenant les premieres cellules hematopoietiques, adherant en foyers it lendothelium [1]. On appelle maintenant ces amas « Hots sanguins », mais Ie terme dhemangioblaste est reste et a meme connu recemment un regain de popularite pour designer une cellule unique it lorigine des cellules hematopoietiques et endotheliales. Cellules endotheliales et cellules hematopoietiques expriment effectivement, depuis les premiers stades du developpement, des marqueurs communs tels que MB1/QHl chez loiseau [2, 3], CD31 chez lhomme [4] ou

An in situ hybridization technique for the study of B19 human parvovirus replication in bone marrow cell cultures

An in situ hybridization technique using digoxigenin labelling was developed to study B19 infection. By using appropriate DNA probes, transcription of structural and non-structural genes was detected in bone marrow cell cultures. Such a simple system is useful to the study of B19-cell interactions in non-permissive cell lines.