Stem Cell Responsiveness to Imatinib in Chronic Myeloid Leukemia.

Chronic myeloid leukemia (CML) is a clonal myeloproliferative disease characterized by the presence of the BCR-ABL fusion gene, which results from the Philadelphia chromosome. Since the introduction of tyrosine kinase inhibitors (TKI) such as imatinib mesylate (IM), the clinical outcomes for patients with CML have improved significantly. However, IM resistance remains the major clinical challenge for many patients, underlining the need to develop new drugs for the treatment of CML. The basis of CML cell resistance to this drug is unclear, but the appearance of additional genetic alterations in leukemic stem cells (LSCs) is the most common cause of patient relapse. However, several groups have identified a rare subpopulation of CD34+ stem cells in adult patients that is present mainly in the bone marrow and is more immature and pluripotent; these cells are also known as very small embryonic-like stem cells (VSELs). The uncontrolled proliferation and a compromised differentiation possibly initiate their transformation to leukemic VSELs (LVSELs). Their nature and possible involvement in carcinogenesis suggest that they cannot be completely eradicated with IM treatment. In this study, we demonstrated that cells from CML patients with the VSELs phenotype (LVSELs) similarly harbor the fusion protein BCR-ABL and are less sensitive to apoptosis than leukemic HSCs after IM treatment. Thus, IM induces apoptosis and reduces the proliferation and mRNA expression of Ki67 more efficiently in LHSCs than in leukemic LVSELs. Finally, we found that the expression levels of some miRNAs are affected in LVSELs. In addition to the tumor suppressor miR-451, both miR-126 and miR-21, known to be responsible for LSC leukemia-initiating capacity, quiescence, and growth, appear to be involved in IM insensitivity of LVSELs CML cell population. Targeting IM-resistant CML leukemic stem cells by acting via the miRNA pathways may represent a promising therapeutic option.

The anti-fibrotic role of mast cells in the liver is mediated by HLA-G and interaction with hepatic stellate cells.

BACKGROUND & AIMS: We have reported a significant association between HLA-G expression or the number of hepatic mast cells and liver fibrosis. Here, we investigated the role of HLA-G and mast cells in liver fibrosis, focusing, in particular, on interactions between human mast and stellate cells. METHODS: Human mast cells (HMC cell line, CD34-derived mast cells, or tissue-derived mast cells) were co-cultured with purified human hepatic stellate cells (HSCs), and collagen I production by HSCs was evaluated. Mast cells and HSCs were characterized by immunocytochemistry. Various conditions were tested: different times in direct or indirect contact, presence or absence of cytokines, addition or not of HLA-G, and presence or absence of specific protease inhibitors. RESULTS: The reciprocal interaction between HSCs and mast cells led to the attraction of mast cells to HSCs in vivo and in vitro, and to a significant decrease in collagen production, at all times of co-culture, following the direct or indirect contact of mast cells with HSCs alone or in the presence of TGF-ß, IFN-α or IL-10. We identified the diffusible factors involved in collagen I degradation as mast cell proteases. Moreover, HLA-G expression increased during the co-culture of HSCs and mast cells, with HLA-G acting on both mast cells and HSCs, to enhance collagen I degradation. CONCLUSIONS: Mast cells play a beneficial, anti-fibrotic role in liver fibrosis, via the HLA-G-mediated decrease of collagen I. These findings are consistent with high levels of cross-communication between mast cells and hepatic stellate cells and the role of HLA-G.

Differential Expression of the Tetraspanin CD9 in Normal and Leukemic Stem Cells.

CD9 plays a crucial role in cellular growth, mobility, and signal transduction, as well as in hematological malignancy. In myeloid neoplasms, CD9 is involved in the altered interactions between leukemic and stromal cells. However, apart from its role in CD34+ progenitors and myeloid and megakaryocytic differentiation, its function in normal and leukemic pluripotent cells has not yet been determined. Very small embryonic-like stem cells (VSELs) are promising pluripotent stem cells found in adult tissues that can be developed for safe and efficient regenerative medicine. VSELs express different surface receptors of the highest importance in cell functioning, including CD9, and can be effectively mobilized after organ injury or in leukemic patients. In the present study, we observed that CD9 is among the most expressed receptors in VSELs under steady-state conditions; however, once the VSELs are expanded, CD9+ VSELs decrease and are more apoptotic. CD9- VSELs had no proliferative improvement in vitro compared to those that were CD9+. Interestingly, the addition of SDF-1 induced CD9 expression on the surface of VSELs, as observed by flow cytometry, and improved their migration. In addition, we observed, in the phenotypically identical VSELs present in the peripheral blood of patients with myeloproliferative neoplasms, compared to healthy subjects, a significantly higher number of CD9+ cells. However, in their hematopoietic stem cell (HSC) counterparts, the expression remained comparable. These results indicate that, likewise, in progenitors and mature cells, CD9 may play an important function in normal and malignant VSELs. This could explain the refractoriness observed by some groups of expanded stem cells to repairing efficiently damaged tissue when used as a source in cell therapies. Understanding the function of the CD9 receptor in normal and malignant CD34+ and VSELs, along with its relationship with the CXCR4/SDF-1 pathway, will enable advances in the field of adult pluripotent cell usage in regenerative medicine and in their role in leukemia.

VSELs Maintain their Pluripotency and Competence to Differentiate after Enhanced Ex Vivo Expansion.

The very small embryonic-like stem cells (VSELs) are known as a subset of adult pluripotent stem cells able to differentiate to all three germ layers. However, their small number and quiescence restrict the possibility of their use in cell therapy. In the present study, we first delineate different subpopulation of VSELs from human cord blood CD34+ cells to define their purity. We next determine genes expression levels in the whole transcriptome of VSELs expressing the pluripotent marker NANOG and control cells under the steady state condition. We found that more than a thousand of genes are downregulated in VSELs, as well as many membrane receptors, cells signaling molecules and CDKs mRNAs. In addition, we observed discordance in some pluripotent genes expression levels with embryonic stem cells (ESCs), which could explain VSELs quiescence. We then evaluate VSELs capacity to expand and differentiate in vitro in specific and appropriate media. After 12 days culture in specific medium containing a pyrimidoindole derivative (UM171), VSELs were significantly expanded for the first time without feeder cells and importantly preserve their capacities to differentiate into hematopoietic and endothelial cells. Interestingly, this stimulation of VSELs self-renewal restores the expression of some downregulated genes known as key regulators of cell proliferation and differentiation. The properties of such pluripotent expanded cells make them a potential candidate in regenerative medicine.

Human Very Small Embryonic-Like Stem Cells Are Present in Normal Peripheral Blood of Young, Middle-Aged, and Aged Subjects.

The purpose of our study was to determine whether the number of human very small embryonic-like stem cells (huVSELs) would vary depending on the age of humans. HuVSELs frequency was evaluated into the steady-state (SS) peripheral blood (PB) of healthy volunteers using flow cytometry analysis. Their numbers were compared with volunteers' age. Blood samples were withdrawn from 28 volunteers (age ranging from 20 to 70 years), who were distributed among three groups of age: "young" (mean age, 27.8 years), "middle" (mean age, 49 years), and "older" (mean age, 64.2 years). Comparing the three groups, we did not observe any statistically significant difference in huVSELs numbers between them. The difference in mRNA expression for PSC markers as SSEA-4, Oct-4, Nanog, and Sox2 between the three groups of age was not statistically significant. A similar frequency of huVSELs into the SS-PB of young, middle-aged, and aged subjects may indicate that the VSELs pool persists all along the life as a reserve for tissue repair in case of minor injury and that there is a continuous efflux of these cells from the BM into the PB.

Identification and isolation from either adult human bone marrow or G-CSF-mobilized peripheral blood of CD34(+)/CD133(+)/CXCR4(+)/ Lin(-)CD45(-) cells, featuring morphological, molecular, and phenotypic characteristics of very small embryonic-like (VSEL) stem cells.

OBJECTIVE: Recently, we demonstrated that normal human bone marrow (hBM)-derived CD34(+) cells, released into the peripheral blood after granulocyte colony-stimulating factor mobilization, contain cell subpopulations committed along endothelial and cardiac differentiation pathways. These subpopulations could play a key role in the regeneration of post-ischemic myocardial lesion after their direct intracardiac delivery. We hypothesized that these relevant cells might be issued from very small embryonic-like stem cells deposited in the BM during ontogenesis and reside lifelong in the adult BM, and that they could be mobilized into peripheral blood by granulocyte colony-stimulating factor. MATERIALS AND METHODS: Samples of normal hBM and leukapheresis products harvested from cancer patients after granulocyte colony-stimulating factor mobilization were analyzed and sorted by multiparameter flow cytometry strategy. Immunofluorescence and reverse transcription quantitative polymerase chain reaction assays were performed to analyze the expression of typical pluripotent stem cells markers. RESULTS: A population of CD34(+)/CD133(+)/CXCR4(+)/Lin(-) CD45(-) immature cells was first isolated from the hBM or from leukapheresis products. Among this population, very small (2-5 µm) cells expressing Oct-4, Nanog, and stage-specific embryonic antigen-4 at protein and messenger RNA levels were identified. CONCLUSIONS: Our study supports the hypothesis that very small embryonic-like stem cells constitute a "mobile" pool of primitive/pluripotent stem cells that could be released from the BM into the peripheral blood under the influence of various physiological or pathological stimuli. In order to fully support that hBM- and leukapheresis product-derived very small embryonic-like stem cells are actually pluripotent, we are currently testing their ability to differentiate in vitro into cells from all three germ layers.