IFATS collection: Identification of hemangioblasts in the adult human adipose tissue.

The stromal-vascular fraction (SVF) of human adipose tissue contains, among other cell types, mesenchymal stem cells and precursors of adipocyte and endothelial cells. Here we show that, in addition, the nonhematopoietic fraction of the SVF has hematopoietic activity, since all types of hematopoietic colony-forming units (CFUs) developed when cultured in methylcellulose-based medium. This hematopoietic activity was restricted to the CD45(-)CD105(+) cell subset, well correlated with KDR(+) cell content, and increased after culture with a combination of early-acting hematopoietic cytokines. Most of the CD45(-)KDR(+)CD105(+) cells were nonadherent and did not express CD31, and this subset included both CD34(-) and CD34(+) cells. Moreover, these nonadherent cells migrated in response to KDR gradient, and when they were cultured in the presence of both hematopoietic and endothelial growth factors, a wave of CFUs was followed by a wave of mixed colonies comprising adherent elongated and nonadherent round hematopoietic cells. These mixed hematopoietic-endothelial (Hem-End) colonies were able to generate secondary Hem-End colonies and exhibited both hematopoietic and endothelial activity, as demonstrated by in vitro functional assays. These findings demonstrate for the first time the existence of primitive mesodermal progenitors within the SVF of human adipose tissue that exhibit in vitro hematopoietic and hemangioblastic activities, susceptible to being used in cell therapy and basic cell research. Disclosure of potential conflicts of interest is found at the end of this article.

Human platelet lysate enhances the proliferative activity of cultured human fibroblast-like cells from different tissues.

Several studies have shown the presence of fibroblast-like cells in the stromal fraction of different tissues with a high proliferative and differentiation potential. Platelet alpha granules contain growth factors released into the environment during activation. The effects of different supplements for culture medium (human serum, bovine serum and platelet lysate) on cultured human fibroblast-like cells from bone marrow, adipose tissue, trabecular bone and dental pulp have been compared. Expression of typical stromal and hematopoietic markers was analyzed and proliferative rates were determined. Flow cytofluorometry showed a homogenous pattern in serial-passaged cells, with a high level of stromal cell-associated markers (CD13, CD90, CD105). The presence of platelet lysate in culture media increased the number of cell generations obtained regardless of cell source. This effect was serum-dependent. Cell-based therapies can benefit by the use of products from human origin for "ex vivo" expansion of multipotent cells.

The functional immaturity of dendritic cells can be relevant to increased tolerance associated with cord blood transplantation.

BACKGROUND: Cord blood (CB) transplants have a significantly lower incidence of graft-versus-host disease (GVHD) compared to marrow or peripheral blood transplants. Because antigen-presenting cells and regulatory T cells (Treg) are involved in transplant tolerance, this study was aimed at analyzing the distribution of dendritic cells (DCs) and CD14+ monocyte-specific subsets in CB and adult peripheral blood (APB) and comparing the ability of DCs from these two blood sources to induce CD4+ Treg. STUDY DESIGN AND METHODS: Myeloid DCs (mDCs), plasmacytoid DCs, the CD14+ cell subsets CD14+CD16+ and CD14+CD209+, and CD4+ T cells were analyzed by fluorescence-activated cell sorting (FACS) in whole blood. To evaluate the functionality of DCs, isolated CD3+ T cells from an adult donor were cultured with allogenic DC-enriched fraction from CB or APB, and CD4+ Treg generation was determined by FACS. Additionally, tumor necrosis factor (TNF)-alpha and interferon (IFN)-alpha release by DCs was measured. RESULTS: CB had a lesser frequency of DCs and specific CD14+ monocyte subsets than APB. After stimulation, monocytes from CB secreted less TNF-alpha than those from APB. Moreover, DCs from CB exhibited a more immature phenotype and had a decreased capacity to release TNF-alpha and IFN-alpha than those derived from APB, but on the contrary, they were efficient inducers of CD4+ T cells with a phenotype of Treg. CONCLUSION: The tolerogenic immunophenotype and diminished functionality of CB DCs can be important to create a microenvironment where Treg develop, that in turn may be relevant to observed lower incidence of chronic GVHD after CB transplantation.

Selective generation of different dendritic cell precursors from CD34+ cells by interleukin-6 and interleukin-3.

There is a growing interest in generating dendritic cells (DCs) for using as vaccines. Several cytokines, especially stem cell factor (SCF) and FLT3-ligand (FL), have been identified as essential to produce large numbers of myeloid precursors and even to increase DC yield obtained by the action of granulocyte-macrophage colony-stimulating factor (GM-CSF) and tumor necrosis factor alpha (TNF-alpha). However, there are few studies on the effect of the early-acting cytokines, commonly used to expand CD34+ progenitor cells, on DC generation. We report here that in the absence of serum, SCF, FL, and thrombopoietin (TPO) plus interleukin-6 (IL-6) and SCF, FL, and TPO plus IL-3 were able to generate CD14+CD1a- and CD14- CD1a+ myeloid DC precursors from CD34+ cells, but IL-6 had an inhibitory effect on the generation of CD14- CD1a+ cells. Both DC precursors differentiated into mature DCs by GM-CSF, IL-4, and TNF-alpha, and DCs obtained from both types of culture exhibited equal allostimulatory capacity. CD1a+ DCs generated could be identified on the basis of DC-specific intracellular adhesion molecule-grabbing nonintegrin (DC-SIGN) expression, a novel C-type lectin receptor expressed on dermal DCs but not on Langerhans cells. In addition, the inclusion of IL-3 to the culture medium induced the appearance of CD13- cells that differentiated into plasmacytoid DC (DC2) on the addition of TNF-alpha, allowing the identification of developmental stages of DC2. Like true plasmacytoid DCs, these cells secreted interferon-alpha after TLR9-specific stimulation with a specific CpG nucleotide.

CD34+CD38- is a good predictive marker of cloning ability and expansion potential of CD34+ cord blood cells.

BACKGROUND: Ex vivo expansion of HPCs is an attractive approach to overcoming the current limitations of human cord blood transplantation. It is important not only to define the optimal culture conditions but also to know the number of progenitor cells that can be obtained. CD34+ cells have a great variability in their cloning capacity and in their ability to expand HPCs. This study was carried out to assess whether this variability could be due to intrinsic or extrinsic factors. STUDY DESIGN AND METHODS: CD34+ cells were analyzed for the expression of CD38, CD133, and CD117 and cultured in serum-free culture medium with four cytokine combinations: SCF plus thrombopoietin plus flt3 ligand (STF), STF plus IL-3, STF plus IL-6, and STF plus IL-6 plus IL-3. After a 1-week culture, the numbers of CD34+ cells and CFUs were determined. RESULTS: The variability observed both in the cloning ability of CD34+ isolated cells and in their expansion capacity was inversely related to the frequency of the more immature CD34+CD38- cells. When more mature CD34+CD38+ cells were present within CD34+-isolated cells, a higher cloning ability, measured as CFUs, and a higher expansion capacity were observed. CONCLUSION: Enumeration of CD34+CD38- cells is correlated with the number of committed progenitors and the capacity of generating CD34+ cells, an important parameter if expansion protocols must be used in clinical transplantation.

IL-6 precludes the differentiation induced by IL-3 on expansion of CD34+ cells from cord blood.

BACKGROUND AND OBJECTIVES: Ex vivo expansion of hematopoietic progenitor cells (HPC) from umbilical cord blood (UCB) is an interesting strategy to obtain a sufficient number of transplantable cells for adults. To define the optimal culture conditions allowing the generation of HPC that retain their proliferative capacity without loss of long-term culture-initiating cells (LTC-IC), the effect of different cytokine combinations on the expansion of CD34+ cells from UCB was assessed. DESIGN AND METHODS: CD34+ cells were cultured in serum-free culture medium with four cytokine combinations: stem cell factor plus thrombopoietin plus flk2/flt3 ligand (STF), STF plus interleukin-3 (IL-3), STF plus interleukin-6 (IL-6) and STF plus IL-6 plus IL-3. After a 1-week culture, the number of CD34+ and CD133+ cells, colony forming units (CFU), LTC-IC and telomerase activity were determined. RESULTS: The addition of IL-6 or IL-3 to the combination of STF significantly enhanced the expansion of CD34+, CD133+ cells and CFU. All cytokine combinations tested induced a slight increase in LTC-IC number except that composed by STF plus IL-3. The greatest induction of telomerase activity was observed with the combination of STF plus IL-3 or plus IL-3 plus IL-6. Decay of the activity along time was observed when the combination of STF plus IL-3 was used, and this effect was reverted by the addition of IL-6. INTERPRETATION AND CONCLUSIONS: Our results demonstrate that the inclusion of IL-6 in a serum-free short-term culture has a beneficial effect on HPC expansion from UCB, and precludes the negative effects induced by IL-3 on LTC-IC expansion and telomerase activity.