Rapid and Efficient Production of Human Functional Mast Cells through a Three-Dimensional Culture of Adipose Tissue-Derived Stromal Vascular Cells.

Mast cells (MC) are innate immune cells involved in many physiological and pathological processes. However, studies of MC function and biology are hampered by the difficulties to obtain human primary MC. To solve this problem, we established a new method to produce easily and rapidly high numbers of MC for in vitro studies using human adipose tissue, which is an abundant and easy access tissue. Stromal vascular fraction of adipose tissue, obtained from human abdominal dermolipectomy, was cultured as spheroids in serum free medium supplemented in stem cell factor. Using this method, we generated, within 3 wk, a highly pure population of connective tissue-type MC expressing MC typical peptidases (tryptase, chymase, and carboxypeptidase-A3) with a yield increasing over time. Stem cell factor was required for this culture, but unlike MC derived from CD34+ cells, this culture did not depend on IL-3 and -6. MC obtained with this method degranulated following FcεRI cross-linking or stimulation by C5a, compound 48/80, and substance P. Interestingly, activation by anti-IgE of both white adipose tissue-MC and MC obtained from peripheral blood-derived CD34+ pluripotent progenitor cells induced the production of PGs as well as proinflammatory cytokines (TNF-α, Il-6, and GM-CSF). In conclusion, we developed a new time saving and reproducible method to produce highly pure and functional human MC in 3 wk from human adipose tissue.

Corrupted adipose tissue endogenous myelopoiesis initiates diet-induced metabolic disease.

Activation and increased numbers of inflammatory macrophages, in adipose tissue (AT) are deleterious in metabolic diseases. Up to now, AT macrophages (ATM) accumulation was considered to be due to blood infiltration or local proliferation, although the presence of resident hematopoietic stem/progenitor cells (Lin-/Sca+/c-Kit+; LSK phenotype) in the AT (AT-LSK) has been reported. By using transplantation of sorted AT-LSK and gain and loss of function studies we show that some of the inflammatory ATM inducing metabolic disease, originate from resident AT-LSK. Transplantation of AT-LSK sorted from high fat diet-fed (HFD) mice is sufficient to induce ATM accumulation, and to transfer metabolic disease in control mice. Conversely, the transplantation of control AT-LSK improves both AT-inflammation and glucose homeostasis in HFD mice. Our results clearly demonstrate that resident AT-LSK are one of the key point of metabolic disease, and could thus constitute a new promising therapeutic target to fight against metabolic disease.

Differential Hematopoietic Activity in White Adipose Tissue Depending on its Localization.

White adipose tissue (WAT) can be found in different locations in the body, and these different adipose deposits exhibit specific physiopathological importance according to the subcutaneous or abdominal locations. We have shown previously the presence of functional hematopoietic stem/progenitor cells (HSPC) in subcutaneous adipose tissue (SCAT). These cells exhibit a specific hematopoietic activity that contributes to the renewal of the immune cell compartment within this adipose deposit. In this study, we investigated whether HSPC can be found in visceral adipose tissue (VAT) and whether a putative difference in in situ hematopoiesis may be related to anatomical location and to site-specific immune cell content in VAT compared to SCAT. Therein, we identified for the first time the presence of HSPC in VAT. Using both in vitro assays and in vivo competitive repopulation experiments with sorted HSPC from VAT or SCAT, we showed that the hematopoietic activity of HSPC was lower in VAT, compared to SCAT. In addition, this altered hematopoietic activity of HSPC in VAT was due to their microenvironment, and may be related to a specific combination of secreted factors and extracellular matrix molecules expressed by adipose derived stromal cells. Our results indicate that WAT specific hematopoietic activity may be generalized to all adipose deposits, although with specificity according to the fat pad location. Considering the abundance of WAT in the body, this emphasizes the potential importance of this hematopoietic activity in physiopathological situations.

An Association between BK Virus Replication in Bone Marrow and Cytopenia in Kidney-Transplant Recipients.

The human polyomavirus BK (BKV) is associated with severe complications, such as ureteric stenosis and polyomavirus-associated nephropathy (PVAN), which often occur in kidney-transplant patients. However, it is unknown if BKV can replicate within bone marrow. The aim of this study was to search for BKV replication within the bone marrow of kidney-transplant patients presenting with a hematological disorder. Seventy-two kidney-transplant patients underwent bone-marrow aspiration for cytopenia. At least one virus was detected in the bone marrow of 25/72 patients (35%), that is, parvovirus B19 alone (n = 8), parvovirus plus Epstein-Barr virus (EBV) (n = 3), cytomegalovirus (n = 4), EBV (n = 2), BKV alone (n = 7), and BKV plus EBV (n = 1). Three of the eight patients who had BKV replication within the bone marrow had no detectable BKV replication in the blood. Neutropenia was observed in all patients with BKV replication in the bone marrow, and blockade of granulocyte maturation was observed. Hematological disorders disappeared in all patients after doses of immunosuppressants were reduced. In conclusion, an association between BKV replication in bone marrow and hematological disorders, especially neutropenia, was observed. Further studies are needed to confirm these findings.

In situ production of innate immune cells in murine white adipose tissue.

White adipose tissue (WAT) is the focus of new interest because of the presence of an abundant and complex immune cell population that is involved in key pathologies such as metabolic syndrome. Based on in vivo reconstitution assays, it is thought that these immune cells are derived from the bone marrow (BM). However, previous studies have shown that WAT exhibits specific hematopoietic activity exerted by an unknown subpopulation of cells. In the present study, we prospectively isolated a peculiar hematopoietic stem/progenitor cell population from murine WAT. The cells are phenotypically similar to BM hematopoietic stem cells and are able to differentiate into both myeloid and lymphoid lineages in vitro. In competitive repopulation assays in vivo, they reconstituted the innate immune compartment in WAT preferentially and more efficiently than BM cells, but did not reconstitute hematopoietic organs. They were also able to give rise to multilineage engraftment in both secondary recipients and in utero transplantation. Therefore, we propose that WAT hematopoietic cells constitute a population of immature cells that are able to renew innate immune cell populations. Considering the amount of WAT in adults, our results suggest that WAT hematopoietic activity controls WAT inflammatory processes and also supports innate immune responses in other organs.

Adipose-derived stromal cells: Their identity and uses in clinical trials, an update.

In adults, adipose tissue is abundant and can be easily sampled using liposuction. Largely involved in obesity and associated metabolic disorders, it is now described as a reservoir of immature stromal cells. These cells, called adipose-derived stromal cells (ADSCs) must be distinguished from the crude stromal vascular fraction (SVF) obtained after digestion of adipose tissue. ADSCs share many features with mesenchymal stem cells derived from bone marrow, including paracrine activity, but they also display some specific features, including a greater angiogenic potential. Their angiogenic properties as well as their paracrine activity suggest a putative tumor-promoting role for ADSCs although contradictory data have been published on this issue. Both SVF cells and ADSCs are currently being investigated in clinical trials in several fields (chronic inflammation, ischemic diseases, etc.). Apart from a phase III trial on the treatment of fistula, most of these are in phase I and use autologous cells. In the near future, the end results of these trials should provide a great deal of data on the safety of ADSC use.

Human adipose-derived stromal cells efficiently support hematopoiesis in vitro and in vivo: a key step for therapeutic studies.

Adipose-derived stromal cells (ADSCs) are close relatives of bone marrow mesenchymal stromal cells (BM-MSCs). The ease of access to subcutaneous fat pad and the abundance of stromal precursors make fat tissue an attractive source of stromal cells for clinicians. However, their ability to support hematopoietic stem cells in vitro and in vivo has not been established definitively. Thus, their usefulness in supporting hematopoietic stem cell engraftment is not as clear as with BM-MSCs. In this article, we show that human ADSCs, cultured with a good manufacturing practice medium, maintain in vitro human early and committed hematopoietic progenitors and support their complete differentiation toward myeloid and lymphoid lineages. Compared with BM-MSCs, ADSCs elicit a more precocious early progenitor formation and faster proliferation and differentiation of hematopoietic progenitors. Further, in vivo, when co-injected in NOD.Cg-Prkdc(scid) Il2(rgtm1Wjl)/SzJ (NSG) mice with a low number of human CD34(+) cells, ADSCs enabled the higher production of immature human hematopoietic progenitors and CD45(+) cells when compared with BM-MSCs. As a whole, our results indicate that human ADSCs, isolated and expanded under clinical-grade conditions, support hematopoiesis in vitro and in vivo and thus provide the rationale for their use in supporting hematopoietic reconstitution in clinical settings.

Adipose tissue as a dedicated reservoir of functional mast cell progenitors.

White adipose tissue (WAT) is a heterogeneous tissue, found in various locations throughout the body, containing mature adipocytes and the stroma-vascular fraction (SVF). The SVF includes a large proportion of immune hematopoietic cells, among which, mast cells that contribute to diet-induced obesity. In this study, we asked whether mast cells present in mice adipose tissue could derive from hematopoietic stem/progenitor cells (HSPC) identified in the tissue. We therefore performed both in vitro and in vivo experiments dedicated to monitoring the progeny of WAT-derived HSPC. The entire study was conducted in parallel with bone marrow-derived cells, considered the gold standard for hematopoietic-lineage studies. Here, we demonstrate that adipose-derived HSPC contain a precursor-cell population committed to the mast cell lineage, and able to efficiently home to peripheral organs such as intestine and skin, where it acquires properties of functional tissue mast cells. Additionally, WAT contains a significant mast cell progenitor population, suggesting that the entire mast cell lineage process take place in WAT. Considering the quantitative importance of WAT in the adult organism and the increasing roles recently assigned to mast cells in physiopathology, WAT may represent an important source of mast cells in physiological and pathological situations.

The emergence of adipocytes.

In mammals, the adipose organ is composed of white adipocytes (primary site in energy storage) and of brown adipocytes (specialized in thermogenesis). Adipocytes arise from mesenchymal stem cells (MSCs) by a sequential pathway of differentiation. MSCs develop either from ectoderm or mesoderm and commit into different undifferentiated precursors, which upon the expression of key transcription factors enter a differentiation program to acquire their specific functions. When triggered by appropriate developmental cues, MSCs become committed to the adipocyte lineage. White adipocytes differentiate from various types of vascular cell types, probably located within the white adipose tissue itself. Brown adipocytes arise from myogenic precursors. The differentiation between white adipocyte and brown adipocyte lineages occurs in the earliest steps of the fetal development, and both phenotypes are acquired independently. A better knowledge of these differentiation pathways allows new therapeutic strategies for reconstruction of damaged conjunctive tissues and for the control or prevention of risks associated with obesity in humans.

Vascular and endothelial regeneration.

Adipose tissue is the final tissue to develop and is strongly involved in energy homeostasis. It can represent up to 50% of body weight in obesity. Beside its metabolic role, endocrine functions appeared to play a key role in interconnecting adipose tissue with other tissues of the organism and in numerous physiological functions. The presence of adipocyte progenitors has long been demonstrated throughout life in the stromal fraction of adipose tissue. Now, it appears that these cells are multipotent and share numerous features with mesenchymal stem cells (MSC) derived from bone marrow. They also display some specificities and a strong pro-angiogenic potential. Altogether, these data emphasize the need to reconsider the potential of adipose tissue. Moreover, since fat pads are easy to sample, numerous and promising perspectives are now opening up in regenerative medicine, particularly in ischemic situations.

Transplantation of adipose derived stromal cells is associated with functional improvement in a rat model of chronic myocardial infarction.

AIMS: To determine the effect of transplantation of undifferentiated and cardiac pre-differentiated adipose stem cells compared with bone marrow mononuclear cells (BM-MNC) in a chronic model of myocardial infarction. METHODS: Ninety-five Sprague-Dawley rats underwent left coronary artery ligation and after 1 month received by direct intramyocardial injection either adipose derived stem cells (ADSC), cardiomyogenic cells (AD-CMG) or BM-MNC from enhanced-Green Fluorescent Protein (eGFP) mice. The control group was treated with culture medium. Heart function was assessed by echocardiography and 18F-FDG microPET. Cell engraftment, differentiation, angiogenesis and fibrosis in the scar tissue were also evaluated by (immuno)histochemistry and immunofluorescence. RESULTS: One month after cell transplantation, ADSC induced a significant improvement in heart function (LVEF 46.3+/-9.6% versus 27.7+/-8% pre-transplant) and tissue viability (64.78+/-7.2% versus 55.89+/-6.3% pre-transplant). An increase in the degree of angiogenesis and a decrease in fibrosis were also detected. Although transplantation of AD-CMG or BM-MNC also had a positive, albeit smaller, effect on angiogenesis and fibrosis in the infarcted hearts, this benefit did not translate into a significant improvement in heart function or tissue viability. CONCLUSION: These results indicate that transplantation of adipose derived cells in chronic infarct provides a superior benefit to cardiac pre-differentiated ADSC and BM-MNC.

Virus-based gene transfer approaches and adipose tissue biology.

The status of adipose tissue changes rapidly. From a simple filler tissue, it successively acquires the status of metabolic active tissue, endocrine tissue, plastic tissue, and finally that of a large reservoir of cells suitable for cell therapy and regenerative medicine. All throughout this story, our knowledge has been largely dependent on genetic tools and gene transfer. Now, the time has come where gene transfer in adipose derived cells can be envisioned, not only for understanding the role or importance of one gene, but also to engineer adipose derived cells for the purpose of therapy by delivering secreted products. In this paper, after a brief overview of adipose tissues, a large part will be devoted to the use of virus-based gene transfer in transducing adipose tissue and cells which reside therein. We also critically review the use of adipose "specific" promoters and the applications already described in the literature.

[Adipose tissue: a subtle and complex cell system]. / Le tissu adipeux: une ecologie cellulaire subtile et complexe.

White adipose tissue (WAT) represents a large amount of all adult tissues. For a long time, it was considered as a poorly active, overgrown and undesirable tissue. It was mainly studied for its involvement in energy metabolism and disorders, as well as for its endocrine functions. WAT is composed of two main populations, matures adipocytes and stroma vascular fraction (SVF) that can be separated easily. The SVF contains two compartments, stromal and hematopoietic that have been recently characterized. The stromal population (or ADAS for Adipose Derived Stromal Cells) presents functional features of, as well as lineage relationship with, macrophages. These stromal cells, that are able to differentiate into adipocytes, also display endothelial potential, and could be considered as vascular progenitors. Differentiation of various adipose-derived cell subsets towards functional cardiomyocytes, osteoblasts, chondrocytes, muscle, hematopoietic and neural cells was also obtained in vitro or in vivo. Adipose tissue thus appears as a complex tissue composed of different cell subsets that could vary according to the nature and the location of fat pads, or to the physiological or pathological status. WAT appears as a very plastic and heterogeneous tissue that is very easy to sample. This represents a great advantage when considering adipose tissue as a potential and suitable source of stem cell for cell therapy. Further investigations in this way have to lead to the emergence of new insights fundamental to progress in our knowledge of adipose tissue biology.

Human subcutaneous adipose cells support complete differentiation but not self-renewal of hematopoietic progenitors.

Adipose tissue is now considered as an endocrine organ implicated in energy regulation, inflammation and immune response, and as a source of multipotent cells with a broad range of differentiation capacities. Some of these cells are of a mesenchymal type which can -- like their bone marrow (BM) counterpart -- support hematopoiesis, since in a previous study we were able to reconstitute lethally irradiated mice by cells isolated from adipose tissue. In the present study, we established that cells derived from the stroma-vascular fraction of human subcutaneous fat pads support the complete differentiation of hematopoietic progenitors into myeloid and B lymphoid cells. However, these cells are unable to maintain the survival and self-renewal of hematopoietic stem cells. These features, similar to those of BM adipocytes, are the opposite of those of other cell types derived from mesenchymal progenitors such as BM myofibroblasts or osteoblasts. Because it is abundant and accessible, adipose tissue could be a convenient source of cells for the short-term reconstitution of hematopoiesis in man.

Immunomodulatory effect of human adipose tissue-derived adult stem cells: comparison with bone marrow mesenchymal stem cells.

Like mesenchymal stem cells from bone marrow (BM-MSCs), adipose tissue-derived adult stem cells (ADAS cells) can differentiate into several lineages and present therapeutical potential for repairing damaged tissues. The use of allogenic stem cells can enlarge their therapeutical interest, provided that the grafted cells could be tolerated. We investigate here, for the first time, the immunosuppressive properties of ADAS cells compared with the well-characterized immunosuppressive properties of BM-MSCs. ADAS cells did not provoke in vitro alloreactivity of incompatible lymphocytes and, moreover, suppressed mixed lymphocyte reaction (MLR) and lymphocyte proliferative response to mitogens. The impairment of inhibition when ADAS cells and BM-MSCs were separated from lymphocytes by a permeable membrane suggests that cell contact is required for a full inhibitory effect. Hepatocyte growth factor is secreted by both stem cells but, similar to interleukin-10 and transforming growth factor-beta (TGF-beta), the levels of which were undetectable in supernatants of MLR inhibited by ADAS cells or BM-MSCs, it did not seem implicated in the stem cell suppressive effect. These findings support that ADAS cells share immunosuppressive properties with BM-MSCs. Therefore, ADAS cell-based reconstructive therapy could employ allogenic cells and because of their immunosuppressive properties, ADAS cells could be an alternative source to BM-MSCs to treat allogenic conflicts.

Human bone marrow adipocytes support complete myeloid and lymphoid differentiation from human CD34 cells.

In humans, the role of bone marrow (BM) adipocytes in supporting haematopoiesis has been questioned. A co-culture system of CD34(+) cells seeded onto either BM undifferentiated mesenchymal stem cells or differentiated adipocytes showed that BM adipocytes did not support the maintenance of immature progenitors but enabled their complete differentiation along the myeloid and lymphoid pathways. These properties appear to be opposite to those of osteoblasts, although both cell types share a common mesenchymal progenitor. These results suggest that stromal cells play a variety of roles in the haematopoietic microenvironment, which could be significant in situations such as osteoporosis or ageing.