Maryse Proulx

Creating capillary networks within human engineered tissues: impact of adipocytes and their secretory products.

The development of tissue-engineered substitutes of substantial volume is closely associated with the need to ensure rapid vascularization upon grafting. Strategies promoting angiogenesis include the in vitro formation of capillary-like networks within engineered substitutes. We generated both connective and adipose tissues based on a cell sheet technology using human adipose-derived stromal cells. This study evaluates the morphology and extent of the capillary networks that developed upon seeding of human microvascular endothelial cells during tissue production. We posited that adipocyte presence/secretory products could modulate the resulting capillary network when compared to connective substitutes. Analyses including confocal imaging of CD31-labeled capillary-like networks indicated slight differences in their morphological appearance. However, the total volume occupied by the networks as well as the frequency distribution of the structures volumes were similar between connective and adipose tissues. The average diameter of the capillary structures tended to be 20% higher in reconstructed adipose tissues. Quantification of pro-angiogenic molecules in conditioned media showed greater amounts of leptin (15×), angiopoietin-1 (3.4×) and HGF (1.7×) secreted from adipose than connective tissues at the time of endothelial cell seeding. However, this difference was attenuated during the following coculture period in endothelial cell-containing media, correlating with the minor differences noted between the networks. Taken together, we developed a protocol allowing reconstruction of both connective and adipose tissues featuring well-developed capillary networks in vitro. We performed a detailed characterization of the network architecture within engineered tissues that is relevant for graft assessment before implantation as well as for in vitro screening of angiogenic modulators using three-dimensional models.

Characterization of In Vitro Engineered Human Adipose Tissues: Relevant Adipokine Secretion and Impact of TNF-α.

Representative modelling of human adipose tissue functions is central to metabolic research. Tridimensional models able to recreate human adipogenesis in a physiological tissue-like context in vitro are still scarce. We describe the engineering of white adipose tissues reconstructed from their cultured adipose-derived stromal precursor cells. We hypothesize that these reconstructed tissues can recapitulate key functions of AT under basal and pro-inflammatory conditions. These tissues, featuring human adipocytes surrounded by stroma, were stable and metabolically active in long-term cultures (at least 11 weeks). Secretion of major adipokines and growth factors by the reconstructed tissues was determined and compared to media conditioned by human native fat explants. Interestingly, the secretory profiles of the reconstructed adipose tissues indicated an abundant production of leptin, PAI-1 and angiopoietin-1 proteins, while higher HGF levels were detected for the human fat explants. We next demonstrated the responsiveness of the tissues to the pro-inflammatory stimulus TNF-α, as reflected by modulation of MCP-1, NGF and HGF secretion, while VEGF and leptin protein expression did not vary. TNF-α exposure induced changes in gene expression for adipocyte metabolism-associated mRNAs such as SLC2A4, FASN and LIPE, as well as for genes implicated in NF-κB activation. Finally, this model was customized to feature adipocytes representative of progressive stages of differentiation, thereby allowing investigations using newly differentiated or more mature adipocytes. In conclusion, we produced tridimensional tissues engineered in vitro that are able to recapitulate key characteristics of subcutaneous white adipose tissue. These tissues are produced from human cells and their neo-synthesized matrix elements without exogenous or synthetic biomaterials. Therefore, they represent unique tools to investigate the effects of pharmacologically active products on human stromal cells, extracellular matrix and differentiated adipocytes, in addition to compounds modulating adipogenesis from precursor cells.

c‐Src tyrosine kinase co‐associates with and phosphorylates signal transducer and activator of transcription 5b which mediates the proliferation of normal human B lymphocytes

c‐Src is the normal human cellular protein homologue of the viral oncogene v‐src. c‐Src activity was reported recently to increase in CD40‐activated human B lymphocytes, suggesting its involvement in proliferation. To elucidate the exact role of c‐Src in this process, we investigated the effects of c‐Src over‐expression on normal B lymphocyte growth. B lymphocytes purified from human peripheral blood were infected with Ad5/F35 vector encoding either a constitutively active c‐Src (c‐Src/dominant‐positive) or a dominant‐negative c‐Src (c‐Src/DN). Little variation of B lymphocytes expansion could be observed between control enhanced yellow fluorescent protein and c‐Src/dominant‐positive‐infected cells. In contrast, over‐expression of c‐Src/DN results in a 40% inhibition of B lymphocyte expansion. These results suggest that DN c‐Src may compete with endogenous c‐Src, resulting in partial inhibition of a transcriptional pathway involved in B lymphocyte proliferation. We demonstrate further that c‐Src can phosphorylate signal transducer and activator of transcription 5b (STAT5b) on tyrosine 699 and that c‐Src and STAT5b co‐associate during B lymphocyte proliferation. These results confirm an important role for c‐Src in the expansion of normal human B lymphocytes in vitro, in which c‐Src may regulate STAT5b in the intracellular signalling pathway important for the proliferation of normal human B lymphocytes.

2-Methoxyestradiol induce the conversion of human peripheral blood memory B lymphocytes into plasma cells.

2-Methoxyestradiol (2ME), an end-metabolite of 17beta-estradiol, is an antiproliferative agent that is currently being tested in clinical trials for cancer treatment. We hereby report that sub-cytotoxic concentrations of 2ME influence the in vitro proliferation of human peripheral blood B lymphocytes. More surprisingly, we have observed that 2ME induces the conversion of CD138(-) B lymphocytes into CD138(+) cells of phenotype similar to immunoglobulin (Ig)-secreting plasma cells. Normal human B lymphocytes expressing CD138 increased in response to 2ME in a dose-dependent fashion, from 2% at baseline up to 31% in cells cultured in the presence of 0.75 microM 2ME. Moreover, most of the converted cells were also CD27(+) and secreted high levels of IgG (151 microg/10(6)cells/24h). IEF studies revealed that conversion occurred in a polyclonal manner. We then exploited this effect of 2ME to gain further insights into the molecular mechanisms that govern changes in transcription factors involved in plasma cells differentiation. Plasma cells generated by 2ME treatment of normal human B lymphocytes expressed elevated levels of IRF4 and reduced levels of Pax5 and Bcl-6. Similarly, levels of XBP-1 and Blimp-1 transcripts were increased. Our results suggest that the differentiation of peripheral blood B lymphocytes into plasma cells requires a similar modulation of transcription factors expression that for tonsil and bone marrow B lymphocytes.

Adipose Stem Cells, Tissue Engineering, and Solid Organ Transplantation and Regeneration

In the past years, adipose tissue has spurred a wide interest as a source of adult multipotent stem cells with the potential to change the landscape of regenerative medicine. These adipose-derived stem/stromal cells (ASCs) are already establishing their effectiveness at improving organ function when delivered for cell therapy. Tissue engineering applications are also progressing at great strides in part due to the unique properties of ASCs. This chapter describes advances in tissue engineering for the production of solid tissues/organs using ASCs as building blocks. It focuses on recent progress achieved for the reconstruction of human adipose tissue as well as new improved skin substitutes. In particular, we will describe how the self-assembly approach of tissue engineering leads to the production of natural soft tissues devoid of exogenous biomaterials. Key considerations pertaining to the engineering of tissues that will sustain long-term regeneration and renewal will also be discussed.