Recently adipose tissue has become a research topic also for the searching for an alternative stem cells source to use in cell based therapies such as tissue engineer. In fact Adipose Stem Cells (ASCs) exhibit an important differentiation potential for several cell lineages such as chondrogenic, osteogenic, myogenic, adipogenic and endothelial cells. ASCs populations isolated using standard methodologies (i.e., based on their adherence ability) are very heterogeneous but very few studies have analysed this aspect. Consequently, several questions are still pending, as for example, on what regard the existence/ or not of distinct ASCs subpopulations. The present study is originally aimed at isolating selected ASCs subpopulations, and to analyse their behaviour towards the heterogeneous population regarding the expression of stem cell markers and also regarding their osteogenic and chondrogenic differentiation potential. Human Adipose derived Stem Cells (hASCs) subpopulations were isolated using immunomagnetic beads coated with several different antibodies (CD29, CD44, CD49d, CD73, CD90, CD 105, Stro-1 and p75) and were characterized by Real Time RT-PCR in order to assess the expression of mesenchymal stem cells markers (CD44, CD73, Stro-1, CD105 and CD90) as well as known markers of the chondrogenic (Sox 9, Collagen II) and osteogenic lineage (Osteopontin, Osteocalcin). The obtained results underline the complexity of the ASCs population demonstrating that it is composed of several subpopulations, which express different levels of ASCs markers and exhibit distinctive differentiation potentials. Furthermore, the results obtained clearly evidence of the advantages of using selected populations in cell-based therapies, such as bone and cartilage regenerative medicine approaches.

The adipose tissue was considered a reserve of energy until the '80s, when it was found that this tissue was involved in the metabolism of sex steroids such as estrogens. From then on, the importance attributed to this tissue radically changed as it was then considered an active organ, involved in important functions of the human body. In 2001, for the first time, the existence of stem cells within this tissue was reported, and since then, this tissue has been gaining an increased importance as a stem cell source for a wide range of potential applications in cell therapies and/or tissue engineering and regenerative medicine strategies, mainly due to its wide availability and easy access. This manuscript provides an overview on adipose stem cells (i.e., adipose tissue-derived stem cells, ASCs) considering the tissue of origin, the niche of the ASCs, and their phenotype in all aspects. In this paper it is also discussed the markers that have been used for the characterization of these cells, their differentiation properties, and their immunological reactivity, reporting studies from 2001 until this date. The ASCs are also compared with bone marrow stem cells (BMSCs), until now considered as the gold standard source of stem cells, underlining the common characteristics and the differences between the stem cells obtained from these two sources, as well as the advantages and disadvantages of their potential use in different applications. Finally, this review will also focus on the potential application of ASCs in tissue engineering applications, particularly in the regeneration of bone and cartilage, commenting on the progress of this approach and future trends of the field.

In this work, the ability of gellan gum hydrogels coupled with autologous cells to regenerate rabbit full-thickness articular cartilage defects was tested. Five study groups were defined: (a) gellan gum with encapsulated chondrogenic predifferentiated rabbit adipose stem cells (ASC þ GF); (b) gellan gum with encapsulated nonchondrogenic predifferentiated rabbit adipose stem cells (ASC); (c) gellan gum with encapsulated rabbit articular chondrocytes (AC) (standard control); (d) gellan gum alone (control); (e) empty defect (control). Fullthickness articular cartilage defects were created and the gellan gum constructs were injected and left for 8 weeks. The macroscopic aspect of the explants showed a progressive increase of similarity with the lateral native cartilage, stable integration at the defect site, more pronouncedly in the cell-loaded constructs. Tissue scoring showed that ASC þ GF exhibited the best results regarding tissue quality progression. Alcian blue retrieved similar results with a better outcome for the cell-loaded constructs. Regarding real-time PCR analyses, ASC þ GF had the best progression with an upregulation of collagen type II and aggrecan, and a downregulation of collagen type I. Gellan gum hydrogels combined with autologous cells constitute a promising approach for the treatment of articular cartilage defects, and adipose derived cells may constitute a valid alternative to currently used articular chondrocytes. ß

Human adipose tissue has been recently recognized as a potential source of stem cells for regenerative medicine applications, including bone tissue engineering (TE). Despite the gathered knowledge regarding the differentiation potential of human adipose tissue-derived stem cells (hASCs), in what concerns the endothelial lineage many uncertainties are still present. The existence of a cell subpopulation within the human adipose tissue that expresses a SSEA-4 marker, usually associated to pluripotency, raises expectations on the differentiation capacity of these cells (SSEA-4 + hASCs). In the present study, the endothelial and osteogenic differentiation potential of the SSEA-4 + hASCs was analyzed, aiming at proposing a single-cell source/ subpopulation for the development of vascularized bone TE constructs. SSEA-4 + hASCs were isolated using immunomagnetic sorting and cultured either in a-MEM, in EGM-2 MV (endothelial growth medium), or in osteogenic medium. SSEA-4 + hASCs cultured in EGM-2 MV formed endothelial cell-like colonies characterized by a cobblestone morphology and expression of CD31, CD34, CD105, and von Willebrand factor as determined by quantitative reverse transcriptase (RT)-polymerase chain reaction, immunofluorescence, and flow cytometry. The endothelial phenotype was also confirmed by their ability to incorporate acetylated lowdensity lipoprotein and to form capillary-like structures when seeded on Matrigel. SSEA-4+ hASCs cultured in a-MEM displayed fibroblastic-like morphology and exhibited a mesenchymal surface marker profile (>90% CD90+ ). After culture in osteogenic conditions, an overexpression of osteogenic-related markers (osteopontin and osteocalcin) was observed both at molecular and protein levels. Matrix mineralization detected by Alizarin Red staining confirmed SSEA-4 + hASCs osteogenic differentiation. Herein, we demonstrate that from a single-cell source, human adipose tissue, and by selecting the appropriate subpopulation it is possible to obtain microvascular-like endothelial cells and osteoblasts, the most relevant cell types for the creation of vascularized bone tissue-engineered constructs.

Adipose stem cells (ASCs) represent a cell population with great potential for tissue engineering applications. Several articles have been published showing the proliferation and differentiation potential, the markers and the wide range of potential applications of these cells. In the majority of these studies the ASCs are isolated using a basic enzymatic procedure, which results in a quite heterogeneous cell population that compromises their proliferation and differentiation. This paper reports the development and optimization of a new isolation/purification method that allows populations of ASCs to be obtained, which significantly reduces (and eventually eliminates) the 'contamination' of other cell types. This method is based on the use of immunomagnetic beads coated with specific antibodies. The first part of the study described here analysed the expression of marker genes for stem cells and the colony-forming unit (CFU) capacity of the cells isolated, while the second part is dedicated to the osteogenic differentiation potential of the isolated cells. The results showed that, using the isolation method based on immunomagnetic beads, it was possible to obtain ASCs and also underline the existence of several subpopulations of stem cells in the adipose tissue.

The first stem cells considered for the reconstruction of bone were bone marrow mesenchymal stem cells (BMSCs). Subsequently, cells with similar marker expression panel and differentiation potential were found in new sources of cells, such as adipose tissue. This source of stem cells has a promising future in tissue-engineering applications, considering the abundance of this tissue in the human body, the easy harvesting and the high number of stem cells that are available from such a small amount of tissue. The isolation of the adipose stem cells is generally performed by means of enzymatic digestion of the tissues, followed by a natural selection of the stem cells based on their capacity to adhere to the culture flasks, leading to a quite heterogeneous population. This constitutes a major drawback for the use of these cells, since the heterogeneity of the cell culture obtained can compromise their proliferation and differentiation potential. In the present study we have analysed the in vitro and in vivo behaviour of two selected subpopulations with high osteogenic potential. For this purpose, ASCs(CD29+) and ASCs (STRO-1+)subpopulations were isolated and in vitro cultured onto a biodegradable polymeric scaffold, using osteogenic medium, before implantation in a nude mice model. The biodegradable polymeric scaffold used is a fibre-mesh structure based on a blend of starch and polycaprolatone (SPCL) that has been successfully used in several bone tissue-engineering studies. The implanted ASCs-scaffold constructs promoted the formation of new bone tissue in nude mice. However, the results obtained show differences in the behaviour of the two ASCs subpopulations under study, particularly regarding their potential to differentiate into the osteogenic lineage, and allowed the indentification of ASCs (STRO-1+) as the best subpopulation for bone tissue-engineering applications.

Bone morphogenetic proteins (BMPs) are cytokines from the TGF-beta superfamily, with important roles during embryonic development and in the induction of bone and cartilage tissue differentiation in the adult body. In this contribution, we report the expression of recombinant human BMP-4, BMP-9, BMP-10, BMP-11 (or growth differentiation factor-11, GDF-11) and BMP-14 (GDF-5), using Escherichia coli pET-25b vector. BMPs were overexpressed, purified by affinity his-tag chromatography and shown to induce the expression of early markers of bone differentiation (e.g. smad-1, smad-5, runx2/cbfa1, dlx5, osterix, osteopontin, bone sialoprotein and alkaline phosphatase) in C2C12 cells and in human adipose stem cells. The described approach is a promising method for producing large amounts of different recombinant BMPs that show potential for novel biomedical applications.

Background: The corpus luteum (CL) is a transient endocrine gland and prostaglandin F2-alpha is considered to be the principal luteolysin in pigs. In this species, the in vivo administration of prostaglandin F2-alpha induces apoptosis in large vessels as early as 6 hours after administration. The presence of the prostaglandin F2-alpha receptor (FPr) on the microvascular endothelial cells (pCL-MVECs) of the porcine corpus luteum has not yet been defined. The aim of the study was to assess FPr expression in pCL-MVECs in the early and mid-luteal phases (ELp, ML-p), and during pregnancy (P-p). Moreover, the effectiveness of prostaglandin F2-alpha treatment in inducing pCL-MVEC apoptosis was tested.