Comparison of cell number, viability, phenotypic profile, clonogenic, and proliferative potential of adipose-derived stem cell populations between centrifuged and noncentrifuged fat.

BACKGROUND: Autologous fat grafting is a widely adopted surgical technique in both the reconstructive and aesthetic fields. This study aimed to compare centrifuged lipoaspirates harvested and refined by the Coleman technique with noncentrifuged lipoaspirates in terms of cell number and viability, phenotypic profile, and clonogenic and proliferative potential of adipose-derived stem cell (ADSC) populations. METHODS: For each patient, both a centrifuged sample using the Coleman's technique and a noncentrifuged sample of adipose tissue were collected. Adipose-derived stem cells from both the centrifuged fraction (CF) and the noncentrifuged fraction (NCF) were isolated. The recovered ADSCs were used to set up flow cytometry analysis, colony-forming units-fibroblast (CFU-F) assays, and ADSC cultures. RESULTS: The number of recovered cells was variable among the different donors but significantly higher in the CF donors. Cell viability, determined by the Trypan Blue dye assay, always exceeded 95 %, in both the CF and NCF fractions. Analysis of the putative ADSC subpopulations showed a significant enrichment of the mesenchymal and endothelial progenitors in CF compared with NCF. No differences in the clonogenic efficiency of the ADSC samples were observed when the same number of cells were plated from each fraction. On the contrary, when equal fat volumes were compared, the colony-forming ability of CF was always significantly higher than that of its NCF counterparts. CONCLUSIONS: This is the first study to comprehensively characterize the impact of Coleman's technique on the quality of lipoaspirates, showing that centrifugation is safe and feasible and does not impair cell viability, can augment the content in ADSC and the frequency of CFU-F, and reduces the number of proinflammatory blood cells.

Electrospun collagen mimicking the reconstituted extracellular matrix improves osteoblastic differentiation onto titanium surfaces.

Titanium and its alloys are the current materials to manufacture oral implants because of their excellent mechanical properties and biocompatibility. However the increasing needs of the patients to receive fast and reliable rehabilitation have forced materials scientists to modified the surface of the materials in order to increase the rate of osseointegration and minimize the times for healing. The presence of a reconstituted extracellular matrix (ECM), constituted of proteins and polysaccharides is a key factor for healing and regeneration of the tissues. The nano-fibrous feature of ECM improves cells proliferation and addresses their phenotype. Electrospinning technique is an efficient processing method to manufacture micro- and nano-sized fibrous structures mimicking the ECM. In this work we describe a method to obtain collagen coating made of nano-fibers onto titanium for oral implant manufacturing, using electrospinning. The obtained collagen coatings showed morphology, structure and chemical composition similar to that of ECM. Moreover the stem cells cultured onto titanium samples coated with electrospun collagen showed faster osteoblastic differentiation and more efficient deposition of mineralized matrix in comparison with those onto uncoated substrates. This effect was amplified using osteogenetic media.

Antiendothelial cell antibodies induce apoptosis of bone marrow endothelial progenitors in systemic sclerosis.

OBJECTIVE: Patients with systemic sclerosis (SSc) have significantly fewer and functionally impaired endothelial progenitor cells (EPC) in peripheral blood and bone marrow; further, endothelial apoptosis seems to play a primary role in the pathogenesis of vascular damage. We investigated whether the failure of bone marrow EPC is related to their apoptotic phenotype and analyzed the possible mechanisms inducing apoptosis. METHODS: The presence of apoptotic cells was investigated in bone marrow aspirates taken from patients with SSc; microvessel density (MVD) and the immunohistochemical expression of vascular endothelial growth factor (VEGF) were also measured in bone marrow biopsies. A correlation between EPC apoptosis and the presence of antiendothelial cell antibodies (AECA) was also investigated. RESULTS: We confirmed the presence of bone marrow EPC dysfunction in SSc, while hematopoiesis was not impaired. Bone marrow studies showed a high percentage of apoptotic progenitors, no signs of fibrosis or an altered MVD, and an increased VEGF index. The patients' bone marrow plasma showed significant titers of AECA, and their presence correlated with that of apoptotic progenitors. These findings were further confirmed by an in vitro assay in which the apoptosis of normal progenitors was induced by the addition of AECA+ purified IgG. CONCLUSION: Our results showed that apoptosis in patients with SSc involves the source compartment of endothelial progenitors and correlates with AECA activity. These findings support the hypothesis that AECA may play a pathogenetic role by affecting the bone marrow EPC machinery that should repair the peripheral vascular lesions.

Anti-L-NGFR and -CD34 monoclonal antibodies identify multipotent mesenchymal stem cells in human adipose tissue.

Stem cells hold great promise in tissue engineering for repairing tissues damaged by disease or injury. Mesenchymal stem cells (MSCs) are multipotent cells able to proliferate and differentiate into multiple mesodermal tissues such as bone, cartilage, muscle, tendon, and fat. We have previously reported that the low-affinity nerve growth factor receptor (L-NGFR or CD271) defines a subset of cells with high proliferative, clonogenic, and multipotential differentiation ability in adult bone marrow (BM). It has been recently shown that adipose tissue is an alternative source of adult multipotent stem cells and human adipose-derived stem cells, selected by plastic adherence (PA hASCs), have been extensively characterized for their functional potentials in vitro. In this study, immunoselected L-NGFR(+) and CD34(+) subpopulations have been analyzed and compared with the PA hASCs. Phenotypic profile of freshly purified subpopulations showed an enrichment in the expression of some stem cell markers; indeed, a great percentage of L-NGFR(+) cells co-expressed CD34 and CD117 antigens, whereas the endothelial-committed progenitor markers KDR and P1H12 were mainly expressed on CD34(+) cells. Differently from PA hASCs, the immunoseparated fractions showed high increments in cell proliferation, and the fibroblast colony-forming activity (CFU-F) was maintained throughout the time of culture. Furthermore, the immunoselected populations showed a greater differentiative potential toward adipocytes, osteoblasts, and chondrocyte-like cells, compared to PA hASCs. Our data suggest that both CD34(+) and L-NGFR(+) hASCs can be considered alternative candidates for tissue engineering and regenerative medicine applications.

Bone marrow endothelial progenitors are defective in systemic sclerosis.

OBJECTIVE: Vascular abnormalities represent the main component of the pathobiology of systemic sclerosis (SSc), progressing from structural derangements of the microcirculation with abortive neoangiogenesis to final vessel loss. Since circulating endothelial progenitor cells (EPCs) are important in the vascular repair process, we undertook this study to examine their numbers in the peripheral blood (PB) of SSc patients and to evaluate whether their status is related to impaired quantitative and/or qualitative aspects of the bone marrow (BM) microenvironment. METHODS: Circulating EPCs from 62 SSc patients were evaluated by flow cytometry and characterized as CD45 negative and CD133 positive. BM EPCs, identified as CD133 positive, were isolated from 14 SSc patients and grown to induce endothelial differentiation. In addition, progenitor numbers and functional properties of hematopoietic and stromal compartments were analyzed by various assays. RESULTS: We found that EPCs were detectable in the PB of patients with SSc, and their number was significantly increased in patients with early-stage disease but not in those with late-stage disease. All of the examined BM samples contained reduced numbers of EPCs and stromal cells, both of which were functionally impaired. Both endothelial and stromal progenitors expressed vascular endothelial growth factor receptor, indicating that BM is strongly induced to differentiate into the endothelial lineage; furthermore, only BM EPCs from patients with early disease led to endothelial differentiation in vitro. CONCLUSION: This study provides the first demonstration that in SSc, there is a complex impairment in the BM microenvironment involving both the endothelial and mesenchymal stem cell compartments and that this impairment might play a role in defective vasculogenesis in scleroderma.

Skeletal muscle differentiation potential of human adult bone marrow cells.

Murine bone marrow (BM) cells have been shown to undergo myogenic differentiation and participate in muscle repair in different muscle regeneration models. In the present paper, we report on a subset of cells (CD45+/desmin+) with myogenic potential being present at very low frequencies in human adult BM. By a simple culture method, we were able to obtain in vitro multinucleated myotubes in up to 20% of the cultures. Myotubes were generated using both BM flushed from rib fragments obtained during thoracotomy and BM derived from iliac crest aspirates. Cells of the different adherent and non-adherent fractions expressed numerous muscle specific markers by immunocytochemistry, real-time RT-PCR, flow cytometry, and Western blot analyses. Moreover, direct injection of whole BM into the right tibialis anterior muscle of immunodeficient mice (NOD/RAG) that had previously been treated with cardiotoxin to induce muscle degeneration, showed a variable but significant level of human cell engraftment (from 0.06 to 0.26% Dys+/FISH+ fibers). These data suggest that cells with skeletal muscle differentiation potential are present in adult human BM can differentiate in vitro and give rise to myogenic cells in vivo in immunodeficient mice after muscle damage. Further improvements might allow new approaches to cell-mediated therapies for muscular diseases.

Isolation of bone marrow mesenchymal stem cells by anti-nerve growth factor receptor antibodies.

OBJECTIVE: Mesenchymal stem cells (MSCs) are a population of multipotent cells that can proliferate and differentiate into multiple mesodermal tissues. We previously reported that monoclonal antibodies to the low-affinity nerve growth factor receptor (alpha-LNGFR) stain bone marrow (BM) mesenchymal cells. We now show that LNGFR antibodies label primitive MSCs with high specificity and purity in adult BM, and compare these cells to those isolated by plastic adherence (PA) and CD45(-)anti-glycophorin A(-) selection. MATERIALS AND METHODS: Low-density mononuclear cells (LD-MNCs) from normal BM were separated by PA or immunomagnetic selection for NGFR(+) or CD45(-)alpha-glycophorin A(-) cells. The three fractions were grown in Iscove's modified Dulbecco medium + 20% fetal bovine serum +/- basic fibroblast growth factor (bFGF) in order to assess their proliferative capacity and evaluate their phenotype during culture. The clonogenic potential of the MSCs was assessed using a colony-forming unit fibroblast (CFU-F) assay, whereas multipotential differentiation was determined after culture in adipocytic and osteoblastic conditioned media. RESULTS: The NGFR(+) mesenchymal cells grown without growth factors showed persistent NGFR expression (rapidly down-regulated after the addition of bFGF) and persistent CFU-F activity. The NGFR(+) fractions were rich in clonogenic precursors: CFU-F median frequency was 1584/1 x 10(6) cells (range 325-13,793) in the NGFR(+) cells and 35/1 x 10(6) cells (range 27-112) in the LD-MNCs. The NGFR(-) fraction never showed any residual CFU-F activity. Compared with the other two fractions, the NGFR(+) cells (+/- bFGF) showed a 1 to 3 log greater expansion in the number of fibroblastic cells and a greater capacity to give rise to adipocyte colonies and induce osteoblastic differentiation, and they had similar effects in supporting the growth of hematopoietic precursors. CONCLUSION: The data suggest that positive selection using low-affinity NGFR antibodies makes it possible to obtain homogeneous multipotent MSCs.