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The Influence of Negative Pressure and of the Harvesting Site on the Characteristics of Human Adipose Tissue-Derived Stromal Cells from Lipoaspirates.

ABSTRACT: Background:Adipose tissue-derived stromal cells (ADSCs) have great potential for cell-based therapies, including tissue engineering. However, various factors can influence the characteristics of isolated ADSCs. Methods:We studied the influence of the harvesting site, i.e., inner thigh (n = 3), outer thigh (n = 3), outer thigh (n = 3), outer thigh (. Results:We revealed higher initial cell yields from the outer thigh region than from the abdomen region. Negative pressure did not influence the cell yields from the outer thigh region, whereas the yields from the abdomen region were higher under high negative pressure than under low negative pressure. In the subsequent passage, in general, no significant relationship was identified between the different negative pressure and ADSC characteristics. No significant difference was observed in the characteristics of thigh ADSCs and abdomen ADSCs. Only on day 1, the diameter was significantly bigger in outer thigh ADSCs than in abdomen ADSCs. Moreover, we noted a tendency of thigh ADSCs (i.e., inner thigh+outer thigh) to reach a higher cell number on day 7. Discussion. The harvesting site and negative pressure can potentially influence initial cell yields from lipoaspirates. However, for subsequent in vitro culturing and for use in tissue engineering, it seems that the harvesting site and the level of negative pressure do not have a crucial or limiting effect on basic ADSC characteristics.in vitro culturing and for use in tissue engineering, it seems that the harvesting site and the level of negative pressure do not have a crucial or limiting effect on basic ADSC characteristics.

SUBMITTER: Travnickova M

PROVIDER: S-EPMC7035580 | biostudies-literature | 2020

REPOSITORIES: biostudies-literature

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The Influence of Negative Pressure and of the Harvesting Site on the Characteristics of Human Adipose Tissue-Derived Stromal Cells from Lipoaspirates.

Travnickova Martina M Pajorova Julia J Zarubova Jana J Krocilova Nikola N Molitor Martin M Bacakova Lucie L

Stem cells international 20200210

<h4>Background</h4>Adipose tissue-derived stromal cells (ADSCs) have great potential for cell-based therapies, including tissue engineering. However, various factors can influence the characteristics of isolated ADSCs.<h4>Methods</h4>We studied the influence of the harvesting site, i.e., inner thigh (<i>n</i> = 3), outer thigh (<i>n</i> = 3), outer thigh (<i>n</i> = 3), outer thigh (.<h4>Results</h4>We revealed higher initial cell yields from the outer thigh region than from the abdomen region. ...[more]

PMID: 32104182

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Project description:The stromal vascular fraction (SVF) of adipose tissue contains an abundant population of multipotent adipose-tissue-derived stem cells (ASCs) that possess the capacity to differentiate into cells of the mesodermal lineage in vitro. For cell-based therapies, an advantageous approach would be to harvest these SVF cells and give them back to the patient within a single surgical procedure, thereby avoiding lengthy and costly in vitro culturing steps. However, this requires SVF-isolates to contain sufficient ASCs capable of differentiating into the desired cell lineage. We have investigated whether the yield and function of ASCs are affected by the anatomical sites most frequently used for harvesting adipose tissue: the abdomen and hip/thigh region. The frequency of ASCs in the SVF of adipose tissue from the abdomen and hip/thigh region was determined in limiting dilution and colony-forming unit (CFU) assays. The capacity of these ASCs to differentiate into the chondrogenic and osteogenic pathways was investigated by quantitative real-time polymerase chain reaction and (immuno)histochemistry. A significant difference (P = 0.0009) was seen in ASC frequency but not in the absolute number of nucleated cells between adipose tissue harvested from the abdomen (5.1 +/- 1.1%, mean +/- SEM) and hip/thigh region (1.2 +/- 0.7%). However, within the CFUs derived from both tissues, the frequency of CFUs having osteogenic differentiation potential was the same. When cultured, homogeneous cell populations were obtained with similar growth kinetics and phenotype. No differences were detected in differentiation capacity between ASCs from both tissue-harvesting sites. We conclude that the yield of ASCs, but not the total amount of nucleated cells per volume or the ASC proliferation and differentiation capacities, are dependent on the tissue-harvesting site. The abdomen seems to be preferable to the hip/thigh region for harvesting adipose tissue, in particular when considering SVF cells for stem-cell-based therapies in one-step surgical procedures for skeletal tissue engineering.

Project description:Recently, substantial attention has been paid toward adipose-derived mesenchymal stem cells (AdMSCs) as a potential therapy in tissue engineering and regenerative medicine applications. Rat AdMSCs (r-AdMSCs) are frequently utilized. However, the influence of the adipose depot site on the multilineage differentiation potential of the r-AdMSCs is still ambiguous. Hence, the main objective of this study was to explore the influence of the adipose tissue harvesting location on the ability of r-AdMSCs to express the stem-cell-related markers and pluripotency genes, as well as their differentiation capacity, for the first time. Herein, we have isolated r-AdMSCs from the inguinal, epididymal, peri-renal, and back subcutaneous fats. Cells were compared in terms of their phenotype, immunophenotype, and expression of pluripotency genes using RT-PCR. Additionally, we investigated their potential for multilineage (adipogenic, osteogenic, and chondrogenic) induction using special stains confirmed by the expression of the related genes using RT-qPCR. All cells could positively express stem cell marker CD 90 and CD 105 with no significant in-between differences. However, they did not express the hematopoietic markers as CD 34 and CD 45. All cells could be induced successfully. However, epididymal and inguinal cells presented the highest capacity for adipogenic and osteogenic differentiation (21.36-fold and 11.63-fold for OPN, 29.69-fold and 26.68-fold for BMP2, and 37.67-fold and 22.35-fold for BSP, respectively, in epididymal and inguinal cells (p < 0.0001)). On the contrary, the subcutaneous cells exhibited a superior potential for chondrogenesis over the other sites (8.9-fold for CHM1 and 5.93-fold for ACAN, (p < 0.0001)). In conclusion, the adipose tissue harvesting site could influence the differentiation capacity of the isolated AdMSCs. To enhance the results of their employment in various regenerative cell-based therapies, it is thus vital to take the collection site selection into consideration.

Project description:The in vivo progenitor of culture-expanded mesenchymal-like adipose-derived stem cells (ADSC) remains elusive, owing in part to the complex organization of stromal cells surrounding the small vessels, and the rapidity with which adipose stromal vascular cells adopt a mesenchymal phenotype in vitro. Immunohistostaining of intact adipose tissue was used to identify three markers (CD31, CD34, and CD146), which together unambiguously discriminate histologically distinct inner and outer rings of vessel-associated stromal cells, as well as capillary and small vessel endothelial cells. These markers were used in multiparameter flow cytometry in conjunction with stem/progenitor markers (CD90 and CD117) to further characterize stromal vascular fraction (SVF) subpopulations. Two mesenchymal and two endothelial populations were isolated by high speed flow cytometric sorting, expanded in short term culture, and tested for adipogenesis. The inner layer of stromal cells in contact with small vessel endothelium (pericytes) was CD146+/alpha-SMA+/CD90+/-/CD34-/CD31-; the outer adventitial stromal ring (designated supra adventitial-adipose stromal cells, SA-ASC) was CD146-/alpha-SMA-/CD90+/CD34+/CD31-. Capillary endothelial cells were CD31+/CD34+/CD90+ (endothelial progenitor), whereas small vessel endothelium was CD31+/CD34-/CD90- (endothelial mature). Flow cytometry confirmed these expression patterns and revealed a CD146+/CD90+/CD34+/CD31- pericyte subset that may be transitional between pericytes and SA-ASC. Pericytes had the most potent adipogenic potential, followed by the more numerous SA-ASC. Endothelial populations had significantly reduced adipogenic potential compared with unsorted expanded SVF cells. In adipose tissue, perivascular stromal cells are organized in two discrete layers, the innermost consisting of CD146+/CD34- pericytes, and the outermost of CD146-/CD34+ SA-ASC, both of which have adipogenic potential in culture. A CD146+/CD34+ subset detected by flow cytometry at low frequency suggests a population transitional between pericytes and SA-ASC.

Project description:BackgroundFat grafting is used to restore breast defects after surgical resection of breast tumors. Supplementing fat grafts with adipose tissue-derived stromal/stem cells (ASCs) is proposed to improve the regenerative/restorative ability of the graft and retention. However, long term safety for ASC grafting in proximity of residual breast cancer cells is unknown. The objective of this study was to determine the impact of human ASCs derived from abdominal lipoaspirates of three donors, on a human breast cancer model that exhibits early metastasis.Methodology/principal findingsHuman MDA-MB-231 breast cancer cells represents "triple negative" breast cancer that exhibits early micrometastasis to multiple mouse organs [1]. Human ASCs were derived from abdominal adipose tissue from three healthy female donors. Indirect co-culture of MDA-MB-231 cells with ASCs, as well as direct co-culture demonstrated that ASCs had no effect on MDA-MB-231 growth. Indirect co-culture, and ASC conditioned medium (CM) stimulated migration of MDA-MB-231 cells. ASC/RFP cells from two donors co-injected with MDA-MB-231/GFP cells exhibited a donor effect for stimulation of primary tumor xenografts. Both ASC donors stimulated metastasis. ASC/RFP cells were viable, and integrated with MDA-MB-231/GFP cells in the tumor. Tumors from the co-injection group of one ASC donor exhibited elevated vimentin, matrix metalloproteinase-9 (MMP-9), IL-8, VEGF and microvessel density. The co-injection group exhibited visible metastases to the lung/liver and enlarged spleen not evident in mice injected with MDA-MB-231/GFP alone. Quantitation of the total area of GFP fluorescence and human chromosome 17 DNA in mouse organs, H&E stained paraffin sections and fluorescent microscopy confirmed multi-focal metastases to lung/liver/spleen in the co-injection group without evidence of ASC/RFP cells.ConclusionsHuman ASCs derived from abdominal lipoaspirates of two donors stimulated metastasis of MDA-MB-231 breast tumor xenografts to multiple mouse organs. MDA-MB-231 tumors co-injected with ASCs from one donor exhibited partial EMT, expression of MMP-9, and increased angiogenesis.

Project description:Total hip arthroplasty (THA) represents one of the commonest surgical procedures in the orthopedic field. Osteointegration of the implant with native bone is essential for an optimal result; thus, the quality of the patient's bone surrounding the implant (i.e., the bone stock) is crucial. However, in some cases, the bone stock is insufficient and needs to be improved with autologous grafts rich in multipotent cells (i.e., from the iliac crest, from the head of the femur, or from the subchondral bone harvested from the acetabulum) or allogenic frozen bone. It is not known if the harvesting site may influence the osteogenic potential of these cells. Thus, our aim was to characterize and compare multipotent cells collected from the bone marrow, acetabular subchondral bone, and trabecular bone on the femoral head with a focus on osteogenic differentiation. The cells from three sources had a fibroblast-like phenotype and expressed surface antigens CD73, CD90, and CD105 and are negative to CD11b, CD34, and CD45. Although all these cells could be induced to differentiate into osteoblasts, chondrocytes, and adipocytes, they displayed different differentiation potentials. In osteogenic differentiation condition, the cells from the acetabulum had the lowest accumulation of calcium deposit while the cells originated from the bone marrow and femur created a considerably increased amount of the deposit. These findings were confirmed by quantitative polymerase chain reaction (qPCR). In chondrogenic and adipogenic conditions, bone marrow cells possessed a predominant differential capacity compared with the others, illustrated by high collagen type II expression together with a cartilage-like lacuna structure and the presence of fat-specific markers, respectively. To our knowledge, this is the first study comparing and demonstrating that the progenitor cells obtained from diverse surgical sites in hip replacement procedure share common characteristics of MSC but differ about plasticity and may provide rational for clinical application in cell therapy and bone grafting. The project number L1033 is registered with ClinicalTrials.gov NCT03369457.

Dataset Information

The Influence of Negative Pressure and of the Harvesting Site on the Characteristics of Human Adipose Tissue-Derived Stromal Cells from Lipoaspirates.

Publications

The Influence of Negative Pressure and of the Harvesting Site on the Characteristics of Human Adipose Tissue-Derived Stromal Cells from Lipoaspirates.

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