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Comparative proteomic analysis of osteogenic differentiated human adipose tissue and bone marrow-derived stromal cells.

ABSTRACT: Mesenchymal stromal cells are promising candidates for regenerative applications upon treatment of bone defects. Bone marrow-derived stromal cells (BMSCs) are limited by yield and donor morbidity but show superior osteogenic capacity compared to adipose-derived stromal cells (ASCs), which are highly abundant and easy to harvest. The underlying reasons for this difference on a proteomic level have not been studied yet. Human ASCs and BMSCs were characterized by FACS analysis and tri-lineage differentiation, followed by an intraindividual comparative proteomic analysis upon osteogenic differentiation. Results of the proteomic analysis were followed by functional pathway analysis. 29 patients were included with a total of 58 specimen analysed. In these, out of 5148 identified proteins 2095 could be quantified in >80% of samples of both cell types, 427 in >80% of ASCs only and 102 in >80% of BMSCs only. 281 proteins were differentially regulated with a fold change of >1.5 of which 204 were higher abundant in BMSCs and 77 in ASCs. Integrin cell surface interactions were the most overrepresented pathway with 5 integrins being among the proteins with highest fold change. Integrin 11a, a known key protein for osteogenesis, could be identified as strongly up-regulated in BMSC confirmed by Western blotting. The integrin expression profile is one of the key distinctive features of osteogenic differentiated BMSCs and ASCs. Thus, they represent a promising target for modifications of ASCs aiming to improve their osteogenic capacity and approximate them to that of BMSCs.

SUBMITTER: Dadras M

PROVIDER: S-EPMC7579700 | biostudies-literature | 2020 Oct

REPOSITORIES: biostudies-literature

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Comparative proteomic analysis of osteogenic differentiated human adipose tissue and bone marrow-derived stromal cells.

Dadras Mehran M May Caroline C Wagner Johannes Maximilian JM Wallner Christoph C Becerikli Mustafa M Dittfeld Stephanie S Serschnitzki Bettina B Schilde Lukas L Guntermann Annika A Sengstock Christina C Köller Manfred M Seybold Dominik D Geßmann Jan J Schildhauer Thomas Armin TA Lehnhardt Marcus M Marcus Katrin K Behr Björn B

Journal of cellular and molecular medicine 20200903 20

Mesenchymal stromal cells are promising candidates for regenerative applications upon treatment of bone defects. Bone marrow-derived stromal cells (BMSCs) are limited by yield and donor morbidity but show superior osteogenic capacity compared to adipose-derived stromal cells (ASCs), which are highly abundant and easy to harvest. The underlying reasons for this difference on a proteomic level have not been studied yet. Human ASCs and BMSCs were characterized by FACS analysis and tri-lineage diffe ...[more]

PMID: 32885592

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Project description:The treatment of bone defects caused by infection, trauma or neoplasms remains a clinical challenge. Autologous bone transplantation is limited by availability, donor site morbidity and surgical risk factors. This has given rise to stromal/stem-cell based therapy. Bone marrow derived stromal cells (BMSCs) have been studied to a large extent and show high regenerative potential but their use is limited by availability, donor site morbidity and the relatively low cell yield as they represent only <0.1% of cell harvested from bone marrow aspirate. At the same time, they are the closest mesenchymal stromal cells for bone tissue engineering given their tissue origin and, unlike other mesenchymal stromal cells, can support the formation of hematopoietic marrow. Adipose tissue derived stromal cells (ASCs) as part of the stromal vascular fraction of adipose tissue can as well undergo osteogenic differentiation but can be additionally isolated in a sufficient quantity from lipoaspirate after liposuction of abundant subcutaneous fat tissue. Here, it has been shown that there are no major differences in regard to proliferation or differentiation capacity of ASCs derived from subcutaneous fat of different anatomical regions. It has been shown that BMSCs are more prone to senescence during expansion and passage than ASCs and that ageing impacts proliferative capabilities of BMSCs more than that of ASCs while it has also been reported that osteogenic differentiation capacity is least impacted by age. Multiple studies have compared the characteristics of these two mesenchymal stromal cells in regard to bone tissue engineering in vitro. Most studies point to inferior extracellular matrix mineralization and lower expression of key osteogenic transcription markers like Runx2 in osteogenic differentiated ASCs compared to BMSCs. On the other hand, a study by Rath et al. found contrary results using particular culturing conditions like 3D bioglass scaffolds. An intraindividual comparison of human MSCs of three donors cultured on decellularized porcine bone confirmed superior osteogenic capacity of BMSCs compared to ASCs. In contrast to BMSCs, ASCs were not able to induce heterogenic ossification in a mouse model. In a sheep tibia defect model application of BMSCs resulted in a significantly higher amount of newly formed bone tissue. Importantly, Osteogenic differentiated ASCs do not support the formation of a hematopoietic marrow. Proteomics enables large-scale analysis of proteins present in a cell type and can be used to identify differentially regulated key proteins in a comparative approach. A comparative proteomic analysis of BMSCs and ASCs by Roche et al. in 2009 identified 556 proteins with 78% of these not being differentially regulated between these two cell populations, regarded as high similarity. Another comparative proteomic study of 2016 by Jeon et al. found 90 differentially regulated proteins out of 3000 total identified proteins. Both studies do not specify a number of different tissue donors and in part using cell lines. Looking for differences upon osteogenic differentiation, transcriptomic comparison of osteogenic differentiated porcine ASCs and BMSCs has been performed, resulting in 21 differentially expressed genes after 21 days of osteogenic culture conditions. Still, it remains unanswered, which are the key distinctive features of osteogenic differentiated ASCs and BMSCs at protein level that might help address the abovementioned weaknesses of ASCs in bone tissue engineering/regeneration for translational research. To overcome this need, an intraindividual comparative DIA based proteomic analysis of osteogenic differentiated human BMSC and ASCs was performed in this study.

Project description:BackgroundBone marrow (BM)- and adipose tissue (AT)-derived mesenchymal stromal cells (MSCs) have shown potential as cell-based therapies for cartilage and bone injuries and are used increasingly in human and veterinary practice to facilitate the treatment of orthopedic conditions. However, human and rodent studies have documented a sharp decline in chondrogenic and osteogenic differentiation potential with increasing donor age, which may be problematic for the important demographic of older orthopedic patients. The aim of this study was to identify the effect of donor age on the chondrogenic and osteogenic differentiation performance of equine BM- and AT-MSCs in vitro. BM- and AT-MSCs and dermal fibroblasts (biological negative control) were harvested from horses in five different age groups (n = 4, N = 60); newborn (0 days), yearling (15-17 months), adult (5-8 years), middle-aged (12-18 years), and geriatric (≥ 22 years). Chondrogenic differentiation performance was assessed quantitatively by measuring pellet size, matrix proteoglycan levels, and gene expression of articular cartilage biomarkers. Osteogenic differentiation performance was assessed quantitatively by measuring alkaline phosphatase activity, calcium deposition, and gene expression of bone biomarkers.ResultsChondrogenic and osteogenic differentiation performance of equine BM- and AT-MSCs declined with increasing donor age. BM-MSCs had a higher chondrogenic differentiation performance. AT-MSCs showed minimal chondrogenic differentiation performance in all age groups. For osteogenesis, alkaline phosphatase activity was also higher in BM-MSCs, but BM-MSCs calcium deposition was affected by donor age earlier than AT-MSCs. Chondrogenic and osteogenic differentiation performance of BM-MSCs exhibited a decline as early as between the newborn and yearling samples. Steady state levels of mRNA encoding growth factors, chondrogenic, and osteogenic biomarkers were lower with increasing donor age in both MSC types.ConclusionsThe data showed that chondrogenic and osteogenic differentiation performance of equine BM-MSCs declined already in yearlings, and that AT-MSCs showed minimal chondrogenic potential, but were affected later by donor age with regards to osteogenesis (calcium deposition). The results highlight the importance of donor age considerations and MSC selection for cell-based treatment of orthopedic injuries and will help inform clinicians on when to implement or potentially cryopreserve cells. Moreover, the study provides molecular targets affected by donor age.

Project description:BackgroundMesenchymal stromal cells (MSC) hold promise for both cell replacement and immune modulation strategies owing to their progenitor and non-progenitor functions, respectively. Characterization of MSC from different sources is an important and necessary step before clinical use of these cells is widely adopted. Little is known about the biology and function of canine MSC compared to their mouse or human counterparts. This knowledge-gap impedes development of canine evidence-based MSC technologies.Hypothesis and objectivesWe hypothesized that canine adipose tissue (AT) and bone marrow (BM) MSC (derived from the same dogs) will have similar differentiation and immune modulatory profiles. Our objectives were to evaluate progenitor and non-progenitor functions as well as other characteristics of AT- and BM-MSC including 1) proliferation rate, 2) cell surface marker expression, 3) DNA methylation levels, 4) potential for trilineage differentiation towards osteogenic, adipogenic, and chondrogenic cell fates, and 5) immunomodulatory potency in vitro.Results1) AT-MSC proliferated at more than double the rate of BM-MSC (population doubling times in days) for passage (P) 2, AT: 1.69, BM: 3.81; P3, AT: 1.80, BM: 4.06; P4, AT: 2.37, BM: 5.34; P5, AT: 3.20, BM: 7.21). 2) Canine MSC, regardless of source, strongly expressed cell surface markers MHC I, CD29, CD44, and CD90, and were negative for MHC II and CD45. They also showed moderate expression of CD8 and CD73 and mild expression of CD14. Minor differences were found in expression of CD4 and CD34. 3) Global DNA methylation levels were significantly lower in BM-MSC compared to AT-MSC. 4) Little difference was found between AT- and BM-MSC in their potential for adipogenesis and osteogenesis. Chondrogenesis was poor to absent for both sources in spite of adding varying levels of bone-morphogenic protein to our standard transforming growth factor (TGF-β3)-based induction medium. 5) Immunomodulatory capacity was equal regardless of cell source when tested in mitogen-stimulated lymphocyte reactions. Priming of MSC with pro-inflammatory factors interferon-gamma and/or tumour necrosis factor did not increase the lymphocyte suppressive properties of the MSC compared to untreated MSC.Conclusions/significanceNo significant differences were found between AT- and BM-MSC with regard to their immunophenotype, progenitor, and non-progenitor functions. Both MSC populations showed strong adipogenic and osteogenic potential and poor chondrogenic potential. Both significantly suppressed stimulated peripheral blood mononuclear cells. The most significant differences found were the higher isolation success and proliferation rate of AT-MSC, which could be realized as notable benefits of their use over BM-MSC.

Project description:Adipose tissue-derived stromal cells (ASCs) play an important role in various therapeutic approaches to bone regeneration. However, such applications become challenging when the obtained cells show a functional disorder, e.g., an impaired osteogenic differentiation potential (ODP). In addition to ASCs, human adipose tissue is also a source for another cell type with therapeutic potential, the dedifferentiated fat cells (DFATs), which can be obtained from mature adipocytes. Here, we for the first time compared the ODPs of each donors ASC and DFAT obtained from the same adipose tissue sample as well as the role of oxidative stress or antioxidative catalase on their osteogenic outcome. Osteogenic potential of ASC and DFAT from nine human donors were compared in vitro. Flow cytometry, staining for calcium accumulation with alizarin red, alkaline phosphatase assay and Western blots were used over an osteogenic induction period of up to 14 days. H2O2 was used to induce oxidative stress and catalase was used as an antioxidative measure. We have found that ASC and DFAT cultures' ODPs are nearly identical. If ASCs from an adipose tissue sample showed good or bad ODP, so did the corresponding DFAT cultures. The inter-individual variability of the donor ODPs was immense with a maximum factor of about 20 and correlated neither with the age nor the sex of the donors of the adipose tissue. Oxidative stress in the form of exogenously added H2O2 led to a significant ODP decrease in both cell types, with this ODP decrease being significantly lower in DFAT cultures than in the corresponding ASC cultures. Regardless of the individual cell culture-specific ODP, however, exogenously applied catalase led to an approx. 2.5-fold increase in osteogenesis in the ASC and DFAT cultures. Catalase appears to be a potent pro-osteogenic factor, at least in vitro. A new finding that points to innovative strategies and therapeutic approaches in bone regeneration. Furthermore, our results show that DFATs behave similarly to ASCs of the same adipose tissue sample with respect to ODPs and could therefore be a very attractive and readily available source of multipotent stem cells in bone regenerative therapies.

Project description:IntroductionOsteoporosis is a disease characterized by low bone mineral density (BMD) and increased risk of fractures. Studies have demonstrated the use of phytoestrogens, or plant-derived estrogens, such as genistein and daidzein, to effectively increase osteogenic activity of bone marrow-derived mesenchymal stem cells (BMSCs). Herein, the effects of daidzein analogs on the osteogenic differentiation efficiency of human BMSC and adipose-derived stromal/stem cells (ASC) were explored.MethodsBMSCs and ASCs underwent osteogenic differentiation in the presence of vehicle, 17?-estradiol (E2), phytoestrogens, or daidzein analogs. Cells were stained for alkaline phosphatase (ALP) enzymatic activity, calcium deposition by alizarin red s, and phosphate mineralization by silver nitrate. Gene expression analysis was conducted on cells treated with daidzein analogs.ResultsCells treated with E2, daidzein, or genistein increased calcium deposition by 1.6-, 1.5-, and 1.4-fold, respectively, relative to vehicle-treated BMSCs and 1.6-, 1.7-, and 1.4-fold relative to vehicle-treated ASCs, respectively. BMSCs treated with daidzein analog 2c, 2g, and 2l demonstrated a 1.6-, 1.6-, and 1.9-fold increase in calcium deposition relative to vehicle-treated BMSCs, respectively, while ASCs treated with daidzein analog 2c, 2g, or 2l demonstrated a 1.7-, 2.0-, and 2.2-fold increase in calcium deposition relative to vehicle-treated ASCs, respectively. Additional analysis with BMSCs and ASCs was conducted in the more efficient compounds: 2g and 2l. ALP activity and phosphate mineralization was increased in 2g- and 2l-treated cells. The analysis of lineage specific gene expression demonstrated increased expression of key osteogenic genes (RUNX2, c-FOS, SPARC, DLX5, SPP1, COL1A1, IGF1, SOST, and DMP1) and earlier induction of these lineage specific genes, following treatment with 2g or 2l, relative to vehicle-treated cells. Estrogen receptor (ER) inhibitor studies demonstrated that ER antagonist fulvestrant inhibited the osteogenic differentiation of 2g in BMSCs and ASCs, while fulvestrant only attenuated the effects of 2l, suggesting that 2l acts by both ER dependent and independent pathways.ConclusionsThese studies provide support for exploring the therapeutic efficacy of daidzein derivatives for the treatment of osteoporosis. Furthermore, the patterns of gene induction differed following treatment with each daidzein analog, suggesting that these daidzein analogs activate distinct ER and non-ER pathways to induce differentiation in BMSCs and ASCs.

Dataset Information

Comparative proteomic analysis of osteogenic differentiated human adipose tissue and bone marrow-derived stromal cells.

Publications

Comparative proteomic analysis of osteogenic differentiated human adipose tissue and bone marrow-derived stromal cells.

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