RANKL from bone marrow adipose lineage cells promotes osteoclast formation and bone loss

Receptor activator of NF‐κB ligand (RANKL) is essential for osteoclast formation and bone remodeling. Nevertheless, the cellular source of RANKL for osteoclastogenesis has not been fully uncovered. Different from peripheral adipose tissue, bone marrow (BM) adipose lineage cells originate from bone marrow mesenchymal stromal cells (BMSCs). Here, we demonstrate that adiponectin promoter‐driven Cre expression (Adipoq^Cre ) can target bone marrow adipose lineage cells. We cross the Adipoq^Cre mice with rankl^fl/fl mice to conditionally delete RANKL from BM adipose lineage cells. Conditional deletion of RANKL increases cancellous bone mass of long bones in mice by reducing the formation of trabecular osteoclasts and inhibiting bone resorption but does not affect cortical bone thickness or resorption of calcified cartilage. Adipoq^Cre; rankl^fl/fl mice exhibit resistance to estrogen deficiency and rosiglitazone (ROS)‐induced trabecular bone loss but show bone loss induced by unloading. BM adipose lineage cells therefore represent an essential source of RANKL for the formation of trabecula osteoclasts and resorption of cancellous bone during remodeling under physiological and pathological conditions. Targeting bone marrow adiposity is a promising way of preventing pathological bone loss.     

Comparative proteomic analysis of human mesenchymal and embryonic stem cells: Towards the definition of a mesenchymal stem cell proteomic signature

Mesenchymal stem cells (MSC) are adult multipotential progenitors which have a high potential in regenerative medicine. They can be isolated from different tissues throughout the body and their homogeneity in terms of phenotype and differentiation capacities is a real concern. To address this issue, we conducted a 2‐DE gel analysis of mesenchymal stem cells isolated from bone marrow (BM), adipose tissue, synovial membrane and umbilical vein wall. We confirmed that BM and adipose tissue derived cells were very similar, which argue for their interchangeable use for cell therapy. We also compared human mesenchymal to embryonic stem cells and showed that umbilical vein wall stem cells, a neo‐natal cell type, were closer to BM cells than to embryonic stem cells. Based on these proteomic data, we could propose a panel of proteins which were the basis for the definition of a mesenchymal stem cell proteomic signature.     

Evidence of specific characteristics and osteogenic potentiality in bone cells from tibia

Human bone cells used for in vitro studies are mainly derived from bone marrow (BM) or trabecular bone (TB). There are no specific markers or procedures for isolation and growth of these cells. To validate the potentiality of these cells, we isolated human mesenchymal stromal cells (MSCs) and osteoblasts (OBs) from the tibial plateau of the same subject, grown in two different media (α-MEM and DMEM/F12) and analyzed for cell growth, proliferation, phenotype and osteogenic potential. We found that OBs grew well in both media tested, but MSCs were able to grow only in α-MEM medium. OBs in DMEM/F12 showed reduced proliferation capability and expressed a low level of alkaline phosphatase (AP), RUNX-2, osteocalcin (OC), bone sialoprotein (BSP), collagen type I (Col.I) compared with OBs in α-MEM but high level of collagen type XV (Col.XV). Compared with MSCs in α-MEM, OBs have an increased ability to proliferate and express more OC and BSP at molecular level but less AP, RUNX-2 and Col.I than MSCs. Time-course experiments to analyze the osteogenic potential of these cells showed that OBs were more efficient than MSCs. However, these cells obtained from tibial plateau showed a different trend of AP, OC and Col.I osteogenic markers compared to control MSCs from the iliac crest. This study shows that bone-adherent OBs grown in α-MEM medium are more efficient for osteogenic differentiation than BM MSCs and contribute to defining their phenotypic and functional characteristics, so providing a rationale for their use in bone tissue engineering or therapeutic purposes.     

Human subcutaneous adipose cells support complete differentiation but not self-renewal of hematopoietic progenitors

Adipose tissue is now considered as an endocrine organ implicated in energy regulation, inflammation and immune response, and as a source of multipotent cells with a broad range of differentiation capacities. Some of these cells are of a mesenchymal type which can -- like their bone marrow (BM) counterpart -- support hematopoiesis, since in a previous study we were able to reconstitute lethally irradiated mice by cells isolated from adipose tissue. In the present study, we established that cells derived from the stroma-vascular fraction of human subcutaneous fat pads support the complete differentiation of hematopoietic progenitors into myeloid and B lymphoid cells. However, these cells are unable to maintain the survival and self-renewal of hematopoietic stem cells. These features, similar to those of BM adipocytes, are the opposite of those of other cell types derived from mesenchymal progenitors such as BM myofibroblasts or osteoblasts. Because it is abundant and accessible, adipose tissue could be a convenient source of cells for the short-term reconstitution of hematopoiesis in man.     

Hypothalamic Proline Rich Polypeptide Regulates Hematopoiesis

The AGAPEPAEPAQPGVY proline-rich polypeptide (PRP-1) was isolated from neurosecretory granules of the bovine neurohypophysis; it is produced by N. supraopticus and N. paraventricularis. It has been shown that PRP-1 has many potentially beneficial biological effects including immunoregulatory, hematopoietic, antimicrobial and anti-neurodegenerative properties. Here we demonstrated that PRP-1 administration influence on redistribution of monocytes, granulocytes and lymphocytes between bone marrow (BM) and peripheral blood and promotes the influx of granulocytes and monocytes/macrophages from BM into peripheral blood and accumulation of immature granulocyte and monocyte in BM and delayed the maturation of T cells in BM. PRP-1 increased colony-forming cell proliferation in rat cells in vivo. In PRP-treated rat BM, the CFU number at day 4, 7 and 14 was considerably increased in comparison with untreated rats BM and no difference was found at day 21 and day 28. We found that PRP-1 enhances erythroid and myeloid colonies formation in human CD34⁺ progenitor cell culture in the presence of different growth factors and down-regulates T cells colony formation and specific surface markers expression during induction of human CD34⁺ progenitor cells differentiation into T lymphocytes lineage. We suggested that the hypothalamic PRP-1 possibly represents an endogenous peptide whose primary functions are to regulate neuronal survival and differentiation and hematopoiesis within neurosecretory hypothalamus—bone marrow humoral axis.     

The efficiency of in vitro isolation and myogenic differentiation of MSCs derived from adipose connective tissue,bone marrow,and skeletal muscle tissue

The objective of the study is to evaluate efficiency of in vitro isolation and myogenic differentiation of mesenchymal stem cells (MSCs) derived from adipose connective tissue (AD-MSCs), bone marrow (BM-MSCs), and skeletal muscle tissue (MC-MSCs). MSCs were isolated from adipose connective tissue, bone marrow, and skeletal muscle tissue of two adult 6-wk-old rats. Cultured MSCs were treated with 5-azacytidine (AZA) to induce myogenic differentiation. Isolated MSCs and differentiated cells were evaluated by immunocytochemistry (ICC), fluorescence-activated cell sorting (FACS), PCR, and RT-PCR. AD-MSCs showed the highest proliferation rate while BM-MSCs had the lowest one. In ICC, isolated MSCs had strong CD90- and CD44-positive expression and negative expression of CD45, CD31, and CD34, while AZA-treated MSCs had strong positive desmin expression. In FACS analysis, AD-MSCs had the highest percentage of CD90- and CD44-positive-expressing cells (99% and 96%) followed by BM-MSCs (97% and 94%) and MC-MSCs (92% and 91%).At 1 wk after incubation with AZA treatment, the peak of myogenin expression reached 93% in differentiated MC-MSCs, 83.3% in BM-MSCs, and 77% in AD-MSCs. MSCs isolated from adipose connective tissue, bone marrow, and skeletal muscle tissue have the same morphology and phenotype, but AD-MSCs were the most easily accessible and had the highest rate of growth on cultivation and the highest percentage of stem cell marker expression. Moreover, although MC-MSCs showed the highest rate of myogenic differentiation potential and expression of myoblast markers, AD-MSCs and BM-MSCs still can be valuable alternatives. The differentiated myoblastic cells could be an available new choice for myoblastic auto-transplantation in regeneration medicine.     

Ex vivo expansion and clonality of CD34+ selected cells from bone marrow and cord blood in a serum-free media

Although umbilical cord blood is increasingly being used in allogeneic marrow transplantation, delayed platelet engraftment is often a concern for cord blood transplant recipients. We evaluated the potential of ex vivo expansion and clonality in CD34+ cells separated from a bone marrow source, and cord blood, in a serum-free Media. The CD34+ cells, selected from bone marrow (BM) and umbilical cord blood (CB), were expanded with hematopoietic growth factors. They were then cultured for burst-forming units of erythrocytes (BFU-E), colony-forming units of granulocytes and monocytes (CFU-GM) and colony-forming units of megakaryocytes (CFU-Mk) at days 0, 4, 7, and 14 under the combination of growth factors, with cell counts. The cytokines included the recombinant human megakaryocyte growth and development (100 ng/ml), interleukin-3 (10 ng/ml), stem cell factor (100 ng/ml), flt-3 ligand (50 ng/ml) and interleukin-11 (200 ng/ml). The CB-selected CD34+ cells showed significantly higher total cell expansion than those from the BM at day 7 (3.0 fold increase than BM), day 14 (2.4 fold), and day 17 (2.6 fold). The colony count of the BFU-E/CFU-E per CD34+ cell at day 0 was 0.14 +/- 0.023 in the CB, which was significantly higher than 0.071 +/- 0.015 in the BM. The CB-selected CD34+ cells produced more BFU-E colonies than the BM on culture days 4, 7, and 14. The BFU-E colonies from the CB cells increased markedly on culture days 4 and 7, with a 4-fold increase at day 14. The colony count of the CFU-Mk per CD34+ cell at day 0 was 0.047 +/- 0.011 in the CB-selected CD34+ cells cultures, which was higher than the 0.026 +/- 0.014 in the BM. The CB-selected CD34+ cells produced more CFU-Mk colonies than the BM on culture days 4, 7 and 14. In conclusion, the ex vivo expansion of the CB cells may be very promising in producing total cellular expansion, CFU-Mk and BFU-E compared with BM, especially at day 7. The ex vivo expansion of the CB may have rationale in making an ex vivo culture for 7 to 14 d.     

Characteristics of human mesenchymal stem cells isolated from bone marrow and adipose tissue

Populations of human mesenchymal stem cells were derived from bone marrow and adipose tissue. Here analysis of six individuals is represented. Cells were isolated, expanded and evaluated by the expression of surface antigens using flow cytometry. These cells displayed similar characteristics for many markers. Cells isolated from bone marrow and adipose tissue were found to be homogeneously positive for CD13, CD44, CD90, CD105, and negative for CD45, CD34, CD31 and CD117. Besides, differences in surface antigene CD10 expression between narrow and adipose tissue-derived cells were detected. All these findings indicate that both bone marrow and adipose tissue are important sources of mesenchymal stem cells, which could be used in cell therapy protocols.     

The effect of medium composition on deposition of collagen type 1 and expression of osteogenic genes in mesenchymal stem cells derived from human adipose tissue and bone marrow

The two mesenchymal stem cell (MSC) populations that have gained most attention in relation to bone tissue engineering are adipose tissue (AT) MSCs and bone marrow (BM) MSCs. The purpose of this study was to investigate the ability of human BM-MSCs and AT-MSCs to survive, proliferate and deposit collagen type 1 when cultured on polycaprolactone nanofiber scaffolds and to ascertain the effect of medium composition on collagen type 1 formation and expression of osteogenic genes. The cells were seeded on polycaprolactone nanofiber scaffolds and cultured in three different types of media that differed by the presence of ascorbic acid, β-glycerophosphate and dexamethasone, that are typical components used for osteogenic differentiation of MSCs in vitro.In summary, AT-MSCs were proliferating significantly faster than BM-MSCs. AT-MSCs also showed better ability to deposit collagen type 1 and had a higher expression of early osteogenic markers, whereas BM-MSCs had higher expression of late osteogenic markers. This suggests that MSCs from diverse sources have different attributes and with respect to osteogenic differentiation, AT-MSCs are more immature compared to BM-MSCs. Collagen formation was depending on medium composition and the organization of collagen type 1 appeared to be influenced by the presence of dexamethasone.     

The utility of human dedifferentiated fat cells in bone tissue engineering in vitro

We compared the osteoblastic differentiation abilities of dedifferentiated fat cells (DFATs) and human bone marrow mesenchymal stem cells (hMSCs) as a cell source for bone regeneration therapies. In addition, the utility of DFATs in bone tissue engineering in vitro was assessed by an alpha-tricalcium phosphate (α-TCP)/collagen sponge (CS). Human DFATs were isolated from the submandibular of a patient by ceiling culture. DFATs and hMSCs at passage 3 were cultured in control medium or osteogenic medium (OM) for 14 days. Runx2 gene expression, alkaline phosphatase (ALP) activity, as well as osteocalcin (OCN) and calcium contents were analyzed to evaluate the osteoblastic differentiation ability of both cell types. DFATs seeded in a α-TCP/CS and cultured in OM for 14 days were analyzed by scanning electron microscopy (SEM) and histologically. Compared with hMSCs, DFATs cultured in OM generally underwent superior osteoblastogenesis by higher Runx2 gene expression at all days tested, as well as higher ALP activity at day 3 and 7, OCN expression at day 14, and calcium content at day 7. In SEM analyses, DFATs seeded in a α-TCP/CS were well spread and covered the α-TCP/CS by day 7. In addition, numerous spherical deposits were found to almost completely cover the α-TCP/CS on day 14. Von Kossa staining showed that DFATs differentiated into osteoblasts in the α-TCP/CS and formed cultured bone by deposition of a mineralized extracellular matrix. The combined use of DFATs and an α-TCP/CS may be an attractive option for bone tissue engineering.     

Higher production of IL-8 in visceral vs. subcutaneous adipose tissue. Implication of nonadipose cells in adipose tissue

IL-8 is released from human adipose tissue. Circulating IL-8 is increased in obese compared with lean subjects and is associated with measures of insulin resistance, development of atherosclerosis, and cardiovascular disease. We studied 1) the production and release of IL-8 in vitro from paired samples of subcutaneous (SAT) and visceral (VAT) adipose tissue and 2) the production of IL-8 from whole adipose tissue, isolated adipocytes, and nonfat cells of adipose tissue. IL-8 release from VAT was fourfold higher than from SAT (P < 0.05), and IL-8 mRNA was twofold higher in VAT compared with SAT (P < 0.01). Dexamethasone (50 nM) attenuated IL-8 production by 50% (P < 0.05), and IL-1beta (2 microg/l) increased IL-8 production up to 15-fold (P < 0.001). IL-8 release from whole SAT explants correlated with body mass index (BMI; r = 0.78; P < 0.001), as did IL-8 release from nonfat cells (r = 0.79; P < 0.001). However, no correlation was found between IL-8 release from the fraction of isolated adipocytes and BMI (r = 0.01). In conclusion, we demonstrated an increased release of IL-8 from VAT compared with SAT. Furthermore, our data suggest that the observed elevation in circulating levels of IL-8 in obese subjects is due primarily to the release of IL-8 from nonfat cells from adipose tissue. The high levels of IL-8 release from human adipose tissue and accumulation of this tissue in obese subjects may account for some of the increase in circulating IL-8 observed in obesity.     

Biologic properties of mesenchymal stem cells derived from bone marrow and adipose tissue

The biologic characteristics of mesenchymal stem cells (MSCs) isolated from two distinct tissues, bone marrow and adipose tissue were evaluated in these studies. MSCs derived from human and non-human primate (rhesus monkey) tissue sources were compared. The data indicate that MSCs isolated from rhesus bone marrow (rBMSCs) and human adipose tissue (hASCs) had more similar biologic properties than MSCs of rhesus adipose tissue (rASCs) and human bone marrow MSCs (hBMSCs). Analyses of in vitro growth kinetics revealed shorter doubling time for rBMSCs and hASCs. rBMSCs and hASCs underwent significantly more population doublings than the other MSCs. MSCs from all sources showed a marked decrease in telomerase activity over extended culture; however, they maintained their mean telomere length. All of the MSCs expressed embryonic stem cell markers, Oct-4, Rex-1, and Sox-2 for at least 10 passages. Early populations of MSCs types showed similar multilineage differentiation capability. However, only the rBMSCs and hASCs retain greater differentiation efficiency at higher passages. Overall in vitro characterization of MSCs from these two species and tissue sources revealed a high level of common biologic properties. However, the results demonstrate clear biologic distinctions, as well.     

Studies on human cord blood progenitor cells

Analysis of agar cultures throughout a 56-day period determined the concentration and cell cycle status of at least 4 different subclasses of hemopoietic colony forming cells (CFC) in human cord blood (CB). Although the concentration of CFC in CB was not significantly different from bone marrow (BM) in day-12 cultures, neutrophil colonies reached their peak on about day 23 in CB cultures and on day 12 in BM cultures. This suggests that the CFC in CB are more primitive than those in BM. In CB cultures, colonies of small cells contained predominantly neutrophils on day 14 and eosinophils on day 35, while the late developing (day 35) colonies of large cells contained mast-cell-like cells (MCL).     

A comparison between osteogenic differentiation of human unrestricted somatic stem cells and mesenchymal stem cells from bone marrow and adipose tissue

To evaluate the potential of three stem cells for cell therapy and tissue engineering applications, the biological behavior and osteogenic capacity of the newly introduced cord-blood-derived, unrestricted somatic stem cells (USSC) were compared with those of mesenchymal stem cells isolated from bone marrow (BM-MSC) and adipose tissue (AT-MSC). There was no significant difference between the rates of proliferation of the three stem cells. During osteogenic differentiation, alkaline phosphatase (ALP) activity peaked on day 7 in USSC compared to BM-MSC which showed the maximum value of ALP activity on day 14. However, BM-MSC had the highest ALP activity and mineralization during osteogenic induction. In addition, AT-MSC showed the lowest capacity for mineralization during differentiation and had the lowest ALP activity on days 7 and 14. Although AT-MSC expressed higher levels of collagen type I, osteonectin and BMP-2 in undifferentiated state, but these genes were expressed higher in BM-MSC during differentiation. BM-MSC also expressed higher levels of ALP, osteocalcin and Runx2 during induction. Taking together, BM-MSC showed the highest capacity for osteogenic differentiation and hold promising potential for bone tissue engineering and cell therapy applications.     

Tissue-specific fatty acid composition,cellularity, and gene expression in diverse cattle breeds

The nutritional quality of beef relates to the fatty acid (FA) composition of bovine adipose tissue. Those molecular mechanisms that induce the differing amounts and composition of fat in cattle breeds according to age at maturity and purpose of production remain unclear. Therefore, this study investigated the composition of total FAs, adipocyte size, and expression of some key genes involved in several adipogenesis and lipogenesis pathways measured in distinct adipose tissue depots from bulls of the genetically diverse cattle breeds Aberdeen Angus (n = 9), Gascon (n = 10), Holstein (n = 9), and Fleckvieh (n = 10). The animals were finished under identical housing and feeding conditions until slaughter at a similar age of 17 months. After slaughter, cod adipose tissue (CAT), subcutaneous adipose tissue (SAT), and M. longissimus lumborum (MLL) samples were collected. The saturated FA proportions were higher (P < .01) in CAT than in SAT across all breeds, whereas monounsaturated FA proportions were consistently higher (P < .001) in SAT compared to CAT and MLL. Aberdeen Angus bulls were distinguished from the other breeds in the proportions of mostly de novo synthesized C14:0, C16:0, C14:1n-5, C16:1n-7, and conjugated linoleic acid (P < .05). Adipocyte size decreased in the order CAT > SAT > MLL, and the largest adipocytes were observed in CAT of Holstein bulls (P < .05). Gene expression differences were more pronounced between adipose tissue depots than between breeds. The expression levels of ACACA, FASN, and SCD1 genes were related to tissue-specific, and to a lesser extent also breed-specific, differences in FA composition.     

The effect of different implant biomaterials on the behavior of canine bone marrow stromal cells during their differentiation into osteoblasts

We investigated the effects of different implant biomaterials on cultured canine bone marrow stromal cells (BMSC) undergoing differentiation into osteoblasts (dBMSC). BMSC were isolated from canine humerus by marrow aspiration, cultured and differentiated on calcium phosphate scaffold (CPS), hydroxyapatite, hydroxyapatite in gel form and titanium mesh. We used the MTT method to determine the effects of osteogenic media on proliferation. The characteristics of dBMSC were assessed using alizarin red (AR), immunocytochemistry and osteoblastic markers including alkaline phosphatase/von Kossa (ALP/VK), osteocalcin (OC) and osteonectin (ON), and ELISA. The morphology of dBMSC on the biomaterials was investigated using inverted phase contrast microscopy and scanning electron microscopy. We detected expression of ALP/VK, AR, OC and ON by day 7 of culture; expression increased from day 14 until day 21. CPS supported the best adhesion, cell spreading, proliferation and differentiation of BMSCs. The effects of the biomaterials depended on their surface properties. Expression of osteoblastic markers showed that canine dBMSCs became functional osteoblasts. Tissue engineered stem cells can be useful clinically for autologous implants for treating bone wounds.     

Characteristic Expression of Extracellular Matrix in Subcutaneous Adipose Tissue Development and Adipogenesis; Comparison with Visceral Adipose Tissue

Adipose tissue is a connective tissue specified for energy metabolism and endocrines, but functional differences between subcutaneous adipose tissue (SAT) and visceral adipose tissue (VAT) have not been fully elucidated. To reveal the physiological role of SAT, we characterized in vivo tissue development and in vitro adipocyte differentiation. In a DNA microarray analysis of SAT and VAT in Wistar rats, functional annotation clusters of extracellular matrix (ECM)-related genes were found in SAT, and major ECM molecules expressed in adipose tissues were profiled. In a histological analysis and quantitative expression analysis, ECM expression patterns could be classified into two types: (i) a histogenesis-correlated type such as type IV and XV collagen, and laminin subunits, (ii) a high-SAT expression type such as type I, III, and V collagen and minor characteristic collagens. Type (i) was related to basal membrane and up-regulated in differentiated 3T3-L1 cells and in histogenesis at depot-specific timings. In contrast, type (ii) was related to fibrous forming and highly expressed in 3T3-L1 preadipocytes. Exceptionally, fibronectin was abundant in developed adipose tissue, although it was highly expressed in 3T3-L1 preadipocytes. The present study showed that adipose tissues site-specifically regulate molecular type and timing of ECM expression, and suggests that these characteristic ECM molecules provide a critical microenvironment, which may affect bioactivity of adipocyte itself and interacts with other tissues. It must be important to consider the depot-specific property for the treatment of obesity-related disorders, dermal dysfunction and for the tissue regeneration.