Osteogenic differentiation of multipotent mesenchymal stromal cells isolated from human bone marrow and subcutaneous adipose tissue

Cellular populations with phenotypes similar to multipotent mesenchymal stromal cells were isolated from two different sources, including human bone marrow (BM) and subcutaneous adipose tissue (SAT). Comparative analysis of the efficiency of differentiation in the direction of osteogenesis has revealed morphological changes confirmed by staining with Alizarin red and von Kossa in bone marrow cells at the 14th day and in adipose tissue cells at the 28th day of cultivation in the medium with inductors. Analysis of expression of the osteopontin, osteocalcin, and bone sialoprotein genes in RT-PCR reactions has detected essential differences in the potential of these cells to differentiate into bone tissue cells. Cells isolated from BM of both the control and experimental groups were positive for octeopontin (OP) on the 14th day. Unlike these cells, in cells isolated from SAT in medium without an inductor, no product of OP gene expression was identified. In the cells subjected to differentiation, OP appeared at day 14. In the BM cells, octeocalcin (OC) was found at the 14th day, while the bone sialoprotein (BS) was found at the 21st day of cultivation in induction medium. In cells isolated from SAT, OC, and BS were not detected, even at the 28th day after the beginning of induction.     

Comparison of mesenchymal stem cells from adipose tissue and bone marrow for ischemic stroke therapy

Background aimsTransplantation of mesenchymal stromal cells (MSC) derived from bone marrow (BM) or adipose tissue is expected to become a cell therapy for stroke. The present study compared the therapeutic potential of adipose-derived stem cells (ASC) with that of BM-derived stem cells (BMSC) in a murine stroke model.MethodsASC and BMSC were isolated from age-matched C57BL/6J mice. These MSC were analyzed for growth kinetics and their capacity to secrete trophic factors and differentiate toward neural and vascular cell lineages in vitro. For in vivo study, ASC or BMSC were administrated intravenously into recipient mice (1 × 10⁵ cells/mouse) soon after reperfusion following a 90-min middle cerebral artery occlusion. Neurologic deficits, the degree of infarction, expression of factors in the brain, and the fate of the injected cells were observed.ResultsASC showed higher proliferative activity with greater production of vascular endothelial cell growth factor (VEGF) and hepatocyte growth factor (HGF) than BMSC. Furthermore, in vitro conditions allowed ASC to differentiate into neural, glial and vascular endothelial cells. ASC administration showed remarkable attenuation of ischemic damage, although the ASC were not yet fully incorporated into the infarct area. Nonetheless, the expression of HGF and angiopoietin-1 in ischemic brain tissue was significantly increased in ASC-treated mice compared with the BMSC group.ConclusionsCompared with BMSC, ASC have great advantages for cell preparation because of easier and safer access to adipose tissue. Taken together, our findings suggest that ASC would be a more preferable source for cell therapy for brain ischemia than BMSC.     

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.     

Neural differentiation ability of mesenchymal stromal cells from bone marrow and adipose tissue: a comparative study

Background aimsThe characteristics, such as morphologic and phenotypic characteristics and neural transdifferentiation ability, of mesenchymal stromal cells (MSC) derived from different origins have yet to be reported for cases isolated from the same individual.MethodsThe proliferation capacity, secretion ability of neurotrophins (NT) and neural differentiation ability in rat MSC isolated from bone marrow (BMSC) and adipose tissue (ADSC) were compared from the same animal.ResultsThe ADSC had a significantly higher proliferation capacity than BMSC according to cell cycle and cumulative population doubling analyses. The proportion of cells expressing neural markers was greater in differentiated ADSC than in differentiated BMSC. Furthermore, the single neurosphere derived from ADSC showed stronger expansion and differentiation abilities than that derived from BMSC. The findings from Western blot lent further support to the immunocytochemical data. The mRNA and protein levels of nerve growth factor (NGF) and brain-derived growth factor (BDNF) expressed in ADSC were significantly higher than those in BMSC at different stages before and following induction.ConclusionsOur study suggests that the proliferation ability of ADSC is superior to that of BMSC. Furthermore, differentiated ADSC expressed higher percentages of neural markers. As one possible alternative source of BMSC, ADSC may have wide potential for treating central nervous system (CNS) diseases.     

Dissimilar differentiation of mesenchymal stem cells from bone marrow, umbilical cord blood, and adipose tissue

Mesenchymal stem cells (MSCs) have been investigated as promising candidates for use in new cell-based therapeutic strategies such as mesenchyme-derived tissue repair. MSCs are easily isolated from adult tissues and are not ethically restricted. MSC-related literature, however, is conflicting in relation to MSC differentiation potential and molecular markers. Here we compared MSCs isolated from bone marrow (BM), umbilical cord blood (UCB), and adipose tissue (AT). The isolation efficiency for both BM and AT was 100%, but that from UCB was only 30%. MSCs from these tissues are morphologically and immunophenotypically similar although their differentiation diverges. Differentiation to osteoblasts and chondroblasts was similar among MSCs from all sources, as analyzed by cytochemistry. Adipogenic differentiation showed that UCB-derived MSCs produced few and small lipid vacuoles in contrast to those of BM-derived MSCs and AT-derived stem cells (ADSCs) (arbitrary differentiation values of 245.57 +/- 943 and 243.89 +/- 145.52 mum(2) per nucleus, respectively). The mean area occupied by individual lipid droplets was 7.37 mum(2) for BM-derived MSCs and 2.36 mum(2) for ADSCs, a finding indicating more mature adipocytes in BM-derived MSCs than in treated cultures of ADSCs. We analyzed FAPB4, ALP, and type II collagen gene expression by quantitative polymerase chain reaction to confirm adipogenic, osteogenic, and chondrogenic differentiation, respectively. Results showed that all three sources presented a similar capacity for chondrogenic and osteogenic differentiation and they differed in their adipogenic potential. Therefore, it may be crucial to predetermine the most appropriate MSC source for future clinical applications.     

Characterization and expression analysis of mesenchymal stem cells from human bone marrow and adipose tissue.

Human mesenchymal stem cells (MSC), that have been reported to be present in bone marrow, adipose tissues, dermis, muscles and peripheral blood, have the potential to differentiate along different lineages including those forming bone, cartilage, fat, muscle and neuron. This differentiation potential makes MSC excellent candidates for cell-based tissue engineering. In this study, we have examined phenotypes and gene expression profile of the human adipose tissue-derived stromal cells (ATSC) in the undifferentiated states, and compared with that of bone marrow stromal cells (BMSC). ATSC were enzymatically released from adipose tissues from adult human donors and were expanded in monolayer with serial passages at confluence. BMSC were harvested from the metaphysis of adult human femur. Flowcytometric analysis showed that ATSC have a marker expression that is similar to that of BMSC. ATSC expressed CD29, CD44, CD90, CD105 and were absent for HLA-DR and c-kit expression. Under appropriate culture conditions, MSC were induced to differentiate to the osteoblast, adipocyte, and chondrogenic lineages. ATSC were superior to BMSC in respect to maintenance of proliferating ability, and microarray analysis of gene expression revealed differentially expressed genes between ATSC and BMSC. The proliferating ability and differentiation potential of ATSC were variable according to the culture condition. The similarities of the phenotypes and the gene expression profiles between ATSC and BMSC could have broad implications for human tissue engineering.     

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.     

Viability and angiogenic activity of mesenchymal stromal cells from adipose tissue and bone marrow under hypoxia and inflammation in vitro

Progenitor stromal cells derived from adipose tissue (ADSC) and bone marrow (BMDSC) hold great promise for use in the cell-based therapy of ischemic diseases. It was demonstrated that these cells secrete a number of angiogenic cytokines that stimulate vascularization. It was demonstrated that ADSC or BMDSC injected intramuscularly or intravenously into the animals with experimental hind-limb ischemia improve vascularization. However, low oxygen levels and inflammation may impair the viability and functional activity of transplanted cells. We have examined ADSC and BMDSC properties in vitro under hypoxic and inflammatory conditions. ADSC and BMDSC derived from Balb/c mice have been cultivated under hypoxia or in the presence of inflammatory cytokines. The viability of cells assessed by annexin V-PE binding and 7AAD storage, as well as by the quantitative TUNEL method, was not changed under hypoxic conditions Cell exposure to inflammatory cytokines induced apoptosis in 70% of cells. Inflammatory cytokines did not stimulate gene expression of angiogenic growth factors. Under hypoxia conditions up-regulation of genes for pro-angiogenic factors and down-regulation of anti-angiogenic genes were more apparent in ADSC. Using angiogenesis models in vitro and in vivo, we demonstrated that stromal cell maintenance under hypoxic conditions increased their ability to stimulate the growth of blood vessels.     

Functional expression of adrenoreceptors in mesenchymal stromal cells derived from the human adipose tissue

Cultured mesenchymal stromal cells (MSCs) from different sources represent a heterogeneous population of proliferating non-differentiated cells that contains multipotent stem cells capable of originating a variety of mesenchymal cell lineages. Despite tremendous progress in MSC biology spurred by their therapeutic potential, current knowledge on receptor and signaling systems of MSCs is mediocre. Here we isolated MSCs from the human adipose tissue and assayed their responsivity to GPCR agonists with Ca^2 + imaging. As a whole, a MSC population exhibited functional heterogeneity. Although a variety of first messengers was capable of stimulating Ca²⁺ signaling in MSCs, only a relatively small group of cells was specifically responsive to the particular GPCR agonist, including noradrenaline. RT-PCR and immunocytochemistry revealed expression of α1B-, α2A-, and β2-adrenoreceptors in MSCs. Their sensitivity to subtype-specific adrenergic agonists/antagonists and certain inhibitors of Ca²⁺ signaling indicated that largely the α2A-isoform coupled to PLC endowed MSCs with sensitivity to noradrenaline. The all-or-nothing dose-dependence was characteristic of responsivity of robust adrenergic MSCs. Noradrenaline never elicited small or intermediate responses but initiated large and quite similar Ca²⁺ transients at all concentrations above the threshold. The inhibitory analysis and Ca²⁺ uncaging implicated Ca²⁺-induced Ca²⁺ release (CICR) in shaping Ca²⁺ signals elicited by noradrenaline. Evidence favored IP₃ receptors as predominantly responsible for CICR. Based on the overall findings, we inferred that adrenergic transduction in MSCs includes two fundamentally different stages: noradrenaline initially triggers a local and relatively small Ca²⁺ signal, which next stimulates CICR, thereby being converted into a global Ca²⁺ signal.     

Functional recovery after transplantation of bone marrow-derived human mesenchymal stromal cells in a rat model of spinal cord injury

Background aimsSpinal cord injury (SCI) is a medically untreatable condition for which stem cells have created hope. Pre-clinical and clinical studies have established that these cells are safe for transplantation. The dose dependency, survivability, route of administration, cell migration to injury site and effect on sensory and motor behavior in an SCI-induced paraplegic model were studied.MethodsA spinal cord contusion injury model was established in rats. Bone marrow (BM) mesenchymal stromal cells (MSC) were tagged to facilitate tracing in vivo. Two different doses (2 and 5 million cells/kg body weight) and two different routes of infusion (site of injury and lumbar puncture) were tested during and after the spinal shock period. The animals were tested post-transplantation for locomotor capacity, motor control, sensory reflex, posture and body position. Stem cell migration was observed 1 month post-transplantation in spinal cord sections.ResultsThe overall results demonstrated that transplantation of BM MSC significantly improved the locomotor and sensory behavior score in the experimental group compared with the sham control group, and these results were dose dependent. All the infused stem cells could be visualized at the site of injury and none was visualized at the injected site. This indicated that the cells had survived in vivo, were probably chemoattracted and had migrated to the lesion site.ConclusionsMSC transplanted with a lumbar puncture method migrate to the site of injury and are the most suitable for SCI healing. These cells demonstrate a dose-dependent effect and promote functional recovery when injected during or after the spinal shock period.     

Secretome analysis of human bone marrow derived mesenchymal stromal cells

As an essential cellular component of the bone marrow (BM) microenvironment mesenchymal stromal cells (MSC) play a pivotal role for the physiological regulation of hematopoiesis, in particular through the secretion of cytokines and chemokines. Mass spectrometry (MS) facilitates the identification and quantification of a large amount of secreted proteins (secretome), but can be hampered by the false-positive identification of contaminating proteins released from dead cells or derived from cell medium. To reduce the likelihood of contaminations we applied an approach combining secretome and proteome analysis to characterize the physiological secretome of BM derived human MSC. Our analysis revealed a secretome consisting of 315 proteins. Pathway analyses of these proteins revealed a high abundance of proteins related to cell growth and/or maintenance, signal transduction and cell communication thereby representing key biological functions of BM derived MSC on protein level. Within the MSC secretome we identified several cytokines and growth factors such as VEGFC, TGF-β1, TGF-β2 and GDF6 which are known to be involved in the physiological regulation of hematopoiesis. By comparing the peptide patterns of secretomes and cell lysates 17 proteins were identified as candidates for proteolytic processing. Taken together, our combined MS work-flow reduced the likelihood of contaminations and enabled us to carve out a specific overview about the composition of the secretome from human BM derived MSC. This methodological approach and the specific secretome signature of BM derived MSC may serve as basis for future comparative analyses of the interplay of MSC and HSPC in patients with hematological malignancies.     

HIF-1基因转染骨髓间充质干细胞治疗SCII的展望

脊髓缺血再灌注损伤(spinal cord ischemia-reperfusion injury,SCII)作为原发性脊髓损伤后的继发性损害是造成神经细胞损伤的一个重要因素,目前基因和细胞移植治疗SCII尚处于探索阶段.低氧诱导因子-1(hypoxia-inducible factor-1,HIF-1)产生并处于缺氧应答的关键环节,在缺血后细胞凋亡及微循环的重建等方面起关键作用,用其转染骨髓间充质干细胞(mesenchymal stem cells,MSCs)进行细胞移植治疗SCII,可有望取得新的突破.     

Proteomic analysis of human mesenchymal stromal cells derived from adipose tissue undergoing osteoblast differentiation

Background aimsStem cells derived from human adipose tissue (ASC) have the capacity for renewal, are easily obtained and have plasticity properties that allow them to differentiate into several cell types, including osteoblast cells. With the aim of understanding the issue of the osteogenic process and finding reliable biomarkers in cells undergoing the osteogeneic differentiation process, this work took advantage of a proteomic approach to identify proteins involved in osteogenesis.MethodsFor this purpose, ASC were analyzed under three conditions: S0, in the absence of stimulation; S1, with 2 weeks of osteogenic medium stimulation; and S2, with 4 weeks of osteogenic medium stimulation. The identification of ASC was carried out by flow cytometry using antibodies specific to known undifferentiated stem cell-surface markers. Cell viability, enzymatic activity, mineral deposition, collagen structure and production and gene analyzes were evaluated for each condition.ResultsPhenotypic modifications were observed during the in vitro osteogenic differentiation process by two-dimensional (2-D) differential image gel electrophoresis (DIGE). The proteins were identified by mass espectrometry in tandem (MS/MS) analyzes using Matrix-assisted laser desorption/ionization with TOF/TOF is a tandem mass spectrometry method where two time-of-flight mass spectrometers are used consecutively (MALDI-TOF/TOF). A total of 51 differentially expressed proteins was identified when comparing the three observed conditions. Sixteen different spots were identified in the S0 stage compared with S2, while 28 different spots were found in S2 compared with S0. S1 expressed seven different spots compared with S0 and S2.ConclusionsThese findings suggest the involvement of several proteins directly related to the osteogenic pathway, which can be used to improve understanding of the osteogenic process.     

Effect of hypoxia on equine mesenchymal stem cells derived from bone marrow and adipose tissue

ABSTRACT: BACKGROUND: Mesenchymal stem cells (MSCs) derived from bone marrow (BM-MSCs) and adipose tissue (AT-MSCs) are being applied to equine cell therapy. The physiological environment in which MSCs reside is hypoxic and does not resemble the oxygen level typically used in in vitro culture (20% O2). This work compares the growth kinetics, viability, cell cycle, phenotype and expression of pluripotency markers in both equine BM-MSCs and AT-MSCs at 5% and 20% O2. RESULTS: At the conclusion of culture, fewer BM-MSCs were obtained in hypoxia than in normoxia as a result of significantly reduced cell division. Hypoxic AT-MSCs proliferated less than normoxic AT-MSCs because of a significantly higher presence of non-viable cells during culture. Flow cytometry analysis revealed that the immunophenotype of both MSCs was maintained in both oxygen conditions. Gene expression analysis using RT-qPCR showed that statistically significant differences were only found for CD49d in BM-MSCs and CD44 in AT-MSCs. Similar gene expression patterns were observed at both 5% and 20% O2 for the remaining surface markers. Equine MSCs expressed the embryonic markers NANOG, OCT4 and SOX2 in both oxygen conditions. Additionally, hypoxic cells tended to display higher expression, which might indicate that hypoxia retains equine MSCs in an undifferentiated state. CONCLUSIONS: Hypoxia attenuates the proliferative capacity of equine MSCs, but does not affect the phenotype and seems to keep them more undifferentiated than normoxic MSCs.     

The immunosuppressive capacity of human mesenchymal stromal cells derived from amnion and bone marrow

Mesenchymal stromal cells derived from amnion (AM-MSCs) can be easily obtained in large quantity by less invasive method in comparison to bone marrow-derived MSCs (BM-MSCs). However, the biological and immunosuppressive properties of AM-MSCs are still poorly characterized. Previous studies demonstrated that BM-MSCs expressed indoleamine 2,3-dioxygenase (IDO) to suppress T-cell responses. This study was designed to address whether IDO contributes to the immunosuppressive function of AM-MSCs. MSCs isolated from amnion were cultured in complete medium similar to BM-MSCs. After culture, AM-MSCs exhibited spindle shape morphology and expressed MSC markers similar to that of BM-MSCs. In addition, AM-MSCs were able to differentiate into adipocytes and osteoblasts. Fascinatingly, AM-MSCs and BM-MSCs exhibited comparable degree of immunosuppressive effect when they were co-cultured with activated T-cells. In addition, IDO secreted by AM-MSCs was responsible for induction of immunosuppressive activities in the same manner as BM-MSCs. Taken together; the results of the present study demonstrate that while AM-MSCs and BM-MSCs show similar immunosuppressive effect, AM-MSCs may have additional advantage over the BM-MSCs in terms of availability. Therefore, AM-MSCs might be considered a potential source for therapeutic applications especially for treatment of immune related diseases.