技术资料

Mesenchymal stem cells (MSC) establish close interactions with bone marrow sinusoids in a putative perivascular niche. These vessels contain a large storage pool of mature nonproliferating neutrophils. Here,we have investigated the effects of human bone marrow MSC on neutrophil survival and effector functions. MSC from healthy donors,at very low MSC:neutrophil ratios (up to 1:500),significantly inhibited apoptosis of resting and interleukin (IL)-8-activated neutrophils and dampened N-formyl-l-methionin-l-leucyl-l-phenylalanine (f-MLP)-induced respiratory burst. The antiapoptotic activity of MSC did not require cell-to-cell contact,as shown by transwell experiments. Antibody neutralization experiments demonstrated that the key MSC-derived soluble factor responsible for neutrophil protection from apoptosis was IL-6,which signaled by activating STAT-3 transcription factor. Furthermore,IL-6 expression was detected in MSC by real-time reverse transcription-polymerase chain reaction and enzyme-linked immunosorbent assay. Finally,recombinant IL-6 was found to protect neutrophils from apoptosis in a dose-dependent manner. MSC had no effect on neutrophil phagocytosis,expression of adhesion molecules,and chemotaxis in response to IL-8,f-MLP,or C5a. These results support the following conclusions: (a) in the bone marrow niche,MSC likely protect neutrophils of the storage pool from apoptosis,preserving their effector functions and preventing the excessive or inappropriate activation of the oxidative metabolism,and (b) a novel mechanism whereby the inflammatory potential of activated neutrophils is harnessed by inhibition of apoptosis and reactive oxygen species production without impairing phagocytosis and chemotaxis has been identified. View Publication

Many different culture systems have been developed for expanding human pluripotent stem cells (hESCs and hiPSCs). In general,4-10 ng/ml of bFGF is supplemented in culture media in feeder-dependent systems regardless of feeder cell types,whereas in feeder-free systems,up to 100 ng/ml of bFGF is required for maintaining long-term culture on various substrates. The amount of bFGF required in native hESCs growth niche is unclear. Here we report using inactivated adipose-derived human mesenchymal stem cells as feeder cells to examine long-term parallel cultures of two hESCs lines (H1 and H9) and one hiPSCs line (DF19-9-7T) in media supplemented with 0,0.4 or 4 ng/ml of bFGF for up to 23 passages,as well as parallel cultures of H9 and DF19 in media supplemented with 4,20 or 100 ng/ml bFGF for up to 13 passages for comparison. Across all cell lines tested,bFGF supplement demonstrated inhibitory effect over growth expansion,single cell colonization and recovery from freezing in a dosage dependent manner. In addition,bFGF exerted differential effects on different cell lines,inducing H1 and DF19 differentiation at 4 ng/ml or higher,while permitting long-term culture of H9 at the same concentrations with no apparent dosage effect. Pluripotency was confirmed for all cell lines cultured in 0,0.4 or 4 ng/ml bFGF excluding H1-4 ng,as well as H9 cultured in 4,20 and 100 ng/ml bFGF. However,DF19 demonstrated similar karyotypic abnormality in both 0 and 4 ng/ml bFGF media while H1 and H9 were karyotypically normal in 0 ng/ml bFGF after long-term culture. Our results indicate that exogenous bFGF exerts dosage and cell line dependent effect on human pluripotent stem cells cultured on mesenchymal stem cells,and implies optimal use of bFGF in hESCs/hiPSCs culture should be based on specific cell line and its culture system. View Publication

Mesenchymal stem cells (MSCs) have a high potential for therapeutic efficacy in treating diverse musculoskeletal injuries and cardiovascular diseases,and for ameliorating the severity of graft-versus-host and autoimmune diseases. While most of these clinical applications require substantial cell quantities,the number of MSCs that can be obtained initially from a single donor is limited. Reports on the derivation of MSC-like cells from pluripotent stem cells (PSCs) are,thus,of interest,as the infinite proliferative capacity of PSCs opens the possibility to generate large amounts of uniform batches of MSCs. However,characterization of such MSC-like cells is currently inadequate,especially with regard to the question of whether these cells are equivalent or identical to MSCs. In this study,we have derived MSC-like cells [induced PSC-derived MSC-like progenitor cells (iMPCs)] using four different methodologies from a newly established induced PSC line reprogrammed from human bone marrow stromal cells (BMSCs),and compared the iMPCs directly with the originating parental BMSCs. The iMPCs exhibited typical MSC/fibroblastic morphology and MSC-typical surface marker profile,and they were capable of differentiation in vitro along the osteogenic,chondrogenic,and adipogenic lineages. However,compared with the parental BMSCs,iMPCs displayed a unique expression pattern of mesenchymal and pluripotency genes and were less responsive to traditional BMSC differentiation protocols. We,therefore,conclude that iMPCs generated from PSCs via spontaneous differentiation represent a distinct population of cells which exhibit MSC-like characteristics. View Publication

Mesenchymal stem cells (MSCs) are being tested in a wide range of human diseases; however,loss of potency and inconsistent quality severely limit their use. To overcome these issues,we have utilized a developmental precursor called the hemangioblast as an intermediate cell type in the derivation of a highly potent and replenishable population of MSCs from human embryonic stem cells (hESCs). This method circumvents the need for labor-intensive hand-picking,scraping,and sorting that other hESC-MSC derivation methods require. Moreover,unlike previous reports on hESC-MSCs,we have systematically evaluated their immunomodulatory properties and in vivo potency. As expected,they dynamically secrete a range of bioactive factors,display enzymatic activity,and suppress T-cell proliferation that is induced by either allogeneic cells or mitogenic stimuli. However,they also display unique immunophenotypic properties,as well as a smaller size and textgreater30,000-fold proliferative capacity than bone marrow-derived MSCs. In addition,this is the first report which demonstrates that hESC-MSCs can inhibit CD83 up-regulation and IL-12p70 secretion from dendritic cells and enhance regulatory T-cell populations induced by interleukin 2 (IL-2). This is also the first report which shows that hESC-MSCs have therapeutic efficacy in two different autoimmune disorder models,including a marked increase in survival of lupus-prone mice and a reduction of symptoms in an autoimmune model of uveitis. Our data suggest that this novel and therapeutically active population of MSCs could overcome many of the obstacles that plague the use of MSCs in regenerative medicine and serve as a scalable alternative to current MSC sources. View Publication

The purpose of this study was to examine the in vitro and in vivo osteogenic potential of human induced pluripotent stem cells (hiPSCs) against that of human bone marrow mesenchymal stem cells (hBMMSCs). Embryoid bodies (EBs),which were formed from undifferentiated hiPSCs,were dissociated into single cells and underwent osteogenic differentiation using the same medium as hBMMSCs for 14 days. Osteoinduced hiPSCs were implanted on the critical-size calvarial defects and long bone segmental defects in rats. The healing of defects was evaluated after 8 weeks and 12 weeks of implantation,respectively. Osteoinduced hiPSCs showed relatively lower and delayed in vitro expressions of the osteogenic marker COL1A1 and bone sialoprotein,as well as a weaker osteogenic differentiation through alkaline phosphatase staining and mineralization through Alizarin red staining compared with hBMMSCs. Calvarial defects treated with osteoinduced hiPSCs had comparable quality of new bone formation,including full restoration of bone width and robust formation of trabeculae,to those treated with hBMMSCs. Both osteoinduced hiPSCs and hBMMSCs persisted in regenerated bone after 8 weeks of implantation. In critical-size long bone segmental defects,osteoinduced hiPSC treatment also led to healing of segmental defects comparable to osteoinduced hBMMSC treatment after 12 weeks. In conclusion,despite delayed in vitro osteogenesis,hiPSCs have an in vivo osteogenic potential as good as hBMMSCs. View Publication

PURPOSE Demonstrate a novel manufacturing method to generate extracellular matrix scaffolds from cardiac fibroblasts (CF-ECM) as a therapeutic mesenchymal stem cell-transfer device. MATERIALS AND METHODS Rat CF were cultured at high-density (˜1.6×10(5)/cm(2)) for 10-14 days. Cell sheets were removed from the culture dish by incubation with EDTA and decellularized with water and peracetic acid. CF-ECM was characterized by mass spectrometry,immunofluorescence and scanning electron microscopy. CF-ECM seeded with human embryonic stem cell derived mesenchymal stromal cells (hEMSCs) were transferred into a mouse myocardial infarction model. 48 hours later,mouse hearts were excised and examined for CF-ECM scaffold retention and cell transfer. RESULTS CF-ECM scaffolds are composed of fibronectin (82%),collagens type I (13%),type III (3.4%),type V (0.2%),type II (0.1%) elastin (1.3%) and 18 non-structural bioactive molecules. Scaffolds remained intact on the mouse heart for 48 hours without the use of sutures or glue. Identified hEMSCs were distributed from the epicardium to the endocardium. CONCLUSIONS High density cardiac fibroblast culture can be used to generate CF-ECM scaffolds. CF-ECM scaffolds seeded with hEMSCs can be maintained on the heart without suture or glue. hEMSC are successfully delivered throughout the myocardium. View Publication

BACKGROUND Cardiovascular cell therapy represents a promising field,with several approaches currently being tested. The advanced therapy medicinal product (ATMP) for the ongoing METHOD clinical study (Bone marrow derived cell therapy in the stable phase of chronic ischemic heart disease") consists of fresh mononuclear cells (MNC) isolated from autologous bone marrow (BM) through density gradient centrifugation on standard Ficoll-Paque. Cells are tested for safety (sterility� View Publication

Expansion of pluripotent stem cells in defined media devoid of animal-derived feeder cells to generate multilayered three-dimensional (3D) bulk preparations or spheroids,rather than two-dimensional (2D) monolayers,is advantageous for many regenerative,biological or disease-modelling studies. Here we show that electrospun polymer matrices comprised of nanofibres that mimic the architecture of the natural fibrous extracellular matrix allow for feeder-free expansion of pluripotent human induced pluripotent stem cells (IPSCs) and human embryonic stem cells (HESCs) into multilayered 3D 'patty-like' spheroid structures in defined xeno-free culture medium. The observation that IPSCs and HESCs readily revert to 2D growth in the absence of the synthetic nanofibre membranes suggests that this 3D expansion behaviour is mediated by the physical microenvironment and artificial niche provided by the nanofibres only. Importantly,we could show that such 3D growth as patties maintained the pluripotency of cells as long as they were kept on nanofibres. The generation of complex multilayered 3D structures consisting of only pluripotent cells on biodegradable nanofibre matrices of the desired shape and size will enable both industrial-scale expansion and intricate organ-tissue engineering applications with human pluripotent stem cells,where simultaneous coupling of differentiation pathways of all germ layers from one stem cell source may be required for organ formation. View Publication

Human induced pluripotent stem cells (hiPSCs) have demonstrated great potential for hyaline cartilage regeneration. However,current approaches for chondrogenic differentiation of hiPSCs are complicated and inefficient primarily due to intermediate embryoid body formation,which is required to generate endodermal,ectodermal,and mesodermal cell lineages. We report a new,straightforward and highly efficient approach for chondrogenic differentiation of hiPSCs,which avoids embryoid body formation. We differentiated hiPSCs directly into mesenchymal stem /stromal cells (MSC) and chondrocytes. hiPSC-MSC-derived chondrocytes showed significantly increased Col2A1,GAG,and SOX9 gene expression compared to hiPSC-MSCs. Following transplantation of hiPSC-MSC and hiPSC-MSC-derived chondrocytes into osteochondral defects of arthritic joints of athymic rats,magnetic resonance imaging studies showed gradual engraftment,and histological correlations demonstrated hyaline cartilage matrix production. Results present an efficient and clinically translatable approach for cartilage tissue regeneration via patient-derived hiPSCs,which could improve cartilage regeneration outcomes in arthritic joints. View Publication

It is well accepted that umbilical cord blood has been a source for hematopoietic stem cells. However,controversy exists as to whether cord blood can serve as a source of mesenchymal stem cells,which can differentiate into cells of different connective tissue lineages such as bone,cartilage,and fat,and little success has been reported in the literature about the isolation of such cells from cord blood. Here we report a novel method to obtain single cell-derived,clonally expanded mesenchymal stem cells that are of multilineage differentiation potential by negative immunoselection and limiting dilution. The immunophenotype of these clonally expanded cells is consistent with that reported for bone marrow mesenchymal stem cells. Under appropriate induction conditions,these cells can differentiate into bone,cartilage,and fat. Surprisingly,these cells were also able to differentiate into neuroglial- and hepatocyte-like cells under appropriate induction conditions and,thus,these cells may be more than mesenchymal stem cells as evidenced by their ability to differentiate into cell types of all 3 germ layers. In conclusion,umbilical cord blood does contain mesenchymal stem cells and should not be regarded as medical waste. It can serve as an alternative source of mesenchymal stem cells to bone marrow. View Publication

Mesenchymal stem cells (MSCs) have the capability for renewal and differentiation into various lineages of mesenchymal tissues. These features of MSCs attract a lot of attention from investigators in the context of cell-based therapies of several human diseases. Despite the fact that bone marrow represents the main available source of MSCs,the use of bone marrow-derived cells is not always acceptable due to the high degree of viral infection and the significant drop in cell number and proliferative/differentiation capacity with age. Thus,the search for possible alternative MSC sources remains to be validated. Umbilical cord blood is a rich source of hematopoietic stem/progenitor cells and does not contain mesenchymal progenitors. However,MSCs circulate in the blood of preterm fetuses and may be successfully isolated and expanded. Where these cells home at the end of gestation is not clear. In this investigation,we have made an attempt to isolate MSCs from the subendothelial layer of umbilical cord vein using two standard methodological approaches: the routine isolation of human umbilical vein endothelial cell protocol and culture of isolated cells under conditions appropriate for bone-marrow-derived MSCs. Our results suggest that cord vasculature contains a high number of MSC-like elements forming colonies of fibroblastoid cells that may be successfully expanded in culture. These MSC-like cells contain no endothelium- or leukocyte-specific antigens but express alpha-smooth muscle actin and several mesenchymal cell markers. Therefore,umbilical cord/placenta stroma could be regarded as an alternative source of MSCs for experimental and clinical needs. View Publication

Germ line mutations in the Adenomatous polyposis coli tumor suppressor gene cause a hereditary form of intestinal tumorigenesis in both mice and man. Here we show that in Apc(Min/+) mice,which carry a heterozygous germ line mutation at codon 850 of Apc,there is progressive loss of immature and mature thymocytes from approximately 80 days of age with complete regression of the thymus by 120 days. In addition,Apc(Min/+) mice show parallel depletion of splenic natural killer (NK) cells,immature B cells,and B progenitor cells in bone marrow due to complete loss of interleukin 7 (IL-7)-dependent B-cell progenitors. Using bone marrow transplantation experiments into wild-type recipients,we have shown that the capacity of transplanted Apc(Min/+) bone marrow cells for T- and B-cell development appears normal. In contrast,although the Apc(Min/+) bone marrow microenvironment supported short-term reconstitution with wild-type bone marrow,Apc(Min/+) animals that received transplants subsequently underwent lymphodepletion. Fibroblast colony-forming unit (CFU-F) colony assays revealed a significant reduction in colony-forming mesenchymal progenitor cells in the bone marrow of Apc(Min/+) mice compared with wild-type animals prior to the onset of lymphodepletion. This suggests that an altered bone marrow microenvironment may account for the selective lymphocyte depletion observed in this model of familial adenomatous polyposis. View Publication