技术资料

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

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 (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

We evaluated the impact of donor age on the efficacy of myocardial cellular therapy for ischemic cardiomyopathy. Characteristics of smooth muscle cells (SMC),bone marrow stromal cells (MSCs),and skeletal muscle cells (SKMCs) from young,adult,and old rats were compared in vitro. Three weeks after coronary ligation,3.5 million SMCs (n = 11) or MSCs (n = 9) from old syngenic rats or culture medium (n = 6) were injected into the ischemic region. Five weeks after implantation,cardiac function was assessed by echocardiography and the Langendorff apparatus. In the in vitro study,the numbers and proliferation of MSCs from fresh bone marrow and SKMCs from fresh tissue but not SMCs were markedly diminished in old animals (P textless 0.05 both groups). SKMCs from old animals did not reach confluence. After treatment with 5-azacytidine (azacitidine),the myogenic potential of old MSCs was decreased compared with young MSCs. In the in vivo study,both SMC and MSC transplantation induced significant angiogenesis compared with media injections (P textless 0.05 both groups). Transplantation of SMCs but not MSCs prevented scar thinning (P = 0.03) and improved ejection fraction and fractional shortening (P textless 0.05). Load-independent indices of cardiac function in a Langendorff preparation confirmed improved function in the aged SMC group (P = 0.01) but not in the MSC group compared with the control group. In conclusion,donor age adversely impacts the efficacy of cellular therapy for myocardial regeneration and is cell-type dependent. SMCs from old donors retain their ability to improve cardiac function after implantation into ischemic myocardium. View Publication