技术资料

Stem cell-based tissue engineering is a promising technology in the effort to create functional tissues of choice. To establish an efficient approach for generating hematopoietic cell lineages directly from embryonic stem (ES) cells and to study the effects of three-dimensional (3D) biomaterials on ES cell differentiation,we cultured mouse ES cells on 3D,highly porous,biomimetic scaffolds. Cell differentiation was evaluated by microscopy and flow cytometry analysis with a variety of hematopoiesis- specific markers. Our data indicate that ES cells differentiated on porous 3D scaffold structures developed embryoid bodies (EBs) similar to those in traditional two-dimensional (2D) cultures; however,unlike 2D differentiation,these EBs integrated with the scaffold and appeared embedded in a network of extracellular matrix. Most significantly,the efficiency of hematopoietic precursor cell (HPC) generation on 3D,as indicated by the expression of various HPC-specific surface markers (CD34,Sca-1,Flk-1,and c-Kit) and colony-forming cell (CFC) assays,was reproducibly increased (about 2-fold) over their 2D counterparts. Comparison of static and dynamic 3D cultures demonstrated that spinner flask technology also contributed to the higher hematopoietic differentiation efficiency of ES cells seeded on scaffolds. Continued differentiation of 3D-derived HPCs into the myeloid lineage demonstrated increased efficiency (2-fold) of generating myeloid compared with differentiation from 2D-derived HPCs. View Publication

Biochemical factors can help reprogram somatic cells into pluripotent stem cells,yet the role of biophysical factors during reprogramming is unknown. Here,we show that biophysical cues,in the form of parallel microgrooves on the surface of cell-adhesive substrates,can replace the effects of small-molecule epigenetic modifiers and significantly improve reprogramming efficiency. The mechanism relies on the mechanomodulation of the cells' epigenetic state. Specifically,decreased histone deacetylase activity and upregulation of the expression of WD repeat domain 5 (WDR5)—a subunit of H3 methyltranferase—by microgrooved surfaces lead to increased histone H3 acetylation and methylation. We also show that microtopography promotes a mesenchymal-to-epithelial transition in adult fibroblasts. Nanofibrous scaffolds with aligned fibre orientation produce effects similar to those produced by microgrooves,suggesting that changes in cell morphology may be responsible for modulation of the epigenetic state. These findings have important implications in cell biology and in the optimization of biomaterials for cell-engineering applications. View Publication

Pancreatic islets are densely packed cellular aggregates containing various hormonal cell types essential for blood glucose regulation. Interactions among these cells markedly affect the glucoregulatory functions of islets along with the surrounding niche and pancreatic tissue-specific geometrical organization. However,stem cell (SC)-derived islets generated in vitro often lack the three-dimensional extracellular microenvironment and peri-vasculature,which leads to the immaturity of SC-derived islets,reducing their ability to detect glucose fluctuations and insulin release. Here,we bioengineer the in vivo-like pancreatic niches by optimizing the combination of pancreatic tissue-specific extracellular matrix and basement membrane proteins and utilizing bioprinting-based geometrical guidance to recreate the spatial pattern of islet peripheries. The bioprinted islet-specific niche promotes coordinated interactions between islets and vasculature,supporting structural and functional features resembling native islets. Our strategy not only improves SC-derived islet functionality but also offers significant potential for advancing research on islet development,maturation,and diabetic disease modeling,with future implications for translational applications. The glucoregulatory functions of pancreatic islets are affected by their surrounding niche and spatial organization. Here,bioengineered stem-cell derived islet niches use bioprinting-based geometrical guidance to promote islet maturation for improved functionality and diabetes research. View Publication

Supplementation of mesenchymal stem cells (MSCs) during hematopoietic stem cell (HSC) transplantation alleviates complications such as graft-versus-host disease,leading to a speedy recovery of hematopoiesis. To meet this clinical demand,a fast MSC expansion method is required. In the present study,we examined the feasibility of using a rotary bioreactor system to expand MSCs from isolated bone marrow mononuclear cells. The cells were cultured in a rotary bioreactor with Myelocult medium containing a combination of supplementary factors,including stem cell factor and interleukin-3 and -6. After 8 days of culture,total cell numbers,Stro-1(+)CD44(+)CD34(-) MSCs,and CD34(+)CD44(+)Stro-1(-) HSCs were increased 9-,29-,and 8-fold,respectively. Colony-forming efficiency-fibroblast per day of the bioreactor-treated cells was 1.44-fold higher than that of the cells without bioreactor treatment. The bioreactor-expanded MSCs showed expression of primitive MSC markers endoglin (SH2) and vimentin,whereas markers associated with lineage differentiation,including osteocalcin (osteogenesis),type II collagen (chondrogenesis),and C/EBP-alpha (CCAAT/enhancer-binding protein-alpha) (adipogenesis),were not detected. Upon induction,the bioreactor-expanded MSCs were able to differentiate into osteoblasts,chondrocytes,and adipocytes. We conclude that the rotary bioreactor with the modified Myelocult medium reported in this study may be used to rapidly expand MSCs. View Publication

Puromycin N-acetyltransferase from Streptomyces alboniger inactivates puromycin by acetylating the amino position of its tyrosinyl moiety. This enzyme has been partially purified by column chromatography through DEAE-cellulose and Affigel Blue and characterized. It has an Mr of 23 000,as determined by gel filtration. In addition to puromycin,the enzyme N-acetylates O-demethylpuromycin,a toxic precursor of the antibiotic,and chryscandin,a puromycin analogue antibiotic. The Km values for puromycin and O-demethylpuromycin are 1.7 and 4.6 microM,respectively. The O-demethylpuromycin O-methyltransferase from S. alboniger,which apparently catalyzes the last step in the biosynthesis of puromycin [Rao,M. M.,Rebello,P. F.,& Pogell,B. M. (1969) J. Biol. Chem. 244,112-118],also O-methylates N-acetyl-O-demethylpuromycin. The Km values of the methylating enzyme for O-demethylpuromycin and N-acetyl-O-demethylpuromycin are 260 and 2.3 microM,respectively. These findings suggest that O-demethylpuromycin,if present in S. alboniger,would be N-acetylated and then O-methylated to be converted into N-acetylpuromycin. It might even be possible that N-acetylation of the puromycin backbone takes place at an earlier precursor. View Publication

Bipolar disorder (BD),characterized by recurrent mood swings between depression and mania,is a highly heritable and devastating mental illness with poorly defined pathophysiology. Recent genome-wide molecular genetic studies have identified several protein-coding genes and microRNAs (miRNAs) significantly associated with BD. Notably,some of the proteins expressed from BD-associated genes function in neuronal synapses,suggesting that abnormalities in synaptic function could be one of the key pathogenic mechanisms of BD. In contrast,however,the role of BD-associated miRNAs in disease pathogenesis remains largely unknown,mainly because of a lack of understanding about their target mRNAs and pathways in neurons. To address this problem,in this study,we focused on a recently identified BD-associated but uncharacterized miRNA,miR-1908-5p. We identified and validated its novel target genes including DLGAP4,GRIN1,STX1A,CLSTN1 and GRM4,which all function in neuronal glutamatergic synapses. Moreover,bioinformatic analyses of human brain expression profiles revealed that the expression levels of miR-1908-5p and its synaptic target genes show an inverse-correlation in many brain regions. In our preliminary experiments,the expression of miR-1908-5p was increased after chronic treatment with valproate but not lithium in control human neural progenitor cells. In contrast,it was decreased by valproate in neural progenitor cells derived from dermal fibroblasts of a BD subject. Together,our results provide new insights into the potential role of miR-1908-5p in the pathogenesis of BD and also propose a hypothesis that neuronal synapses could be a key converging pathway of some BD-associated protein-coding genes and miRNAs. View Publication

According to the current model of adult hematopoiesis,differentiation of pluripotential hematopoietic stem cells into common myeloid- and lymphoid-committed progenitors establishes an early separation between the myeloid and lymphoid lineages. This report describes a rare and previously unidentified CD45R-CD19+ B cell progenitor population in postnatal bone marrow that can also generate macrophages. In addition to the definition of this B-lineage intermediate,the data indicate that a developmental relationship between the B and macrophage lineages is retained during postnatal hematopoiesis. View Publication

The compound BIRB796 inhibits the stress-activated protein kinases p38alpha and p38beta and is undergoing clinical trials for the treatment of inflammatory diseases. Here we report that BIRB796 also inhibits the activity and the activation of SAPK3/p38gamma. This occurs at higher concentrations of BIRB796 than those that inhibit p38alpha and p38beta and at lower concentrations than those that inhibit the activation of JNK isoforms. We also show that at these concentrations,BIRB796 blocks the stress-induced phosphorylation of the scaffold protein SAP97,further establishing that this is a physiological substrate of SAPK3/p38gamma. Our results demonstrate that BIRB796,in combination with SB203580,a compound that inhibits p38alpha and p38beta,but not the other p38 isoforms,can be used to identify physiological substrates of SAPK3/p38gamma as well as those of p38alpha and p38beta. View Publication

Bispecific antibodies directed against tumor-associated target antigens and to surface receptors mediating T-cell activation,such as the TCR/CD3 complex and the costimulatory receptor CD28,are capable of mediating T-cell activation resulting in tumor cell killing. In this study,we used the B-cell-associated antigens CD19 and CD20 as target structures on human leukemic cells. We found that a combination of bispecific antibody fragments (bsFab2) with target x CD3 and target x CD28 specificity induces vigorous autologous T-cell activation and killing of malignant cells in peripheral blood and bone marrow cultures from patients with chronic lymphocytic leukemia and follicular lymphoma. The bsFab2 targeting CD20 were considerably more effective than those binding to CD19. The colony-forming capacity of treated bone marrow was impaired due to large amounts of tumor necrosis factor alpha produced during bsFab2-induced T-cell activation. Neutralizing tumor necrosis factor alpha antibodies were found to reverse this negative effect without affecting T-cell activation and tumor cell killing. CD20 x CD28 bsFab2,when used alone rather than in combination,markedly improved the recognition of leukemic cells by allogeneic T cells. Therefore,these reagents may be capable of enhancing the immunogenicity of leukemic cells in general and,in particular,of increasing the antileukemic activity of allogeneic donor buffy coat cells in relapsed bone marrow transplanted patients. View Publication

The molecular mechanisms by which tumor cells metastasize to bone are likely to involve invasion,cell adhesion to bone,and the release of soluble mediators from tumor cells that stimulate osteoclast-mediated bone resorption. Bisphosphonates (BPs) are powerful inhibitors of the osteoclast activity and are,therefore,used in the treatment of patients with osteolytic metastases. However,an added beneficial effect of BPs may be direct antitumor activity. We previously reported that BPs inhibit breast and prostate carcinoma cell adhesion to bone (Boissier et al.,Cancer Res.,57: 3890-3894,1997). Here,we provided evidence that BP pretreatment of breast and prostate carcinoma cells inhibited tumor cell invasion in a dose-dependent manner. The order of potency for four BPs in inhibiting tumor cell invasion was: zoledronate textgreater ibandronate textgreater NE-10244 (active pyridinium analogue of risedronate) textgreater clodronate. In addition,NE-58051 (the inactive pyridylpropylidene analogue of risedronate) had no inhibitory effect,whereas NE-10790 (a phosphonocarboxylate analogue of risedronate in which one of the phosphonate groups is substituted by a carboxyl group) inhibited tumor cell invasion to an extent similar to that observed with NE-10244,indicating that the inhibitory activity of BPs on tumor cells involved the R2 chain of the molecule. BPs did not induce apoptosis in tumor cells,nor did they inhibit tumor cell migration at concentrations that did inhibit tumor cell invasion. However,although BPs did not interfere with the production of matrix metalloproteinases (MMPs) by tumor cells,they inhibited their proteolytic activity. The inhibitory effect of BPs on MMP activity was completely reversed in the presence of an excess of zinc. In addition,NE-10790 did not inhibit MMP activity,suggesting that phosphonate groups of BPs are responsible for the chelation of zinc and the subsequent inhibition of MMP activity. In conclusion,our results provide evidence for a direct cellular effect of BPs in preventing tumor cell invasion and an inhibitory effect of BPs on the proteolytic activity of MMPs through zinc chelation. These results suggest,therefore,that BPs may be useful agents for the prophylactic treatment of patients with cancers that are known to preferentially metastasize to bone. View Publication

Characterization and modeling of biological neural networks has emerged as a field driving significant advancements in our understanding of brain function and related pathologies. As of today,pharmacological treatments for neurological disorders remain limited,pushing the exploration of promising alternative approaches such as electroceutics. Recent research in bioelectronics and neuromorphic engineering have fostered the development of the new generation of neuroprostheses for brain repair. However,achieving their full potential necessitates a deeper understanding of biohybrid interaction. In this study,we present a novel real-time,biomimetic,cost-effective and user-friendly neural network capable of real-time emulation for biohybrid experiments. Our system facilitates the investigation and replication of biophysically detailed neural network dynamics while prioritizing cost-efficiency,flexibility and ease of use. We showcase the feasibility of conducting biohybrid experiments using standard biophysical interfaces and a variety of biological cells as well as real-time emulation of diverse network configurations. We envision our system as a crucial step towards the development of neuromorphic-based neuroprostheses for bioelectrical therapeutics,enabling seamless communication with biological networks on a comparable timescale. Its embedded real-time functionality enhances practicality and accessibility,amplifying its potential for real-world applications in biohybrid experiments. Beaubois et al. introduce a real-time biomimetic neural network for biohybrid experiments,providing a tool to study closed-loop applications for neuroscience and neuromorphic-based neuroprostheses. View Publication

Histologic variant (HV) subtypes of bladder cancer are clinically aggressive tumors that are more resistant to standard therapy compared to conventional urothelial carcinoma (UC). Little is known about the transcriptional programs that account for their biological differences. Here we show using single cell analysis that HVs harbor a tumor cell state characterized by expression of MUC16 (CA125),MUC4,and KRT24 . This cell state is enriched in metastases,predicted to be highly resistant to chemotherapy,and linked with poor survival. We also find enriched expression of TM4SF1,a transmembrane protein,in HV tumor cells. Chimeric antigen receptor (CAR) T cells engineered against TM4SF1 protein demonstrated in vitro and in vivo activity against bladder cancer cell lines in a TM4SF1 expression-dependent manner,highlighting its potential as a therapeutic target. Subject terms: Bladder cancer,Tumour biomarkers,Targeted therapies View Publication