技术资料

The heart wall tissue,or the myocardium,is one of the main targets in cardiovascular disease prevention and treatment. Animal models have not been sufficient in mimicking the human myocardium as evident by the very low clinical translation rates of cardiovascular drugs. Additionally,current in vitro models of the human myocardium possess several shortcomings such as lack of physiologically relevant co-culture of myocardial cells,lack of a 3D biomimetic environment,and the use of non-human cells. In this study,we address these shortcomings through the design and manufacture of a myocardium-on-chip (MOC) using 3D cell-laden hydrogel constructs and human induced pluripotent stem cell (hiPSC) derived myocardial cells. The MOC utilizes 3D spatially controlled co-culture of hiPSC derived cardiomyocytes (iCMs) and hiPSC derived endothelial cells (iECs) integrated among iCMs as well as in capillary-like side channels,to better mimic the microvasculature seen in native myocardium. We first fully characterized iCMs using immunostaining,genetic,and electrochemical analysis and iECs through immunostaining and alignment analysis to ensure their functionality,and then seeded these cells sequentially into the MOC device. We showed that iECs could be cultured within the microfluidic device without losing their phenotypic lineage commitment,and align with the flow upon physiological level shear stresses. We were able to incorporate iCMs within the device in a spatially controlled manner with the help of photocrosslinkable polymers. The iCMs were shown to be viable and functional within the device up to 7 days,and were integrated with the iECs. The iCMs and iECs in this study were derived from the same hiPSC cell line,essentially mimicking the myocardium of an individual human patient. Such devices are essential for personalized medicine studies where the individual drug response of patients with different genetic backgrounds can be tested in a physiologically relevant manner. View Publication

Many breast cancer deaths result from tumors acquiring resistance to available therapies. Thus,new therapeutic agents are needed for targeting drug-resistant breast cancers. Drug-refractory breast cancers include HER2+ tumors that have acquired resistance to HER2-targeted antibodies and kinase inhibitors,and Triple-Negative" Breast Cancers (TNBCs) that lack the therapeutic targets Estrogen Receptor� View Publication

The spinal cord consists of multiple neuronal cell types that are critical to motor control and arise from distinct progenitor domains in the developing neural tube. Excitatory V2a interneurons in particular are an integral component of central pattern generators that control respiration and locomotion; however,the lack of a robust source of human V2a interneurons limits the ability to molecularly profile these cells and examine their therapeutic potential to treat spinal cord injury (SCI). Here,we report the directed differentiation of CHX10(+) V2a interneurons from human pluripotent stem cells (hPSCs). Signaling pathways (retinoic acid,sonic hedgehog,and Notch) that pattern the neural tube were sequentially perturbed to identify an optimized combination of small molecules that yielded ∼25% CHX10(+) cells in four hPSC lines. Differentiated cultures expressed much higher levels of V2a phenotypic markers (CHX10 and SOX14) than other neural lineage markers. Over time,CHX10(+) cells expressed neuronal markers [neurofilament,NeuN,and vesicular glutamate transporter 2 (VGlut2)],and cultures exhibited increased action potential frequency. Single-cell RNAseq analysis confirmed CHX10(+) cells within the differentiated population,which consisted primarily of neurons with some glial and neural progenitor cells. At 2 wk after transplantation into the spinal cord of mice,hPSC-derived V2a cultures survived at the site of injection,coexpressed NeuN and VGlut2,extended neurites textgreater5 mm,and formed putative synapses with host neurons. These results provide a description of V2a interneurons differentiated from hPSCs that may be used to model central nervous system development and serve as a potential cell therapy for SCI. View Publication

The authors surveyed whole-exome and RNA-sequencing data from 252 unique pluripotent stem cell lines,some of which are in the pipeline for clinical use,and found that approximately 5{\%} of cell lines had acquired mutations in the TP53 gene that allow mutant cells to rapidly outcompete non-mutant cells,but do not prevent differentiation. View Publication

Exosomes are cell-derived nanovesicles that hold promise as living vehicles for intracellular delivery of therapeutics to mammalian cells. This potential,however,is undermined by the lack of effective methods to load exosomes with therapeutic proteins and to facilitate their uptake by target cells. Here,we demonstrate how a vesicular stomatitis virus glycoprotein (VSVG) can both load protein cargo onto exosomes and increase their delivery ability via a pseudotyping mechanism. By fusing a set of fluorescent and luminescent reporters with VSVG,we show the successful targeting and incorporation of VSVG fusions into exosomes by gene transfection and fluorescence tracking. We subsequently validate our system by live cell imaging of VSVG and its participation in endosomes/exosomes that are ultimately released from transfected HEK293 cells. We show that VSVG pseudotyping of exosomes does not affect the size or distributions of the exosomes,and both the full-length VSVG and the VSVG without the ectodomain are shown to integrate into the exosomal membrane,suggesting that the ectodomain is not required for protein loading. Finally,exosomes pseudotyped with full-length VSVG are internalized by multiple-recipient cell types to a greater degree compared to exosomes loaded with VSVG without the ectodomain,confirming a role of the ectodomain in cell tropism. In summary,our work introduces a new genetically encoded pseudotyping platform to load and enhance the intracellular delivery of therapeutic proteins via exosome-based vehicles to target cells. View Publication

The high risk of neonatal death from sepsis is thought to result from impaired responses by innate immune cells; however,the clinical observation of hyperinflammatory courses of neonatal sepsis contradicts this concept. Using transcriptomic,epigenetic and immunological approaches,we demonstrated that high amounts of the perinatal alarmins S100A8 and S100A9 specifically altered MyD88-dependent proinflammatory gene programs. S100 programming prevented hyperinflammatory responses without impairing pathogen defense. TRIF-adaptor-dependent regulatory genes remained unaffected by perinatal S100 programming and responded strongly to lipopolysaccharide,but were barely expressed. Steady-state expression of TRIF-dependent genes increased only gradually during the first year of life in human neonates,shifting immune regulation toward the adult phenotype. Disruption of this critical sequence of transient alarmin programming and subsequent reprogramming of regulatory pathways increased the risk of hyperinflammation and sepsis. Collectively these data suggest that neonates are characterized by a selective,transient microbial unresponsiveness that prevents harmful hyperinflammation in the delicate neonate while allowing for sufficient immunological protection. View Publication

Recent studies using defined transcription factors to convert skin fibroblasts into chondrocytes have raised the question of whether osteo-chondroprogenitors expressing SOX9 and RUNX2 could also be generated during the course of the reprogramming process. Here,we demonstrated that doxycycline-inducible expression of reprogramming factors (KLF4 [K] and c-MYC [M]) for 6 days were sufficient to convert murine fibroblasts into SOX9(+)/RUNX2(+) cellular aggregates and together with SOX9 (S) promoted the conversion efficiency when cultured in a defined stem cell medium,mTeSR. KMS-reprogrammed cells possess gene expression profiles akin to those of native osteo-chondroprogenitors with elevated osteogenic properties and can differentiate into osteoblasts and chondrocytes in vitro,but form bone tissue upon transplantation under the skin and in the fracture site of mouse tibia. Altogether,we provide a reprogramming strategy to enable efficient derivation of osteo-chondrogenic cells that may hold promise for cell replacement therapy not limited to cartilage but also for bone tissues. View Publication

Genome-wide association studies (GWASs) have identified many disease-associated variant alleles,but understanding whether and how different genes/loci interact requires a platform for probing how the variant alleles act mechanistically. Isogenic mutant human embryonic stem cells (hESCs) provide an unlimited resource to derive and study human disease-relevant cells. Here,we focused on CDKAL1,linked by GWASs to diabetes. Through transcript profiling,we find that expression of the metallothionein (MT) gene family,also linked by GWASs to diabetes,is significantly downregulated in CDKAL1(-/-) cells that have been differentiated to insulin-expressing pancreatic beta-like cells. Forced MT1E expression rescues both hypersensitivity of CDKAL1 mutant cells to glycolipotoxicity and pancreatic beta-cell dysfunction in vitro and in vivo. MT1E functions at least in part through relief of ER stress. This study establishes an isogenic hESC-based platform to study the interaction of GWAS-identified diabetes gene variants and illuminate the molecular network impacting disease progression. View Publication

Under appropriate conditions in culture,embryonic stem cells will differentiate and form embryoid bodies that have been shown to contain cells of the hematopoietic,endothelial,muscle and neuronal lineages. Many aspects of the lineage-specific differentiation programs observed within the embryoid bodies reflect those found in the embryo,indicating that this model system provides access to early cell populations that develop in a normal fashion. Recent studies involving the differentiation of genetically altered embryonic stem cells highlight the potential of this in vitro differentiation system for defining the function of genes in early development. View Publication

Ligand-dependent chimeric Cre recombinases are powerful tools to induce specific DNA rearrangements in cultured cells and in mice. We report here the construction and characterization of a series of chimeric recombinases,each consisting of Cre fused to a mutated human oestrogen receptor (ER) ligand-binding domain (LBD). Two new ligand-dependent recombinases which contain either the G400V/M543A/L544A or the G400V/L539A/L540A triple mutation of the human ER LBD are efficiently induced by the synthetic ER antagonists 4-hydroxytamoxifen (OHT) and ICI 182,780 (ICI),respectively,but are insensitive to 17 beta-oestradiol (E2). Both chimeric recombinases should be useful for efficient spatio-temporally controlled site-directed somatic mutagenesis. View Publication

Adult-derived hippocampal progenitors generate neurons,astrocytes,and oligodendrocytes in vitro and following grafting into the adult brain. Although these progenitors have a considerable capacity for in vitro self renewal,it is not known if each lineage is generated by separate committed precursors or by multipotent stem cells. By genetic marking,we have followed individual cells through the process of proliferative expansion,commitment,and differentiation. All three lineages are generated by single marked cells and the relative proportions of each lineage can be strongly influenced by environmental cues. Differentiation is accompanied by a characteristic progression of lineage-specific markers and can be potentiated by retinoic acid,elevated cyclic AMP,or neurotrophic factors. The ability to genetically mark and clone normal diploid hippocampal progenitors provides the first definitive evidence that multipotent neural stem cells exist outside of the adult striatal subventricular zone and supports the hypothesis that FGF-2-responsive neural stem cells may be broadly distributed in the adult brain. View Publication

We have identified three non-cross-reacting anti-human CD44 monoclonal antibodies that have significant positive or negative (or no) effects on normal human haemopoiesis in the long-term culture (LTC) system. These effects manifested as increases or decreases in the number of LTC-initiating cells (LTC-IC),and the number of colony-forming cells (CFC) recovered from cultures in which either unseparated or highly purified CD34+ CD38- normal marrow cells were placed on pre-established normal marrow feeder layers in the presence or absence of each antibody. The effects seen were rapid and sustained,and dependent on the presence of a preformed feeder layer. Interestingly,the same anti-CD44 antibodies had no effect on the maintenance of leukaemic (Ph+) progenitors (from patients with chronic myeloid leukaemia) when these cells were cultured on preformed feeder layers established from normal marrow. CD44 appears to be part of a mechanism by which stromal elements can regulate primitive normal haemopoietic cells but not their leukaemic (Ph+) counterparts. View Publication