技术资料

Although hematopoietic stem cells (HSC) are the best characterized and the most clinically used adult stem cells,efforts are still needed to understand how to best ex vivo expand these cells. Here we present our unexpected finding that OCT4 is involved in the enhancement of cytokine-induced expansion capabilities of human cord blood (CB) HSC. Activation of OCT4 by Oct4-activating compound 1 (OAC1) in CB CD34(+) cells enhanced ex vivo expansion of HSC,as determined by a rigorously defined set of markers for human HSC,and in vivo short-term and long-term repopulating ability in NSG mice. Limiting dilution analysis revealed that OAC1 treatment resulted in 3.5-fold increase in the number of SCID repopulating cells (SRCs) compared with that in day 0 uncultured CD34(+) cells and 6.3-fold increase compared with that in cells treated with control vehicle. Hematopoietic progenitor cells,as assessed by in vitro colony formation,were also enhanced. Furthermore,we showed that OAC1 treatment led to OCT4-mediated upregulation of HOXB4. Consistently,siRNA-mediated knockdown of HOXB4 expression suppressed effects of OAC1 on ex vivo expansion of HSC. Our study has identified the OCT4-HOXB4 axis in ex vivo expansion of human CB HSC. View Publication

Severe congenital neutropenia (SCN) is often associated with inherited heterozygous point mutations in ELANE,which encodes neutrophil elastase (NE). However,a lack of appropriate models to recapitulate SCN has substantially hampered the understanding of the genetic etiology and pathobiology of this disease. To this end,we generated both normal and SCN patient-derived induced pluripotent stem cells (iPSCs),and performed genome editing and differentiation protocols that recapitulate the major features of granulopoiesis. Pathogenesis of ELANE point mutations was the result of promyelocyte death and differentiation arrest,and was associated with NE mislocalization and activation of the unfolded protein response/ER stress (UPR/ER stress). Similarly,high-dose G-CSF (or downstream signaling through AKT/BCL2) rescues the dysgranulopoietic defect in SCN patient-derived iPSCs through C/EBP$$-dependent emergency granulopoiesis. In contrast,sivelestat,an NE-specific small-molecule inhibitor,corrected dysgranulopoiesis by restoring normal intracellular NE localization in primary granules; ameliorating UPR/ER stress; increasing expression of CEBPA,but not CEBPB; and promoting promyelocyte survival and differentiation. Together,these data suggest that SCN disease pathogenesis includes NE mislocalization,which in turn triggers dysfunctional survival signaling and UPR/ER stress. This paradigm has the potential to be clinically exploited to achieve therapeutic responses using lower doses of G-CSF combined with targeting to correct NE mislocalization. View Publication

Cardiomyocytes derived from human induced pluripotent stem cells (iPSC) show great promise as autologous donor cells to treat heart disease. A major technical obstacle to this approach is that available induction methods often produce heterogeneous cell population with low percentage of cardiomyocytes. Here we describe a cardiac enrichment approach using non-integrating adeno-associated virus (AAV). We first examined several AAV serotypes for their ability to selectively transduce iPSC-derived cardiomyocytes. Result showed that AAV1 demonstrated the highest in vitro transduction efficiency among seven widely used serotypes. Next differentiated iPSC derivatives were transduced with drug-selectable AAV1 expressing neomycin resistance gene. Selection with G418 enriched the cardiac cell fraction from 27% to 57% in two weeks. Compared to other enrichment strategies such as integrative genetic selection,mitochondria labeling or surface marker cell sorting,this simple AAV method described herein bypasses antibody or dye labeling. These findings provide proof-of-concept for large-scale cardiomyocyte enrichment by exploiting AAV's intrinsic tissue tropism. View Publication

Tissue morphogenesis and organ formation are the consequences of biochemical and biophysical cues that lead to cellular spatial patterning in development. To model such events in vitro,we use PEG-patterned substrates to geometrically confine human pluripotent stem cell colonies and spatially present mechanical stress. Modulation of the WNT/β-catenin pathway promotes spatial patterning via geometric confinement of the cell condensation process during epithelial-mesenchymal transition,forcing cells at the perimeter to express an OCT4+ annulus,which is coincident with a region of higher cell density and E-cadherin expression. The biochemical and biophysical cues synergistically induce self-organizing lineage specification and creation of a beating human cardiac microchamber confined by the pattern geometry. These highly defined human cardiac microchambers can be used to study aspects of embryonic spatial patterning,early cardiac development and drug-induced developmental toxicity. View Publication

Pluripotent stem cell (PSC) usage in heart regenerative medicine requires producing enriched cardiomyocytes (CMs) with mature phenotypes in a defined medium. However,current methods are typically performed in 2D environments that produce immature CMs. Here we report a simple,growth factor-free 3D culture system to rapidly and efficiently generate 85.07 ± 1.8% of spontaneously contractile cardiac spheres (scCDSs) using 3D-cultured human and monkey PSC-spheres. Along with small molecule-based 3D induction,this protocol produces CDSs of up to 95.7% CMs at a yield of up to 237 CMs for every input pluripotent cell,is effective for human and monkey PSCs,and maintains 81.03 ± 12.43% of CDSs in spontaneous contractibility for over three months. These CDSs displayed CM ultrastructure,calcium transient,appropriate pharmacological responses and CM gene expression profiles specific for maturity. Furthermore,3D-derived CMs displayed more mature phenotypes than those from a parallel 2D-culture. The system is compatible to large-scaly produce CMs for disease study,cell therapy and pharmaceutics screening. View Publication

Regulatory T cells (Tregs) are physiologically designed to prevent autoimmune disease and maintain self-tolerance. In tumour microenvironments,their presence is related to a poor prognosis,and they influence the therapeutic outcome due to their capacity to suppress the immune response by cell-cell contact and to release immunosuppressive cytokines. In this study,we demonstrate that Treg immunosuppressive activity can be modulated by the cross-linking between the CD45RA expressed by Tregs and the C-type lectin MGL. This specific interaction strongly decreases the immunosuppressive activity of Tregs,restoring the proliferative capacity of co-cultured T lymphocytes. This effect can be attributed to changes in CD45RA and TCR signalling through the inhibition of Lck and inactivation of Zap-70,an increase in the Foxp3 methylation status and,ultimately,the reduced production of suppressive cytokines. These results indicate a role of MGL as an immunomodulator within the tumour microenvironment interfering with Treg functions,suggesting its possible use in the design of anticancer vaccines. View Publication

Stem cells integrate inputs from multiple sources. Stem cell niches provide signals that promote stem cell maintenance,while differentiated daughter cells are known to provide feedback signals to regulate stem cell replication and differentiation. Recently,stem cells have been shown to regulate themselves using an autocrine mechanism. The existence of a 'stem cell niche' was first postulated by Schofield in 1978 to define local environments necessary for the maintenance of haematopoietic stem cells. Since then,an increasing body of work has focused on defining stem cell niches. Yet little is known about how progenitor cell and differentiated cell numbers and proportions are maintained. In the airway epithelium,basal cells function as stem/progenitor cells that can both self-renew and produce differentiated secretory cells and ciliated cells. Secretory cells also act as transit-amplifying cells that eventually differentiate into post-mitotic ciliated cells . Here we describe a mode of cell regulation in which adult mammalian stem/progenitor cells relay a forward signal to their own progeny. Surprisingly,this forward signal is shown to be necessary for daughter cell maintenance. Using a combination of cell ablation,lineage tracing and signalling pathway modulation,we show that airway basal stem/progenitor cells continuously supply a Notch ligand to their daughter secretory cells. Without these forward signals,the secretory progenitor cell pool fails to be maintained and secretory cells execute a terminal differentiation program and convert into ciliated cells. Thus,a parent stem/progenitor cell can serve as a functional daughter cell niche. View Publication

BACKGROUND Haemolytic infection lyses red blood cells,releasing haemoglobin (Hb) into the plasma. Although recent studies showed that immune cells recognize redox-active cytotoxic extracellular Hb (metHb) bound to pathogen-associated molecular patterns (PAMPs),currently available information is limited to experiments performed in defined conditions using single cell lines. Therefore,a systemic approach targeting primary whole blood cells is required to better understand the cellular immune defence against metHb and PAMPs,when under a haemolytic infection. METHODS We investigated how human white blood cells,including neutrophils,respond to metHb and lipoteichoic acid (LTA) by measuring reactive oxygen species (ROS),signalling mediators (ERK and p38),NF-κB,cytokines,elastase secretion and cell activation markers. FINDINGS metHb activates NF-κB in TLR2-expressing HEK293 cells but not in normal or TLR9-expressing HEK293 cells. Treatment of isolated neutrophils with metHb increased production of ROS and expressions of IL-8,TNFα,and CD11b,which were further enhanced by metHb + LTA complex. While LTA stimulated the survival of neutrophils,it caused apoptotic cell death when complexed with metHb. The activation of neutrophils by metHb + LTA was subdued by the presence of other types of white blood cells. INTERPRETATION metHb and metHb + LTA complex are ligands of TLR2,inducing an unconventional TLR signalling pathway. Neutrophils are a highly sensitive cell type to metHb + LTA complex. During a haemolytic infection,white blood cells in the vicinity crosstalk to modulate neutrophil TLR-signalling induced by metHb and LTA. View Publication

In the CNS,oligodendrocytes act as the myelinating cells. Oligodendrocytes have been identified to be key players in several neurodegenerative disorders. This protocol describes a robust,fast and reproducible differentiation protocol to generate human oligodendrocytes from pluripotent stem cells (PSCs) using a chemically defined,growth factor-rich medium. Within 8 d,PSCs differentiate into paired box 6-positive (PAX6(+)) neural stem cells,which give rise to OLIG2(+) progenitors by day 12. Oligodendrocyte lineage transcription factor 2-positive (OLIG2(+)) cells begin to express the transcription factor NKX2.2 around day 18,followed by SRY-box 10 (SOX10) around day 40. Oligodendrocyte progenitor cells (OPCs) that are positive for the cell surface antigen recognized by the O4 antibody (O4(+)) appear around day 50 and reach,on average,43% of the cell population after 75 d of differentiation. O4(+) OPCs can be isolated by cell sorting for myelination studies,or they can be terminally differentiated to myelin basic protein-positive (MBP(+)) oligodendrocytes. This protocol also describes an alternative strategy for markedly reducing the length and the costs of the differentiation and generating ∼30% O4(+) cells after only 55 d of culture. View Publication

The biological effects of low-dose ionizing radiation (LDIR) exposure in humans are not comprehensively understood,generating a high degree of controversy in published literature. The earliest stages of human development are known to be among the most sensitive to stress exposures,especially genotoxic stresses. However,the risks stemming from exposure to LDIR,particularly within the clinical diagnostic relevant dose range,have not been directly evaluated in human embryonic stem cells (hESCs). Here,we describe the dynamics of the whole genome transcriptional responses of different hESC lines to both LDIR and,as a reference,high-dose IR (HDIR). We found that even doses as low as 0.05 Gy could trigger statistically significant transient changes in a rather limited subset of genes in all hESCs lines examined. Gene expression signatures of hESCs exposed to IR appear to be highly dose-,time-,and cell line-dependent. We identified 50 genes constituting consensus gene expression signature as an early response to HDIR across all lines of hESC examined. We observed substantial differences in biological pathways affected by either LDIR or HDIR in hESCs,suggesting that the molecular mechanisms underpinning the responses of hESC may fundamentally differ depending on radiation doses. View Publication

PURPOSE To investigate the effects and mechanisms of fasudil hydrochloride (fasudil) on and in alkali burn-induced corneal neovascularization (CNV) in mice. METHODS To observe the effect of fasudil,mice with alkali-burned corneas were treated with either fasudil eye drops or phosphate-buffered saline (PBS) four times per day for 14 consecutive days. After injury,CNV and corneal epithelial defects were measured. The production of reactive oxygen species (ROS) and heme oxygenase-1(HO-1) was measured. The infiltration of polymorphonuclear neutrophils (PMNs) and the mRNA expressions of CNV-related genes were analyzed on day 14. RESULTS The incidence of CNV was significantly lower after treatment with 100 μM and 300 μM fasudil than with PBS,especially with 100 μM fasudil. Meanwhile,the incidences of corneal epithelial defects was lower (n=15,all ptextless0.01). After treatment with 100 μM fasudil,the intensity of DHE fluorescence was reduced in the corneal epithelium and stroma than with PBS treatment (n=5,all ptextless0.01),and the number of filtrated PMNs decreased. There were significant differences between the expressions of VEGF,TNF-a,MMP-8,and MMP-9 in the 100 μM fasudil group and the PBS group (n=8,all ptextless0.05). The production of HO-1 protein in the 100 μM fasudil group was 1.52±0.34 times more than in the PBS group (n=5 sample,ptextless0.05). CONCLUSIONS 100 μM fasudil eye drops administered four times daily can significantly inhibit alkali burn-induced CNV and promote the healing of corneal epithelial defects in mice. These effects are attributed to a decrease in inflammatory cell infiltration,reduction of ROS,and upregulation of HO-1 protein after fasudil treatment. View Publication

Reprogramming of patient cells to human induced pluripotent stem cells (hiPSC) has facilitated in vitro disease modeling studies aiming at deciphering the molecular and cellular mechanisms that contribute to disease pathogenesis and progression. To fully exploit the potential of hiPSC for biomedical applications,technologies that enable the standardized generation and expansion of hiPSC from large numbers of donors are required. Paralleled automated processes for the expansion of hiPSC could provide an opportunity to maximize the generation of hiPSC collections from patient cohorts while minimizing hands-on time and costs. In order to develop a simple method for the parallel expansion of human pluripotent stem cells (hPSC) we established a protocol for their cultivation as undifferentiated aggregates in a bench-top bioreactor system (BioLevitator™). We show that long-term expansion (10 passages) of hPSCs either in mTeSR or E8 medium preserved a normal karyotype,three-germ-layer differentiation potential and high expression of pluripotency-associated markers. The system enables the expansion from low inoculation densities (0.3 × 10(5) cells/mL) and provides a simplified,cost-efficient and time-saving method for the provision of hiPSC at midi-scale. Implementation of this protocol in cell production schemes has the potential to advance cell manufacturing in many areas of hiPSC-based medical research. View Publication