技术资料

Epiblast stem cells (EpiSCs) are pluripotent cells derived from post-implantation late epiblasts in vitro. EpiSCs are incapable of contributing to chimerism,indicating that EpiSCs are less pluripotent and represent a later developmental pluripotency state compared with inner cell mass stage murine embryonic stem cells (mESCs). Using a chemical approach,we found that blockage of the TGFβ pathway or inhibition of histone demethylase LSD1 with small molecule inhibitors induced dramatic morphological changes in EpiSCs toward mESC phenotypes with simultaneous activation of inner cell mass-specific gene expression. However,full conversion of EpiSCs to the mESC-like state with chimerism competence could be readily generated only with the combination of LSD1,ALK5,MEK,FGFR,and GSK3 inhibitors. Our results demonstrate that appropriate synergy of epigenetic and signaling modulations could convert cells at the later developmental pluripotency state to the earlier mESC-like pluripotency state,providing new insights into pluripotency regulation. View Publication

Hematopoietic stem and progenitor cells (HSPCs) are critical for the treatment of blood diseases in clinic. However,the limited source of HSPCs severely hinders their clinical application. In the embryo,hematopoietic stem cells (HSCs) arise from hemogenic endothelial (HE) cells lining the major arteries in vivo. In this work,by engineering vascular niche endothelial cells (VN-ECs),we generated functional HSPCs in vitro from ECs at various sites,including the aorta-gonad-mesonephros (AGM) region and the placenta. Firstly,we converted mouse embryonic HE cells from the AGM region (aHE) into induced HSPCs (iHSPCs),which have the abilities for multilineage differentiation and self-renewal. Mechanistically,we found that VN-ECs can promote the generation of iHSPCs via secretion of CX3CL1 and IL1A. Next,through VN-EC co-culture,we showed that placental HE (pHE) cells,a type of extra-embryonic HE cells,were successfully converted into iHSPCs (pHE-iHSPCs),which have multilineage differentiation capacity,but exhibit limited self-renewal ability. Furthermore,comparative transcriptome analysis of aHE-iHSPCs and pHE-iHSPCs showed that aHE-iHSPCs highly expressed HSC-specific and self-renewal-related genes. Moreover,experimental validation showed that retinoic acid (RA) treatment promoted the transformation of pHE cells into iHSPCs that have self-renewal ability. Collectively,our results suggested that pHE cells possess the potential to transform into self-renewing iHSPCs through RA treatment,which will facilitate the clinical application of placental endothelial cells in hematopoietic cell generation. Subject terms: Haematopoietic stem cells,Haematopoietic stem cells View Publication

CD4+ T cells recognize antigens through their T cell receptors (TCRs); however,additional signals involving costimulatory receptors,for example,CD28,are required for proper T cell activation. Alternative costimulatory receptors have been proposed,including members of the Toll-like receptor (TLR) family,such as TLR5 and TLR2. To understand the molecular mechanism underlying a potential costimulatory role for TLR5,we generated detailed molecular maps and logical models for the TCR and TLR5 signaling pathways and a merged model for cross-interactions between the two pathways. Furthermore,we validated the resulting model by analyzing how T cells responded to the activation of these pathways alone or in combination,in terms of the activation of the transcriptional regulators CREB,AP-1 (c-Jun),and NF-kappaB (p65). Our merged model accurately predicted the experimental results,showing that the activation of TLR5 can play a similar role to that of CD28 activation with respect to AP-1,CREB,and NF-kappaB activation,thereby providing insights regarding the cross-regulation of these pathways in CD4+ T cells. View Publication

Haplo-cord stem cell transplantation combines the infusion of CD34 selected hematopoietic progenitors from a haplo-identical donor with an umbilical cord blood (UCB) graft from an unrelated donor and allows faster count recovery,with low rates of disease recurrence and chronic graft-versus-host disease (GVHD). But the contribution of the umbilical cord blood graft to long-term transplant outcome remains unclear. We analyzed 39 recipients of haplo-cord transplants with acute myeloid leukemia (AML) and myelodysplastic syndrome (MDS),engrafted and in remission at 2 months. Median age was 66 (18-72) and all had intermediate,high,or very-high risk disease. Less than 20% UCB chimerism in the CD33 lineage was associated with an increased rate of disease recurrence (54% versus 11% p textless 0.0001) and decrease in one year progression-free (20% versus 55%,p = 0.004) and overall survival (30% versus 62%,p = 0.02). Less than 100% UCB chimerism in the CD3 lineage was associated with increase rate of disease recurrence (46% versus 12%,p = 0.007). Persistent haplo-chimerism in the CD3 lineage was associated with an increased rate of disease recurrence (40% versus 15%,p = 0.009) Chimerism did not predict for treatment related mortality. The cumulative incidence of acute GVHD by day 100 was 43%. The cumulative incidence of moderate/severe chronic GVHD was only 5%. Engraftment of the umbilical cord blood grafts provides powerful graft-versus-leukemia (GVL) effects which protect against disease recurrence and is associated with low risk of chronic GVHD. Engraftment of CD34 selected haplo-identical cells can lead to rapid development of circulating T-cells,but when these cells dominate,GVL-effects are limited and rates of disease recurrence are high. View Publication

The small number of hematopoietic stem and progenitor cells in cord blood units limits their widespread use in human transplant protocols. We identified a family of chemically related small molecules that stimulates the expansion ex vivo of human cord blood cells capable of reconstituting human hematopoiesis for at least 6 months in immunocompromised mice. The potent activity of these newly identified compounds,UM171 being the prototype,is independent of suppression of the aryl hydrocarbon receptor,which targets cells with more-limited regenerative potential. The properties of UM171 make it a potential candidate for hematopoietic stem cell transplantation and gene therapy. View Publication

The cornea is the transparent outermost surface of the eye,consisting of a stratified epithelium,a collagenous stroma and an innermost single-cell layered endothelium and providing 2/3 of the refractive power of the eye. Multiple diseases of the cornea arise from genetic defects where the ultimate phenotype can be influenced by cross talk between the cell types and the extracellular matrix. Cell culture modeling of diseases can benefit from cornea organoids that include multiple corneal cell types and extracellular matrices. Here we present human iPS cell-derived organoids through sequential rounds of differentiation programs. These organoids share features of the developing cornea,harboring three distinct cell types with expression of key epithelial,stromal and endothelial cell markers. Cornea organoid cultures provide a powerful 3D model system for investigating corneal developmental processes and their disruptions in diseased conditions. View Publication

Splice-site mutations in the beta-globin gene can lead to aberrant transcripts and decreased functional beta-globin,causing beta-thalassemia. Triplex-forming DNA oligonucleotides (TFOs) and peptide nucleic acids (PNAs) have been shown to stimulate recombination in reporter gene loci in mammalian cells via site-specific binding and creation of altered helical structures that provoke DNA repair. We have designed a series of triplex-forming PNAs that can specifically bind to sequences in the human beta-globin gene. We demonstrate here that these PNAs,when cotransfected with recombinatory donor DNA fragments,can promote single base-pair modification at the start of the second intron of the beta-globin gene,the site of a common thalassemia-associated mutation. This single base pair change was detected by the restoration of proper splicing of transcripts produced from a green fluorescent protein-beta-globin fusion gene. The ability of these PNAs to induce recombination was dependent on dose,sequence,cell-cycle stage,and the presence of a homologous donor DNA molecule. Enhanced recombination,with frequencies up to 0.4%,was observed with use of the lysomotropic agent chloroquine. Finally,we demonstrate that these PNAs were effective in stimulating the modification of the endogenous beta-globin locus in human cells,including primary hematopoietic progenitor cells. This work suggests that PNAs can be effective tools to induce heritable,site-specific modification of disease-related genes in human cells. View Publication

Autosomal recessive osteopetrosis (ARO) is a rare genetic disease,characterized by increased bone density due to defective osteoclast function. Most of the cases are due to TCIRG1 gene mutation,leading to severe bone phenotype and death in the first years of life. The standard therapy is the hematopoietic stem cell transplantation (HSCT),but its success is limited by several constraints. Conversely,gene therapy (GT) could minimize the immune-mediated complications of allogeneic HSCT and offer a prompt treatment to these patients. The Tcirg1 -defective oc/oc mouse model displays a short lifespan and high bone density,closely mirroring the human condition. In this work,we exploited the oc/oc neonate mice to optimize the critical steps for a successful therapy. First,we showed that lentiviral vector GT can revert the osteopetrotic bone phenotype,allowing long-term survival and reducing extramedullary haematopoiesis. Then,we demonstrated that plerixafor-induced mobilization can further increase the high number of HSPCs circulating in peripheral blood,facilitating the collection of adequate numbers of cells for therapeutic purposes. Finally,pre-transplant non-genotoxic conditioning allowed the stable engraftment of HSPCs,albeit at lower level than conventional total body irradiation,and led to long-term survival and correction of bone phenotype,in the absence of acute toxicity. These results will pave the way to the implementation of an effective GT protocol,reducing the transplant-related complication risks in the very young and severely affected ARO patients. View Publication

The abnormal trafficking of CD34+ cells is a unique characteristic of primary myelofibrosis (PMF). We have further studied the behavior of PMF CD34+ cells by examining their homing to the marrow and the spleens of nonobese diabetic/severe combined immunodeficient (NOD/SCID) mice. Following the infusion of PMF and normal granulocyte colony-stimulating factor-mobilized peripheral blood (mPB) CD34+ cells into NOD/SCID mice,reduced numbers of PMF CD34+ cells and granulocyte-macrophage colony-forming unit (CFU-GM) compared with mPB were detected in the marrow of these mice,whereas similar numbers of PMF and mPB CD34+ cells and CFU-GM homed to their spleens. The abnormal homing of PMF CD34+ cells was associated with reduced expression of CXCR4,but was not related to the presence of JAK2V617F. The sequential treatment of PMF CD34+ cells with the chromatin-modifying agents 5-aza-2'-deoxycytidine (5azaD) and trichostatin A (TSA),but not treatment with small molecule inhibitors of JAK2,resulted in the generation of increased numbers of CD34+CXCR4+ cells,which was accompanied by enhanced homing of PMF CD34+ cells to the marrow but not the spleens of NOD/SCID mice. Following 5azaD/TSA treatment,JAK2V617F-negative PMF hematopoietic progenitor cells preferentially homed to the marrow but not the spleens of recipient mice. Our data suggest that PMF CD34+ cells are characterized by a reduced ability to home to the marrow but not the spleens of NOD/SCID mice and that this homing defect can be corrected by sequential treatment with chromatin-modifying agents. View Publication

Canine leukocyte adhesion deficiency (CLAD) represents the canine counter-part of the human disease leukocyte adhesion deficiency (LAD). Defects in the leukocyte integrin CD18 adhesion molecule in both CLAD and LAD lead to recurrent,life-threatening bacterial infections. We evaluated ex vivo retroviral-mediated gene therapy in CLAD using 2 nonmyeloablative conditioning regimens--200 cGy total body irradiation (TBI) or 10 mg/kg busulfan--with or without posttransplantation immunosuppression. In 6 of 11 treated CLAD dogs,therapeutic levels of CD18(+) leukocytes were achieved. Conditioning with either TBI or busulfan allowed long-term engraftment,and immunosuppression was not required for efficacy. The percentage of CD18(+) leukocytes in the peripheral blood progressively increased over 6 to 8 months after infusion to levels ranging from 1.26% to 8.37% at 1-year follow-up in the 6 dogs. These levels resulted in reversal or moderation of the severe CLAD phenotype. Linear amplification-mediated polymerase chain reaction assays indicated polyclonality of insertion sites. These results describe ex vivo hematopoietic stem cell gene transfer in a disease-specific,large animal model using 2 clinically applicable conditioning regimens,and they provide support for the use of nonmyeloablative conditioning regimens in preclinical protocols of retroviral-mediated gene transfer for nonmalignant hematopoietic diseases such as LAD. View Publication

Inter-alpha inhibitor protein (IalphaIp) is an endogenous serine protease inhibitor in human plasma. Circulating IalphaIp levels were lower in 51 patients with severe sepsis than in healthy volunteers. Mean levels were 688+/-295 mg/L in patients with severe sepsis who survived (n=32),486+/-193 mg/L in patients with sepsis who died (n=19),and 872+/-234 mg/L in control subjects (n=25). IalphaIp levels were lower in patients with shock versus those without (540+/-246 [n=33] vs. 746+/-290 [n=18] mg/L; P=.0102). IalphaIp levels were inversely correlated with 28-day mortality rates and Acute Physiology and Chronic Health Evaluation II scores and directly correlated with antithrombin III,protein C,and protein S levels. The administration of IalphaIp (30 mg/kg body weight intravenously) increased the 50% lethal dose in mice by 100-fold after an intravenous challenge of Escherichia coli. Thus,human IalphaIp may be a useful predictive marker and potential therapeutic agent in sepsis. View Publication

To further clarify the transformation from monoclonal gammopathy of undetermined significance (MGUS) to plasma cell myeloma (PCM),we compared interphase fluorescence in situ hybridization (FISH) patterns in 381 MGUS and 301 PCM patients. According to the World Health Organization and the International Myeloma Working Group,a threshold of 10% of bone marrow plasma cells separated MGUS from PCM. After magnetic activated cell sorting for CD138(+) cells,FISH succeeded in 272 of 301 (90.4%) PCM,but in only 302 of 381 (79.3%) MGUS cases (P textless 0.001). Cytogenetic alterations were more frequent in PCM (237 of 272; 87.1%) than MGUS (169 of 302; 56.0%; P = 0.0002). PCM showed a median of two cytogenetic alterations (range,0-9) and MGUS one (range,0-6). Considering only cases with a yield of plasma cells allowing five or more FISH probes,del(13)(q14) was found in 99 of 251 (39.3%) PCM but in only 59 of 267 (22.1%) MGUS (P = 0.0001),del(17p) in 15 PCM (6.0%) and in 6 MGUS (2.2%) patients (P = 0.029). A t(4;14)/IGH-FGFR3 was detected in 28 PCM (11.1%) and 5 MGUS (1.9%; P textless 0.001). The t(11;14)/IGH-CCND1 and the t(14;16)/IGH-MAF showed no significant differences. Cytomorphology detected higher numbers of plasma cells than multiparameter flow cytometry (median ratio 4.25). This study underlines the genetic heterogeneity of MGUS similar to PCM. Genetic analysis might contribute to more diversified monitoring strategies for MGUS patients. View Publication