技术资料

The proliferation and differentiation effects of the synthetic retinoid TTNPB and of 13-cis retinoic acid (RA) on hemopoietic progenitors from bone marrow of myelodysplastic syndrome (MDS) patients were compared. The addition of TTNPB or RA to culture plates containing MDS patient's marrow cells stimulated myeloid colony (CFU-C) growth and caused a significant increase in granulocytic colonies (CFU-G). In the presence of RA the increase in CFU-G was statistically insignificant. Cellular differentiation studies in liquid suspension culture revealed that the two retinoic acid analogues cause a marked decrease in immature granulocytes and an increase in mature granulocytes. There was further an increase in the number of cells that reacted positively with monoclonal antibodies (McAb) binding specifically to granulocytes (B4,3,B13,9 and Leu M4) and a decrease in the percentage of cells reacting with the McAb against Ia-like determinants. These findings indicate that TTNPB is as active as RA in stimulating the growth of hemopoietic progenitors from MDS patients and in enhancing granulocytic differentiation in liquid culture. View Publication

Twenty-four patients with acute promyelocytic leukemia (APL) were treated with all-trans retinoic acid (45 to 100 mg/m2/day). Of these,eight cases had been either nonresponsive or resistant to previous chemotherapy; the other 16 cases were previously untreated. All patients attained complete remission without developing bone marrow hypoplasia. Bone marrow suspension cultures were studied in 15 of the 24 patients. Fourteen of these patients had morphological maturation in response to the retinoic acid (1 mumol/L). Chloroacetate esterase and alpha-naphthyl acetate esterase staining as well as electronmicroscopic examination confirmed that retinoic acid-induced cells differentiated to granulocytes with increased functional maturation (as measured by nitroblue tetrazolium reduction,NBT). The single nonresponder to retinoic acid in vitro was resistant to treatment with retinoic acid but attained complete remission after addition of low-dose cytosine arabinoside (ara-C). During the course of therapy,none of the patients showed any abnormalities in the coagulation parameters we measured,suggesting an absence of any subclinical disseminated intravascular coagulation. The only side effects consisted of mild dryness of the lips and skin,with occasional headaches and digestive symptoms. Eight patients have relapsed after 2 to 5 months of complete remission. The others remain in complete remission at 1+ to 11+ months and are still being followed up. We conclude that all-trans retinoic acid is an effective inducer for attaining complete remission in APL. View Publication

A liquid culture system is described whereby proliferation of haemopoietic stem cells (CFU-S),production of granulocyte precursor cells (CFU-C),and extensive granulopoiesis can be maintained in vetro for several months. Such cultures consist of adherent and non-adherent populations of cells. The adherent population contains phagocytic mononuclear cells,epithelial" cells� View Publication

Ca2+-dependent myosin phosphorylation by Ca2+/calmodulin-dependent myosin light chain kinase (MLC-kinase) and protein kinase C were studied using selective inhibitors,isoquinolinesulfonamide derivatives. Both protein kinases were potently inhibited by 1-(8-chloro-5-isoquinolinesulfonyl)piperazine (HA-156) and its derivatives. Kinetic analysis indicated that HA-156 inhibited both enzymes competitively with respect to ATP,and Ki values of HA-156 for MLC-kinase and protein kinase C were 7.3 and 7.2 microM,respectively. To clarify molecular mechanisms of the isoquinolinesulfonamides to inhibit the Ca2+-dependent protein kinases,we examined the structure-activity relationships of HA-156 and its derivatives. The dechlorinated analogues,HA-100 and HA-142,markedly decreased the affinity for MLC-kinase,suggesting that the inhibitory effect of isoquinolinesulfonamide derivatives depends upon hydrophobicity of the compounds. There is a good correlation between MLC-kinase inhibition and hydrophobicity determined by reverse phase chromatography. In contrast,HA-140 and HA-142 showed weak inhibition of protein kinase C,suggesting that the electron density of the nitrogen in the isoquinoline ring of the compounds correlates with the potency to inhibit protein kinase C activity. These pairs of isoquinolinesulfonamides will aid in elucidating the biological roles of Ca2+-dependent myosin phosphorylation in intact cells. HA-156 and HA-140 inhibited myosin light chain phosphorylation in platelets exposed to collagen,whereas HA-142 and HA-100 did not,significantly. These isoquinolinesulfonamide derivatives should prove to be useful tools for distinguishing between the biological functions of Ca2+-activated,phospholipid-dependent,and Ca2+/calmodulin-dependent myosin light chain phosphorylation,in vivo. View Publication

Total-body exposure to radiation causes widespread tissue injury. Damage to the hematopoietic and intestinal stem cell compartments is particularly lethal and mitigators of this damage are critical in providing effective treatment. Parabiosis radiation experiments,in which the vasculatures of two rodents are anastomosed prior to irradiation of one of the animals,have shown that there is a circulating factor that protects mice from radiation-induced intestinal death. Recently reported studies have suggested that growth differentiation factor 11 (GDF11) is responsible for the rejuvenation of stem cells observed in parabiosis experiments involving aging mice. In this study,we investigated the efficacy of GDF11 as a potential mitigator of radiation-induced damage to intestinal stem cells. In ex vivo cultures of intestinal organoids,the number of cells expressing the stem cell marker Lgr5 was increased after irradiation and GDF11 supplementation. Further ex vivo studies to assess stem cell function,measured by the ability to grow new crypt-like structures,did not show increased stem cell activity in response to GDF11 treatment. In addition,GDF11 was unable to improve survival of mice subjected to total-abdominal irradiation. These data demonstrate that GDF11 does not mitigate radiation damage to intestinal stem cells. View Publication

Specifically ablating genes in human induced pluripotent stem cells (iPSCs) allows for studies of gene function as well as disease mechanisms in disorders caused by loss-of-function (LOF) mutations. While techniques exist for engineering such lines,we have developed and rigorously validated a method of simultaneous iPSC reprogramming while generating CRISPR/Cas9-dependent insertions/deletions (indels). This approach allows for the efficient and rapid formation of genetic LOF human disease cell models with isogenic controls. The rate of mutagenized lines was strikingly consistent across experiments targeting four different human epileptic encephalopathy genes and a metabolic enzyme-encoding gene,and was more efficient and consistent than using CRISPR gene editing of established iPSC lines. The ability of our streamlined method to reproducibly generate heterozygous and homozygous LOF iPSC lines with passage-matched isogenic controls in a single step provides for the rapid development of LOF disease models with ideal control lines,even in the absence of patient tissue. View Publication

Fibrodysplasia ossificans progressiva (FOP) is a rare and intractable disease characterized by extraskeletal bone formation through endochondral ossification. Patients with FOP harbor point mutations in ACVR1,a type I receptor for BMPs. Although mutated ACVR1 (FOP-ACVR1) has been shown to render hyperactivity in BMP signaling,we and others have uncovered a mechanism by which FOP-ACVR1 mistransduces BMP signaling in response to Activin-A,a molecule that normally transduces TGF-β signaling. Although Activin-A evokes enhanced chondrogenesis in vitro and heterotopic ossification (HO) in vivo,the underlying mechanisms have yet to be revealed. To this end,we developed a high-throughput screening (HTS) system using FOP patient-derived induced pluripotent stem cells (FOP-iPSCs) to identify pivotal pathways in enhanced chondrogenesis that are initiated by Activin-A. In a screen of 6,809 small-molecule compounds,we identified mTOR signaling as a critical pathway for the aberrant chondrogenesis of mesenchymal stromal cells derived from FOP-iPSCs (FOP-iMSCs). Two different HO mouse models,an FOP model mouse expressing FOP-ACVR1 and an FOP-iPSC-based HO model mouse,revealed critical roles for mTOR signaling in vivo. Moreover,we identified ENPP2,an enzyme that generates lysophosphatidic acid,as a linker of FOP-ACVR1 and mTOR signaling in chondrogenesis. These results uncovered the crucial role of the Activin-A/FOP-ACVR1/ENPP2/mTOR axis in FOP pathogenesis. View Publication

Intestinal tuft cells are one of 4 secretory cell linages in the small intestine and the source of IL-25,a critical initiator of the type 2 immune response to parasite infection. When Raptor,a critical scaffold protein for mammalian target of rapamycin complex 1 (mTORC1),was acutely deleted in intestinal epithelium via Tamoxifen injection in Tritrichomonas muris (Tm) infected mice,tuft cells,IL-25 in epithelium and IL-13 in the mesenchyme were significantly reduced,but Tm burden was not affected. When Tm infected mice were treated with rapamycin,DCLK1 and IL-25 expression in enterocytes and IL-13 expression in mesenchyme were diminished. After massive small bowel resection,tuft cells and Tm were diminished due to the diet used postoperatively. The elimination of Tm and subsequent re-infection of mice with Tm led to type 2 immune response only in WT,but Tm colonization in both WT and Raptor deficient mice. When intestinal organoids were stimulated with IL-4,tuft cells and IL-25 were induced in both WT and Raptor deficient organoids. In summary,our study reveals that enterocyte specific Raptor is required for initiating a type 2 immune response which appears to function through the regulation of mTORC1 activity. View Publication

Mutations in WD40-repeat protein 62 (WDR62) are commonly associated with primary microcephaly and other developmental cortical malformations. We used human pluripotent stem cells (hPSC) to examine WDR62 function during human neural differentiation and model early stages of human corticogenesis. Neurospheres lacking WDR62 expression showed decreased expression of intermediate progenitor marker,TBR2,and also glial marker,S100β. In contrast,inhibition of c-Jun N-terminal kinase (JNK) signalling during hPSC neural differentiation induced upregulation of WDR62 with a corresponding increase in neural and glial progenitor markers,PAX6 and EAAT1,respectively. These findings may signify a role of WDR62 in specifying intermediate neural and glial progenitors during human pluripotent stem cell differentiation. View Publication

The human iPSC cell line,RP2-FiPS4F1 (RCPFi001-A),derived from dermal fibroblasts from the patient with retinitis pigmentosa caused by the mutation of the gene PRPF8,was generated by non-integrative reprogramming technology using OCT3/4,SOX2,CMYC and KLF4 reprogramming factors. View Publication

Peripheral blood mononuclear cells (PBMCs) were collected from a clinically characterized patient with autism spectrum disorder (ASD). The PMBCs were reprogrammed with the human OSKM transcription factors using the Sendai-virus delivery system. The pluripotency of transgene-free iPSCs was verified by immunocytochemistry for pluripotency markers and by spontaneous in vitro differentiation towards the 3 germ layers. Furthermore,the iPSC line showed normal karyotype. Our model might offer a good platform to study the pathomechanism of ASD,also for drug testing,early biomarker discovery and gene therapy studies. View Publication

Type I and type III interferons (IFNs) are crucial components of the first-line antiviral host response. While specific receptors for both IFN types exist,intracellular signaling shares the same Jak-STAT pathway. Due to its receptor expression,IFN-λ responsiveness is restricted mainly to epithelial cells. Here,we display IFN-stimulated gene induction at the single cell level to comparatively analyze the activities of both IFN types in intestinal epithelial cells and mini-gut organoids. Initially,we noticed that the response to both types of IFNs at low concentrations is based on a single cell decision-making determining the total cell intrinsic antiviral activity. We identified histone deacetylase (HDAC) activity as a crucial restriction factor controlling the cell frequency of IFN-stimulated gene (ISG) induction upon IFN-λ but not IFN-β stimulation. Consistently,HDAC blockade confers antiviral activity to an elsewise non-responding subpopulation. Second,in contrast to the type I IFN system,polarization of intestinal epithelial cells strongly enhances their ability to respond to IFN-λ signaling and raises the kinetics of gene induction. Finally,we show that ISG induction in mini-gut organoids by low amounts of IFN is characterized by a scattered heterogeneous responsiveness of the epithelial cells and HDAC activity fine-tunes exclusively IFN-λ activity. This study provides a comprehensive description of the differential response to type I and type III IFNs and demonstrates that cell polarization in gut epithelial cells specifically increases IFN-λ activity. View Publication