技术资料

Numerous publications have described the importance of bone morphogenetic protein (BMP) signaling in the specification of hematopoietic tissue in developing embryos. Here we investigate the full role of canonical BMP signaling in both adult and fetal liver hematopoiesis using conditional knockout strategies because conventional disruption of components of the BMP signaling pathway result in early death of the embryo. By targeting both Smad1 and Smad5,we have generated a double-knockout mouse with complete disruption of canonical BMP signaling. Interestingly,concurrent deletion of Smad1 and Smad5 results in death because of extrahematopoietic pathologic changes in the colon. However,Smad1/Smad5-deficient bone marrow cells can compete normally with wild-type cells and display unaffected self-renewal and differentiation capacity when transplanted into lethally irradiated recipients. Moreover,although BMP receptor expression is increased in fetal liver,fetal liver cells deficient in both Smad1 and Smad5 remain competent to long-term reconstitute lethally irradiated recipients in a multilineage manner. In conclusion,canonical BMP signaling is not required to maintain either adult or fetal liver hematopoiesis,despite its crucial role in the initial patterning of hematopoiesis in early embryonic development. View Publication

It is well known that hepatic stellate cells (HSC) develop into cells,which are thought to contribute to liver fibrogenesis. Recent data suggest that HSC are progenitor cells with the capacity to differentiate into cells of endothelial and hepatocyte lineages. The present study shows that beta-catenin-dependent canonical Wnt signaling is active in freshly isolated HSC of rats. Mimicking of the canonical Wnt pathway in cultured HSC by TWS119,an inhibitor of the glycogen synthase kinase 3beta,led to reduced beta-catenin phosphorylation,induced nuclear translocation of beta-catenin,elevated glutamine synthetase production,impeded synthesis of alpha-smooth muscle actin and Wnt5a,but promoted the expression of glial fibrillary acidic protein,Wnt10b,and paired-like homeodomain transcription factor 2c. In addition,canonical Wnt signaling lowered DNA synthesis and hindered HSC from entering the cell cycle. The findings demonstrate that beta-catenin-dependent Wnt signaling maintains the quiescent state of HSC and,similar to stem and progenitor cells,influences their developmental fate. View Publication

Genetic evidence indicates that Wnt signaling is critically involved in bone homeostasis. In this study,we investigated the functions of canonical Wnts on differentiation of adult multipotent human mesenchymal stem cells (hMSCs) in vitro and in vivo. We observe differential sensitivities of hMSCs to Wnt inhibition of osteogenesis versus adipogenesis,which favors osteoblastic commitment under binary in vitro differentiation conditions. Wnt inhibition of osteogenesis is associated with decreased expression of osteoblastic transcription factors and inhibition of c-Jun N-terminal kinase and p38 mitogen-activated protein kinase activation,which are involved in osteogenic differentiation. An hMSC subpopulation exhibits high endogenous Wnt signaling,the inhibition of which enhances osteogenic and adipogenic differentiation in vitro. In an in vivo bone formation model,high levels of Wnt signaling inhibit de novo bone formation by hMSCs. However,hMSCs with exogenous expression of Wnt1 but not stabilized beta-catenin markedly stimulate bone formation by naive hMSCs,arguing for an important role of a canonical Wnt gradient in hMSC osteogenesis in vivo. View Publication

Encapsulation of donor islets using a hydrogel material is a well-studied strategy for islet transplantation,which protects donor islets from the host immune response. Replacement of donor islets by human embryonic stem cell (hESC) derived islets will also require a means of immune-isolating hESCs by encapsulation. However,a critical consideration of hESC differentiation is the effect of surrounding biophysical environment,in this case capsule biophysical properties,on differentiation. The objective of this study,thus,was to evaluate the effect of capsule properties on growth,viability,and differentiation of encapsulated hESCs throughout pancreatic induction. It was observed that even in the presence of soluble chemical cues for pancreatic induction,substrate properties can significantly modulate pancreatic differentiation,hence necessitating careful tuning of capsule properties. Capsules in the range of 4-7. kPa supported cell growth and viability,whereas capsules of higher stiffness suppressed cell growth. While an increase in capsule stiffness enhanced differentiation at the intermediate definitive endoderm (DE) stage,increased stiffness strongly suppressed pancreatic progenitor (PP) induction. Signaling pathway analysis indicated an increase in pSMAD/pAKT levels with substrate stiffness likely the cause of enhancement of DE differentiation. In contrast,sonic hedgehog inhibition was more efficient under softer gel conditions,which is necessary for successful PP differentiation. Statement of Significance: Cell replacement therapy for type 1 diabetes (T1D),affecting millions of people worldwide,requires the immunoisolation of insulin-producing islets by encapsulation with a semi-impermeable material. Due to the shortage of donor islets,human pluripotent stem cell (hPSC) derived islets are an attractive alternative. However,properties of the encapsulating substrate are known to influence hPSC cell fate. In this work,we determine the effect of substrate stiffness on growth and pancreatic fate of encapsulated hPSCs. We precisely identify the range of substrate properties conducive for pancreatic cell fate,and also the mechanism by which substrate properties modify the cell signaling pathways and hence cell fate. Such information will be critical in driving regenerative cell therapy for long term treatment of T1D. View Publication

Embryonic stem (ES) cells have been available from inbred mice since 1981 but have not been validated for other rodents. Failure to establish ES cells from a range of mammals challenges the identity of cultivated stem cells and our understanding of the pluripotent state. Here we investigated derivation of ES cells from the rat. We applied molecularly defined conditions designed to shield the ground state of authentic pluripotency from inductive differentiation stimuli. Undifferentiated cell lines developed that exhibited diagnostic features of ES cells including colonization of multiple tissues in viable chimeras. Definitive ES cell status was established by transmission of the cell line genome to offspring. Derivation of germline-competent ES cells from the rat paves the way to targeted genetic manipulation in this valuable biomedical model species. Rat ES cells will also provide a refined test-bed for functional evaluation of pluripotent stem cell-derived tissue repair and regeneration. View Publication

BACKGROUND The incidence and number of circulating tumor cells (CTCs) in the peripheral blood of colorectal cancer patients are lower than in other cancer types,which may point to a particular biology of colorectal cancer affecting CTC detection. METHODS We detected CTCs in the peripheral and mesenteric blood of colorectal cancer patients by use of 2 independent technologies on the basis of different biological properties of colon cancer cells. Seventy-five patients diagnosed with localized (M0,n = 60) and metastatic (M1,n = 15) colorectal cancer were included. Peripheral and mesenteric blood samples were collected before tumor resection. We performed CTC enumeration with an EpCAM-independent enrichment method followed by the Epispot assay that detected only viable CK19-releasing CTCs. In parallel,we used the FDA-cleared EpCAM-dependent CellSearch® as the reference method. RESULTS The enumeration of CK19-releasing cells by the CK19-Epispot assay revealed viable CTCs in 27 of 41 (65.9%) and 41 of 74 (55.4%) (P = 0.04) patients in mesenteric and peripheral blood,respectively,whereas CellSearch detected CTCs in 19 of 34 (55.9%) and 20 of 69 (29.0%) (P = 0.0046) patients. In mesenteric blood,medians of 4 (range 0-247) and 2.7 CTCs (range 0-286) were found with Epispot and CellSearch (P = 0.2),respectively,whereas in peripheral blood,Epispot and CellSearch detected a median of 1.2 (range 0-92) and 0 CTCs (range 0-147) (P = 0.002). CONCLUSIONS A considerable portion of viable CTCs detectable by the Epispot assay are trapped in the liver as the first filter organ in CRC patients. View Publication

Coxsackie virus and adenovirus receptor,CXADR (CAR),is present during embryogenesis and is involved in tissue regeneration,cancer and intercellular adhesion. We investigated the expression of CAR in human preimplantation embryos and embryonic stem cells (hESC) to identify its role in early embryogenesis and differentiation. CAR protein was ubiquitously present during preimplantation development. It was localised in the nucleus of uncommitted cells,from the cleavage stage up to the precursor epiblast,and corresponded with the presence of soluble CXADR3/7 splice variant. CAR was displayed on the membrane,involving in the formation of tight junction at compaction and blastocyst stages in both outer and inner cells,and CAR corresponded with the full-length CAR-containing transmembrane domain. In trophectodermal cells of hatched blastocysts,CAR was reduced in the membrane and concentrated in the nucleus,which correlated with the switch in RNA expression to the CXADR4/7 and CXADR2/7 splice variants. The cells in the outer layer of hESC colonies contained CAR on the membrane and all the cells of the colony had CAR in the nucleus,corresponding with the transmembrane CXADR and CXADR4/7. Upon differentiation of hESC into cells representing the three germ layers and trophoblast lineage,the expression of CXADR was downregulated. We concluded that CXADR is differentially expressed during human preimplantation development. We described various CAR expressions: i) soluble CXADR marking undifferentiated blastomeres; ii) transmembrane CAR related with epithelial-like cell types,such as the trophectoderm (TE) and the outer layer of hESC colonies; and iii) soluble CAR present in TE nuclei after hatching. The functions of these distinct forms remain to be elucidated. View Publication

Chimeric antigen receptor (CAR)-engineered T and NK cells can cause durable remission of B-cell malignancies; however,limited persistence restrains the full potential of these therapies in many patients. The FAS ligand (FAS-L)/FAS pathway governs naturally-occurring lymphocyte homeostasis,yet knowledge of which cells express FAS-L in patients and whether these sources compromise CAR persistence remains incomplete. Here,we constructed a single-cell atlas of diverse cancer types to identify cellular subsets expressing FASLG,the gene encoding FAS-L. We discovered that FASLG is limited primarily to endogenous T cells,NK cells,and CAR-T cells while tumor and stromal cells express minimal FASLG. To establish whether CAR-T/NK cell survival is regulated through FAS-L,we performed competitive fitness assays using lymphocytes modified with or without a FAS dominant negative receptor (ΔFAS). Following adoptive transfer,ΔFAS-expressing CAR-T and CAR-NK cells became enriched across multiple tissues,a phenomenon that mechanistically was reverted through FASLG knockout. By contrast,FASLG was dispensable for CAR-mediated tumor killing. In multiple models,ΔFAS co-expression by CAR-T and CAR-NK enhanced antitumor efficacy compared with CAR cells alone. Together,these findings reveal that CAR-engineered lymphocyte persistence is governed by a FAS-L/FAS auto-regulatory circuit. View Publication

Chimeric antigen receptor (CAR)-T cells are engineered to identify and eliminate cells expressing a target antigen. Current manufacturing protocols vary between commercial CAR-T cell products warranting an assessment of these methods to determine which approach optimally balances successful manufacturing capacity and product efficacy. One difference between commercial product manufacturing methods is whether T cell engineering begins with fresh (unfrozen) patient cells or cells that have been cryopreserved prior to manufacture. Starting with frozen PBMC material allows for greater manufacturing flexibility,and the possibility of collecting and storing blood from patients prior to multiple lines of therapy. We prospectively analyzed if second generation anti-CD19 CAR-T cells with either CD28 or 4-1BB co-stimulatory domains have different phenotype or function when prepared side-by-side using fresh or cryopreserved PBMCs. We found that cryopreserved PBMC starting material is associated with slower CAR-T cell expansion during manufacture but does not affect phenotype. We also demonstrate that CAR-T cell activation,cytokine production and in vitro anti-tumor cytotoxicity were not different when CAR-T cells were manufactured from fresh or cryopreserved PBMC. As CAR-T cell therapy expands globally,the need for greater flexibility around the timing of manufacture will continue to grow. This study helps support the concept that cryopreservation of PBMCs could be the solution to these issues without compromising the quality of the final CAR-T product. View Publication

Molecular mimicry between microbial and self-components is postulated as the mechanism that accounts for the antigen and tissue specificity of immune responses in postinfectious autoimmune diseases. Little direct evidence exists,and research in this area has focused principally on T cell-mediated,antipeptide responses,rather than on humoral responses to carbohydrate structures. Guillain-Barré syndrome,the most frequent cause of acute neuromuscular paralysis,occurs 1-2 wk after various infections,in particular,Campylobacter jejuni enteritis. Carbohydrate mimicry [Galbeta1-3GalNAcbeta1-4(NeuAcalpha2-3)Galbeta1-] between the bacterial lipooligosaccharide and human GM1 ganglioside is seen as having relevance to the pathogenesis of Guillain-Barré syndrome,and conclusive evidence is reported here. On sensitization with C. jejuni lipooligosaccharide,rabbits developed anti-GM1 IgG antibody and flaccid limb weakness. Paralyzed rabbits had pathological changes in their peripheral nerves identical with those present in Guillain-Barré syndrome. Immunization of mice with the lipooligosaccharide generated a mAb that reacted with GM1 and bound to human peripheral nerves. The mAb and anti-GM1 IgG from patients with Guillain-Barré syndrome did not induce paralysis but blocked muscle action potentials in a muscle-spinal cord coculture,indicating that anti-GM1 antibody can cause muscle weakness. These findings show that carbohydrate mimicry is an important cause of autoimmune neuropathy. View Publication

OBJECTIVE: We examined the effect of the vasoactive agents carbon monoxide (CO) and nitric oxide (NO) : n the phosphorylation and intracellular redistribution of vasodilator-stimulated phosphoprotein (VASP),a critical actin motor protein required for cell migration that also controls vasodilation and platelet aggregation. RESEARCH DESIGN AND METHODS: We examined the effect of donor-released CO and NO in endothelial progenitor cells (EPCs) and platelets from nondiabetic and diabetic subjects and in human microvascular endothelial cells (HMECs) cultured under low (5.5 mmol/l) or high (25 mmol/l) glucose conditions. VASP phosphorylation was evaluated using phosphorylation site-specific antibodies. RESULTS: In control platelets,CO selectively promotes phosphorylation at VASP Ser-157,whereas NO promotes phosphorylation primarily at Ser-157 and also at Ser-239,with maximal responses at 1 min with both agents on Ser-157 and at 15 min on Ser-239 with NO treatment. In diabetic platelets,neither agent resulted in VASP phosphorylation. In nondiabetic EPCs,NO and CO increased phosphorylation at Ser-239 and Ser-157,respectively,but this response was markedly reduced in diabetic EPCs. In endothelial cells cultured under low glucose conditions,both CO and NO induced phosphorylation at Ser-157 and Ser-239; however,this response was completely lost when cells were cultured under high glucose conditions. In control EPCs and in HMECs exposed to low glucose,VASP was redistributed to filopodia-like structures following CO or NO exposure; however,redistribution was dramatically attenuated under high glucose conditions. CONCLUSIONS: Vasoactive gases CO and NO promote cytoskeletal changes through site- and cell type-specific VASP phosphorylation,and in diabetes,blunted responses to these agents may lead to reduced vascular repair and tissue perfusion. View Publication

Cardiomyocytes (CMs) generated from human pluripotent stem cells (hPSCs) are a potential cell source for regenerative therapies,drug discovery and disease modeling. All these applications require a routine supply of relatively large quantities of in vitro-generated CMs. This protocol describes a suspension culture-based strategy for the generation of hPSC-CMs as cell-only aggregates,which facilitates process development and scale-up. Aggregates are formed for 4 d in hPSC culture medium followed by 10 d of directed differentiation by applying chemical Wnt pathway modulators. The protocol is applicable to static multiwell formats supporting fast adaptation to specific hPSC line requirements. We also demonstrate how to apply the protocol using stirred tank bioreactors at a 100-ml scale,providing a well-controlled upscaling platform for CM production. In bioreactors,the generation of 40-50 million CMs per differentiation batch at textgreater80% purity without further lineage enrichment can been achieved within 24 d. View Publication