技术资料

Mitochondria are critical to neurogenesis,but the mechanisms of mitochondria in neurogenesis have not been well explored. We fully characterized mitochondrial alterations and function in relation to the development of human induced pluripotent stem cell (hiPSC)-derived dopaminergic (DA) neurons. Following directed differentiation of hiPSCs to DA neurons,mitochondria in these neurons exhibit pronounced changes during differentiation,including mature neurophysiology characterization and functional synaptic network formation. Inhibition of mitochondrial respiratory chains via application of complex IV inhibitor KCN (potassium cyanide) or complex I inhibitor rotenone restricted neurogenesis of DA neurons. These results demonstrated the direct importance of mitochondrial development and bioenergetics in DA neuronal differentiation. Our study also provides a neurophysiologic model of mitochondrial involvement in neurogenesis,which will enhance our understanding of the role of mitochondrial dysfunctions in neurodegenerative diseases. View Publication

An immunochromatographic assay (Campy-ICA) using a newly generated single monoclonal antibody against a 15-kDa cell surface protein of Campylobacter jejuni was developed. When cell suspensions of 86 C. jejuni strains and 27 Campylobacter coli strains were treated with a commercially available bacterial protein extraction reagent and the resulting extracts were tested with the Campy-ICA,they all yielded positive results. The minimum detectable limits for the C. jejuni strains ranged from 1.8 x 10(4) to 8.2 x 10(5) CFU/ml of cell suspension,and those for the C. coli strains ranged from 1.4 x 10(5) to 4.6 x 10(6) CFU/ml of cell suspension. All 26 non-Campylobacter species tested yielded negative results with the Campy-ICA. To evaluate the ability of the Campy-ICA to detect C. jejuni and C. coli in human stool specimens,suspensions of 222 stool specimens from patients with acute gastroenteritis were treated with the bacterial protein extraction reagent,and the resulting extracts were tested with the Campy-ICA. The Campy-ICA results showed a sensitivity of 84.8% (28 of 33 specimens) and a specificity of 100% (189 of 189 specimens) compared to the results of isolation of C. jejuni and C. coli from the stool specimens by a bacterial culture test. The Campy-ICA was simple to perform and was able to detect Campylobacter antigen in a fecal extract within 15 min. These results suggest that Campy-ICA testing of fecal extracts may be useful as a simple and rapid adjunct to stool culture for detecting C. jejuni and C. coli in human stool specimens. View Publication

BACKGROUND It is clinically important to maintain high viability and potency of umbilical cord blood units (CBUs) for transplantation during thawing. In the absence of a standard thawing protocol,this study was designed to develop one based on the consensus practice of transplant centers and address the shortage of dextran 40 thawing solution. STUDY DESIGN AND METHODS Frozen CBU aliquots were thawed using dextran 40 thawing solution while manipulating temperature and volume of diluent and mode of dilution. The effects of these on CD45+ and CD34+ cell viability were measured through annexin V and SYTOX green staining. The developed protocol was then used to compare dextran 40 and PLASMA-LYTE A thawing solutions and finally tested on whole CBUs. RESULTS Step-by-step investigations resulted in the development of a protocol that thaws and dilutes CBUs with room temperature diluent to five times the original volume using two sequential dilutions separated by equilibration times. PLASMA-LYTE A diluent provided superior viability of CD45+ and CD34+ cells than dextran 40 and recovered more colony-forming units. However,both diluents were equally effective in maintaining stability of the thawed CBU for 4 hours. Moreover,the stem cell-enriched CD34+CD38- subpopulations appeared more resistant to cryoinjuries than their CD34+CD38+ counterpart. CONCLUSION The developed thawing protocol recovers viable CD45+ and CD34+ cells above the standard thresholds and maintains CBU potency. PLASMA-LYTE A for thawing solution proved to be an efficient alternative to dextran 40. Finally,greater dilution should be avoided to maintain the viability of CD45+ cells and maximize graft cell dose. View Publication

BACKGROUND Microglia,the principle immune cells of the brain,play important roles in neuronal development,homeostatic function and neurodegenerative disease. Recent genetic studies have further highlighted the importance of microglia in neurodegeneration with the identification of disease risk polymorphisms in many microglial genes. To better understand the role of these genes in microglial biology and disease,we,and others,have developed methods to differentiate microglia from human induced pluripotent stem cells (iPSCs). While the development of these methods has begun to enable important new studies of microglial biology,labs with little prior stem cell experience have sometimes found it challenging to adopt these complex protocols. Therefore,we have now developed a greatly simplified approach to generate large numbers of highly pure human microglia. RESULTS iPSCs are first differentiated toward a mesodermal,hematopoietic lineage using commercially available media. Highly pure populations of non-adherent CD43+ hematopoietic progenitors are then simply transferred to media that includes three key cytokines (M-CSF,IL-34,and TGF$\beta$-1) that promote differentiation of homeostatic microglia. This updated approach avoids the prior requirement for hypoxic incubation,complex media formulation,FACS sorting,or co-culture,thereby significantly simplifying human microglial generation. To confirm that the resulting cells are equivalent to previously developed iPSC-microglia,we performed RNA-sequencing,functional testing,and transplantation studies. Our findings reveal that microglia generated via this simplified method are virtually identical to iPS-microglia produced via our previously published approach. To also determine whether a small molecule activator of TGF$\beta$ signaling (IDE1) can be used to replace recombinant TGF$\beta$1,further reducing costs,we examined growth kinetics and the transcriptome of cells differentiated with IDE1. These data demonstrate that a microglial cell can indeed be produced using this alternative approach,although transcriptional differences do occur that should be considered. CONCLUSION We anticipate that this new and greatly simplified protocol will enable many interested labs,including those with little prior stem cell or flow cytometry experience,to generate and study human iPS-microglia. By combining this method with other advances such as CRISPR-gene editing and xenotransplantation,the field will continue to improve our understanding of microglial biology and their important roles in human development,homeostasis,and disease. View Publication

Hodgkin Lymphoma (HL) is a malignancy that frequently affects young adults. Although,there are effective treatments not every patient responds,necessitating the development of novel therapeutic approaches,especially for relapsed and refractory cases. The two TNF receptor family members CD30 and CD137 are expressed on Hodgkin and Reed Sternberg (HRS) cells,the malignant cells in HL. We found that this co-expression is specific for HRS cells. Based on this discovery we developed a bispecific antibody that binds preferentially to the CD30,CD137-double positive HRS cells. The CD30,CD137 bispecific antibody gets internalized into HRS cells opening up the possibility to use it as a carrier for a toxin. This antibody also induces antibody-dependent,cell-mediated cytotoxicity in CD30,CD137-double positive HRS cells. The enhances specificity of the CD30,CD137 bispecific antibody to HRS cells makes it a promising candidate for development as a novel HL treatment. View Publication

OBJECTIVE In this work we set to develop and to validate a new in vivo frameless orthotopic Diffuse Intrinsic Pontine Glioma (DIPG) model based in the implantation of a guide-screw system. METHODS It consisted of a guide-screw also called bolt,a Hamilton syringe with a 26-gauge needle and an insulin-like 15-gauge needle. The guide screw is 2.6 mm in length and harbors a 0.5 mm central hole which accepts the needle of the Hamilton syringe avoiding a theoretical displacement during insertion. The guide-screw is fixed on the mouse skull according to the coordinates: 1mm right to and 0.8 mm posterior to lambda. To reach the pons the Hamilton syringe is adjusted to a 6.5 mm depth using a cuff that serves as a stopper. This system allows delivering not only cells but also any kind of intratumoral chemotherapy,antibodies or gene/viral therapies. RESULTS The guide-screw was successfully implanted in 10 immunodeficient mice and the animals were inoculated with DIPG human cell lines during the same anesthetic period. All the mice developed severe neurologic symptoms and had a median overall survival of 95 days ranging the time of death from 81 to 116 days. Histopathological analysis confirmed tumor into the pons in all animals confirming the validity of this model. CONCLUSION Here we presented a reproducible and frameless DIPG model that allows for rapid evaluation of tumorigenicity and efficacy of chemotherapeutic or gene therapy products delivered intratumorally to the pons. View Publication

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Human immunodeficiency virus type 1 (HIV-1) vectors transduce rhesus blood cells poorly due to a species-specific block by TRIM5alpha and APOBEC3G,which target HIV-1 capsid and viral infectivity factor (Vif),respectively. We sought to develop a lentiviral vector capable of transducing both human and rhesus blood cells by combining components of both HIV-1 and simian immunodeficiency virus (SIV),including SIV capsid (sCA) and SIV Vif. A chimeric HIV-1 vector including sCA (chiHIV) was superior to the conventional SIV in transducing a human blood cell line and superior to the conventional HIV-1 vector in transducing a rhesus blood cell line. Among human CD34(+) hematopoietic stem cells (HSCs),the chiHIV and HIV-1 vectors showed similar transduction efficiencies; in rhesus CD34(+) HSCs,the chiHIV vector yielded superior transduction rates. In in vivo competitive repopulation experiments with two rhesus macaques,the chiHIV vector demonstrated superior marking levels over the conventional HIV-1 vector in all blood lineages (first rhesus,15 to 30% versus 1 to 5%; second rhesus,7 to 15% versus 0.5 to 2%,respectively) 3 to 7 months postinfusion. In summary,we have developed an HIV-1-based lentiviral vector system that should allow comprehensive preclinical testing of HIV-1-based therapeutic vectors in the rhesus macaque model with eventual clinical application. View Publication

Peripheral blood mononuclear cells (PBMCs) were collected from a clinically diagnosed 64-year old male Parkinson's disease (PD) patient with N551K variant in the LRRK2 gene. The PMBCs were reprogrammed with the human OSKM transcription factors using the Sendai-virus reprogramming system. The transgene-free iPSC showed pluripotency confirmed by immunofluorescent staining for pluripotency markers and differentiated into the 3 germ layers in vivo. The iPSC line also showed normal karyotype. This cellular model can complement in vivo PD models for pathophysiological studies and drug screening. View Publication

Breast cancer is the most common cancer in women diagnosed in the U.S. and worldwide. Obesity increases breast cancer risk without clear underlying molecular mechanisms. Our studies demonstrate that circulating adipose fatty acid binding protein (A-FABP,or FABP4) links obesity-induced dysregulated lipid metabolism and breast cancer risk,thus potentially offering a new target for breast cancer treatment. We immunized FABP4 knockout mice with recombinant human FABP4 and screened hybridoma clones with specific binding to FABP4. The potential effects of antibodies on breast cancer cells in vitro were evaluated using migration,invasion,and limiting dilution assays. Tumor progression in vivo was evaluated in various types of tumorigenesis models including C57BL/6 mice,Balb/c mice,and SCID mice. The phenotype and function of immune cells in tumor microenvironment were characterized with multi-color flow cytometry. Tumor stemness was detected by ALDH assays. To characterize antigen-antibody binding capacity,we determined the dissociation constant of selected anti-FABP4 antibodies via surface plasmon resonance. Further analyses in tumor tissue were performed using 10X Genomics Visium spatial single cell technology. Herein,we report the generation of humanized monoclonal antibodies blocking FABP4 activity for breast cancer treatment in mouse models. One clone,named 12G2,which significantly reduced circulating levels of FABP4 and inhibited mammary tumor growth,was selected for further characterization. After confirming the therapeutic efficacy of the chimeric 12G2 monoclonal antibody consisting of mouse variable regions and human IgG1 constant regions,16 humanized 12G2 monoclonal antibody variants were generated by grafting its complementary determining regions to selected human germline sequences. Humanized V9 monoclonal antibody showed consistent results in inhibiting mammary tumor growth and metastasis by affecting tumor cell mitochondrial metabolism. Our current evidence suggests that targeting FABP4 with humanized monoclonal antibodies may represent a novel strategy for the treatment of breast cancer and possibly other obesity- associated diseases. The online version contains supplementary material available at 10.1186/s13058-024-01873-y. View Publication

5-Fluorouracil (5-FU) is a small,very membrane permeable drug that is poorly retained within the aqueous compartment of liposomal nanoparticles (LNP). To address this problem a novel method relying on formation of a ternary complex comprising copper,low molecular weight polyethylenimine (PEI) and 5-FU has been developed. More specifically,in the presence of entrapped copper and PEI,externally added 5-FU can be efficiently encapsulated (textgreater95%) in DSPC/Chol (1,2-Distearoyl-sn-Glycero-3-Phosphocholine/cholesterol; 55:45 mol%) liposomes (130-170 nm) to achieve drug-to-lipid ratios of 0.1 (mol:mol). Drug release studies completed using this LNP formulation of 5-FU demonstrated significant improvements in drug retention in vitro and in vivo. Plasma concentrations of 5-FU were 7- to 23-fold higher when the drug was administered intravenously to mice as the LNP 5-FU formulation compared to free 5-FU. Further,the therapeutic effects of the LNP 5-FU formulation,as determined in a HT-29 subcutaneous colorectal cancer model where treatment was given QDx5,was greater than that which could be achieved with free 5-FU when compared at equivalent doses. This is the first time an active loading method has been described for 5-FU. The use of ternary metal complexation strategy to encapsulate therapeutic agents may define a unique platform for preparation of LNP drug formulations. View Publication

Neutrophil breach of the mucosal surface is a common pathological consequence of infection. We present an advanced co-culture model to explore neutrophil transepithelial migration utilizing airway mucosal barriers differentiated from primary human airway basal cells and examined by advanced imaging. Human airway basal cells were differentiated and cultured at air-liquid interface (ALI) on the underside of 3 μm pore-sized transwells,compatible with the study of transmigrating neutrophils. Inverted ALIs exhibit beating cilia and mucus production,consistent with conventional ALIs,as visualized by micro-optical coherence tomography (μOCT). μOCT is a recently developed imaging modality with the capacity for real time two- A nd three-dimensional analysis of cellular events in marked detail,including neutrophil transmigratory dynamics. Further,the newly devised and imaged primary co-culture model recapitulates key molecular mechanisms that underlie bacteria-induced neutrophil transepithelial migration previously characterized using cell line-based models. Neutrophils respond to imposed chemotactic gradients,and migrate in response to Pseudomonas aeruginosa infection of primary ALI barriers through a hepoxilin A3-directed mechanism. This primary cell-based co-culture system combined with μOCT imaging offers significant opportunity to probe,in great detail,micro-anatomical and mechanistic features of bacteria-induced neutrophil transepithelial migration and other important immunological and physiological processes at the mucosal surface. View Publication