搜索结果: 'methocult media formulations for mouse hematopoietic cells serum containing'

BACKGROUND {\&} AIMS The nuclear receptor subfamily 0 group B member 2 (NR0B2,also called SHP) is expressed at high levels in the liver and intestine. Postprandial fibroblast growth factor 19 (human FGF19,mouse FGF15) signaling increases the transcriptional activity of SHP. We studied the functions of SHP and FGF19 in the intestines of mice,including their regulation of expression of the cholesterol transporter NPC1L1 )NPC1-like intracellular cholesterol transporter 1) and cholesterol absorption. METHODS We performed histologic and biochemical analyses of intestinal tissues from C57BL/6 and SHP-knockout mice and performed RNA-sequencing analyses to identify genes regulated by SHP. The effects of fasting and refeeding on intestinal expression of NPC1L1 were examined in C57BL/6,SHP-knockout,and FGF15-knockout mice. Mice were given FGF19 daily for 1 week; fractional cholesterol absorption,cholesterol and bile acid (BA) levels,and composition of BAs were measured. Intestinal organoids were generated from C57BL/6 and SHP-knockout mice,and cholesterol uptake was measured. Luciferase reporter assays were performed with HT29 cells. RESULTS We found that the genes that regulate lipid and ion transport in intestine,including NPC1L1,were up-regulated and that cholesterol absorption was increased in SHP-knockout mice compared with C57BL/6 mice. Expression of NPC1L1 was reduced in C57BL/6 mice after refeeding after fasting but not in SHP-knockout or FGF15-knockout mice. SHP-knockout mice had altered BA composition compared with C57BL/6 mice. FGF19 injection reduced expression of NPC1L1,decreased cholesterol absorption,and increased levels of hydrophilic BAs,including tauro-$\alpha$- and -$\beta$-muricholic acids; these changes were not observed in SHP-knockout mice. SREBF2 (sterol regulatory element binding transcription factor 2),which regulates cholesterol,activated transcription of NPC1L1. FGF19 signaling led to phosphorylation of SHP,which inhibited SREBF2 activity. CONCLUSIONS Postprandial FGF19 and SHP inhibit SREBF2,which leads to repression of intestinal NPC1L1 expression and cholesterol absorption. Strategies to increase FGF19 signaling to activate SHP might be developed for treatment of hypercholesterolemia. View Publication

A broad range of hematopoietic stem cells and progenitors reside within a fraction of umbilical cord blood (UCB) that exhibits low light scatter properties (SSC(lo)) and high expression of aldehyde dehydrogenase (ALDH(br)). Many SSC(lo) ALDH(br) cells coexpress CD34; however,other cells express either ALDH or CD34. To investigate the developmental potential of these cell subsets,purified ALDH(br) CD34+,ALDH(neg) CD34+,and ALDH(br) CD34(neg) UCB cells were characterized within a variety of in vivo and in vitro assays. Primitive progenitors capable of multilineage development were monitored in long- and short-term repopulation assays performed on nonobese diabetic/severe combined immunodeficiency (NOD/SCID) mice,and in primary and secondary long-term culture assays. These progenitors were highly enriched within the ALDH(br) CD34+ fraction. This cell fraction also enriched short-term myeloid progenitors that were detected in vitro. By comparison,ALDH(neg) CD34+ cells contained few primitive progenitors and had diminished short-term myeloid potential but exhibited enhanced short-term natural killer (NK) cell development in vitro. The ALDH(br) CD34(neg) cells were not efficiently supported by any of the assays used. These studies suggested that in particular the expression of ALDH delineated distinct CD34+ stem cell and progenitor compartments. The differential expression of ALDH may provide a means to explore normal and malignant processes associated with myeloid and lymphoid development. View Publication

In the United States alone,there are approximately 500,000 burn injuries that require medical treatment every year. Limitations of current treatments necessitate the development of new methods that can be applied quicker,result in faster wound regeneration,and yield skin that is cosmetically similar to undamaged skin. The development of new hydrogel biomaterials and bioprinting deposition technologies has provided a platform to address this need. Herein we evaluated characteristics of twelve hydrogels to determine their suitability for bioprinting applications. We chose hydrogels that are either commercially available,or are commonly used for research purposes. We evaluated specific hydrogel properties relevant to bioprinting applications,specifically; gelation time,swelling or contraction,stability,biocompatibility and printability. Further,we described regulatory,commercial and financial aspects of each of the hydrogels. While many of the hydrogels screened may exhibit characteristics suitable for other applications,UV-crosslinked Extracel,a hyaluronic acid-based hydrogel,had many of the desired properties for our bioprinting application. Taken together with commercial availability,shelf life,potential for regulatory approval and ease of use,these materials hold the potential to be further developed into fast and effective wound healing treatments. View Publication

BACKGROUND Lymphoid neoplasms,including multiple myeloma (MM),non-Hodgkin lymphoma (NHL),and NK/T cell neoplasms,are a major cause of blood cancer morbidity and mortality. CD38 (cyclic ADP ribose hydrolase) is a transmembrane glycoprotein expressed on the surface of plasma cells and MM cells. The high expression of CD38 across MM and other lymphoid malignancies and its restricted expression in normal tissues make CD38 an attractive target for immunotherapy. CD38-targeting antibodies,like daratumumab,have been approved for the treatment of MM and tested against lymphoma and leukemia in multiple clinical trials. METHODS We generated chimeric antigen receptor (CAR) T cells targeting CD38 and tested its cytotoxicity against multiple CD38high and CD38low lymphoid cancer cells. We evaluated the synergistic effects of all-trans retinoic acid (ATRA) and CAR T cells or daratumumab against cancer cells and xenograft tumors. RESULTS CD38-CAR T cells dramatically inhibited the growth of CD38high MM,mantle cell lymphoma (MCL),Waldenstrom's macroglobulinemia (WM),T-cell acute lymphoblastic leukemia (T-ALL),and NK/T-cell lymphoma (NKTCL) in vitro and in mouse xenografts. ATRA elevated CD38 expression in multiple CD38low cancer cells and enhanced the anti-tumor activity of daratumumab and CD38-CAR T cells in xenograft tumors. CONCLUSIONS These findings may expand anti-CD38 immunotherapy to a broad spectrum of lymphoid malignancies and call for the incorporation of ATRA into daratumumab or other anti-CD38 immunological agents for cancer therapy. View Publication

By mimicking embryonic development of the hematopoietic system,we have developed an optimized in vitro differentiation protocol for the generation of precursors of hematopoietic lineages and primitive hematopoietic cells from human embryonic stem cells (ESC) and induced pluripotent stem cells (iPSCs). Factors such as cytokines,extra cellular matrix components,and small molecules as well as the temporal association and concentration of these factors were tested on seven different human ESC and iPSC lines. We report the differentiation of up to 84% human CD45+ cells (average 41% ± 16%,from seven pluripotent lines) from the differentiation culture,including significant numbers of primitive CD45+/CD34+ and CD45+/CD34+/CD38- hematopoietic progenitors. Moreover,the numbers of hematopoietic progenitor cells generated,as measured by colony forming unit assays,were comparable to numbers obtained from fresh umbilical cord blood mononuclear cell isolates on a per CD45+ cell basis. Our approach demonstrates highly efficient generation of multipotent hematopoietic progenitors with among the highest efficiencies reported to date (CD45+/CD34+) using a single standardized differentiation protocol on several human ESC and iPSC lines. Our data add to the cumulating evidence for the existence of an in vitro derived precursor to the hematopoietic stem cell (HSC) with limited engrafting ability in transplanted mice but with multipotent hematopoietic potential. Because this protocol efficiently expands the preblood precursors and hematopoietic progenitors,it is ideal for testing novel factors for the generation and expansion of definitive HSCs with long-term repopulating ability. View Publication

BackgroundHematopoietic stem cell (HSC) regeneration underlies hematopoietic recovery from myelosuppression,which is a life-threatening side effect of cytotoxicity. HSC niche is profoundly disrupted after myelosuppressive injury,while if and how the niche is reshaped and regulates HSC regeneration are poorly understood.MethodsA mouse model of radiation injury-induced myelosuppression was built by exposing mice to a sublethal dose of ionizing radiation. The dynamic changes in the number,distribution and functionality of HSCs and megakaryocytes were determined by flow cytometry,immunofluorescence,colony assay and bone marrow transplantation,in combination with transcriptomic analysis. The communication between HSCs and megakaryocytes was determined using a coculture system and adoptive transfer. The signaling mechanism was investigated both in vivo and in vitro,and was consolidated using megakaryocyte-specific knockout mice and transgenic mice.ResultsMegakaryocytes become a predominant component of HSC niche and localize closer to HSCs after radiation injury. Meanwhile,transient insulin-like growth factor 1 (IGF1) hypersecretion is predominantly provoked in megakaryocytes after radiation injury,whereas HSCs regenerate paralleling megakaryocytic IGF1 hypersecretion. Mechanistically,HSCs are particularly susceptible to megakaryocytic IGF1 hypersecretion,and mTOR downstream of IGF1 signaling not only promotes activation including proliferation and mitochondrial oxidative metabolism of HSCs,but also inhibits ferritinophagy to restrict HSC ferroptosis. Consequently,the delicate coordination between proliferation,mitochondrial oxidative metabolism and ferroptosis ensures functional HSC expansion after radiation injury. Importantly,punctual IGF1 administration simultaneously promotes HSC regeneration and hematopoietic recovery after radiation injury,representing a superior therapeutic approach for myelosuppression.ConclusionsOur study identifies megakaryocytes as a last line of defense against myelosuppressive injury and megakaryocytic IGF1 as a novel niche signal safeguarding HSC regeneration.Supplementary InformationThe online version contains supplementary material available at 10.1186/s12964-024-01651-5. View Publication

Alzheimer’s disease (AD) is a neurodegenerative disorder characterized by the accumulation of amyloid-β (Aβ) plaques and neurofibrillary tangles composed of hyperphosphorylated tau protein. The combination of biomarkers is crucial for AD diagnosis. The triggering receptor expressed on myeloid cells 2 (TREM2),a receptor expressed on microglia,is important in AD pathogenesis. Impairment of TREM2 function aggravates the toxic effects of amyloid plaques,and its activation has been shown to reduce Aβ burden and memory deficits. Increased levels of soluble TREM2 (sTREM2) in blood and cerebrospinal fluid is associated with AD. Therefore,TREM2 could serve as a non-invasive biomarker for AD. In this study,we developed a preclinical immunoassay to detect TREM2 for AD diagnosis. Highly sensitive and specific TREM2 antibodies were produced using the hybridoma technique. The three optimized immunoassays exhibited lower limit of quantitation (LLOQ) of 0.474,0.807,and 0.415 ng/mL,respectively. These preclinical immunoassays showed high sensitivity and specificity. The sandwich enzyme-linked immunosorbent assay (ELISA) could potentially be used for AD diagnosis. View Publication

Transplantation-associated stress can compromise the hematopoietic potential of hematopoietic stem cells (HSCs). As a consequence,HSCs may undergo exhaustion" in serial transplant recipients� View Publication

Hematopoietic stem cell (HSC) engraftment is a multistep process involving HSC homing to bone marrow,self-renewal,proliferation,and differentiation to mature blood cells. Here,we show that loss of p190-B RhoGTPase activating protein,a negative regulator of Rho GTPases,results in enhanced long-term engraftment during serial transplantation. This effect is associated with maintenance of functional HSC-enriched cells. Furthermore,loss of p190-B led to marked improvement of HSC in vivo repopulation capacity during ex vivo culture without altering proliferation and multilineage differentiation of HSC and progeny. Transcriptional analysis revealed that p190-B deficiency represses the up-regulation of p16(Ink4a) in HSCs in primary and secondary transplantation recipients,providing a possible mechanism of p190-B-mediated HSC functions. Our study defines p190-B as a critical transducer element of HSC self-renewal activity and long-term engraftment,thus suggesting that p190-B is a target for HSC-based therapies requiring maintenance of engraftment phenotype. View Publication

Genital herpes,caused by herpes simplex virus type 2 (HSV-2),is one of the most prevalent sexually transmitted diseases worldwide and a risk factor for acquiring human immunodeficiency virus. Although many vaccine candidates have shown promising results in animal models,they have failed to be effective in human trials. In this study,a humanized mouse strain was evaluated as a potential preclinical model for studying human immune responses to HSV-2 infection and vaccination. Immunodeficient mouse strains were examined for their abilities to develop human innate and adaptive immune cells after transplantation of human umbilical cord stem cells. A RAG2(-/-) gammac(-/-) mouse strain with a BALB/c background was chosen as the most appropriate model and was then examined for its ability to mount innate and adaptive immune responses to intravaginal HSV-2 infection and immunization. After primary infection,human cells in the lymph nodes were able to generate a protective innate immune response and produce gamma interferon (IFN-gamma). After intravaginal immunization and infection,human T cells and NK cells were found in the genital tract and iliac lymph nodes. In addition,human T cells in the spleen,lymph nodes,and vaginal tract were able to respond to stimulation with HSV-2 antigens by replicating and producing IFN-gamma. Human B cells were also able to produce HSV-2-specific immunoglobulin G. These adaptive responses were also shown to be protective and reduce local viral replication in the genital tract. This approach provides a means for studying human immune responses in vivo using a small-animal model and may become an important preclinical tool. View Publication

Regulatory T (Treg)-based cell therapy holds promise for autoimmune and inflammatory diseases,yet challenges remain regarding the functional stability and persistence of transferred Tregs. Here we engineer Tregs to express a partial agonist form of IL-2 (IL-2pa) to enhance persistence while avoiding toxicity from excessive signaling. Mouse Tregs expressing wild-type IL-2 (Tregs-IL2wt) have only a transient growth advantage,limited by toxicity from likely excessive signaling. By contrast,mouse Tregs-IL2pa exhibit sustained expansion,long-term survival in immunocompetent mice for over a year,and bystander expansion of endogenous Tregs. Tregs-IL2pa maintain a stable activated phenotype,Treg-specific demethylation,and a diverse TCR repertoire. In vivo,prophylactic transfer of Tregs-IL2pa ameliorates multi-organ autoimmunity in a Treg depletion-induced mouse autoimmune model. Lastly,compared with control Treg,human Tregs-IL2pa show enhanced survival in the IL-2-depleted environment of immune-deficient mice and improved control of xenogeneic graft-versus-host disease. Our results thus show that IL-2pa self-sufficiency enhances the stability,durability and efficacy of Treg therapies in preclinical settings. Subject terms: Cell delivery,Regulatory T cells,Autoimmune diseases,Interleukins View Publication

Symptomatic dengue virus infection ranges in disease severity from an influenza-like illness to life-threatening shock. One model of the mechanism underlying severe disease proposes that weakly neutralizing,dengue serotype cross-reactive antibodies induced during a primary infection facilitate virus entry into Fc receptor-bearing cells during a subsequent secondary infection,increasing viral replication and the release of cytokines and vasoactive mediators,culminating in shock. This process has been termed antibody-dependent enhancement of infection and has significantly hindered vaccine development. Much of our understanding of this process has come from studies using mouse monoclonal antibodies (MAbs); however,antibody responses in mice typically exhibit less complexity than those in humans. A better understanding of the humoral immune response to natural dengue virus infection in humans is sorely needed. Using a high-efficiency human hybridoma technology,we isolated 37 hybridomas secreting human MAbs to dengue viruses from 12 subjects years or even decades following primary or secondary infection. The majority of the human antibodies recovered were broadly cross-reactive,directed against either envelope or premembrane proteins,and capable of enhancement of infection in vitro; few exhibited serotype-specific binding or potent neutralizing activity. Memory B cells encoding enhancing antibodies predominated in the circulation,even two or more decades following infection. Mapping the epitopes and activity of naturally occurring dengue antibodies should prove valuable in determining whether the enhancing and neutralizing activity of antibodies can be separated. Such principles could be used in the rational design of vaccines that enhance the induction of neutralizing antibodies,while lowering the risk of dengue shock syndrome. View Publication