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

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

MicroRNAs (miRNAs) are pivotal for regulation of hematopoiesis but their critical targets remain largely unknown. Here,we show that ectopic expression of miR-17,-20,-93 and -106,all AAAGUGC seed-containing miRNAs,increases proliferation,colony outgrowth and replating capacity of myeloid progenitors and results in enhanced P-ERK levels. We found that these miRNAs are endogenously and abundantly expressed in myeloid progenitors and down-regulated in mature neutrophils. Quantitative proteomics identified sequestosome 1 (SQSTM1),an ubiquitin-binding protein and regulator of autophagy-mediated protein degradation,as a major target for these miRNAs in myeloid progenitors. In addition,we found increased expression of Sqstm1 transcripts during CSF3-induced neutrophil differentiation of 32D-CSF3R cells and an inverse correlation of SQSTM1 protein levels and miR-106 expression in AML samples. ShRNA-mediated silencing of Sqstm1 phenocopied the effects of ectopic miR-17/20/93/106 expression in hematopoietic progenitors in vitro and in mice. Further,SQSTM1 binds to the ligand-activated colony-stimulating factor 3 receptor (CSF3R) mainly in the late endosomal compartment,but not in LC3 positive autophagosomes. SQSTM1 regulates CSF3R stability and ligand-induced mitogen-activated protein kinase signaling. We demonstrate that AAAGUGC seed-containing miRNAs promote cell expansion,replating capacity and signaling in hematopoietic cells by interference with SQSTM1-regulated pathways. View Publication

Vascular endothelial growth factor (VEGF) and stem cell factor (SCF) act as growth factors for the hemangioblast,an embryonic progenitor of the hematopoietic and endothelial lineages. Because thrombopoietin (TPO) and its receptor,c-Mpl,regulate primitive hematopoietic populations,including bone marrow hematopoietic stem cells,we investigated whether TPO acts on the hemangioblasts that derive from differentiation of embryonic stem cells in vitro. Reverse transcriptase polymerase chain reaction analysis detected expression of c-Mpl beginning on day 3 of embryoid body differentiation when the hemangioblast first arises. In assays of the hemangioblast colony-forming cell (BL-CFC),TPO alone supported BL-CFC formation and nearly doubled the number of BL-CFC when added together with VEGF and SCF. When replated under the appropriate conditions,TPO-stimulated BL-CFC gave rise to secondary hematopoietic colonies,as well as endothelial cells,confirming their nature as hemangioblasts. Addition of a neutralizing anti-VEGF antibody did not block TPO enhancement of BL-CFC formation,suggesting that TPO acts independently of VEGF. These results establish that Mpl signaling plays a role in the earliest stages of hematopoietic development and that TPO represents a third growth factor influencing hemangioblast formation. View Publication

Concomitant inhibition of multiple cancer-driving kinases is an established strategy to improve the durability of clinical responses to targeted therapies. The difficulty of discovering kinase inhibitors with an appropriate multitarget profile has,however,necessitated the application of combination therapies,which can pose major clinical development challenges. Epigenetic reader domains of the bromodomain family have recently emerged as new targets for cancer therapy. Here we report that several clinical kinase inhibitors also inhibit bromodomains with therapeutically relevant potencies and are best classified as dual kinase-bromodomain inhibitors. Nanomolar activity on BRD4 by BI-2536 and TG-101348,which are clinical PLK1 and JAK2-FLT3 kinase inhibitors,respectively,is particularly noteworthy as these combinations of activities on independent oncogenic pathways exemplify a new strategy for rational single-agent polypharmacological targeting. Furthermore,structure-activity relationships and co-crystal structures identify design features that enable a general platform for the rational design of dual kinase-bromodomain inhibitors. View Publication

Previous studies in the mouse indicated that ARID3A plays a critical role in the first cell fate decision required for generation of trophectoderm (TE). Here,we demonstrate that ARID3A is widely expressed during mouse and human placentation and essential for early embryonic viability. ARID3A localizes to trophoblast giant cells and other trophoblast-derived cell subtypes in the junctional and labyrinth zones of the placenta. Conventional Arid3a knockout embryos suffer restricted intrauterine growth with severe defects in placental structural organization. Arid3a null placentas show aberrant expression of subtype-specific markers as well as significant alteration in cytokines,chemokines and inflammatory response-related genes,including previously established markers of human placentation disorders. BMP4-mediated induction of trophoblast stem (TS)-like cells from human induced pluripotent stem cells results in ARID3A up-regulation and cytoplasmic to nuclear translocation. Overexpression of ARID3A in BMP4-mediated TS-like cells up-regulates TE markers,whereas pluripotency markers are down-regulated. Our results reveal an essential,conserved function for ARID3A in mammalian placental development through regulation of both intrinsic and extrinsic developmental programs. View Publication

While a third of the world carries the burden of tuberculosis,disease control has been hindered by a lack of tools,including a rapid,point-of-care diagnostic and a protective vaccine. In many infectious diseases,antibodies (Abs) are powerful biomarkers and important immune mediators. However,in Mycobacterium tuberculosis (Mtb) infection,a discriminatory or protective role for humoral immunity remains unclear. Using an unbiased antibody profiling approach,we show that individuals with latent tuberculosis infection (Ltb) and active tuberculosis disease (Atb) have distinct Mtb-specific humoral responses,such that Ltb infection is associated with unique Ab Fc functional profiles,selective binding to FcγRIII,and distinct Ab glycosylation patterns. Moreover,compared to Abs from Atb,Abs from Ltb drove enhanced phagolysosomal maturation,inflammasome activation,and,most importantly,macrophage killing of intracellular Mtb. Combined,these data point to a potential role for Fc-mediated Ab effector functions,tuned via differential glycosylation,in Mtb control. View Publication

We have examined a number of reagents for their ability to modulate activity of the Hh signaling pathway during embryonic development of Xenopus. In particular we have focused on regulation of events occurring during tailbud stages and later. Two inducible protein reagents based on the Gli1 and Gli3 transcription factors were generated and the activity of these proteins was compared to the Hh signaling pathway inhibitor,cyclopamine,and the activators,Smoothened agonist (SAG) and purmorphamine (PMA). Effectiveness of reagents was assayed using both molecular biological techniques and biological readouts. We found that the small molecule modulators of the Hh pathway were highly specific and effective and produced results generally superior to the more conventional protein reagents for examination of later stage developmental processes. View Publication

Intracellular delivery of macromolecules is a challenge in research and therapeutic applications. Existing vector-based and physical methods have limitations,including their reliance on exogenous materials or electrical fields,which can lead to toxicity or off-target effects. We describe a microfluidic approach to delivery in which cells are mechanically deformed as they pass through a constriction 30–80% smaller than the cell diameter. The resulting controlled application of compression and shear forces results in the formation of transient holes that enable the diffusion of material from the surrounding buffer into the cytosol. The method has demonstrated the ability to deliver a range of material,such as carbon nanotubes,proteins,and siRNA,to 11 cell types,including embryonic stem cells and immune cells. When used for the delivery of transcription factors,the microfluidic devices produced a 10-fold improvement in colony formation relative to electroporation and cell-penetrating peptides. Indeed,its ability to deliver structurally diverse materials and its applicability to difficult-to-transfect primary cells indicate that this method could potentially enable many research and clinical applications. View Publication

The immune system is replenished by self-renewing hematopoietic stem cells (HSCs) that produce multipotent progenitors (MPPs) with little renewal capacity. E-proteins,the widely expressed basic helix-loop-helix transcription factors,contribute to HSC and MPP activity,but their specific functions remain undefined. Using quantitative in vivo and in vitro approaches,we show that E47 is dispensable for the short-term myeloid differentiation of HSCs but regulates their long-term capabilities. E47-deficient progenitors show competent myeloid production in short-term assays in vitro and in vivo. However,long-term myeloid and lymphoid differentiation is compromised because of a progressive loss of HSC self-renewal that is associated with diminished p21 expression and hyperproliferation. The activity of E47 is shown to be cell-intrinsic. Moreover,E47-deficient HSCs and MPPs have altered expression of genes associated with cellular energy metabolism,and the size of the MPP pool but not downstream lymphoid precursors in bone marrow or thymus is rescued in vivo by antioxidant. Together,these observations suggest a role for E47 in the tight control of HSC proliferation and energy metabolism,and demonstrate that E47 is not required for short-term myeloid differentiation. View Publication

The stem cell factor (SCF)/Kit system has served as a classic model in deciphering molecular signaling events in the hematopoietic compartment,and Kit expression is a most critical marker for hematopoietic stem cells (HSCs) and progenitors. However,it remains to be elucidated how Kit expression is regulated in HSCs. Herein we report that a cytoplasmic tyrosine phosphatase Shp2,acting downstream of Kit and other RTKs,promotes Kit gene expression,constituting a Kit-Shp2-Kit signaling axis. Inducible ablation of PTPN11/Shp2 resulted in severe cytopenia in BM,spleen,and peripheral blood in mice. Shp2 removal suppressed the functional pool of HSCs/progenitors,and Shp2-deficient HSCs failed to reconstitute lethally irradiated recipients because of defects in homing,self-renewal,and survival. We show that Shp2 regulates coordinately multiple signals involving up-regulation of Kit expression via Gata2. Therefore,this study reveals a critical role of Shp2 in maintenance of a functional HSC/progenitor pool in adult mammals,at least in part through a kinase-phosphatase-kinase cascade. View Publication