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

There is an urgent need for an efficient approach to obtain a large-scale and renewable source of functional human vascular smooth muscle cells (VSMCs) to establish robust,patient-specific tissue model systems for studying the pathogenesis of vascular disease,and for developing novel therapeutic interventions. Here,we have derived a large quantity of highly enriched functional VSMCs from human induced pluripotent stem cells (hiPSC-VSMCs). Furthermore,we have engineered 3D tissue rings from hiPSC-VSMCs using a facile one-step cellular self-assembly approach. The tissue rings are mechanically robust and can be used for vascular tissue engineering and disease modeling of supravalvular aortic stenosis syndrome. Our method may serve as a model system,extendable to study other vascular proliferative diseases for drug screening. Thus,this report describes an exciting platform technology with broad utility for manufacturing cell-based tissues and materials for various biomedical applications. View Publication

The $\beta$-haemoglobinopathies,such as sickle cell disease and $\beta$-thalassaemia,are caused by mutations in the $\beta$-globin (HBB) gene and affect millions of people worldwide. Ex vivo gene correction in patient-derived haematopoietic stem cells followed by autologous transplantation could be used to cure $\beta$-haemoglobinopathies. Here we present a CRISPR/Cas9 gene-editing system that combines Cas9 ribonucleoproteins and adeno-associated viral vector delivery of a homologous donor to achieve homologous recombination at the HBB gene in haematopoietic stem cells. Notably,we devise an enrichment model to purify a population of haematopoietic stem and progenitor cells with more than 90{\%} targeted integration. We also show efficient correction of the Glu6Val mutation responsible for sickle cell disease by using patient-derived stem and progenitor cells that,after differentiation into erythrocytes,express adult $\beta$-globin (HbA) messenger RNA,which confirms intact transcriptional regulation of edited HBB alleles. Collectively,these preclinical studies outline a CRISPR-based methodology for targeting haematopoietic stem cells by homologous recombination at the HBB locus to advance the development of next-generation therapies for $\beta$-haemoglobinopathies. View Publication

Glioblastoma is the most common form of primary malignant brain tumour. These tumours are highly proliferative and infiltrative resulting in a median patient survival of only 14 months from diagnosis. The current treatment regimens are ineffective against the small population of cancer stem cells residing in the tumourigenic niche; however,a new therapeutic approach could involve the removal of these cells from the microenvironment that maintains the cancer stem cell phenotype. We have isolated multipotent sphere-forming cells from human high grade glioma (glioma sphere-forming cells (GSCs)) to investigate the adhesive and migratory properties of these cells in vitro. We have focused on the role of two closely related metalloproteinases ADAM10 and ADAM17 due to their high expression in glioblastoma and GSCs and their ability to activate cytokines and growth factors. Here,we report that ADAM10 and ADAM17 inhibition selectively increases GSC,but not neural stem cell,migration and that the migrated GSCs exhibit a differentiated phenotype. We also observed a correlation between nestin,a stem/progenitor marker,and fibronectin,an extracellular matrix protein,expression in high grade glioma tissues. GSCs adherence on fibronectin is mediated by α5β1 integrin,where fibronectin further promotes GSC migration and is an effective candidate for in vivo cancer stem cell migration out of the tumourigenic niche. Our results suggest that therapies against ADAM10 and ADAM17 may promote cancer stem cell migration away from the tumourigenic niche resulting in a differentiated phenotype that is more susceptible to treatment. View Publication

Ex vivo CRISPR gene editing in haematopoietic stem and progenitor cells has opened potential treatment modalities for numerous diseases. The current process uses electroporation,sometimes followed by virus transduction. While this complex manipulation has resulted in high levels of gene editing at some genetic loci,cellular toxicity was observed. We have developed a CRISPR nanoformulation based on colloidal gold nanoparticles with a unique loading design capable of cellular entry without the need for electroporation or viruses. This highly monodispersed nanoformulation avoids lysosomal entrapment and localizes to the nucleus in primary human blood progenitors without toxicity. Nanoformulation-mediated gene editing is efficient and sustained with different CRISPR nucleases at multiple loci of therapeutic interest. The engraftment kinetics of nanoformulation-treated primary cells in humanized mice are better relative to those of non-treated cells,with no differences in differentiation. Here we demonstrate non-toxic delivery of the entire CRISPR payload into primary human blood progenitors. View Publication

The oral mucosa plays an important role in maintaining oral and systemic health by protecting the body from harmful environmental stimuli and pathogens. Current reconstructed human gingiva models (RhG) serve as valuable testing platforms for safety and efficacy testing of dental materials,however they lack important phenotypic characteristics typical of the gingival epithelium. We aimed to determine whether incorporating induced pluripotent stem cells (iPSCs) into the hydrogel of a cell-line RhG (reconstructed epithelium on fibroblast-populated-hydrogel) would improve its phenotype. Immortalized human gingival fibroblasts were resuspended with and without iPSCs in collagen-fibrin hydrogels and gingival keratinocytes were seeded on top of the hydrogels to construct RhGs. RhGs were cultured at air-liquid interface for 1,2,4 and 6 weeks and extensively characterized by immunohistochemistry. In situ hybridization for X and Y chromosomes was conducted to identify female iPSCs and male fibroblasts in the RhGs. iPSC-RhGs showed increased epithelial thickening,rete ridge formation,increased cell proliferation and normalized expression of differentiation markers (keratins,involucrin,loricrin,SKALP/elafin) compared to standard RhGs,resulting in an epithelial phenotype very similar to the native gingiva. An increase in apoptotic cells was detected in iPSC-RhGs after 1 week air-exposed culture,and no iPSCs were detected in the hydrogels after 2 weeks air-exposed culture. The increase in apoptotic iPSCs after 1 week air-exposed culture correlated with an increase in keratinocyte proliferation responsible for the superior phenotype observed at 2 weeks. View Publication

Transfer of therapeutic genes to human hematopoietic stem cells (HSCs) using complex vectors at clinically relevant efficiencies remains a major challenge. Recently we described a stable retroviral vector that sustains long-term expression of green fluorescent protein (GFP) and a human beta-globin gene in the erythroid progeny of transduced murine HSCs. We now report the efficient transduction of primitive human CD34(+) fetal liver or cord blood cells with this vector and expression of the beta-globin transgene in the erythroid progeny of these human cells for at least 2 months. After growth factor prestimulation and then a 2- to 3-day exposure to the virus,35% to 55% GFP(+) progeny were seen in assays of transduced colony-forming cells,primitive erythroid precursors that generate large numbers of glycophorin A(+) cells in 3-week suspension cultures,and 6-week long-term culture-initiating cells. In immunodeficient mice injected with unselected infected cells,5% to 15% of the human cells regenerated in the marrow (including the erythroid cells) were GFP(+) 3 and 6 weeks after transplantation. Importantly,the numbers of GFP(+) human lymphoid and either granulopoietic or erythroid cells in individual mice 6 weeks after transplantation were significantly correlated,indicative of the initial transduction of human multipotent cells with in vivo repopulating activity. Expression of the transduced beta-globin gene in human cells obtained directly from the mice or after their differentiation into erythroid cells in vitro was demonstrated by reverse transcriptase-polymerase chain reaction using specific primers. These experiments represent a significant step toward the realization of a gene therapy approach for human beta-globin gene disorders. View Publication

Regulatory T cells (Treg) play an important role in regulating immune homeostasis in health and disease. Traditionally their suppressive function has been assayed by mixing purified cell populations,which does not provide an accurate picture of a physiologically relevant response. To overcome this limitation,we here develop ‘single cell suppression profiling of human Tregs’ (scSPOT). scSPOT uses a 52-marker CyTOF panel,a cell division detection algorithm,and a whole PBMC system to assess the effect of Tregs on all other cell types simultaneously. In this head-to-head comparison,we find Tregs having the clearest suppressive effects on effector memory CD8 T cells through partial division arrest,cell cycle inhibition,and effector molecule downregulation. Additionally,scSPOT identifies a Treg phenotypic split previously observed in viral infection and propose modes of action by the FDA-approved drugs Ipilimumab and Tazemetostat. scSPOT is thus scalable,robust,widely applicable,and may be used to better understand Treg immunobiology and screen for therapeutic compounds. Traditional regulatory T cell (Tregs) assays utilize mixture of purified cell population. Here the authors develop a ‘single cell suppression profiling of human Tregs’ (scSPOT) with 52-marker CyTOF panel,a cell division detection algorithm,and a whole PBMC system to assess Treg suppressive function on all cell types simultaneously. View Publication

The homeobox gene Hoxa-9 is normally expressed in primitive bone marrow cells,and overexpression of Hoxa-9 markedly expands hematopoietic stem cells,suggesting a function in early hematopoiesis. We present evidence for major functional defects in Hoxa-9-/- hematopoietic stem cells. Hoxa-9-/- marrow cells have normal numbers of immunophenotypic stem cells (Lin(-)c-kit(+)flk-2(-)Sca-1+ [KLFS] cells). However,sublethally irradiated Hoxa-9-/- mice develop persistent pancytopenia,indicating unusual sensitivity to ionizing irradiation. In competitive transplantation assays,Hoxa-9-/- cells showed an 8-fold reduction in multilineage long-term repopulating ability,a defect not seen in marrow cells deficient for the adjacent Hoxa-10 gene. Single-cell cultures of KLFS cells showed a 4-fold reduction in large high-proliferation potential colonies. In liquid cultures,Hoxa-9-deficient Lin(-)Sca-1(+) cells showed slowed proliferation (a 5-fold reduction in cell numbers at day 8) and delayed emergence of committed progenitors (a 5-fold decrease in colony-forming cells). Slowing of proliferation was accompanied by a delay in myeloid maturation,with a decrease in Gr-1hiMac-1hi cells at the end of the culture. Retroviral transduction with a Hoxa-9 expression vector dramatically enhanced the cytokine-driven proliferation and in vivo engraftment of Hoxa-9-/- marrow cells. Hoxa-9 appears to be specifically required for normal hematopoietic stem cell function both in vitro and in vivo. View Publication

Ectopic delivery of HOXB4 elicits the expansion of engrafting hematopoietic stem cells (HSCs). We hypothesized that inhibition of tumor necrosis factor-alpha (TNF-alpha) signaling may be central to the self-renewal signature of HOXB4. Because HSCs derived from Fanconi anemia (FA) knockout mice are hypersensitive to TNF-alpha,we studied Fancc(-/-) HSCs to determine the physiologic effects of HOXB4 on TNF-alpha sensitivity and the relationship of these effects to the engraftment defect of FA HSCs. Overexpression of HOXB4 reversed the in vitro hypersensitivity to TNF-alpha of Fancc(-/-) HSCs and progenitors (P) and partially rescued the engraftment defect of these cells. Coexpression of HOXB4 and the correcting FA-C protein resulted in full correction compared with wild-type (WT) HSCs. Ectopic expression of HOXB4 resulted in a reduction in both apoptosis and reactive oxygen species in Fancc(-/-) but not WT HSC/P. HOXB4 overexpression was also associated with a significant reduction in surface expression of TNF-alpha receptors on Fancc(-/-) HSC/P. Finally,enhanced engraftment was seen even when HOXB4 was expressed in a time-limited fashion during in vivo reconstitution. Thus,the HOXB4 engraftment signature may be related to its effects on TNF-alpha signaling,and this pathway may be a molecular target for timed pharmacologic manipulation of HSC during reconstitution. View Publication

Non-small cell lung cancer (NSCLC) is known for high relapse rates despite resection in early stages. Here,we present the results of a phase I clinical trial in which a dendritic cell (DC) vaccine targeting patient-individual neoantigens is evaluated in patients with resected NSCLC. Vaccine manufacturing is feasible in six of 10 enrolled patients. Toxicity is limited to grade 1–2 adverse events. Systemic T cell responses are observed in five out of six vaccinated patients,with T cell responses remaining detectable up to 19 months post vaccination. Single-cell analysis indicates that the responsive T cell population is polyclonal and exhibits the near-entire spectrum of T cell differentiation states,including a naive-like state,but excluding exhausted cell states. Three of six vaccinated patients experience disease recurrence during the follow-up period of 2 years. Collectively,these data support the feasibility,safety,and immunogenicity of this treatment in resected NSCLC. View Publication