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

Realizing the potential of human pluripotent stem cell (hPSC)-based therapy requires the development of defined scalable culture systems with efficient expansion,differentiation and isolation protocols. We report an engineered 3D microfiber system that efficiently supports long-term hPSCs self-renewal under chemically defined conditions. The unique feature of this system lies in the application of a 3D ECM-like environment in which cells are embedded,that affords: (i) uniform high cell loading density in individual cell-laden constructs (∼10 7 cells/ml); (ii) quick recovery of encapsulated cells (textless10min at 37°C) with excellent preservation of cell viability and 3D multicellular structure; (iii) direct cryopreservation of the encapsulated cells in situ in the microfibers with textgreater17-fold higher cell viability compared to those cultured on Matrigel surface; (iv) long-term hPSC propagation under chemically defined conditions. Four hPSC lines propagated in the microfibrous scaffold for 10 consecutive passages were capable of maintaining an undifferentiated phenotype as demonstrated by the expression of stem cell markers and stable karyotype invitro and the ability to form derivatives of the three germ layers both invitro and invivo. Our 3D microfibrous system has the potential for large-scale cultivation of transplantable hESCs and derivatives for clinical applications. textcopyright 2011 Elsevier Ltd. View Publication

Molecular- and cellular-based therapies have the potential to reduce obesity-associated disease. In response to cold,beige adipocytes form in subcutaneous white adipose tissue and convert energy stored in metabolic substrates to heat,making them an attractive therapeutic target. We developed a robust method to generate a renewable source of human beige adipocytes from induced pluripotent stem cells (iPSCs). Developmentally,these cells are derived from FOXF1+ mesoderm and progress through an expandable mural-like mesenchymal stem cell (MSC) to form mature beige adipocytes that display a thermogenically active profile. This includes expression of uncoupling protein 1 (UCP1) concomitant with increased uncoupled respiration. With this method,dysfunctional adipogenic precursors can be reprogrammed and differentiated into beige adipocytes with increased thermogenic function and anti-diabetic secretion potential. This resource can be used to (1) elucidate mechanisms that underlie the control of beige adipogenesis and (2) generate material for cellular-based therapies that target metabolic syndrome in humans. View Publication

New approach methodologies (NAMs) for predicting embryotoxicity and developmental toxicity are urgently needed for generating human relevant data,while reducing turnover time and costs,and alleviating ethical concerns related to the use of animal models. We have previously developed the PluriLum assay,a NKX2.5-reporter gene 3D model using human-induced pluripotent stem cells (hiPSCs) that are genetically modified to enable the assessment of adverse effects of chemicals on the early-stage embryo. Aiming at improving the predictive value of the PluriLum assay for future screening purposes,we sought to introduce standardization steps to the protocol,improving the overall robustness of the PluriLum assay,as well as a shortening of the assay protocol. First,we showed that the initial size of embryoid bodies (EBs) is crucial for a proper differentiation into cardiomyocytes and overall reproducibility of the assay. When the starting diameter of the EBs exceeds 500 µm,robust differentiation can be anticipated. In terms of reproducibility,exposure to the fungicide epoxiconazole at smaller initial diameters resulted in a larger variation of the derived data,compared to more reliable concentration–response curves obtained using spheroids with larger initial diameters. We further investigated the ideal length of the differentiation protocol,resulting in a shortening of the PluriLum assay by 24 h to 7 days. Following exposure to the teratogens all-trans and 13-cis retinoic acid,both cardiomyocyte contraction and measurement of NKX2.5-derived luminescence were recorded with a similar or increased sensitivity after 6 days of differentiation when compared to the original 7 days. Finally,we have introduced an efficient step for enzymatic dissociation of the EBs at assay termination. This allows for an even splitting of the individual EBs and testing of additional endpoints other than the NKX2.5-luciferase reporter,which was demonstrated in this work by the simultaneous assessment of ATP levels. In conclusion,we have introduced standardizations and streamlined the PluriLum assay protocol to improve its suitability as a NAM for screening of a large number of chemicals for developmental toxicity testing. View Publication

Brain Microphysiological Systems,including neural organoids derived from human induced pluripotent stem cells,offer a unique lens to study the intricate workings of the human brain. This paper investigates the foundational elements of learning and memory in neural organoids by quantifying immediate early gene expression in response to chemical modulation,input-specific short- and long-term synaptic plasticity,neuronal network dynamics,connectivity,and criticality to demonstrate the utility of these organoids in basic science research. Neural organoids showed synapse formation,glutamatergic and GABAergic receptor expression,immediate early gene expression basally and evoked,functional connectivity,criticality,and synaptic plasticity in response to theta-burst stimulation. In addition,pharmacological interventions on GABAergic and glutamatergic receptors and input-specific theta-burst stimulation further shed light on the capacity of neural organoids to mirror synaptic modulation,specifically short- and long-term potentiation and depression,demonstrating their potential as tools for studying neurophysiological and neurological processes and informing therapeutic strategies for diseases. Neural organoids exhibit key aspects of learning and memory,including input-specific synaptic plasticity,basal and evoked immediate early gene expression,and critical network dynamics,highlighting their value in modeling human neurophysiology. View Publication

Long-term management for leukemia is challenging due to the painful and invasive procedure of bone marrow (BM) biopsy. At present,non-invasive liquid (blood) biopsy is not utilized for leukemia,due to lower counts of leukemia blast cells in the blood. Here,we described a robust system for the simultaneous detection and enrichment of rare blast cells. Enrichment of blast cells was achieved from blood with a one-step microfluidic blast cell biochip (BCB) sorting system,without specific targeting of proteins by antibodies. Non-target cells encountered a differential net force as compared to stiffer blast cells and were removed. The efficiency of the BCB promotes high detection sensitivity (1 in 106 cells) even from patients with minimal residual disease. The procedure was validated using actual blast cells from patients with various types of leukemia. Outcomes were compared to current evaluation standards,such as flow cytometry,using BM aspirates. Blast cell detection efficiency was higher in 55.6{\%} of the patients using the BCB as compared to flow cytometry,despite the lower concentrations of blast cells in liquid biopsy. These studies promote early-stage detection and routine monitoring for minimal residual disease in patients. View Publication

Transplantation of photoreceptor precursor cells (PPCs) differentiated from human embryonic stem cells (hESCs) is a promising approach to treat common blinding diseases such as age-related macular degeneration and retinitis pigmentosa. However,existing PPC generation methods are inefficient. To enhance differentiation protocols for rapid and high-yield production of PPCs,we focused on optimizing the handling of the cells by including feeder-independent growth of hESCs,using size-controlled embryoid bodies (EBs),and addition of triiodothyronine (T3) and taurine to the differentiation medium,with subsequent removal of undifferentiated cells via negative cell-selection. Our novel protocol produces higher yields of PPCs than previously reported while reducing the time required for differentiation,which will help understand retinal diseases and facilitate large-scale preclinical trials. View Publication

The prevalence of hearing loss after damage to the mammalian cochlea has been thought to be due to a lack of spontaneous regeneration of hair cells,the primary receptor cells for sound. Here,we show that supporting cells,which surround hair cells in the normal cochlear epithelium,differentiate into new hair cells in the neonatal mouse following ototoxic damage. Using lineage tracing,we show that new hair cells,predominantly outer hair cells,arise from Lgr5-expressing inner pillar and third Deiters cells and that new hair cell generation is increased by pharmacological inhibition of Notch. These data suggest that the neonatal mammalian cochlea has some capacity for hair cell regeneration following damage alone and that Lgr5-positive cells act as hair cell progenitors in the cochlea. View Publication

We demonstrated previously that mouse hepatic stellate cells (HSCs) suppress T cells via programmed death-ligand 1 (PD-L1),but it remains unknown whether they exert any effects on B cells,the other component of the adaptive immune system. In this study,we found that mouse HSCs directly inhibited B cells and that PD-L1 was also integrally involved. We found that HSCs inhibited the upregulation of activation markers on activated B cells,as well as the proliferation of activated B cells and their cytokine/Ig production in vitro,and that pharmaceutically or genetically blocking the interaction of PD-L1 with programmed cell death protein 1 impaired the ability of HSCs to inhibit B cells. To test the newly discovered B cell-inhibitory activity of HSCs in vivo,we developed a protocol of intrasplenic artery injection to directly deliver HSCs into the spleen. We found that local delivery of wild-type HSCs into the spleens of mice that had been immunized with 4-hydroxy-3-nitrophenylacetyl-Ficoll,a T cell-independent Ag,significantly suppressed Ag-specific IgM and IgG production in vivo,whereas splenic artery delivery of PD-L1-deficient HSCs failed to do so. In conclusion,in addition to inhibiting T cells,mouse HSCs concurrently inhibit B cells via PD-L1. This direct B cell-inhibitory activity of HSCs should contribute to the mechanism by which HSCs maintain the liver's immune homeostasis. View Publication

BACKGROUND Many groups have generated insulin-secreting cells from hESCs/iPSCs in multiple differentiation stages by mimicking the developmental processes. However,these cells do not always secrete glucose responsive insulin,one of the most important characteristics of pancreatic $$ cells. We focused on the importance of endodermal differentiation from human iPSCs in order to obtain functional pancreatic $$ cells. METHODS We established a 6-stage protocol for the differentiation process from hiPSCs to pancreatic $$ cells using defined culture media without feeders or serum. We examined the effect of CHIR99021,the selective inhibitor of GSK-3$$,in the presence of Activin,FGF2,and BMP4 during definitive endodermal induction by immunostaining for SOX17 and FOXA2. We also compared the insulin secretion at the last stage between monolayer culture and spheroid culture conditions. Cultured cells were transplanted under the kidney capsules of STZ-induced diabetic NOD-SCID mice,and blood glucose levels were measured. Immunohistochemical analysis was performed 4 weeks and 12 weeks after transplantation. RESULTS Addition of CHIR99021 in the presence of Activin,FGF2,and BMP4 for 2 days improved the viability of the endodermal cells,keeping the high positive rate of SOX17. Spheroid formation after the endocrine progenitor stage showed more efficient insulin secretion than monolayer culture did. After cell transplantation,diabetic mice showed lowered blood glucose levels,and we detected islet-like structures in vivo. CONCLUSION We generated functional pancreatic $$ cells from human iPS cells. Induction of definitive endoderm and spheroid formation might be key steps for producing them. View Publication

Embryonic stem (ES) cells are permanent pluripotent stem cell lines established from pre-implantation mouse embryos. There is currently great interest in the potential therapeutic applications of analogous cells derived from human embryos. The isolation of ES cells is commonly presented as a straightforward transfer of cells in the early embryo into culture. In reality,however,continuous expansion of pluripotent cells does not occur in vivo,and in vitro is the exception rather than the norm. Both genetic and epigenetic factors influence the ability to derive ES cells. We have tracked the expression of a key marker and determinant of pluripotency,the transcription factor Oct-4,in primary cultures of mouse epiblasts and used this to assay the effect of experimental manipulations on the maintenance of a pluripotent cell compartment. We find that expression of Oct-4 is often lost prior to overt cytodifferentiation of the epiblast. The rate and extent of Oct-4 extinction varies with genetic background. We report that treatment with the MAP kinase/ERK kinase inhibitor PD98059,which suppresses activation of the mitogen-activated protein kinases Erk1 and Erk2,results in increased persistence of Oct-4-expressing cells. Oct-4 expression is also relatively sustained in cultures of diapause embryos and of isolated inner cell masses. Combination of all three conditions allowed the derivation of germline-competent ES cells from the normally refractory CBA mouse strain. These findings suggest that the genesis of an ES cell is a relatively complex process requiring epigenetic modulation of key gene expression over a brief time-window. Procedures that extend this time-window and/or directly regulate the critical genes should increase the efficiency of ES cell derivation. View Publication

Neural stem cells (NSCs) possess immunosuppressive characteristics,but effects of NSCs on human dendritic cells (DCs),the most important antigen presenting cells,are less well studied. We used an in vitro approach to evaluate the effects of human NSCs on differentiation of human blood CD14+ monocytes into DCs. NSCs derived from H1 human embryonic stem cells (hESC-NSCs) and human ReNcell NSC line,as well as human bone marrow derived mesenchymal stem cells (MSCs),were tested. We observed that in response to treatment with interleukin-4 and granulocyte macrophage colony-stimulating factor CD14+ monocytes co-cultured with NSCs were able to down-regulate CD14 and up-regulate the differentiation marker CD1a,whereas MSC co-culture strongly inhibited CD1a expression and supported prolonged expression of CD14. A similar difference between NSCs and MSCs was noted when lipopolysaccharides were included to induce maturation of monocyte-derived DCs. However,when effects on the function of derived DCs were investigated,NSCs suppressed the elevation of the DC maturation marker CD83,although not the up-regulation of costimulatory molecules CD80,CD86 and CD40,and impaired the functional capacity of the derived DCs to stimulate alloreactive T cells. We did not observe any obvious difference between hESC-NSCs and ReNcell NSCs in inhibiting DC maturation and function. Our data suggest that although human NSCs are less effective than human MSCs in suppressing monocyte differentiation into DCs,these stem cells can still affect the function of DCs,ultimately regulating specific immune responses. View Publication

Mesenchymal stem cells (MSC) are part of the bone marrow that provides signals supporting survival and growth of bystander hematopoietic stem cells (HSC). MSC modulate also the immune response,as they inhibit proliferation of lymphocytes. In order to investigate whether MSC can support survival of T cells,we investigated MSC capacity of rescuing T lymphocytes from cell death induced by different mechanisms. We observed that MSC prolong survival of unstimulated T cells and apoptosis-prone thymocytes cultured under starving conditions. MSC rescued T cells from activation induced cell death (AICD) by downregulation of Fas receptor and Fas ligand on T cell surface and inhibition of endogenous proteases involved in cell death. MSC dampened also Fas receptor mediated apoptosis of CD95 expressing Jurkat leukemic T cells. In contrast,rescue from AICD was not associated with a significant change of Bcl-2,an inhibitor of apoptosis induced by cell stress. Accordingly,MSC exhibited a minimal capacity of rescuing Jurkat cells from chemically induced apoptosis,a process disrupting the mitochondrial membrane potential regulated by Bcl-2. These results suggest that MSC interfere with the Fas receptor regulated process of programmed cell death. Overall,MSC can inhibit proliferation of activated T cells while supporting their survival in a quiescent state,providing a model of their activity inside the HSC niche. Disclosure of potential conflicts of interest is found at the end of this article. View Publication