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

The high proliferation rate of embryonic stem cells (ESCs) is thought to arise partly from very low expression of p21. However,how p21 is suppressed in ESCs has been unclear. We found that p53 binds to the p21 promoter in human ESCs (hESCs) as efficiently as in differentiated human mesenchymal stem cells,however it does not promote p21 transcription in hESCs. We observed an enrichment for both the repressive histone H3K27me3 and activating histone H3K4me3 chromatin marks at the p21 locus in hESCs,suggesting it is a suppressed,bivalent domain which overrides activation by p53. Reducing H3K27me3 methylation in hESCs rescued p21 expression,and ectopic expression of p21 in hESCs triggered their differentiation. Further,we uncovered a subset of bivalent promoters bound by p53 in hESCs that are similarly induced upon differentiation in a p53-dependent manner,whereas p53 promotes the transcription of other target genes which do not show an enrichment of H3K27me3 in ESCs. Our studies reveal a unique epigenetic strategy used by ESCs to poise undesired p53 target genes,thus balancing the maintenance of pluripotency in the undifferentiated state with a robust response to differentiation signals,while utilizing p53 activity to maintain genomic stability and homeostasis in ESCs. View Publication

Induced pluripotent stem cells (iPSCs) have the ability to differentiate in any specialized somatic cell type,which makes them an attractive tool for a wide variety of scientific approaches,including regenerative medicine. However,their pluripotent state and their growth in compact colonies render them difficult to access and,therefore,restrict delivery of specific agents for cell manipulation. Thus,our investigation focus was set on the evaluation of the capability of Layer-by-Layer (LbL) designed microcarriers to serve as a potential drug delivery system to iPSCs,as they offer several appealing advantages. Most notably,these carriers allow for the transport of active agents in a protected environment and for a rather specific delivery through surface modifications. As we could show,charge and mode of LbL carrier application as well as the size of the iPSC colonies determine the interaction with and the uptake rate by iPSCs. None of the examined conditions had an influence on iPSC colony properties such as colony morphology and size or maintenance of pluripotent properties. An overall interaction rate of LbL carriers with iPSCs of up to 20 % was achieved. Those data emphasize the applicability of LbL carriers for stem cell research. Additionally,the potential use of LbL carriers as a promising delivery tool for iPSCs was contrasted to viral particles and liposomes. The identified differences among those delivery tools have substantiated our major conclusion that LbL carrier uptake rate is influenced by characteristic features of the iPSC colonies (most notably colony size) in addition to their surface charges. View Publication

The demand of high cell numbers for applications in cellular therapies and drug screening requires the development of scalable platforms capable to generating highly pure populations of tissue-specific cells from human pluripotent stem cells. In this work,we describe the scaling-up of an aggregate-based culture system for neural induction of human induced pluripotent stem cells (hiPSCs) under chemically-defined conditions. A combination of non-enzymatic dissociation and rotary agitation was successfully used to produce homogeneous populations of hiPSC aggregates with an optimal (140 μm) and narrow distribution of diameters (coefficient of variation of 21.6%). Scalable neural commitment of hiPSCs as 3D aggregates was performed in 50 mL spinner flasks,and the process was optimized using a factorial design approach,involving parameters such as agitation rate and seeding density. We were able to produce neural progenitor cell cultures,that at the end of a 6-day neural induction process contained less than 3% of Oct4-positive cells and that,after replating,retained more than 60% of Pax6-positive neural cells. The results here presented should set the stage for the future generation of a clinically relevant number of human neural progenitors for transplantation and other biomedical applications using controlled,automated and reproducible large-scale bioreactor culture systems. View Publication

Human pluripotent stem cells (hPSCs) - including embryonic stem cells (hESCs) and induced pluripotent stem cells (hiPSCs) - are very promising candidates for cell therapies,tissue engineering,high throughput pharmacology screens,and toxicity testing. These applications require large numbers of high quality cells; however,scalable production of human pluripotent stem cells and their derivatives at a high density and under well-defined conditions has been a challenge. We recently reported a simple,efficient,fully defined,scalable,and good manufacturing practice (GMP) compatible 3D culture system based on a thermoreversible hydrogel for hPSC expansion and differentiation. Here,we describe additional design rationale and characterization of this system. For instance,we have determined that culturing hPSCs as a suspension in a liquid medium can exhibit lower volumetric yields due to cell agglomeration and possible shear force-induced cell loss. By contrast,using hydrogels as 3D scaffolds for culturing hPSCs reduces aggregation and may insulate from shear forces. Additionally,hydrogel-based 3D culture systems can support efficient hPSC expansion and differentiation at a high density if compatible with hPSC biology. Finally,there are considerable opportunities for future development to further enhance hydrogel-based 3D culture systems for producing hPSCs and their progeny. View Publication

The shortage of human organs and tissues for transplant has led to significant interest in xenotransplantation of pig tissues for human patients. However,transplantation of pig organs results in an acute immune rejection,leading to death of the organ within minutes. The $\$-1,3-galactosyltransferase (GALT) gene has been knocked out in pigs to reduce rejection,yet additional genes need to be modified to ultimately make pig tissue immunocompatible with humans. The development of pig induced pluripotent stem cells (piPSCs) from GALT knockout (GALT-KO) tissue would provide an excellent cell source for complex genetic manipulations (e.g.,gene targeting) that often require highly robust and proliferative cells. In this report,we generated GALT-KO piPSCs by the overexpression of POU5F1,SOX2,NANOG,LIN28,KLF-4,and C-MYC reprogramming genes. piPSCs showed classical stem cell morphology and characteristics,expressing integrated reprogramming genes in addition to the pluripotent markers AP,SSEA1,and SSEA4. GALT-KO piPSCs were highly proliferative and possessed doubling times and telomerase activity similar to human embryonic stem cells. These results demonstrated successful reprogramming of GALT-KO fibroblasts into GALT-KO piPSCs. GALT-KO piPSCs are potentially an excellent immortal cell source for the generation of pigs with complex genetic modifications for xenotransplantation,somatic cell nuclear transfer,or chimera formation. View Publication

The field of human induced pluripotent stem cells (hiPSCs) has seen significant progress since the discovery of reprogramming somatic cells using the transcription factors Oct4,Sox2,Klf4,and c-Myc. hiPSCs are similar to embryonic stem cells in a primed state of pluripotency and have the potential to differentiate into any adult human cell type,offering a versatile tool for research and potential therapeutic applications. However,the efficiency of differentiation protocols for generating complex structures with multiple cell types,Like kidney organoids,remains a challenge. This study investigates the impact of treating hiPSCs with a low-dose dimethyl sulfoxide to enhance kidney organoid differentiation using the stepwise 2D monolayer-based protocol developed by Morizane et al. 2017. We found that treating hiPSCs with 1–2% DMSO affects gene expression of pluripotent transcription factors,the epigenetic landscape,and hiPSC colony morphology. Our findings also suggest DMSO treatment enhances the expression of the key metanephric mesenchyme nephron progenitor marker,SIX2 after 9 days of kidney organoid differentiation and helps improve hiPSC differentiation protocol efficiency toward the development of tubular kidney organoids. Further research is needed to fully elucidate the mechanisms underlying these effects and refine the differentiation process for potential in vitro research applications in biomedical research and drug development. View Publication

BackgroundSpinal ventral root avulsion results in massive motoneuron degeneration with poor prognosis and high costs. In this study,we compared different isoforms of basic fibroblast growth factor 2 (FGF2),overexpressed in stably transfected Human embryonic stem cells (hESCs),following motor root avulsion and repair with a heterologous fibrin biopolymer (HFB).MethodsIn the present work,hESCs bioengineered to overexpress 18,23,and 31 kD isoforms of FGF2,were used in combination with reimplantation of the avulsed roots using HFB. Statistical analysis was conducted using GraphPad Prism software with one-way or two-way ANOVA,followed by Tukey’s or Dunnett’s multiple comparison tests. Significance was set at *p < 0.05,**p < 0.01,***p < 0.001,and ****p < 0.0001.ResultsFor the first set of experiments,rats underwent avulsion of the ventral roots with local administration of HFB and engraftment of hESCs expressing the above-mentioned FGF2 isoforms. Analysis of motoneuron survival,glial reaction,and synaptic coverage,two weeks after the lesion,indicated that therapy with hESCs overexpressing 31 kD FGF2 was the most effective. Consequently,the second set of experiments was performed with that isoform,so that ventral root avulsion was followed by direct spinal cord reimplantation. Motoneuron survival,glial reaction,synaptic coverage,and gene expression were analyzed 2 weeks post-lesion; while the functional recovery was evaluated by the walking track test and von Frey test for 12 weeks. We showed that engraftment of hESCs led to significant neuroprotection,coupled with immunomodulation,attenuation of astrogliosis,and preservation of inputs to the rescued motoneurons. Behaviorally,the 31 kD FGF2 - hESC therapy enhanced both motor and sensory recovery.ConclusionTransgenic hESCs were an effective delivery platform for neurotrophic factors,rescuing axotomized motoneurons and modulating glial response after proximal spinal cord root injury,while the 31 kD isoform of FGF2 showed superior regenerative properties over other isoforms in addition to the significant functional recovery.Supplementary InformationThe online version contains supplementary material available at 10.1186/s13287-024-03676-6. View Publication

Mesenchymal stem cells (MSC) establish close interactions with bone marrow sinusoids in a putative perivascular niche. These vessels contain a large storage pool of mature nonproliferating neutrophils. Here,we have investigated the effects of human bone marrow MSC on neutrophil survival and effector functions. MSC from healthy donors,at very low MSC:neutrophil ratios (up to 1:500),significantly inhibited apoptosis of resting and interleukin (IL)-8-activated neutrophils and dampened N-formyl-l-methionin-l-leucyl-l-phenylalanine (f-MLP)-induced respiratory burst. The antiapoptotic activity of MSC did not require cell-to-cell contact,as shown by transwell experiments. Antibody neutralization experiments demonstrated that the key MSC-derived soluble factor responsible for neutrophil protection from apoptosis was IL-6,which signaled by activating STAT-3 transcription factor. Furthermore,IL-6 expression was detected in MSC by real-time reverse transcription-polymerase chain reaction and enzyme-linked immunosorbent assay. Finally,recombinant IL-6 was found to protect neutrophils from apoptosis in a dose-dependent manner. MSC had no effect on neutrophil phagocytosis,expression of adhesion molecules,and chemotaxis in response to IL-8,f-MLP,or C5a. These results support the following conclusions: (a) in the bone marrow niche,MSC likely protect neutrophils of the storage pool from apoptosis,preserving their effector functions and preventing the excessive or inappropriate activation of the oxidative metabolism,and (b) a novel mechanism whereby the inflammatory potential of activated neutrophils is harnessed by inhibition of apoptosis and reactive oxygen species production without impairing phagocytosis and chemotaxis has been identified. View Publication

Increasing evidence suggests that the deposition of amyloid plaques,composed primarily of the amyloid-beta protein (Abeta),within the cerebrovasculature is a frequent occurrence in Alzheimer's disease and may play a significant role in disease progression. Accordingly,the pathogenic mechanisms by which Abeta can alter vascular function may have therapeutic implications. Despite observations that Abeta elicits a number of physiological responses in endothelial cells,ranging from alteration of protein expression to cell death,the Abeta species accountable for these responses remains unexplored. In the current study,we show that isolated soluble Abeta aggregation intermediates activate human brain microvascular endothelial cells for both adhesion and subsequent transmigration of monocyte cells in the absence of endothelial cell death and monolayer disruption. In contrast,unaggregated Abeta monomer and mature Abeta fibril fail to induce any change in endothelial adhesion or transmigration. Correlations between average Abeta aggregate size and observed increases in adhesion illustrate that smaller soluble aggregates are more potent activators of endothelium. These results support previous studies demonstrating heightened neuronal activity of soluble Abeta aggregates,including Abeta-derived diffusible ligands,oligomers,and protofibrils,and further show that soluble aggregates also selectively exhibit activity in a vascular cell model. View Publication

Adoptive cell therapy is an emerging treatment strategy for a number of serious diseases. Regulatory T (Treg) cells represent 1 cell type of particular interest for therapy of inflammatory conditions,as they are responsible for controlling unwanted immune responses. Initial clinical trials of adoptive transfer of Treg cells in patients with graft-versus-host disease were shown to be safe. However,obtaining sufficient numbers of highly pure and functional Treg cells with minimal contamination remains a challenge. We developed a novel approach to isolate untouched" human Treg cells from healthy donors on the basis of negative selection using the surface markers CD49d and CD127. This procedure� View Publication

Proof of drug-target engagement in physiologically-relevant contexts is a key pillar of successful therapeutic target validation. We developed two orthogonal technologies,the cellular thermal shift assay (CETSA) and a covalent chemical probe reporter approach (harnessing sulfonyl fluoride tyrosine labeling and subsequent click chemistry) to measure the occupancy of the mRNA-decapping scavenger enzyme DcpS by a small molecule inhibitor in live cells. Enzyme affinity determined using isothermal dose response fingerprinting (ITDRFCETSA) and the concentration required to occupy 50% of the enzyme (OC50) using the chemical probe reporter assay were very similar. In this case,the chemical probe method worked well due to the long offset kinetics of the reversible inhibitor (determined using a fluorescent dye-tagged probe). This work suggests that CETSA could become the first choice assay to determine in-cell target engagement due to its simplicity. View Publication

Induced pluripotent stem cells (iPSCs) offer an unlimited resource of cells to be used for the study of underlying molecular biology of disease,therapeutic drug screening,and transplant-based regenerative medicine. However,methods for the directed differentiation of skeletal muscle for these purposes remain scarce and incomplete. Here,we present a novel,small molecule-based protocol for the generation of multinucleated skeletal myotubes using eight independent iPSC lines. Through combinatorial inhibition of phosphoinositide 3-kinase (PI3K) and glycogen synthase kinase 3β (GSK3β) with addition of bone morphogenic protein 4 (BMP4) and fibroblast growth factor 2 (FGF2),we report up to 64% conversion of iPSCs into the myogenic program by day 36 as indicated by MYOG+ cell populations. These cells began to exhibit spontaneous contractions as early as 34 days in vitro in the presence of a serum-free medium formulation. We used this protocol to obtain iPSC-derived muscle cells from frontotemporal dementia (FTD) patients harboring C9orf72 hexanucleotide repeat expansions (rGGGGCC),sporadic FTD,and unaffected controls. iPSCs derived from rGGGGCC carriers contained RNA foci but did not vary in differentiation efficiency when compared to unaffected controls nor display mislocalized TDP-43 after as many as 120 days in vitro. This study presents a rapid,efficient,and transgene-free method for generating multinucleated skeletal myotubes from iPSCs and a resource for further modeling the role of skeletal muscle in amyotrophic lateral sclerosis and other motor neuron diseases. SIGNIFICANCE Protocols to produce skeletal myotubes for disease modeling or therapy are scarce and incomplete. The present study efficiently generates functional skeletal myotubes from human induced pluripotent stem cells using a small molecule-based approach. Using this strategy,terminal myogenic induction of up to 64% in 36 days and spontaneously contractile myotubes within 34 days were achieved. Myotubes derived from patients carrying the C9orf72 repeat expansion show no change in differentiation efficiency and normal TDP-43 localization after as many as 120 days in vitro when compared to unaffected controls. This study provides an efficient,novel protocol for the generation of skeletal myotubes from human induced pluripotent stem cells that may serve as a valuable tool in drug discovery and modeling of musculoskeletal and neuromuscular diseases. View Publication