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

Mutation of highly conserved residues in transcription factors may affect protein-protein or protein-DNA interactions,leading to gene network dysregulation and human disease. Human mutations in GATA4,a cardiogenic transcription factor,cause cardiac septal defects and cardiomyopathy. Here,iPS-derived cardiomyocytes from subjects with a heterozygous GATA4-G296S missense mutation showed impaired contractility,calcium handling,and metabolic activity. In human cardiomyocytes,GATA4 broadly co-occupied cardiac enhancers with TBX5,another transcription factor that causes septal defects when mutated. The GATA4-G296S mutation disrupted TBX5 recruitment,particularly to cardiac super-enhancers,concomitant with dysregulation of genes related to the phenotypic abnormalities,including cardiac septation. Conversely,the GATA4-G296S mutation led to failure of GATA4 and TBX5-mediated repression at non-cardiac genes and enhanced open chromatin states at endothelial/endocardial promoters. These results reveal how disease-causing missense mutations can disrupt transcriptional cooperativity,leading to aberrant chromatin states and cellular dysfunction,including those related to morphogenetic defects. View Publication

Self-renewal of human embryonic stem cells (ESCs) is promoted by FGF and TGFbeta/Activin signaling,and differentiation is promoted by BMP signaling,but how these signals regulate genes critical to the maintenance of pluripotency has been unclear. Using a defined medium,we show here that both TGFbeta and FGF signals synergize to inhibit BMP signaling; sustain expression of pluripotency-associated genes such as NANOG,OCT4,and SOX2; and promote long-term undifferentiated proliferation of human ESCs. We also show that both TGFbeta- and BMP-responsive SMADs can bind with the NANOG proximal promoter. NANOG promoter activity is enhanced by TGFbeta/Activin and FGF signaling and is decreased by BMP signaling. Mutation of putative SMAD binding elements reduces NANOG promoter activity to basal levels and makes NANOG unresponsive to BMP and TGFbeta signaling. These results suggest that direct binding of TGFbeta/Activin-responsive SMADs to the NANOG promoter plays an essential role in sustaining human ESC self-renewal. View Publication

Primitive erythroid cells,the first red blood cells produced in the mammalian embryo,are necessary for embryonic survival. Erythropoietin and its receptor EpoR,are absolutely required for survival of late-stage definitive erythroid progenitors in the fetal liver and adult bone marrow. Epo- and Epor-null mice die at E13.5 with a lack of definitive erythrocytes. However,the persistence of circulating primitive erythroblasts raises questions about the role of erythropoietin/EpoR in primitive erythropoiesis. Using Epor-null mice and a novel primitive erythroid 2-step culture we found that erythropoietin is not necessary for specification of primitive erythroid progenitors. However,Epor-null embryos develop a progressive,profound anemia by E12.5 as primitive erythroblasts mature as a synchronous cohort. This anemia results from reduced primitive erythroblast proliferation associated with increased p27 expression,from advanced cellular maturation,and from markedly elevated rates of apoptosis associated with an imbalance in pro- and anti-apoptotic gene expression. Both mouse and human primitive erythroblasts cultured without erythropoietin also undergo accelerated maturation and apoptosis at later stages of maturation. We conclude that erythropoietin plays an evolutionarily conserved role in promoting the proliferation,survival,and appropriate timing of terminal maturation of primitive erythroid precursors. View Publication

Populations of cells create local environments that lead to emergent heterogeneity. This is particularly evident with human pluripotent stem cells (hPSCs): microenvironmental heterogeneity limits hPSC cell fate control. We developed a high-throughput platform to screen hPSCs in configurable microenvironments in which we optimized colony size,cell density and other parameters to achieve rapid and robust cell fate responses to exogenous cues. We used this platform to perform single-cell protein expression profiling,revealing that Oct4 and Sox2 costaining discriminates pluripotent,neuroectoderm,primitive streak and extraembryonic cell fates. We applied this Oct4-Sox2 code to analyze dose responses of 27 developmental factors to obtain lineage-specific concentration optima and to quantify cell line–specific endogenous signaling pathway activation and differentiation bias. We demonstrated that short-term responses predict definitive endoderm induction efficiency and can be used to rescue differentiation of cell lines reticent to cardiac induction. This platform will facilitate high-throughput hPSC-based screening and quantification of lineage-induction bias. View Publication

Cardiac differentiation of human induced pluripotent stem cells is readily achievable,yet derivation of mature cardiomyocytes has been a recognized limitation. Here,a mesoderm priming approach was engineered to boost the maturation of cardiomyocyte progeny derived from pluripotent stem cells under standard cardiac differentiation conditions. Functional and structural hallmarks of maturity were assessed through multiparametric evaluation of cardiomyocytes derived from induced pluripotent stem cells following transfection of the mesoderm transcription factor Brachyury prior to initiation of lineage differentiation. Transfection with Brachyury resulted in earlier induction of a cardiopoietic state as hallmarked by early upregulation of the cardiac-specific transcription factors NKX2.5,GATA4,TBX20. Enhanced sarcomere maturity following Brachyury conditioning was documented by an increase in the proportion of cells expressing the ventricular isoform of myosin light chain and an increase in sarcomere length. Mesoderm primed cells displayed increased reliance on mitochondrial respiration as determined by increased mitochondrial size and a greater basal oxygen consumption rate. Further,Brachyury priming drove maturation of calcium handling enabling transfected cells to maintain calcium transient morphology at higher external field stimulation rates and augmented both calcium release and sequestration kinetics. In addition,transfected cells displayed a more mature action potential morphology with increased depolarization and repolarization kinetics. Derived cells transfected with Brachyury demonstrated increased toxicity response to doxorubicin as determined by a compromise in calcium transient morphology. Thus,Brachyury pre-treatment here achieved a streamlined strategy to promote maturity of human pluripotent stem cell-derived cardiomyocytes establishing a generalizable platform ready for deployment. View Publication

Peptide-based supramolecular nanostructures offer a versatile platform with substantial promise for clinical translation in regenerative medicine. These systems allow for the incorporation of biologically active sequences and can be engineered to modulate tissue-specific parameters such as stiffness,diffusivity,and biodegradability. We developed here a bioactive supramolecular nanostructure containing a peptide designed based on glial cell-derived neurotrophic factor. These nanostructures form scaffolds that mimic important trophic effects provided by this growth factor on iPSC-derived human dopaminergic neurons. Our in vitro data show that the nanostructures promote cell viability,confer neuroprotection against 6-hydroxydopamine toxicity,enhance neuronal morphology,facilitate electrophysiological maturation,and induce genes involved in neuronal survival. We also found that the scaffold promoted axonal extension in midbrain human organoids. These findings suggest that the supramolecular system could be useful to improve outcomes in cell-based therapies for Parkinson’s disease,where progressive dopaminergic degeneration is a hallmark. View Publication

Microsporidia are single-celled intracellular parasites that cause opportunistic diseases in humans. Encephalitozoon intestinalis is a prevalent human-infecting species that invades the small intestine. Macrophages are potential reservoirs of infection,and dissemination to other organ systems is also observed. The macrophage response to infection and the developmental trajectory of the parasite are not well studied. Here we use single cell RNA sequencing to investigate transcriptional changes in both the parasite and the host during E. intestinalis infection of human macrophages in vitro. The parasite undergoes large transcriptional changes throughout the life cycle,providing a blueprint for parasite development. While a small population of infected macrophages mount a response,most remain transcriptionally unchanged,suggesting that the majority of parasites may avoid host detection. The stealthy microsporidian lifestyle likely allows these parasites to harness macrophages for replication. Together,our data provide insights into the host response in primary human macrophages and the E. intestinalis developmental program. Microsporidia such as Encephalitozoon intestinalis are single-celled intracellular parasites that cause opportunistic infections and disease in humans involving infection of macrophages. Here the authors infect human macrophages with E. intestinalis,in vitro and use single cell transcriptomics to assess the consequences of cellular infection compared to bystander effects on macrophages and provide insights into the E. intestinalis developmental program. View Publication

The corneal epithelium acts as a barrier to pathogens entering the eye; corneal epithelial cells are continuously renewed by uni-potent,quiescent limbal stem cells (LSCs) located at the limbus,where the cornea transitions to conjunctiva. There has yet to be a consensus on LSC markers and their transcriptome profile is not fully understood,which may be due to using cadaveric tissue without an intact stem cell niche for transcriptomics. In this study,we addressed this problem by using single nuclei RNA sequencing (snRNAseq) on healthy human limbal tissue that was immediately snap-frozen after excision from patients undergoing cataract surgery. We identified the quiescent LSCs as a sub-population of corneal epithelial cells with a low level of total transcript counts. Moreover,TP63,KRT15,CXCL14,and ITGβ4 were found to be highly expressed in LSCs and transiently amplifying cells (TACs),which constitute the corneal epithelial progenitor populations at the limbus. The surface markers SLC6A6 and ITGβ4 could be used to enrich human corneal epithelial cell progenitors,which were also found to specifically express the putative limbal progenitor cell markers MMP10 and AC093496.1. View Publication

Astrocytes are essential for the formation and maintenance of neural networks. However,a major technical challenge for investigating astrocyte function and disease-related pathophysiology has been the limited ability to obtain functional human astrocytes. Despite recent advances in human pluripotent stem cell (hPSC) techniques,primary rodent astrocytes remain the gold standard in coculture with human neurons. We demonstrate that a combination of leukemia inhibitory factor (LIF) and bone morphogenetic protein-4 (BMP4) directs hPSC-derived neural precursor cells to a highly pure population of astroglia in 28 d. Using single-cell RNA sequencing,we confirm the astroglial identity of these cells and highlight profound transcriptional adaptations in cocultured hPSC-derived astrocytes and neurons,consistent with their further maturation. In coculture with human neurons,multielectrode array recordings revealed robust network activity of human neurons in a coculture with hPSC-derived or rat astrocytes [3.63 ± 0.44 min −1 (hPSC-derived),2.86 ± 0.64 min −1 (rat); p = 0.19]. In comparison,we found increased spike frequency within network bursts of human neurons cocultured with hPSC-derived astrocytes [56.31 ± 8.56 Hz (hPSC-derived),24.77 ± 4.04 Hz (rat); p < 0.01],and whole-cell patch-clamp recordings revealed an increase of postsynaptic currents [2.76 ± 0.39 Hz (hPSC-derived),1.07 ± 0.14 Hz (rat); p < 0.001],consistent with a corresponding increase in synapse density [14.90 ± 1.27/100 μm 2 (hPSC-derived),8.39 ± 0.63/100 μm 2 (rat); p < 0.001]. Taken together,we show that hPSC-derived astrocytes compare favorably with rat astrocytes in supporting human neural network activity and maturation,providing a fully human platform for investigating astrocyte function and neuronal-glial interactions. View Publication

Brain organoids have been proposed as suitable human brain model candidates for a variety of applications. However,the lack of appropriate maturation limits the transferability of such functional tools. Here,we present a method to facilitate neuronal maturation by integrating astrocyte-secreted factors into hPSC-derived 2D and 3D neural culture systems. We demonstrate that protein- and nutrient-enriched astrocyte-conditioned medium (ACM) accelerates neuronal differentiation with enlarged neuronal layer and the overproduction of deep-layer cortical neurons. We captured the elevated changes in the functional activity of neuronal networks within ACM-treated organoids using comprehensive electrophysiological recordings. Furthermore,astrocyte-secreted cues can induce lipid droplet accumulation in neural cultures,offering protective effects in neural differentiation to withstand cellular stress. Together,these data indicate the potential of astrocyte secretions to promote neural maturation. Subject terms: Neurological models,Neuronal development View Publication