RESUMEN
PURPOSE: We established the genetic etiology of a syndromic neurodevelopmental condition characterized by variable cognitive impairment, recognizable facial dysmorphism, and a constellation of extra-neurological manifestations. METHODS: We performed phenotypic characterization of 6 participants from 4 unrelated families presenting with a neurodevelopmental syndrome and used exome sequencing to investigate the underlying genetic cause. To probe relevance to the neurodevelopmental phenotype and craniofacial dysmorphism, we established two- and three-dimensional human stem cell-derived neural models and generated a stable cachd1 zebrafish mutant on a transgenic cartilage reporter line. RESULTS: Affected individuals showed mild cognitive impairment, dysmorphism featuring oculo-auriculo abnormalities, and developmental defects involving genitourinary and digestive tracts. Exome sequencing revealed biallelic putative loss-of-function variants in CACHD1 segregating with disease in all pedigrees. RNA sequencing in CACHD1-depleted neural progenitors revealed abnormal expression of genes with key roles in Wnt signaling, neurodevelopment, and organ morphogenesis. CACHD1 depletion in neural progenitors resulted in reduced percentages of post-mitotic neurons and enlargement of 3D neurospheres. Homozygous cachd1 mutant larvae showed mandibular patterning defects mimicking human facial dysmorphism. CONCLUSION: Our findings support the role of loss-of-function variants in CACHD1 as the cause of a rare neurodevelopmental syndrome with facial dysmorphism and multisystem abnormalities.
Asunto(s)
Anomalías Múltiples , Anomalías Craneofaciales , Anomalías Musculoesqueléticas , Trastornos del Neurodesarrollo , Animales , Humanos , Anomalías Múltiples/genética , Anomalías Craneofaciales/genética , Discapacidad Intelectual/genética , Anomalías Musculoesqueléticas/genética , Trastornos del Neurodesarrollo/genética , Fenotipo , Síndrome , Pez Cebra/genéticaRESUMEN
Human genome variation contributes to diversity in neurodevelopmental outcomes and vulnerabilities; recognizing the underlying molecular and cellular mechanisms will require scalable approaches. Here, we describe a "cell village" experimental platform we used to analyze genetic, molecular, and phenotypic heterogeneity across neural progenitor cells from 44 human donors cultured in a shared in vitro environment using algorithms (Dropulation and Census-seq) to assign cells and phenotypes to individual donors. Through rapid induction of human stem cell-derived neural progenitor cells, measurements of natural genetic variation, and CRISPR-Cas9 genetic perturbations, we identified a common variant that regulates antiviral IFITM3 expression and explains most inter-individual variation in susceptibility to the Zika virus. We also detected expression QTLs corresponding to GWAS loci for brain traits and discovered novel disease-relevant regulators of progenitor proliferation and differentiation such as CACHD1. This approach provides scalable ways to elucidate the effects of genes and genetic variation on cellular phenotypes.
Asunto(s)
Células-Madre Neurales , Infección por el Virus Zika , Virus Zika , Humanos , Células-Madre Neurales/metabolismo , Diferenciación Celular/genética , Encéfalo/metabolismo , Virus Zika/metabolismo , Expresión Génica , Proteínas de la Membrana/metabolismo , Proteínas de Unión al ARN/metabolismoRESUMEN
Mutations in N-glycanase 1 (NGLY1), which deglycosylates misfolded glycoproteins for degradation, can cause NGLY1 deficiency in patients and their abnormal fetal development in multiple organs, including microcephaly and other neurological disorders. Using cerebral organoids (COs) developed from human embryonic stem cells (hESCs) and induced pluripotent stem cells (hiPSCs), we investigate how NGLY1 dysfunction disturbs early brain development. While NGLY1 loss had limited impact on the undifferentiated cells, COs developed from NGLY1-deficient hESCs showed defective formation of SATB2-positive upper-layer neurons, and attenuation of STAT3 and HES1 signaling critical for sustaining radial glia. Bulk and single-cell transcriptomic analysis revealed premature neuronal differentiation accompanied by downregulation of secreted and transcription factors, including TTR, IGFBP2, and ID4 in NGLY1-deficient COs. NGLY1 malfunction also dysregulated ID4 and enhanced neuronal differentiation in CO transplants developed in vivo. NGLY1-deficient CO cells were more vulnerable to multiple stressors; treating the deficient cells with recombinant TTR reduced their susceptibility to stress from proteasome inactivation, likely through LRP2-mediated activation of MAPK signaling. Expressing NGLY1 led to IGFBP2 and ID4 upregulation in CO cells developed from NGLY1-deficiency patient's hiPSCs. In addition, treatment with recombinant IGFBP2 enhanced ID4 expression, STAT3 signaling, and proliferation of NGLY1-deficient CO cells. Overall, our discoveries suggest that dysregulation of stress responses and neural precursor differentiation underlies the brain abnormalities observed in NGLY1-deficient individuals.
Asunto(s)
Organoides , Péptido-N4-(N-acetil-beta-glucosaminil) Asparagina Amidasa/metabolismo , Complejo de la Endopetidasa Proteasomal , Glicoproteínas/metabolismo , Humanos , Neurogénesis , Organoides/metabolismo , Péptido-N4-(N-acetil-beta-glucosaminil) Asparagina Amidasa/genética , Complejo de la Endopetidasa Proteasomal/metabolismoRESUMEN
Until the discovery of human embryonic stem cells and human induced pluripotent stem cells, biotechnology companies were severely limited in the number of human tissues that they could model in large-scale in vitro studies. Until this point, companies have been limited to immortalized cancer lines or a small number of primary cell types that could be extracted and expanded. Nowadays, protocols continue to be developed in the stem cell field, enabling researchers to model an ever-growing library of cell types in controlled, large-scale screens. One differentiation method in particular- cerebral organoids- shows substantial potential in the field of neuroscience and developmental neurobiology. Cerebral organoid technology is still in an early phase of development, and there are several challenges that are currently being addressed by academic and industrial researchers alike. Here we briefly describe some of the early adopters of cerebral organoids, several of the challenges that they are likely facing, and various technologies that are currently being implemented to overcome them.
Asunto(s)
Descubrimiento de Drogas/métodos , Drogas en Investigación/farmacología , Modelos Biológicos , Enfermedades Neurodegenerativas/tratamiento farmacológico , Fármacos Neuroprotectores/farmacología , Organoides/efectos de los fármacos , Sistemas CRISPR-Cas , Diferenciación Celular , Corteza Cerebral/efectos de los fármacos , Corteza Cerebral/metabolismo , Corteza Cerebral/patología , Drogas en Investigación/química , Humanos , Células Madre Pluripotentes Inducidas/citología , Células Madre Pluripotentes Inducidas/efectos de los fármacos , Células Madre Pluripotentes Inducidas/metabolismo , Aprendizaje Automático , Mutación , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/metabolismo , Enfermedades Neurodegenerativas/genética , Enfermedades Neurodegenerativas/metabolismo , Enfermedades Neurodegenerativas/patología , Neuronas/citología , Neuronas/efectos de los fármacos , Neuronas/metabolismo , Fármacos Neuroprotectores/química , Organoides/metabolismo , Organoides/patología , Análisis de Secuencia de ARN/métodos , Análisis de la Célula Individual/métodosRESUMEN
We develop CellSIUS (Cell Subtype Identification from Upregulated gene Sets) to fill a methodology gap for rare cell population identification for scRNA-seq data. CellSIUS outperforms existing algorithms for specificity and selectivity for rare cell types and their transcriptomic signature identification in synthetic and complex biological data. Characterization of a human pluripotent cell differentiation protocol recapitulating deep-layer corticogenesis using CellSIUS reveals unrecognized complexity in human stem cell-derived cellular populations. CellSIUS enables identification of novel rare cell populations and their signature genes providing the means to study those populations in vitro in light of their role in health and disease.
Asunto(s)
Análisis de la Célula Individual/métodos , Transcriptoma , Algoritmos , Línea Celular , Humanos , Neuronas/citologíaRESUMEN
Human pluripotent stem cells (hPSCs) generate a variety of disease-relevant cells that can be used to improve the translation of preclinical research. Despite the potential of hPSCs, their use for genetic screening has been limited by technical challenges. We developed a scalable and renewable Cas9 and sgRNA-hPSC library in which loss-of-function mutations can be induced at will. Our inducible mutant hPSC library can be used for multiple genome-wide CRISPR screens in a variety of hPSC-induced cell types. As proof of concept, we performed three screens for regulators of properties fundamental to hPSCs: their ability to self-renew and/or survive (fitness), their inability to survive as single-cell clones, and their capacity to differentiate. We identified the majority of known genes and pathways involved in these processes, as well as a plethora of genes with unidentified roles. This resource will increase the understanding of human development and genetics. This approach will be a powerful tool to identify disease-modifying genes and pathways.
Asunto(s)
Sistemas CRISPR-Cas/genética , Pruebas Genéticas/métodos , Genoma/genética , Células Madre Pluripotentes/metabolismo , HumanosRESUMEN
CRISPR/Cas9 has revolutionized our ability to engineer genomes and conduct genome-wide screens in human cells1-3. Whereas some cell types are amenable to genome engineering, genomes of human pluripotent stem cells (hPSCs) have been difficult to engineer, with reduced efficiencies relative to tumour cell lines or mouse embryonic stem cells3-13. Here, using hPSC lines with stable integration of Cas9 or transient delivery of Cas9-ribonucleoproteins (RNPs), we achieved an average insertion or deletion (indel) efficiency greater than 80%. This high efficiency of indel generation revealed that double-strand breaks (DSBs) induced by Cas9 are toxic and kill most hPSCs. In previous studies, the toxicity of Cas9 in hPSCs was less apparent because of low transfection efficiency and subsequently low DSB induction3. The toxic response to DSBs was P53/TP53-dependent, such that the efficiency of precise genome engineering in hPSCs with a wild-type P53 gene was severely reduced. Our results indicate that Cas9 toxicity creates an obstacle to the high-throughput use of CRISPR/Cas9 for genome engineering and screening in hPSCs. Moreover, as hPSCs can acquire P53 mutations14, cell replacement therapies using CRISPR/Cas9-enginereed hPSCs should proceed with caution, and such engineered hPSCs should be monitored for P53 function.
Asunto(s)
Proteína 9 Asociada a CRISPR/metabolismo , Sistemas CRISPR-Cas/genética , Ingeniería Genética , Células Madre Pluripotentes/metabolismo , Proteína p53 Supresora de Tumor/metabolismo , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/genética , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/metabolismo , Roturas del ADN de Doble Cadena , Eliminación de Gen , Humanos , ARN Guía de Kinetoplastida/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Transcripción Genética , Receptor fas/genética , Receptor fas/metabolismoRESUMEN
The recent emergence of Zika virus (ZIKV) in susceptible populations has led to an abrupt increase in microcephaly and other neurodevelopmental conditions in newborn infants. While mosquitos are the main route of viral transmission, it has also been shown to spread via sexual contact and vertical mother-to-fetus transmission. In this latter case of transmission, due to the unique viral tropism of ZIKV, the virus is believed to predominantly target the neural progenitor cells (NPCs) of the developing brain. Here a method for modeling ZIKV infection, and the resulting microcephaly, that occur when human cerebral organoids are exposed to live ZIKV is described. The organoids display high levels of virus within their neural progenitor population, and exhibit severe cell death and microcephaly over time. This three-dimensional cerebral organoid model allows researchers to conduct species-matched experiments to observe and potentially intervene with ZIKV infection of the developing human brain. The model provides improved relevance over standard two-dimensional methods, and contains human-specific cellular architecture and protein expression that are not possible in animal models.
Asunto(s)
Encéfalo/virología , Células-Madre Neurales/virología , Organoides/virología , Infección por el Virus Zika/virología , Encéfalo/patología , Humanos , Células-Madre Neurales/patologíaRESUMEN
Zika virus (ZIKV) can cross the placental barrier, resulting in infection of the fetal brain and neurological defects including microcephaly. The cellular tropism of ZIKV and the identity of attachment factors used by the virus to gain access to key cell types involved in pathogenesis are under intense investigation. Initial studies suggested that ZIKV preferentially targets neural progenitor cells (NPCs), providing an explanation for the developmental phenotypes observed in some pregnancies. The AXL protein has been nominated as a key attachment factor for ZIKV in several cell types including NPCs. However, here we show that genetic ablation of AXL has no effect on ZIKV entry or ZIKV-mediated cell death in human induced pluripotent stem cell (iPSC)-derived NPCs or cerebral organoids. These findings call into question the utility of AXL inhibitors for preventing birth defects after infection and suggest that further studies of viral attachment factors in NPCs are needed.
Asunto(s)
Cerebro/patología , Eliminación de Gen , Células-Madre Neurales/metabolismo , Células-Madre Neurales/virología , Neuroprotección , Organoides/virología , Proteínas Proto-Oncogénicas/genética , Proteínas Tirosina Quinasas Receptoras/genética , Infección por el Virus Zika/prevención & control , Muerte Celular , Técnicas de Inactivación de Genes , Humanos , Células-Madre Neurales/patología , Organoides/metabolismo , Organoides/patología , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Tirosina Quinasas Receptoras/metabolismo , Infección por el Virus Zika/patología , Tirosina Quinasa del Receptor AxlRESUMEN
Although phase separation is a simple method of preparing tissue engineering scaffolds, it suffers from organic solvent residual in the scaffold. Searching for nontoxic solvents and developing effective solvent removal methods are current challenges in scaffold fabrication. In this study, thermoplastic polyurethane (TPU) scaffolds containing carbon nanotubes (CNTs) or nanofibrillated cellulose fibers (NFCs) were prepared using low toxicity dimethyl sulfoxide (DMSO) as a solvent. The effects of two solvent removal approaches on the final scaffold morphology were studied. The freeze drying method caused large pores, with small pores on the pore walls, which created connections between the pores. Meanwhile, the leaching and freeze drying method led to interconnected fine pores with smaller pore diameters. The nucleation effect of CNTs and the phase separation behavior of NFCs in the TPU solution resulted in significant differences in the microstructures of the resulting scaffolds. The mechanical performance of the nanocomposite scaffolds with different morphologies was investigated. Generally, the scaffolds with a fine pore structure showed higher compressive properties, and both the CNTs and NFCs improved the compressive properties of the scaffolds, with greater enhancement found in TPU/NFC nanocomposite scaffolds. In addition, all scaffolds showed good sustainability under cyclical load bearing, and the biocompatibility of the scaffolds was verified via 3T3 fibroblast cell culture.
Asunto(s)
Celulosa/química , Nanotubos de Carbono/química , Poliuretanos/química , Andamios del Tejido , Células 3T3 , Animales , Materiales Biocompatibles/química , Dimetilsulfóxido , Liofilización , Ratones , Porosidad , Solventes , Ingeniería de TejidosRESUMEN
Several studies have reported reprogramming of fibroblasts into induced cardiomyocytes; however, reprogramming into proliferative induced cardiac progenitor cells (iCPCs) remains to be accomplished. Here we report that a combination of 11 or 5 cardiac factors along with canonical Wnt and JAK/STAT signaling reprogrammed adult mouse cardiac, lung, and tail tip fibroblasts into iCPCs. The iCPCs were cardiac mesoderm-restricted progenitors that could be expanded extensively while maintaining multipotency to differentiate into cardiomyocytes, smooth muscle cells, and endothelial cells in vitro. Moreover, iCPCs injected into the cardiac crescent of mouse embryos differentiated into cardiomyocytes. iCPCs transplanted into the post-myocardial infarction mouse heart improved survival and differentiated into cardiomyocytes, smooth muscle cells, and endothelial cells. Lineage reprogramming of adult somatic cells into iCPCs provides a scalable cell source for drug discovery, disease modeling, and cardiac regenerative therapy.
Asunto(s)
Proliferación Celular , Técnicas de Reprogramación Celular/métodos , Reprogramación Celular , Fibroblastos/metabolismo , Mioblastos Cardíacos/metabolismo , Factores de Transcripción/biosíntesis , Animales , Supervivencia Celular , Fibroblastos/citología , Ratones , Ratones Transgénicos , Mioblastos Cardíacos/citología , Factores de Transcripción/genéticaRESUMEN
The mechanical properties of vascular tissues affect hemodynamics and can alter disease progression. The uniaxial tensile test is a simple and effective method for determining the stress-strain relationship in arterial tissue ex vivo. To enable calculation of strain, stretch can be measured directly with image tracking of markers on the tissue or indirectly from the distance between the grips used to hold the specimen. While the imaging technique is generally considered more accurate, it also requires more analysis, and the grip distance method is more widely used. The purpose of this study is to compare the stretch of the testing specimen calculated from the grip distance method to that obtained from the imaging method for canine descending aortas and large proximal pulmonary arteries. Our results showed a significant difference in stretch between the two methods; however, this difference was consistently less than 2%. Therefore, the grip distance method is an accurate approximation of the stretch in large elastic arteries in the uniaxial tensile test.
Asunto(s)
Aorta Torácica , Elasticidad , Ensayo de Materiales , Arteria Pulmonar , Resistencia a la Tracción , Animales , Perros , Estrés MecánicoRESUMEN
Fabrication of small diameter vascular grafts plays an important role in vascular tissue engineering. In this study, thermoplastic polyurethane (TPU)/graphene oxide (GO) scaffolds were fabricated via electrospinning at different GO contents as potential candidates for small diameter vascular grafts. In terms of mechanical and surface properties, the tensile strength, Young's modulus, and hydrophilicity of the scaffolds increased with an increase of GO content while plasma treatment dramatically improved the scaffold hydrophilicity. Mouse fibroblast (3T3) and human umbilical vein endothelial cells (HUVECs) were cultured on the scaffolds separately to study their biocompatibility and potential to be used as vascular grafts. It was found that cell viability for both types of cells, fibroblast proliferation, and HUVEC attachment were the highest at a 0.5wt.% GO loading whereas oxygen plasma treatment also enhanced HUVEC viability and attachment significantly. In addition, the suture retention strength and burst pressure of tubular TPU/GO scaffolds containing 0.5wt.% GO were found to meet the requirements of human blood vessels, and endothelial cells were able to attach to the inner surface of the tubular scaffolds. Platelet adhesion tests using mice blood indicated that vascular scaffolds containing 0.5% GO had low platelet adhesion and activation. Therefore, the electrospun TPU/GO tubular scaffolds have the potential to be used in vascular tissue engineering.
Asunto(s)
Grafito , Poliuretanos , Andamios del Tejido , Células 3T3 , Animales , Plaquetas/citología , Adhesión Celular , Células Endoteliales de la Vena Umbilical Humana , Humanos , Ratones , Óxidos , Espectrometría Raman , Resistencia a la TracciónRESUMEN
Poly(ethylene glycol) (PEG)-grafted cellulose nanocrystals (CNCs) were successfully synthesized and incorporated into poly(lactic acid) (PLA) as a reinforcing filler to produce nanocomposite scaffolds consisting of CNC-g-PEG and PLA using an electrospinning technique. Morphological, thermal, mechanical, and wettability properties as well as preliminary biocompatibility using human mesenchymal stem cells (hMSCs) of PLA/CNC and PLA/CNC-g-PEG nanocomposite scaffolds were characterized and compared. The average diameter of the electrospun nanofibers decreased with increased filler loading level, due to the increased conductivity of the electrospun solutions. DSC results showed that both the glass transition temperature and cold crystallization temperature decreased progressively with higher CNC-g-PEG loading level, suggesting that improved interfacial adhesion between CNCs and PLA was achieved by grafting PEG onto the CNCs. Wettability of the electrospun nanofibers was not affected with the addition of CNCs or CNC-g-PEG and indicating that the fillers tended to stay inside of the fiber matrix under electrical field. The tensile strength of the composite fiber mats was effectively improved by the addition of up to 5% CNC-g-PEG up to 5wt.%. In addition, the cell culture results showed that PLA/CNC-g-PEG composite nanofibers exhibited improved biocompatibility to hMSCs, which revealed the potential application of this nanocomposite as the scaffolds in bone tissue engineering.
Asunto(s)
Huesos/citología , Celulosa/química , Nanocompuestos , Nanopartículas/química , Polietilenglicoles/química , Ingeniería de Tejidos , Andamios del Tejido , Materiales BiocompatiblesRESUMEN
Unidirectionally and orthogonally aligned thermoplastic polyurethane (TPU) nanofibers were electrospun using a custom-built electrospinning device. The unidirectionally aligned fibers were collected using two parallel copper plates, and the orthogonally aligned fibers were collected using two orthogonal sets of parallel copper plates with alternate negative connections. Carbon nanotubes (CNT) and polyacrylic acid (PAA) were added to modify the polymer solution. It was found that both CNT and PAA were capable of increasing solution conductivity. The TPU/PAA fiber showed the highest degree of fiber orientation with more than 90% of the fibers having an orientation angle between -10° and 10° for unidirectionally aligned fibers, and for orthogonally aligned fibers, the orientation angle of 50% fibers located between -10° and 10° and 48% fibers located between 80° and 100°. Viability assessment of 3T3 fibroblasts cultured on TPU/PAA fibers suggested that the material was cytocompatible. The cells' orientation and migration direction closely matched the fibers' orientation. The cell migration velocity and distance were both enhanced with the guidance of fibers compared with cells cultured on random fibers and common tissue culture plastic. Controlling cell migration velocity and directionality may provide ways to influence differentiation and gene expression and systems that would allow further exploration of wound repair and metastatic cell behavior.
Asunto(s)
Resinas Acrílicas/química , Movimiento Celular , Nanofibras/química , Nanotubos de Carbono/química , Células 3T3 , Animales , RatonesRESUMEN
Soft and hard thermoplastic polyurethane (TPU) and their blends were electrospun to fabricate nanofibrous scaffolds with various properties in order to investigate the substrate property effects on cellular response. The scaffolds were characterized with Fourier transform infrared spectroscopy, thermogravimetric analysis, scanning electron microscopy, water contact angle tests, and protein absorption tests. It was found that the hard segment content in the scaffold increased with the hard TPU ratio, which resulted in improved hydrophobicity and decreased over all protein absorption. 3T3 fibroblasts were cultured on those scaffolds to investigate the cellular response. On soft TPU scaffolds, the cells formed were round in shape and aggregated into clusters. However, on hard TPU scaffolds, the cells exhibited a spindle shape and spread out on the scaffolds, indicating preferred cell-substrate interaction. The cell viability and proliferation of cells on hard scaffolds were higher than on soft scaffolds and on 50% hard/50% soft scaffolds.
Asunto(s)
Forma de la Célula , Nanofibras/química , Poliuretanos/química , Andamios del Tejido/química , Células 3T3 , Animales , RatonesRESUMEN
Patients with mammographically dense breast tissue have a greatly increased risk of developing breast cancer. Dense breast tissue contains more stromal collagen, which contributes to increased matrix stiffness and alters normal cellular responses. Stromal collagen within and surrounding mammary tumors is frequently aligned and reoriented perpendicular to the tumor boundary. We have shown that aligned collagen predicts poor outcome in breast cancer patients, and postulate this is because it facilitates invasion by providing tracks on which cells migrate out of the tumor. However, the mechanisms by which alignment may promote migration are not understood. Here, we investigated the contribution of matrix stiffness and alignment to cell migration speed and persistence. Mechanical measurements of the stiffness of collagen matrices with varying density and alignment were compared with the results of a 3D microchannel alignment assay to quantify cell migration. We further interpreted the experimental results using a computational model of cell migration. We find that collagen alignment confers an increase in stiffness, but does not increase the speed of migrating cells. Instead, alignment enhances the efficiency of migration by increasing directional persistence and restricting protrusions along aligned fibers, resulting in a greater distance traveled. These results suggest that matrix topography, rather than stiffness, is the dominant feature by which an aligned matrix can enhance invasion through 3D collagen matrices.
Asunto(s)
Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Colágeno/metabolismo , Línea Celular Tumoral , Movimiento Celular , Matriz Extracelular/metabolismo , Femenino , Geles , Humanos , Modelos BiológicosRESUMEN
In this study, human embryonic stem cell-derived cardiomyocytes were seeded onto controlled two-dimensional micropatterned features, and an improvement in sarcomere formation and cell alignment was observed in specific feature geometries. High-resolution photolithography techniques and microcontact printing were utilized to produce features of various rectangular geometries, with areas ranging from 2500 µm(2) to 160,000 µm(2). The microcontact printing method was used to pattern non-adherent poly(ethylene glycol) regions on gold coated glass slides. Matrigel and fibronectin extracellular matrix (ECM) proteins were layered onto the gold-coated glass slides, providing a controlled geometry for cell adhesion. We used small molecule-based differentiation and an antibiotic purification step to produce a pure population of immature cardiomyocytes from H9 human embryonic stem cells (hESCs). We then seeded this pure population of human cardiomyocytes onto the micropatterned features of various sizes and observed how the cardiomyocytes remodeled their myofilament structure in response to the feature geometries. Immunofluorescence was used to measure α-actinin expression, and phalloidin stains were used to detect actin presence in the patterned cells. Analysis of nuclear alignment was also used to determine how cell direction was influenced by the features. The seeded cells showed clear alignment with the features, dependent on the width rather than the overall aspect ratio of the features. It was determined that features with widths between 30 µm and 80 µm promoted highly aligned cardiomyocytes with a dramatic increase in sarcomere alignment relative to the long axis of the pattern. This creation of highly-aligned cell aggregates with robust sarcomere structures holds great potential in advancing cell-based pharmacological studies, and will help researchers to understand the means by which ECM geometries can affect myofilament structure and maturation in hESC-derived cardiomyocytes.
Asunto(s)
Células Madre Embrionarias/citología , Miocitos Cardíacos/citología , Sarcómeros/metabolismo , Ingeniería de Tejidos/métodos , Andamios del Tejido/química , Adhesión Celular , Diferenciación Celular , Línea Celular , Colágeno/química , Combinación de Medicamentos , Células Madre Embrionarias/metabolismo , Fibronectinas/química , Oro/química , Humanos , Laminina/química , Miocitos Cardíacos/metabolismo , Proteoglicanos/química , Propiedades de SuperficieRESUMEN
Polylactic acid (PLA) and thermoplastic polyurethane (TPU) are two kinds of biocompatible and biodegradable polymers that can be used in biomedical applications. PLA has rigid mechanical properties while TPU possesses flexible mechanical properties. Blended TPU/PLA tissue engineering scaffolds at different ratios for tunable properties were fabricated via twin screw extrusion and microcellular injection molding techniques for the first time. Multiple test methods were used to characterize these materials. Fourier transform infrared spectroscopy (FTIR) confirmed the existence of the two components in the blends; differential scanning calorimetry (DSC) and dynamic mechanical analysis (DMA) confirmed the immiscibility between the TPU and PLA. Scanning electron microscopy (SEM) images verified that, at the composition ratios studied, PLA was dispersed as spheres or islands inside the TPU matrix and that this phase morphology further influenced the scaffold's microstructure and surface roughness. The blends exhibited a large range of mechanical properties that covered several human tissue requirements. 3T3 fibroblast cell culture showed that the scaffolds supported cell proliferation and migration properly. Most importantly, this study demonstrated the feasibility of mass producing biocompatible PLA/TPU scaffolds with tunable microstructures, surface roughnesses, and mechanical properties that have the potential to be used as artificial scaffolds in multiple tissue engineering applications.