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

Classical lissencephaly is a genetic neurological disorder associated with mental retardation and intractable epilepsy,and Miller-Dieker syndrome (MDS) is the most severe form of the disease. In this study,to investigate the effects of MDS on human progenitor subtypes that control neuronal output and influence brain topology,we analyzed cerebral organoids derived from control and MDS-induced pluripotent stem cells (iPSCs) using time-lapse imaging,immunostaining,and single-cell RNA sequencing. We saw a cell migration defect that was rescued when we corrected the MDS causative chromosomal deletion and severe apoptosis of the founder neuroepithelial stem cells,accompanied by increased horizontal cell divisions. We also identified a mitotic defect in outer radial glia,a progenitor subtype that is largely absent from lissencephalic rodents but critical for human neocortical expansion. Our study,therefore,deepens our understanding of MDS cellular pathogenesis and highlights the broad utility of cerebral organoids for modeling human neurodevelopmental disorders. View Publication

The essential functions of Polycomb Repressive Complex 1 (PRC1) in development and gene silencing are thought to involve long non-coding RNAs (lncRNAs),but few specific lncRNAs that guide PRC1 activity are known. We screened for lncRNAs which co-precipitate with PRC1 from chromatin and found candidates that impact Polycomb Group protein (PcG)-regulated gene expression in vivo. A novel lncRNA from this screen,CAT7,regulates expression and PcG binding at the MNX1 locus during early neuronal differentiation. CAT7 contains a unique tandem repeat domain which shares high sequence similarity to a non-syntenic zebrafish analog,cat7l. Defects caused by interference of cat7l RNA during zebrafish embryogenesis were rescued by human CAT7 RNA,enhanced by interference of a PRC1 component,and suppressed by interference of a known PRC1 target gene,demonstrating cat7l genetically interacts with a PRC1. We propose a model whereby PRC1 acts in concert with specific lncRNAs,and that CAT7/cat7l represent convergent lncRNAs that independently evolved to tune PRC1 repression at individual loci. View Publication

AIM: Commercial regenerative medicine will require large quantities of clinical-specification human cells. The cost and quality of manufacture is notoriously difficult to control due to highly complex processes with poorly defined tolerances. As a step to overcome this,we aimed to demonstrate the use of 'quality-by-design' tools to define the operating space for economic passage of a scalable human embryonic stem cell production method with minimal cell loss. MATERIALS & METHODS: Design of experiments response surface methodology was applied to generate empirical models to predict optimal operating conditions for a unit of manufacture of a previously developed automatable and scalable human embryonic stem cell production method. RESULTS & CONCLUSION: Two models were defined to predict cell yield and cell recovery rate postpassage,in terms of the predictor variables of media volume,cell seeding density,media exchange and length of passage. Predicted operating conditions for maximized productivity were successfully validated. Such 'quality-by-design' type approaches to process design and optimization will be essential to reduce the risk of product failure and patient harm,and to build regulatory confidence in cell therapy manufacturing processes. View Publication

AIMS/HYPOTHESIS: Islet transplantation is a promising cell therapy for patients with diabetes,but it is currently limited by the reliance upon cadaveric donor tissue. We previously demonstrated that human embryonic stem cell (hESC)-derived pancreatic progenitor cells matured under the kidney capsule in a mouse model of diabetes into glucose-responsive insulin-secreting cells capable of reversing diabetes. However,the formation of cells resembling bone and cartilage was a major limitation of that study. Therefore,we developed an improved differentiation protocol that aimed to prevent the formation of off-target mesoderm tissue following transplantation. We also examined how variation within the complex host environment influenced the development of pancreatic progenitors in vivo.backslashnbackslashnMETHODS: The hESCs were differentiated for 14 days into pancreatic progenitor cells and transplanted either under the kidney capsule or within Theracyte (TheraCyte,Laguna Hills,CA,USA) devices into diabetic mice.backslashnbackslashnRESULTS: Our revised differentiation protocol successfully eliminated the formation of non-endodermal cell populations in 99% of transplanted mice and generated grafts containing textgreater80% endocrine cells. Progenitor cells developed efficiently into pancreatic endocrine tissue within macroencapsulation devices,despite lacking direct contact with the host environment,and reversed diabetes within 3 months. The preparation of cell aggregates pre-transplant was critical for the formation of insulin-producing cells in vivo and endocrine cell development was accelerated within a diabetic host environment compared with healthy mice. Neither insulin nor exendin-4 therapy post-transplant affected the maturation of macroencapsulated cells.backslashnbackslashnCONCLUSIONS/INTERPRETATION: Efficient differentiation of hESC-derived pancreatic endocrine cells can occur in a macroencapsulation device,yielding glucose-responsive insulin-producing cells capable of reversing diabetes. View Publication

Recent advances in developmental biology and stem cell technology have led to the engineering of functional organs in a dish. However,the limited size of these organoids and absence of a large circulatory system poses limits to its clinical translation. To overcome these issues,decellularized whole kidney scaffolds with native microstructure and extracellular matrix (ECM) are employed for kidney bioengineering,using human-induced pluripotent-stem-cell-derived renal progenitor cells and endothelial cells. To demonstrate ECM-guided cellular assembly,the present work is focused on generating the functional unit of the kidney,the glomerulus. In the repopulated organ,the presence of endothelial cells broadly upregulates the expression level of genes related to renal development. When the cellularized native scaffolds are implanted in SCID mice,glomeruli assembly can be achieved by co-culture of the renal progenitors and endothelial cells. These individual glomerular units are shown to be functional in the context of the whole organ using a simulated bio-reactor set-up with urea and creatinine excretion and albumin reabsorption. Our results indicate that the repopulation of decellularized native kidney using clinically relevant,expandable patient-specific renal progenitors and endothelial cells may be a viable approach for the generation of a functional whole kidney. View Publication

While distinct stem cell phenotypes follow global changes in chromatin marks,single-cell chromatin technologies are unable to resolve or predict stem cell fates. We propose the first such use of optical high content nanoscopy of histone epigenetic marks (epi-marks) in stem cells to classify emergent cell states. By combining nanoscopy with epi-mark textural image informatics,we developed a novel approach,termed EDICTS (Epi-mark Descriptor Imaging of Cell Transitional States),to discern chromatin organizational changes,demarcate lineage gradations across a range of stem cell types and robustly track lineage restriction kinetics. We demonstrate the utility of EDICTS by predicting the lineage progression of stem cells cultured on biomaterial substrates with graded nanotopographies and mechanical stiffness,thus parsing the role of specific biophysical cues as sensitive epigenetic drivers. We also demonstrate the unique power of EDICTS to resolve cellular states based on epi-marks that cannot be detected via mass spectrometry based methods for quantifying the abundance of histone post-translational modifications. Overall,EDICTS represents a powerful new methodology to predict single cell lineage decisions by integrating high content super-resolution nanoscopy and imaging informatics of the nuclear organization of epi-marks. View Publication

Abnormal differentiation of myeloid cells is one of the hallmarks of cancer. However,the molecular mechanisms of this process remain elusive. In this study,we investigated the effect of tumor-derived factors on Janus kinase (Jak)/STAT signaling in myeloid cells during their differentiation into dendritic cells. Tumor cell conditioned medium induced activation of Jak2 and STAT3,which was associated with an accumulation of immature myeloid cells. Jak2/STAT3 activity was localized primarily in these myeloid cells,which prevented the differentiation of immature myeloid cells into mature dendritic cells. This differentiation was restored after removal of tumor-derived factors. Inhibition of STAT3 abrogated the negative effects of these factors on myeloid cell differentiation,and overexpression of STAT3 reproduced the effects of tumor-derived factors. Thus,this is a first demonstration that tumor-derived factors may affect myeloid cell differentiation in cancer via constitutive activation of Jak2/STAT3. View Publication

We showed that resistance to severe hypoxia defines hierarchical levels within normal hematopoietic populations and that hypoxia modulates the balance between generation of progenitors and maintenance of hematopoietic stem cells (HSC) in favor of the latter. This study deals with the effects of hypoxia (0.1% oxygen) in vitro on Friend's murine erythroleukemia (MEL) cells,addressing the question of whether a clonal leukemia cell population comprise functionally different cell subsets characterized by different hypoxia resistance. To identify leukemia stem cells (LSC),we used the Culture Repopulating Ability (CRA) assay we developed to quantify in vitro stem cells capable of short-term reconstitution (STR). Hypoxia strongly inhibited the overall growth of MEL cell population,which,despite its clonality,comprised progenitors characterized by markedly different hypoxia-resistance. These included hypoxia-sensitive colony-forming cells and hypoxia-resistant STR-type LSC,capable of repopulating secondary liquid cultures of CRA assays,confirming what was previously shown for normal hematopoiesis. STR-type LSC were found capable not only of surviving in hypoxia but also of being mostly in cycle,in contrast with the fact that almost all hypoxia-surviving cells were growth-arrested and with what we previously found for HSC. However,quiescent LSC were also detected,capable of delayed culture repopulation with the same efficiency as STR-like LSC. The fact that even quiescent LSC,believed to sustain minimal residual disease in vivo,were found within the MEL cells indicates that all main components of leukemia cell populations may be present within clonal cell lines,which are therefore suitable to study the sensitivity of individual components to treatments. Disclosure of potential conflicts of interest is found at the end of this article. View Publication

There is a foundational need for quality control tools in stem cell laboratories engaged in basic research,regenerative therapies,and toxicological studies. These tools require automated methods for evaluating cell processes and quality during in vitro passaging,expansion,maintenance,and differentiation. In this paper,an unbiased,automated high-content profiling toolkit,StemCellQC,is presented that non-invasively extracts information on cell quality and cellular processes from time-lapse phase-contrast videos. Twenty four (24) morphological and dynamic features were analyzed in healthy,unhealthy,and dying human embryonic stem cell (hESC) colonies to identify those features that were affected in each group. Multiple features differed in the healthy versus unhealthy/dying groups,and these features were linked to growth,motility,and death. Biomarkers were discovered that predicted cell processes before they were detectable by manual observation. StemCellQC distinguished healthy and unhealthy/dying hESC colonies with 96% accuracy by non-invasively measuring and tracking dynamic and morphological features over 48 hours. Changes in cellular processes can be monitored by StemCellQC and predictions can be made about the quality of pluripotent stem cell colonies. This toolkit reduced the time and resources required to track multiple pluripotent stem cell colonies and eliminated handling errors and false classifications due to human bias. StemCellQC provided both user-specified and classifier-determined analysis in cases where the affected features are not intuitive or anticipated. Video analysis algorithms allowed assessment of biological phenomena using automatic detection analysis,which can aid facilities where maintaining stem cell quality and/or monitoring changes in cellular processes are essential. In the future StemCellQC can be expanded to include other features,cell types,treatments,and differentiating cells. View Publication

Sickle cell anemia affects millions of people worldwide and is an emerging global health burden. As part of a large NIH-funded NextGen Consortium,we generated a diverse,comprehensive,and fully characterized library of sickle-cell-disease-specific induced pluripotent stem cells (iPSCs) from patients of different ethnicities,β-globin gene (HBB) haplotypes,and fetal hemoglobin (HbF) levels. iPSCs stand to revolutionize the way we study human development,model disease,and perhaps eventually,treat patients. Here,we describe this unique resource for the study of sickle cell disease,including novel haplotype-specific polymorphisms that affect disease severity,as well as for the development of patient-specific therapeutics for this phenotypically diverse disorder. As a complement to this library,and as proof of principle for future cell- and gene-based therapies,we also designed and employed CRISPR/Cas gene editing tools to correct the sickle hemoglobin (HbS) mutation. View Publication

High maternal blood glucose levels caused by diabetes mellitus can irreversibly lead to maldevelopment of the growing fetus with specific effects on the skeleton. To date,it remains controversial at which stage embryonic development is affected. Specifically during embryonic bone development,it is unclear whether diminished bone mineral density is caused by reduced osteoblast or rather enhanced osteoclast function. Therefore,the aim of this study was to characterize the growth as well as the skeletal differentiation capability of pluripotent embryonic stem cells (ESCs),which may serve as an in vitro model for all stages of embryonic development,when cultured in diabetic levels of D-glucose (4.5 g/L) versus physiological levels (1.0 g/L). Results showed that cells cultivated in physiological glucose gave rise to a higher number of colonies with an undifferentiated character as compared to cells grown in diabetic glucose concentrations. In contrast,these cultures were characterized by slightly decreased expression of proteins associated with the stem cell state. Furthermore,differentiation of ESCs into osteoblasts and osteoclasts was favored in physiological glucose concentrations,demonstrated by an increased matrix calcification,enhanced expression of cell-type-specific mRNAs,as well as activity of the cell-type-specific enzymes,alkaline,and tartrate resistant acidic phosphatase. In fact,this pattern was noted in murine as well as in primate ESCs. Our study suggests that an interplay between both the osteoblast and the osteoclast lineage is needed for proper skeletal development to occur,which seems impaired in hyperglycemic conditions. View Publication

Efficient approaches for the precise genetic engineering of stem cells can enhance both basic and applied stem cell research. Adeno-associated virus (AAV) vectors have demonstrated high-efficiency gene delivery and gene targeting to numerous cell types,and AAV vectors developed specifically for gene delivery to stem cells have further increased gene targeting frequency compared to plasmid construct techniques. This chapter details the production and purification techniques necessary to generate adeno-associated viral vectors for use in high-efficiency gene targeting of adult or pluripotent stem cell applications. Culture conditions used to achieve high gene targeting frequencies in rat neural stem cells and human pluripotent stem cells are also described. View Publication