scholarly journals Neuronal differentiation strategies: insights from single-cell sequencing and machine learning

Development ◽  
2020 ◽  
Vol 147 (23) ◽  
pp. dev193631
Author(s):  
Nikolaos Konstantinides ◽  
Claude Desplan
Keyword(s):  
Machine Learning ◽  
Single Cell ◽  
Neuronal Differentiation ◽  
Cell Types ◽  
Specific Cell ◽  
Single Cell Sequencing ◽  
Differentiated Cells ◽  
History Of ◽  
Induced Pluripotent

ABSTRACTNeuronal replacement therapies rely on the in vitro differentiation of specific cell types from embryonic or induced pluripotent stem cells, or on the direct reprogramming of differentiated adult cells via the expression of transcription factors or signaling molecules. The factors used to induce differentiation or reprogramming are often identified by informed guesses based on differential gene expression or known roles for these factors during development. Moreover, differentiation protocols usually result in partly differentiated cells or the production of a mix of cell types. In this Hypothesis article, we suggest that, to overcome these inefficiencies and improve neuronal differentiation protocols, we need to take into account the developmental history of the desired cell types. Specifically, we present a strategy that uses single-cell sequencing techniques combined with machine learning as a principled method to select a sequence of programming factors that are important not only in adult neurons but also during differentiation.

scholarly journals CellMap: Characterizing the types and composition of iPSC-derived cells from RNA-seq data

2021 ◽  
Author(s):  
Zhengyu Ouyang ◽  
Nathanael Bourgeois ◽  
Eugenia Lyashenko ◽  
Paige Cundiff ◽  
Patrick F Cullen ◽  
...  
Keyword(s):  
Single Cell ◽  
Induced Pluripotent Stem Cell ◽  
Cell Types ◽  
Model Systems ◽  
Rna Seq ◽  
Cell Type ◽  
Fine Grained ◽  
Single Nucleus ◽  
Induced Pluripotent

Induced pluripotent stem cell (iPSC) derived cell types are increasingly employed as in vitro model systems for drug discovery. For these studies to be meaningful, it is important to understand the reproducibility of the iPSC-derived cultures and their similarity to equivalent endogenous cell types. Single-cell and single-nucleus RNA sequencing (RNA-seq) are useful to gain such understanding, but they are expensive and time consuming, while bulk RNA-seq data can be generated quicker and at lower cost. In silico cell type decomposition is an efficient, inexpensive, and convenient alternative that can leverage bulk RNA-seq to derive more fine-grained information about these cultures. We developed CellMap, a computational tool that derives cell type profiles from publicly available single-cell and single-nucleus datasets to infer cell types in bulk RNA-seq data from iPSC-derived cell lines.

scholarly journals Defining totipotency using criteria of increasing stringency

2020 ◽  
Author(s):  
Eszter Posfai ◽  
John Paul Schell ◽  
Adrian Janiszewski ◽  
Isidora Rovic ◽  
Alexander Murray ◽  
...  
Keyword(s):  
Stem Cells ◽  
Embryonic Stem Cells ◽  
Single Cell ◽  
Pluripotent Stem Cells ◽  
Embryonic Stem ◽  
Cell Types ◽  
Differentiated Cells ◽  
Totipotent Cell

AbstractTotipotency is the ability of a single cell to give rise to all the differentiated cells that build the conceptus, yet how to capture this property in vitro remains incompletely understood. Defining totipotency relies upon a variety of assays of variable stringency. Here we describe criteria to define totipotency. We illustrate how distinct criteria of increasing stringency can be used to judge totipotency by evaluating candidate totipotent cell types in the mouse, including early blastomeres and expanded or extended pluripotent stem cells. Our data challenge the notion that expanded or extended pluripotent states harbor increased totipotent potential relative to conventional embryonic stem cells under in vivo conditions.

scholarly journals Single-cell sequencing of developing human gut reveals transcriptional links to childhood Crohn’s disease

2020 ◽  
Author(s):  
Rasa Elmentaite ◽  
Alexander Ross ◽  
Kylie R. James ◽  
Daniel Ortmann ◽  
Tomas Gomes ◽  
...  
Keyword(s):  
Crohn’S Disease ◽  
Crohn's Disease ◽  
Single Cell ◽  
Ex Vivo ◽  
Cell Types ◽  
Gut Development ◽  
Human Gut ◽  
Single Cell Sequencing ◽  
Computational Analyses

SummaryHuman gut development requires the orchestrated interaction of various differentiating cell types. Here we generate an in-depth single-cell map of the developing human intestine at 6–10 weeks post-conception, a period marked by crypt-villus formation. Our analysis reveals the transcriptional profile of cycling epithelial precursor cells, which are distinct from LGR5-expressing cells. We use computational analyses to show that these cells contribute to differentiated cell subsets directly and indirectly via the generation of LGR5-expressing stem cells and receive signals from the surrounding mesenchymal cells. Furthermore, we draw parallels between the transcriptomes of ex vivo tissues and in vitro fetal organoids, revealing the maturation of organoid cultures in a dish. Lastly, we compare scRNAseq profiles from paediatric Crohn’s disease epithelium alongside matched healthy controls to reveal disease associated changes in epithelial composition. Contrasting these with the fetal profiles reveals re-activation of fetal transcription factors in Crohn’s disease epithelium. Our study provides a unique resource, available at www.gutcellatlas.org, and underscores the importance of unravelling fetal development in understanding disease.

scholarly journals Human embryoid bodies model basal lamina assembly and muscular dystrophy

2019 ◽  
Author(s):  
Alec R. Nickolls ◽  
Michelle M. Lee ◽  
Kristen Zukosky ◽  
Barbara S. Mallon ◽  
Carsten G. Bönnemann
Keyword(s):  
Basal Lamina ◽  
Cancer Metastasis ◽  
Cell Types ◽  
Embryoid Bodies ◽  
Patient Specific ◽  
Muscular Dystrophies ◽  
Specific Cell ◽  
Induced Pluripotent ◽  
Supplement Therapy

AbstractThe basal lamina is a specialized sheet of dense extracellular matrix (ECM), linked to the plasma membrane of specific cell types in their tissue context, that serves as a structural scaffold for organ genesis and maintenance. Disruption of the basal lamina and its functions is central to many disease processes, including cancer metastasis, kidney disease, eye disease, muscular dystrophies, and specific types of brain malformation. The latter three pathologies occur in the dystroglycanopathies, which are caused by dysfunction of the ECM receptor dystroglycan. However, opportunities to study the basal lamina in various human disease tissues are restricted due to its limited accessibility. Here, we report the generation of embryoid bodies from human induced pluripotent stem cells to model basal lamina formation. Embryoid bodies cultured via this protocol mimic pre-gastrulation embryonic development, consisting of an epithelial core surrounded by a basal lamina and a peripheral layer of ECM-secreting endoderm. In dystroglycanopathy patient embryoid bodies, electron and fluorescence microscopy revealed ultrastructural basal lamina defects and reduced ECM assembly. By starting from patient-derived cells, these results establish a method for the in vitro synthesis of patient-specific basal lamina and recapitulate disease-relevant ECM defects seen in muscular dystrophies. Finally, we applied this system to evaluate an experimental ribitol supplement therapy on genetically diverse dystroglycanopathy patient samples.

scholarly journals NFIL3 mutations alter immune homeostasis and sensitise for arthritis pathology

2018 ◽  
Vol 78 (3) ◽  
pp. 342-349 ◽  
Author(s):  
Susan Schlenner ◽  
Emanuela Pasciuto ◽  
Vasiliki Lagou ◽  
Oliver Burton ◽  
Teresa Prezzemolo ◽  
...  
Keyword(s):  
Immune System ◽  
Single Cell ◽  
Immune Cell ◽  
Cell Types ◽  
Monozygotic Twin ◽  
Preclinical Model ◽  
Potential Contribution ◽  
Mutant Form ◽  
Single Cell Sequencing

ObjectivesNFIL3 is a key immunological transcription factor, with knockout mice studies identifying functional roles in multiple immune cell types. Despite the importance of NFIL3, little is known about its function in humans.MethodsHere, we characterised a kindred of two monozygotic twin girls with juvenile idiopathic arthritis at the genetic and immunological level, using whole exome sequencing, single cell sequencing and flow cytometry. Parallel studies were performed in a mouse model.ResultsThe patients inherited a novel p.M170I in NFIL3 from each of the parents. The mutant form of NFIL3 demonstrated reduced stability in vitro. The potential contribution of this mutation to arthritis susceptibility was demonstrated through a preclinical model, where Nfil3-deficient mice upregulated IL-1β production, with more severe arthritis symptoms on disease induction. Single cell sequencing of patient blood quantified the transcriptional dysfunctions present across the peripheral immune system, converging on IL-1β as a pivotal cytokine.ConclusionsNFIL3 mutation can sensitise for arthritis development, in mice and humans, and rewires the innate immune system for IL-1β over-production.

scholarly journals Single-Cell Sequencing Applications in the Inner Ear

2021 ◽  
Vol 9 ◽  
Author(s):  
Mingxuan Wu ◽  
Mingyu Xia ◽  
Wenyan Li ◽  
Huawei Li
Keyword(s):  
Gene Expression ◽  
Single Cell ◽  
Inner Ear ◽  
Signaling Pathways ◽  
Cell Types ◽  
Specific Cell ◽  
Tissue Heterogeneity ◽  
Single Cell Sequencing ◽  
Cell Groups ◽  
Different Cell Types

Genomics studies face specific challenges in the inner ear due to the multiple types and limited amounts of inner ear cells that are arranged in a very delicate structure. However, advances in single-cell sequencing (SCS) technology have made it possible to analyze gene expression variations across different cell types as well as within specific cell groups that were previously considered to be homogeneous. In this review, we summarize recent advances in inner ear research brought about by the use of SCS that have delineated tissue heterogeneity, identified unknown cell subtypes, discovered novel cell markers, and revealed dynamic signaling pathways during development. SCS opens up new avenues for inner ear research, and the potential of the technology is only beginning to be explored.

338 IN VITRO GENERATION OF LENTOID BODIES FROM INDUCED PLURIPOTENT STEM CELLS OF TRANSGENIC MICE

2015 ◽  
Vol 27 (1) ◽  
pp. 257
Author(s):  
T. Anand ◽  
D. Kumar ◽  
T. R. Talluri ◽  
H. Niemann ◽  
W. A. Kues
Keyword(s):  
Stem Cells ◽  
Cell Types ◽  
Ips Cells ◽  
Sleeping Beauty ◽  
Specific Cell ◽  
Fluorescence Excitation ◽  
Ips Cell ◽  
Developmental Potential ◽  
Induced Pluripotent

Pluripotent cells have the developmental potential to generate all adult cell types, so ocular diseases resulting from the failure of specific cell types could be potentially treatable through the transplantation of differentiated cells derived from stem cells. The present study was conducted with the aim of generating a cataract model. We attempted to derive the induced pluripotent stem (iPS) cells from fibroblast cells of transgenic (crytom) mice carrying a transgenic construct-alphaA crystallin promoter driving the tandem dimer (td) Tomato marker transgene, integrated in the genome. The 4- to 6-week-old female crytom mice were selected, superovulated, and mated. The fetuses were recovered and examined on various different days (10.5 to 15.5 days postfertilization), and the reporter expression was found to be initiated 12.5 days postfertilization and the intensity was increased thereafter. The expression of tdTomato was confirmed in the fetuses by Western blotting. Murine embryonic fibroblast (MEF) cultures were generated and electroporated with a reprogramming transposon cassette carrying Yamanaka factors (OCT4, SOX2, KLF4, and MYC) and Sleeping Beauty transposase to generate iPS cells which were picked up and clonally expanded. The cells were confirmed by PCR for tdTomato in the genome and characterised for the expression of Oct4 and cryAB by immunofluorescence. The iPS cells were also injected into the nude CD1 mice to test for teratoma formation. The generated cells were allowed to differentiate spontaneously on 3 different types (viz. P19, NTERA, and STO) of cell lines as feeders, in the absence of LIF, and cells were expected to fluoresce if differentiated to eye lens lineage. After long-term cultures, the iPS cells were found to differentiate and form lentoid bodies which expressed tdTomato. Thus, alphaA crystallin-tdTomato construct was allowed following lens cell formation by specific fluorescence excitation in a spatial and temporal manner. The employment of cell type-specific reporters for establishing and optimizing targeted differentiation in vitro seems to be an efficient and generally applicable approach for developing differentiation protocols for desired cell populations. Hence a transgenic murine iPS cell line was generated which exhibited potential to be used as a model for eye cataracts and other eye abnormalities.

scholarly journals Human cerebral organoids recapitulate gene expression programs of fetal neocortex development

2015 ◽  
Vol 112 (51) ◽  
pp. 15672-15677 ◽  
Author(s):  
J. Gray Camp ◽  
Farhath Badsha ◽  
Marta Florio ◽  
Sabina Kanton ◽  
Tobias Gerber ◽  
...  
Keyword(s):  
Gene Expression ◽  
Single Cell ◽  
Neuronal Differentiation ◽  
Cortical Development ◽  
Cell Types ◽  
Fetal Tissue ◽  
Neural Progenitor Cell ◽  
Neural Progenitor

Cerebral organoids—3D cultures of human cerebral tissue derived from pluripotent stem cells—have emerged as models of human cortical development. However, the extent to which in vitro organoid systems recapitulate neural progenitor cell proliferation and neuronal differentiation programs observed in vivo remains unclear. Here we use single-cell RNA sequencing (scRNA-seq) to dissect and compare cell composition and progenitor-to-neuron lineage relationships in human cerebral organoids and fetal neocortex. Covariation network analysis using the fetal neocortex data reveals known and previously unidentified interactions among genes central to neural progenitor proliferation and neuronal differentiation. In the organoid, we detect diverse progenitors and differentiated cell types of neuronal and mesenchymal lineages and identify cells that derived from regions resembling the fetal neocortex. We find that these organoid cortical cells use gene expression programs remarkably similar to those of the fetal tissue to organize into cerebral cortex-like regions. Our comparison of in vivo and in vitro cortical single-cell transcriptomes illuminates the genetic features underlying human cortical development that can be studied in organoid cultures.

scholarly journals Unchain My Heart: Integrins at the Basis of iPSC Cardiomyocyte Differentiation

2019 ◽  
Vol 2019 ◽  
pp. 1-20 ◽  
Author(s):  
Rosaria Santoro ◽  
Gianluca Lorenzo Perrucci ◽  
Aoife Gowran ◽  
Giulio Pompilio
Keyword(s):  
Cell Fate ◽  
Pluripotent Stem Cells ◽  
Cellular Response ◽  
Cell Types ◽  
Specific Cell ◽  
Cardiomyocyte Differentiation ◽  
Cellular Models ◽  
Cellular Machinery ◽  
Induced Pluripotent

The cellular response to the extracellular matrix (ECM) microenvironment mediated by integrin adhesion is of fundamental importance, in both developmental and pathological processes. In particular, mechanotransduction is of growing importance in groundbreaking cellular models such as induced pluripotent stem cells (iPSC), since this process may strongly influence cell fate and, thus, augment the precision of differentiation into specific cell types, e.g., cardiomyocytes. The decryption of the cellular machinery starting from ECM sensing to iPSC differentiation calls for new in vitro methods. Conveniently, engineered biomaterials activating controlled integrin-mediated responses through chemical, physical, and geometrical designs are key to resolving this issue and could foster clinical translation of optimized iPSC-based technology. This review introduces the main integrin-dependent mechanisms and signalling pathways involved in mechanotransduction. Special consideration is given to the integrin-iPSC linkage signalling chain in the cardiovascular field, focusing on biomaterial-based in vitro models to evaluate the relevance of this process in iPSC differentiation into cardiomyocytes.

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