scholarly journals Injury-Free In Vivo Delivery and Engraftment into the Cornea Endothelium Using Extracellular Matrix Shrink-Wrapped Cells

2021 ◽  
Author(s):  
Rachelle N. Palchesko ◽  
Yiqin Du ◽  
Moira L. Geary ◽  
Santiago Carrasquilla ◽  
Daniel J. Shiwarski ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Single Cell ◽  
Single Cells ◽  
Tissue Level ◽  
Cell Junctions ◽  
Small Island ◽  
Corneal Endothelial Cells ◽  
Corneal Blindness

AbstractCell injection has emerged as a widespread approach for therapeutic delivery of healthy cells into diseased and damaged tissues to achieve regeneration. However, cell retention, viability and integration at the injection site has generally been poor, driving the need for improved approaches. Additionally, it is unknown how efficiently single cells can integrate and repair tissue level function. Here we have developed a technique to address these issues by engineering islands of interconnected cells on ECM nanoscaffolds that can be non-destructively released from the surface via thermal dissolution of the underlying thermo-responsive polymer. Upon dissolution of the polymer, the ECM nanoscaffold shrink-wraps around the small island of cells, creating a small patch of cells that maintain their cell-cell junctions and cytoskeletal structure throughout collection, centrifugation and injection that we have termed μMonolayers. These μMonolayers were made with corneal endothelial cells, as a model system, as single cell injections of corneal endothelial cells have been used with some success clinically to treat corneal blindness. In vitro our μMonolayers exhibited increased integration compared to single cells into low density corneal endothelial monolayers and in vivo into the high-density healthy rabbit corneal endothelium. These results indicate that this technique could be used to increase the integration of healthy cells into existing tissues to treat not only corneal blindness, but also other conditions such as cystic fibrosis, myocardial infarction, diabetes, etc.One Sentence SummarySmall monolayers of interconnected endothelial cells are shrinkwrapped in a thin layer of ECM and exhibit enhanced adhesion and integration in vivo compared to single cell suspensions.

scholarly journals Single-Cell RNA Sequencing Reveals Renal Endothelium Heterogeneity and Metabolic Adaptation to Water Deprivation

2019 ◽  
Vol 31 (1) ◽  
pp. 118-138 ◽  
Author(s):  
Sébastien J. Dumas ◽  
Elda Meta ◽  
Mila Borri ◽  
Jermaine Goveia ◽  
Katerina Rohlenova ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Endothelial Cell ◽  
Oxidative Phosphorylation ◽  
Single Cell ◽  
Rna Sequencing ◽  
Water Deprivation ◽  
Metabolic Adaptation ◽  
Single Cell Rna Sequencing

BackgroundRenal endothelial cells from glomerular, cortical, and medullary kidney compartments are exposed to different microenvironmental conditions and support specific kidney processes. However, the heterogeneous phenotypes of these cells remain incompletely inventoried. Osmotic homeostasis is vitally important for regulating cell volume and function, and in mammals, osmotic equilibrium is regulated through the countercurrent system in the renal medulla, where water exchange through endothelium occurs against an osmotic pressure gradient. Dehydration exposes medullary renal endothelial cells to extreme hyperosmolarity, and how these cells adapt to and survive in this hypertonic milieu is unknown.MethodsWe inventoried renal endothelial cell heterogeneity by single-cell RNA sequencing >40,000 mouse renal endothelial cells, and studied transcriptome changes during osmotic adaptation upon water deprivation. We validated our findings by immunostaining and functionally by targeting oxidative phosphorylation in a hyperosmolarity model in vitro and in dehydrated mice in vivo.ResultsWe identified 24 renal endothelial cell phenotypes (of which eight were novel), highlighting extensive heterogeneity of these cells between and within the cortex, glomeruli, and medulla. In response to dehydration and hypertonicity, medullary renal endothelial cells upregulated the expression of genes involved in the hypoxia response, glycolysis, and—surprisingly—oxidative phosphorylation. Endothelial cells increased oxygen consumption when exposed to hyperosmolarity, whereas blocking oxidative phosphorylation compromised endothelial cell viability during hyperosmotic stress and impaired urine concentration during dehydration.ConclusionsThis study provides a high-resolution atlas of the renal endothelium and highlights extensive renal endothelial cell phenotypic heterogeneity, as well as a previously unrecognized role of oxidative phosphorylation in the metabolic adaptation of medullary renal endothelial cells to water deprivation.

The Basic Helix-Loop-Helix TAL1/SCL and Its Partners E47 and LMO2 Act Upstream of the VE-Cadherin Gene in Endothelial Cells.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 1795-1795
Author(s):  
Virginie Deleuze ◽  
Elias Chalhoub ◽  
Rawan El-Hajj ◽  
Christiane Dohet ◽  
Mikael Le Clech ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Ectopic Expression ◽  
Cell Junctions ◽  
Small Interfering Rnas ◽  
Hek 293 ◽  
Basic Helix Loop Helix ◽  
Helix Loop Helix ◽  
Cell Cell

Abstract The basic helix-loop-helix protein TAL-1/SCL, essential for the formation of the hematopoietic system, is also required for vascular development and more particularly for embryonic angiogenesis. We previously reported that TAL-1 acts as a positive factor for post-natal angiogenesis by stimulating endothelial morphogenesis. To understand how TAL-1 modulates angiogenesis, we investigated the functional consequences of TAL-1 silencing, mediated by small-interfering RNAs, in human primary endothelial cells (ECs). We found that TAL-1 knockdown impaired in vitro EC tubulomorphogenesis (in 2-D on Matrigel or 3-D in collagen I gel), with the notable absence of cell-cell contacts, a prerequisite for morphogenesis initiation. This cellular deficiency was associated with a dramatic reduction in the vascular-endothelial (VE)-cadherin at intercellular junctions, the major component of endothelial adherens junctions. In contrast, PECAM (or CD31) was present at cell-cell junctions at the same levels as control cells. Importantly, silencing of two known TAL-1-partners in hematopoietic cells, E47 or LMO2, produce the same effects as TAL-1. Accordingly, silencing of TAL-1, as well as E47 and LMO2, provoked down-regulation of VE-cadherin at both the mRNA and protein levels. Transient transfection experiments in HUVECs showed that TAL-1 and E47 regulate the VE-cadherin promoter through a specialized E-box element. Finally, endogenous VE-cadherin transcription could be directly activated in non-endothelial HEK-293 cells that neither express TAL-1 or LMO2, by the sole concomitant ectopic expression of TAL-1, E47 and LMO2. Overall, our data demonstrate that a multiprotein complex containing at least TAL-1, LMO2 and E47 act upstream of the VE-cadherin gene. We are currently performing chromatin immunoprecipitation (ChIP) to investigate whether the TAL-1-containing complex binds in vivo the VE-cadherin promoter. This study identifies VE-cadherin as an upstream TAL-1-target gene in the endothelial lineage, and provides a first clue in TAL-1 function in the control of angiogenesis.

scholarly journals Single Cell RNA-Seq Characterises Pre-Leukemic Transformation Driven By CEBPA N321D in the Hoxb8-FL Cell Line

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 3887-3887
Author(s):  
Moosa Qureshi ◽  
Fernando Calero-Nieto ◽  
Iwo Kucinski ◽  
Sarah Kinston ◽  
George Giotopoulos ◽  
...  
Keyword(s):  
Dendritic Cell ◽  
Single Cell ◽  
Cell Line ◽  
Single Cells ◽  
Mutant Form ◽  
Rna Seq ◽  
Wild Type ◽  
Leukemic Transformation

Abstract The C/EBPα transcription factor plays a pivotal role in myeloid differentiation and E2F-mediated cell cycle regulation. Although CEBPA mutations are common in acute myeloid leukaemia (AML), little is known regarding pre-leukemic alterations caused by mutated CEBPA. Here, we investigated early events involved in pre-leukemic transformation driven by CEBPA N321D in the LMPP-like cell line Hoxb8-FL (Redecke et al., Nat Methods 2013), which can be maintained in vitro as a self-renewing LMPP population using Flt3L and estradiol, as well as differentiated both in vitro and in vivo into myeloid and lymphoid cell types. Hoxb8-FL cells were retrovirally transduced with Empty Vector (EV), wild-type CEBPA (CEBPA WT) or its N321D mutant form (CEBPA N321D). CEBPA WT-transduced cells showed increased expression of cd11b and SIRPα and downregulation of c-kit, suggesting that wild-type CEBPA was sufficient to promote differentiation even under LMPP growth conditions. Interestingly, we did not observe the same phenotype in CEBPA N321D-transduced cells. Upon withdrawal of estradiol, both EV and CEBPA WT-transduced cells differentiated rapidly into a conventional dendritic cell (cDC) phenotype by day 7 and died within 12 days. By contrast, CEBPA N321D-transduced cells continued to grow for in excess of 56 days, with an initial cDC phenotype but by day 30 demonstrating a plasmacytoid dendritic cell precursor phenotype. CEBPA N321D-transduced cells were morphologically distinct from EV-transduced cells. To test leukemogenic potential in vivo, we performed transplantation experiments in lethally irradiated mice. Serial monitoring of peripheral blood demonstrated that Hoxb8-FL derived cells had disappeared by 4 weeks, and did not reappear. However, at 6 months CEBPA N321D-transduced cells could still be detected in bone marrow in contrast to EV-transduced cells but without any leukemic phenotype. To identify early events involved in pre-leukemic transformation, the differentiation profiles of EV, CEBPA WT and CEBPA N321D-transduced cells were examined with single cell RNA-seq (scRNA-seq). 576 single cells were taken from 3 biological replicates at days 0 and 5 post-differentiation, and analysed using the Automated Single-Cell Analysis Pipeline (Gardeux et al., Bioinformatics 2017). Visualisation by t-SNE (Fig 1) demonstrated: (i) CEBPA WT-transduced cells formed a distinct cluster at day 0 before withdrawal of estradiol; (ii) CEBPA N321D-transduced cells separated from EV and CEBPA WT-transduced cells after 5 days of differentiation, (iii) two subpopulations could be identified within the CEBPA N321D-transduced cells at day 5, with a cluster of five CEBPA N321D-transduced single cells distributed amongst or very close to the day 0 non-differentiated cells. Differential expression analysis identified 224 genes upregulated and 633 genes downregulated specifically in the CEBPA N321D-transduced cells when compared to EV cells after 5 days of differentiation. This gene expression signature revealed that CEBPA N321D-transduced cells switched on a HSC/MEP/CMP transcriptional program and switched off a myeloid dendritic cell program. Finally, in order to further dissect the effect of the N321D mutation, the binding profile of endogenous and CEBPA N321D was compared by ChIP-seq before and after 5 days of differentiation. Integration with scRNA-seq data identified 160 genes specifically downregulated in CEBPA N321D-transduced cells which were associated with the binding of the mutant protein. This list of genes included genes previously implicated in dendritic cell differentiation (such as NOTCH2, JAK2), as well as a number of genes not previously implicated in the evolution of AML, representing potentially novel therapeutic targets. Disclosures No relevant conflicts of interest to declare.

scholarly journals Single-cell RNA-seq revealed the role of Alkbh5 in reproduction

2021 ◽  
Author(s):  
Xiaozhong Shen ◽  
Gangcai Xie
Keyword(s):  
Single Cell ◽  
Messenger Rna ◽  
Single Cells ◽  
Rna Seq ◽  
Single Cell Sequencing ◽  
Differential Gene ◽  
Higher Eukaryotes

AbstractN(6)-methyladenosine (m(6)a) is the most common internal modification of messenger RNA (mRNA) in higher eukaryotes. According to previous literature reports, alkbh5, as another demethylase in mammals, can reverse the expression of m(6)a gene in vivo and in vitro. In order to reveal the effect of Alkbh5 deletion on the level of single cells in the testis during spermatogenesis in mice, the data were compared using single-cell sequencing. In this article, we discussed the transcription profile and cell type identification of mouse testis, the expression of mitochondrial and ribosomal genes in mice, the analysis of differential gene expression, and the effects of Alkbh5 deletion, and try to explain the role and influence of Alkbh5 on reproduction at the level of single-cell sequencing.

scholarly journals Extracellular Vesicles Derived From Human Corneal Endothelial Cells Inhibit Proliferation of Human Corneal Endothelial Cells

2021 ◽  
Author(s):  
Mohit Parekh ◽  
Hefin Rhys ◽  
Tiago Ramos ◽  
Stefano Ferrari ◽  
Sajjad Ahmad
Keyword(s):  
Endothelial Cells ◽  
Extracellular Vesicles ◽  
Ex Vivo ◽  
Treatment Options ◽  
Proliferative Capacity ◽  
Corneal Endothelial Cells ◽  
Human Corneal Endothelial Cells ◽  
The Media

Abstract Corneal endothelial cells (CEnCs) are a monolayer of hexagonal cells that are responsible for maintaining the function and transparency of the cornea. Damage or dysfunction of CEnCs could lead to blindness. Human CEnCs (HCEnCs) have shown limited proliferative capacity in vivo hence, their maintenance is crucial. Extracellular vesicles (EVs), are responsible for inter- and intra-cellular communication, proliferation, cell-differentiation, migration, and many other complex biological processes. Therefore, we investigated the effect of EVs (derived from human corneal endothelial cell line – HCEC-12) on corneal endothelial cells. HCEC-12 cells were starved with serum-depleted media for 72 hours. The media was ultracentrifuged at 100,000xg to isolate the EVs. EV counting, characterization, internalization and localization were performed using NanoSight, flow cytometry, Dil labelling and confocal microscopy respectively. HCEC-12 and HCEnCs were cultured with media supplemented with EVs. Extracted EVs showed a homogeneous mixture of exosomes and microvesicles. Cells with EVs decreased the proliferation rate; increased apoptosis and cell size; showed poor wound healing response in vitro and on ex vivo human, porcine, and rabbit CECs. Thirteen miRNAs were found in the EV sample using next generation sequencing. We observed that increased cellular uptake of EVs by CECs limit the proliferative capacity of HCEnCs. These preliminary data may help in understanding the pathology of corneal endothelial dysfunction and provide further insights in the development of future therapeutic treatment options.

scholarly journals Single Cell Clones Derived from a Patient's AML Xenograft Display Genetic and Functional Heterogeneity

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 1450-1450
Author(s):  
Christina Maria Zeller ◽  
Daniel Richter ◽  
Binje Vick ◽  
Tobias Herold ◽  
Johannes Bagnoli ◽  
...  
Keyword(s):  
Single Cell ◽  
Single Cells ◽  
Multiple Hypothesis Testing ◽  
Targeted Sequencing ◽  
Functional Heterogeneity ◽  
Nras Mutation ◽  
In Vivo Experiments ◽  
Cell Clones

Introduction: Acute myeloid leukemia (AML) shows substantial genetic and epigenetic heterogeneity, even within an individual patient. Due to treatment resistance and ability to induce relapse, adverse subclones present a major clinical challenge in determining the patient's prognosis. Here, we aimed at characterizing the genetic and functional heterogeneity within a single AML patient, and at identifying adverse subclones that result in therapy failure or give rise to relapse. Methods: Leukemic cells from an AML patient at first and second relapse were transplanted into immuno-compromised mice to generate patient-derived xenografts (PDX). PDX AML cells allowed serial transplantation and genetic engineering by lentiviruses. To distinguish single cells and generate PDX AML clones derived from a single cell (single cell clones, SCC), cells were transduced with a genetic barcode and transplanted into recipient mice near leukemia initiating cell frequency. Resulting SCC were genetically marked to express recombinant fluorochromes to enable flow cytometry analysis. All SCC were characterized for known subclonal mutations of the AML patient by targeted sequencing. Additionally, transcriptome and methylome analysis were performed by SCRB-seq and methylation array, respectively. Results: We successfully generated thirteen serially transplantable PDX SCC from a single AML patient, expressing combinations of up to four fluorochromes to enable competitive in vitro and in vivo experiments. In targeted sequencing, we found that SCC originated from at least four genetically distinct AML subclones and were distinguished by mutations in KRAS (4/13), NRAS (5/13), EZH2 (2/13) or EZH2 and NRAS (2/13). While the NRAS mutation was detected in a minority of bulk cells over serial passages (<10%) from both the first and second relapse, 50% of SCC carried the NRAS mutation. This indicates that NRAS mutated AML cells have an increased stem cell capacity upon transplantation of low cell numbers. Transcriptome analysis revealed 442 genes as differentially expressed between SCC and up to four biological replicates after adjustment for multiple hypothesis testing. Unsupervised clustering demonstrated a strong correlation of gene expression profiles with the respective genotype. In competitive in vivo experiments, homing capability was comparable between the four genetically distinct subclones. However, 2/2 EZH2-mutated SCC overgrew all other clones showing a clear growth advantage within two weeks of in vivo growth. The EZH2-mutated SCC (2/2) were resistant towards in vivo treatment with Cytarabine, whereas the KRAS (4/4), NRAS (5/5) mutated as well as the EZH2 and NRAS double-mutated SCC (2/2) responded to treatment. Conclusion: Taken together, we experimentally prove the existence of genetically and functionally diverse subclones within an individual AML sample. Our approach allows not only genetic, but also functional in vitro and in vivo characterization of adverse subclones. Our approach can be used to identify novel therapeutic approaches in order to specifically target the most adverse cells within patients' AML sample. Disclosures Metzeler: Celgene: Honoraria, Research Funding; Daiichi Sankyo: Honoraria; Otsuka: Honoraria.

scholarly journals Biomaterials for corneal endothelial cell culture and tissue engineering

2021 ◽  
Vol 12 ◽  
pp. 204173142199053
Author(s):  
Mohit Parekh ◽  
Vito Romano ◽  
Kareem Hassanin ◽  
Valeria Testa ◽  
Rintra Wongvisavavit ◽  
...  
Keyword(s):  
Tissue Engineering ◽  
Endothelial Cells ◽  
Standard Treatment ◽  
Corneal Endothelial Cell ◽  
Corneal Tissue ◽  
Corneal Endothelial Cells ◽  
Endothelial Cell Culture ◽  
Corneal Transplants

The corneal endothelium is the posterior monolayer of cells that are responsible for maintaining overall transparency of the avascular corneal tissue via pump function. These cells are non-regenerative in vivo and therefore, approximately 40% of corneal transplants undertaken worldwide are a result of damage or dysfunction of endothelial cells. The number of available corneal donor tissues is limited worldwide, hence, cultivation of human corneal endothelial cells (hCECs) in vitro has been attempted in order to produce tissue engineered corneal endothelial grafts. Researchers have attempted to recreate the current gold standard treatment of replacing the endothelial layer with accompanying Descemet’s membrane or a small portion of stroma as support with tissue engineering strategies using various substrates of both biologically derived and synthetic origin. Here we review the potential biomaterials that are currently in development to support the transplantation of a cultured monolayer of hCECs.

scholarly journals Vimentin fibers orient traction stress

2017 ◽  
Vol 114 (20) ◽  
pp. 5195-5200 ◽  
Author(s):  
Nancy Costigliola ◽  
Liya Ding ◽  
Christoph J. Burckhardt ◽  
Sangyoon J. Han ◽  
Edgar Gutierrez ◽  
...  
Keyword(s):  
Cell Migration ◽  
Single Cell ◽  
Graph Matching ◽  
Single Cells ◽  
Mechanical Strain ◽  
Migration Direction ◽  
Human Foreskin Fibroblasts ◽  
And Migration

The intermediate filament vimentin is required for cells to transition from the epithelial state to the mesenchymal state and migrate as single cells; however, little is known about the specific role of vimentin in the regulation of mesenchymal migration. Vimentin is known to have a significantly greater ability to resist stress without breaking in vitro compared with actin or microtubules, and also to increase cell elasticity in vivo. Therefore, we hypothesized that the presence of vimentin could support the anisotropic mechanical strain of single-cell migration. To study this, we fluorescently labeled vimentin with an mEmerald tag using TALEN genome editing. We observed vimentin architecture in migrating human foreskin fibroblasts and found that network organization varied from long, linear bundles, or “fibers,” to shorter fragments with a mesh-like organization. We developed image analysis tools employing steerable filtering and iterative graph matching to characterize the fibers embedded in the surrounding mesh. Vimentin fibers were aligned with fibroblast branching and migration direction. The presence of the vimentin network was correlated with 10-fold slower local actin retrograde flow rates, as well as spatial homogenization of actin-based forces transmitted to the substrate. Vimentin fibers coaligned with and were required for the anisotropic orientation of traction stresses. These results indicate that the vimentin network acts as a load-bearing superstructure capable of integrating and reorienting actin-based forces. We propose that vimentin's role in cell motility is to govern the alignment of traction stresses that permit single-cell migration.

Single-Cell Analysis of Lymphoid-Primed Multipotent Progenitors (LMPPs) Reveal Alterations in Lineage Commitment during Aging

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 244-244
Author(s):  
Sneha Borikar ◽  
Vivek Philip ◽  
Jennifer J. Trowbridge
Keyword(s):  
Single Cell ◽  
Progenitor Cells ◽  
Cell Fate ◽  
Progenitor Cell ◽  
Conflicts Of Interest ◽  
Single Cells ◽  
Differentiation Potential ◽  
Increased Risk

Abstract During aging, the hematopoietic compartment undergoes lineage skewing, biased toward myeloid differentiation at the expense of lymphoid differentiation. This skewing clinically presents as impaired adaptive immunity and an increased risk of myeloproliferative disorders. However, little is known of the regulatory mechanisms underlying these changes in differentiation potential due in part to the inadequacy of current analytic techniques to evaluate lineage potency of individual progenitor cells. Recent demonstration that long-lived hematopoietic progenitor cells drive steady-state hematopoiesis has shifted focus onto the progenitor cell compartment to understand clonal dynamics of native hematopoiesis. Here, we critically assess the functional and molecular alterations in the multipotent progenitor cell pool with aging at the single-cell level. We developed novel in vitro and in vivo assays to define the heterogeneity of the LMPP population and test cell-fate potential from single cells. Our results demonstrate, for the first time, distinct, intrinsic lineage potential of single in vitro LMPPs at the cellular and molecular level. We find that clonal alterations in the lymphoid-primed multipotent progenitor (LMPP) compartment contributes to the functional alterations in hematopoiesis observed during aging. Unbiased single-cell transcriptome analysis reveals that true multipotential clones and lymphoid-restricted clones are reduced with aging, while bipotential and myeloid-restricted clones are modestly expanded. Furthermore, myeloid-restricted clones gain myc driver signatures, molecularly identifying clones emerging during aging that are susceptible to transformation. Our study reveals that aging alters the clonal composition of multipotential progenitor cells, directly contributing to the global loss of the lymphoid compartment and increased susceptibility to myeloid transformation. Disclosures No relevant conflicts of interest to declare.

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