Nanofiber curvature with Rho GTPase activity increases mouse embryonic fibroblast random migration velocity

2022 ◽  
Author(s):  
Daniel T Bowers ◽  
Justin L Brown
Keyword(s):  
Stem Cell ◽  
Flat Surface ◽  
Rho Gtpase ◽  
Kinase Inhibitor ◽  
Single Cells ◽  
Stem Cell Differentiation ◽  
Migration Velocity ◽  
Embryonic Cell ◽  
Small Gtpase ◽  
Curved Surface

Abstract Mechanotransduction arises from information encoded in the shape of materials such as curvature. It induces activation of small GTPase signaling affecting cell phenotypes including differentiation. We carried out a set of preliminary experiments to test the hypothesis that curvature (1/radius) would also affect cell motility due to signal pathway crosstalk. High molecular weight poly (methyl methacrylate) straight nanofibers were electrospun with curvature ranging from 41 to 1 μm−1 and collected on a passivated glass substrate. The fiber curvature increased mouse mesenchymal stem cell aspect ratio (P < 0.02) and decreased cell area (P < 0.01). Despite little effect on some motility patterns such as polarity and persistence, we found selected fiber curvatures can increase normalized random fibroblastic mouse embryonic cell (MEF) migration velocity close to 2.5 times compared with a flat surface (P < 0.001). A maximum in the velocity curve occurred near 2.5 μm−1 and may vary with the time since initiation of attachment to the surface (range of 0–20 h). In the middle range of fiber curvatures, the relative relationship to curvature was similar regardless of treatment with Rho-kinase inhibitor (Y27632) or cdc42 inhibitor (ML141), although it was decreased on most curvatures (P < 0.05). However, below a critical curvature threshold MEFs may not be able to distinguish shallow curvature from a flat surface, while still being affected by contact guidance. The preliminary data in this manuscript suggested the large low curvature fibers were interpreted in a manner similar to a non-curved surface. Thus, curvature is a biomaterial construct design parameter that should be considered when specific biological responses are desired. Statement of integration, innovation, and insight  Replacement of damaged or diseased tissues that cannot otherwise regenerate is transforming modern medicine. However, the extent to which we can rationally design materials to affect cellular outcomes remains low. Knowing the effect of material stiffness and diameter on stem cell differentiation, we investigated cell migration and signaling on fibrous scaffolds. By investigating diameters across orders of magnitude (50–2000 nm), we identified a velocity maximum of ~800 nm. Furthermore, the results suggest large fibers may not be interpreted by single cells as a curved surface. This work presents insight into the design of constructs for engineering tissues.

scholarly journals Transformative opportunities for single-cell proteomics

2018 ◽  
Author(s):  
Harrison Specht ◽  
Nikolai Slavov
Keyword(s):  
Mass Spectrometry ◽  
Stem Cell ◽  
Cell Differentiation ◽  
Single Cell ◽  
Single Cells ◽  
Biological Systems ◽  
Stem Cell Differentiation ◽  
New Age ◽  
Technological Developments

Many pressing medical challenges - such as diagnosing disease, enhancing directed stem cell differentiation, and classifying cancers - have long been hindered by limitations in our ability to quantify proteins in single cells. Mass-spectrometry (MS) is poised to transcend these limitations by developing powerful methods to routinely quantify thousands of proteins and proteoforms across many thousands of single cells. We outline specific technological developments and ideas that can increase the sensitivity and throughput of single cell MS by orders of magnitude and usher in this new age. These advances will transform medicine and ultimately contribute to understanding biological systems on an entirely new level.

scholarly journals Bioconductor workflow for single-cell RNA sequencing: Normalization, dimensionality reduction, clustering, and lineage inference

F1000Research ◽  
2017 ◽  
Vol 6 ◽  
pp. 1158 ◽  
Author(s):  
Fanny Perraudeau ◽  
Davide Risso ◽  
Kelly Street ◽  
Elizabeth Purdom ◽  
Sandrine Dudoit
Keyword(s):  
Stem Cell ◽  
Cell Differentiation ◽  
Dimensionality Reduction ◽  
Single Cell ◽  
Single Cells ◽  
Differential Expression Analysis ◽  
Stem Cell Differentiation ◽  
Cell Types ◽  
Cell Clustering ◽  
Cell Transcriptome

Novel single-cell transcriptome sequencing assays allow researchers to measure gene expression levels at the resolution of single cells and offer the unprecendented opportunity to investigate at the molecular level fundamental biological questions, such as stem cell differentiation or the discovery and characterization of rare cell types. However, such assays raise challenging statistical and computational questions and require the development of novel methodology and software. Using stem cell differentiation in the mouse olfactory epithelium as a case study, this integrated workflow provides a step-by-step tutorial to the methodology and associated software for the following four main tasks: (1) dimensionality reduction accounting for zero inflation and over dispersion and adjusting for gene and cell-level covariates; (2) cell clustering using resampling-based sequential ensemble clustering; (3) inference of cell lineages and pseudotimes; and (4) differential expression analysis along lineages.

scholarly journals Defining Parameters Attributing to the Role of Osteomacs in Regulating Stem Cell Function and the Hematopoietic Niche

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 2576-2576
Author(s):  
Safa F. Mohamad ◽  
Joydeep Ghosh ◽  
Andrea M. Gunawan ◽  
Rachel Blosser ◽  
Malgorzata Kamocka ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Stem Cell ◽  
Cell Function ◽  
Single Cells ◽  
Stem Cell Differentiation ◽  
Cell Renewal ◽  
Hematopoietic Stem ◽  
Hematopoietic Niche ◽  
Hematopoietic Activity

Abstract Networking between hematopoietic stem cells (HSC) and cells of the hematopoietic niche is critical for the maintenance of stem cell renewal and function. HSC maintenance in the hematopoietic niche is considered to be the product of intimate interactions between cellular and soluble elements of the niche and stem cells. Among the cellular components of the niche participating in this function are a group of specialized bone-resident macrophages known as osteomacs (OM). Previously, we established the importance of osteoblasts (OB) in hematopoiesis and quite recently, we described the importance of OM and their interactions with OB and megakaryocytes (MK) in sustaining HSC function. We have also illustrated that CD166 is a critical functional marker of stem cell function and competence of the hematopoietic niche. Interestingly, immature OB which are CD166+ mediate the highest level of hematopoietic enhancing activity. We report here the importance of CD166 on calvarie-resident OM (identified as CD45+F4/80+ cells) and outline how these cells require cooperation from MK to increase CD166 expression and sustain HSC function. Bone resident-osteomacs, which are phenotypically similar but functionally different from bone marrow-derived macrophages, were collected by the enzymatic digestion of neonatal calvarial cells (NCC) or long bones of adult mice. Transplantation assays indicated that OM are relatively radioresistant and survive several weeks post lethal radiation. However, they eventually deplete and are replenished by progeny of donor HSC. To understand the importance of OM-OB-MK interactions in maintaining HSC function in the niche, we performed 3D cytometry on fixed and stained bone marrow sections that revealed intimate spatial interactions between OM, OB, MK and HSC. To assess changes in gene expression observed due to these interactions, we cultured NCC for 16hr in the absence or presence of MK prepared from fetal liver followed by sorting out OM from each group. These cells were then captured as single cells and sequenced to identify potential targets through which OM enhanced hematopoietic activity. Strikingly, several genes involved in the hematopoietic stem cell differentiation pathway including lmo2, fli1 and ikzf1 were upregulated in OM cultured in the presence of MK. Other genes that were upregulated were embigin and PF-4, both of which have been implicated in the maintenance of HSC function. Interestingly, OM express embigin, angiogenin and IL-18 mRNA similar to proximal osteolineage cells which we previously described as HSC regulators. To investigate changes at the translational level, we performed single cell proteomics using CyTOF. NCC were cultured for 2 days in the absence and presence of MK followed by staining for a panel of 29 surface and intracellular markers. Expression of markers such as CD166, embigin, mac-2 and STAT3 amongst others was elevated on OM cultured with MK compared to OM cultured without. These data informed our decision to focus our future investigations on CD166 and embigin. Next CD166+OM and CD166-OM were isolated by cell sorting and used in co-culture assays with OB to support the production of clonogenic cells in vitro. Only the CD166+ fraction of OM maintained hematopoietic activity similar to unsorted OM, implicating CD166 as one of the mediators of OM function. These results were validated using recombinant CD166 protein to substitute for OM function. Under these conditions, recombinant CD166 supported the hematopoietic enhancing activity of OB in the absence of OM. Recombinant Angiogenin and IL-18 were unable to augment the CD166-mediated support of hematopoiesis. Interestingly, CD166 knockout OM were unable to mediate the same hematopoietic enhancing activity observed with WT OM regardless of the presence or absence of MK in culture. In vivo transplantation studies to corroborate these findings have been initiated and are very early to yield meaningful conclusions. These data demonstrate that CD166 is one of the important mediators through which OM maintain HSC function. However, CD166-OM mediated HSC function is only maintained in conjunction with OB-MK interactions. Our data indicate the importance of crosstalk between OM, OB and MK which leads to the expression of novel mediators such as CD166 to support HSC function. Disclosures No relevant conflicts of interest to declare.

scholarly journals Ultra-multiplexed analysis of single-cell dynamics reveals logic rules in differentiation

2019 ◽  
Vol 5 (4) ◽  
pp. eaav7959 ◽  
Author(s):  
Ce Zhang ◽  
Hsiung-Lin Tu ◽  
Gengjie Jia ◽  
Tanzila Mukhtar ◽  
Verdon Taylor ◽  
...  
Keyword(s):  
Stem Cell ◽  
Cell Differentiation ◽  
Single Cell ◽  
Cell Fate ◽  
Single Cell Analysis ◽  
Single Cells ◽  
Critical Role ◽  
Stem Cell Differentiation ◽  
Cell Fate Decisions ◽  
Cellular Microenvironments

Dynamical control of cellular microenvironments is highly desirable to study complex processes such as stem cell differentiation and immune signaling. We present an ultra-multiplexed microfluidic system for high-throughput single-cell analysis in precisely defined dynamic signaling environments. Our system delivers combinatorial and time-varying signals to 1500 independently programmable culture chambers in week-long live-cell experiments by performing nearly 106 pipetting steps, where single cells, two-dimensional (2D) populations, or 3D neurospheres are chemically stimulated and tracked. Using our system and statistical analysis, we investigated the signaling landscape of neural stem cell differentiation and discovered “cellular logic rules” that revealed the critical role of signal timing and sequence in cell fate decisions. We find synergistic and antagonistic signal interactions and show that differentiation pathways are highly redundant. Our system allows dissection of hidden aspects of cellular dynamics and enables accelerated biological discovery.

scholarly journals Citrate Mediates Crosstalk between Mitochondria and the Nucleus to Promote Human Mesenchymal Stem Cell In Vitro Osteogenesis

Cells ◽  
2020 ◽  
Vol 9 (4) ◽  
pp. 1034
Author(s):  
Claudia Morganti ◽  
Massimo Bonora ◽  
Saverio Marchi ◽  
Letizia Ferroni ◽  
Chiara Gardin ◽  
...  
Keyword(s):  
Stem Cell ◽  
Mesenchymal Stem Cell ◽  
Early Stage ◽  
Single Cells ◽  
Stem Cell Differentiation ◽  
Human Mesenchymal Stem Cell ◽  
Differentiation Process ◽  
Contact Sites ◽  
Citrate Transporter

Citrate, generated in the mitochondria, is a key metabolite that might link metabolism with signaling, chromatin structure and transcription to orchestrate mesenchymal stem cells (MSCs) fate determination. Based on a detailed morphological analysis of 3D reconstruction of mitochondria and nuclei in single cells, we identified contact sites between these organelles that drastically increase in volume and number during the early stage of mesenchymal stem cell differentiation. These contact sites create a microdomain that facilitates exchange of signals from mitochondria to the nucleus. Interestingly, we found that the citrate derived from mitochondria is necessary for osteogenic lineage determination. Indeed, inhibition of the citrate transporter system dramatically affected osteogenesis, reduced citrate levels that could be converted in α-ketoglutarate, and consequently affected epigenetic marker H3K9me3 associated with the osteogenesis differentiation process. These findings highlight that mitochondrial metabolites play key regulatory roles in the MSCs differentiation process. Further in-depth investigation is needed to provide novel therapeutic strategies in the field of regenerative medicine.

scholarly journals Transformative opportunities for single-cell proteomics

2018 ◽  
Author(s):  
Harrison Specht ◽  
Nikolai Slavov
Keyword(s):  
Mass Spectrometry ◽  
Stem Cell ◽  
Cell Differentiation ◽  
Single Cell ◽  
Single Cells ◽  
Biological Systems ◽  
Stem Cell Differentiation ◽  
New Age ◽  
Technological Developments

Many pressing medical challenges - such as diagnosing disease, enhancing directed stem cell differentiation, and classifying cancers - have long been hindered by limitations in our ability to quantify proteins in single cells. Mass-spectrometry (MS) is poised to transcend these limitations by developing powerful methods to routinely quantify thousands of proteins and proteoforms across many thousands of single cells. We outline specific technological developments and ideas that can increase the sensitivity and throughput of single cell MS by orders of magnitude and usher in this new age. These advances will transform medicine and ultimately contribute to understanding biological systems on an entirely new level.

scholarly journals Routinely quantifying single cell proteomes: A new age in quantitative biology and medicine

2018 ◽  
Author(s):  
Harrison Specht ◽  
Nikolai Slavov
Keyword(s):  
Mass Spectrometry ◽  
Stem Cell ◽  
Cell Differentiation ◽  
Single Cell ◽  
Single Cells ◽  
Biological Systems ◽  
Stem Cell Differentiation ◽  
New Age ◽  
Quantitative Biology ◽  
Technological Developments

Many pressing medical challenges -- such as diagnosing disease, enhancing directed stem cell differentiation, and classifying cancers -- have long been hindered by limitations in our ability to quantify proteins in single cells. Mass-spectrometry (MS) is poised to transcend these limitations by developing powerful methods to routinely quantify thousands of proteins and proteoforms across many thousands of single cells. We outline specific technological developments and ideas that can increase the sensitivity and throughput of single cell MS by orders of magnitude and usher in this new age. These advances will transform medicine and ultimately contribute to understanding biological systems on an entirely new level.

scholarly journals Universal Microfluidic System for Analysis and Control of Cell Dynamics

2017 ◽  
Author(s):  
Ce Zhang ◽  
Hsiung-Lin Tu ◽  
Gengjie Jia ◽  
Tanzila Mukhtar ◽  
Verdon Taylor ◽  
...  
Keyword(s):  
Stem Cell ◽  
Cell Differentiation ◽  
Single Cells ◽  
Stem Cell Differentiation ◽  
Microfluidic System ◽  
Cell Dynamics ◽  
Point Analysis ◽  
Wide Range ◽  
Microfluidic Cell Culture ◽  
Cellular Logic

SUMMARYDynamical control of the cellular microenvironment is highly desired for quantitative studies of stem cells and immune signaling. Here, we present an automated microfluidic system for high-throughput culture, differentiation and analysis of a wide range of cells in precisely defined dynamic microenvironments recapitulating cellular niches. This system delivers complex, time-varying biochemical signals to 1,500 independently programmable cultures containing either single cells, 2-D populations, or 3-D organoids, and dynamically stimulates adherent or non-adherent cells while tracking and retrieving them for end-point analysis. Using this system, we investigated the signaling landscape of neural stem cell differentiation under combinatorial and dynamic stimulation with growth factors. Experimental and computational analyses identified “cellular logic rules” for stem cell differentiation, and demonstrated the importance of signaling sequence and timing in brain development. This universal platform greatly enhances capabilities of microfluidic cell culture, allows dissection of previously hidden aspects of cellular dynamics, and enables accelerated biological discovery.

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