Publications by authors named "Todd C McDevitt"

93 Publications

The NIH Somatic Cell Genome Editing program.

Nature 2021 Apr 7;592(7853):195-204. Epub 2021 Apr 7.

Department of Neurosurgery, Yale University, New Haven, CT, USA.

The move from reading to writing the human genome offers new opportunities to improve human health. The United States National Institutes of Health (NIH) Somatic Cell Genome Editing (SCGE) Consortium aims to accelerate the development of safer and more-effective methods to edit the genomes of disease-relevant somatic cells in patients, even in tissues that are difficult to reach. Here we discuss the consortium's plans to develop and benchmark approaches to induce and measure genome modifications, and to define downstream functional consequences of genome editing within human cells. Central to this effort is a rigorous and innovative approach that requires validation of the technology through third-party testing in small and large animals. New genome editors, delivery technologies and methods for tracking edited cells in vivo, as well as newly developed animal models and human biological systems, will be assembled-along with validated datasets-into an SCGE Toolkit, which will be disseminated widely to the biomedical research community. We visualize this toolkit-and the knowledge generated by its applications-as a means to accelerate the clinical development of new therapies for a wide range of conditions.
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http://dx.doi.org/10.1038/s41586-021-03191-1DOI Listing
April 2021

SARS-CoV-2 infection of human iPSC-derived cardiac cells reflects cytopathic features in hearts of patients with COVID-19.

Sci Transl Med 2021 Mar 15. Epub 2021 Mar 15.

Gladstone Institutes, San Francisco, CA 94158, USA.

Although coronavirus disease 2019 (COVID-19) causes cardiac dysfunction in up to 25% of patients, its pathogenesis remains unclear. Exposure of human induced pluripotent stem cell (iPSC)-derived heart cells to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) revealed productive infection and robust transcriptomic and morphological signatures of damage, particularly in cardiomyocytes. Transcriptomic disruption of structural genes corroborates adverse morphologic features, which included a distinct pattern of myofibrillar fragmentation and nuclear disruption. Human autopsy specimens from patients with COVID-19 reflected similar alterations, particularly sarcomeric fragmentation. These striking cytopathic features in cardiomyocytes provide insights into SARS-CoV-2-induced cardiac damage, offer a platform for discovery of potential therapeutics, and raise concerns about the long-term consequences of COVID-19 in asymptomatic as well as severe cases.
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http://dx.doi.org/10.1126/scitranslmed.abf7872DOI Listing
March 2021

Spinal Interneurons as Gatekeepers to Neuroplasticity after Injury or Disease.

J Neurosci 2021 Feb 20;41(5):845-854. Epub 2021 Jan 20.

Department of Neurobiology and Anatomy, and the Marion Murray Spinal Cord Research Center, Drexel University, Philadelphia, Pennsylvania, 19129

Spinal interneurons are important facilitators and modulators of motor, sensory, and autonomic functions in the intact CNS. This heterogeneous population of neurons is now widely appreciated to be a key component of plasticity and recovery. This review highlights our current understanding of spinal interneuron heterogeneity, their contribution to control and modulation of motor and sensory functions, and how this role might change after traumatic spinal cord injury. We also offer a perspective for how treatments can optimize the contribution of interneurons to functional improvement.
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http://dx.doi.org/10.1523/JNEUROSCI.1654-20.2020DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7880285PMC
February 2021

Dimethyloxalylglycine, a small molecule, synergistically increases the homing and angiogenic properties of human mesenchymal stromal cells when cultured as 3D spheroids.

Biotechnol J 2021 Jan 20:e2000389. Epub 2021 Jan 20.

Department of Bioengineering, iBB-Institute for Bioengineering and Biosciences, Instituto Superior Técnico, Universidade de Lisboa, Lisboa, Portugal.

Strategies aiming at increasing the survival and paracrine activity of human mesenchymal stromal cells (MSCs) are of utmost importance to achieve the full therapeutic potential of these cells. Herein, we propose both physical and biochemical strategies to enhance the survival, homing, angiogenic, and immunomodulatory properties of MSCs in vitro. To that purpose, we compared the effect of exposing either 2D monolayer or 3D spheroids of MSCs to (i) hypoxia (2% O ) or to (ii) a hypoxic-mimetic small molecule, dimethyloxalylglycine (DMOG), with cells cultured at 21% O . 3D-cultured MSC spheroids evidenced higher survival upon exposure to oxidative stress and expressed higher levels of factors involved in tissue repair processes, namely tumor necrosis factor-stimulated gene-6, matrix metalloproteinase-2, and vascular endothelial growth factor. MSCs cultured as 3D spheroids and further exposed to hypoxia or hypoxic-mimetic conditions provided by DMOG synergistically favored the expression of the cell surface marker C-X-C chemokine receptor type-4, involved in homing processes to injured tissues, and adhesion to extracellular matrix components as fibronectin. These results highlight the role of ex vivo preconditioning approaches, presenting a novel strategy that combine biochemical stimuli with 3D spheroid organization of MSCs to maximize their tissue regeneration potential.
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http://dx.doi.org/10.1002/biot.202000389DOI Listing
January 2021

Mouse gastruloids take heart.

Nat Rev Cardiol 2021 Apr;18(4):233-234

Gladstone Institutes, San Francisco, CA, USA.

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http://dx.doi.org/10.1038/s41569-020-00501-4DOI Listing
April 2021

Engineering the Spatiotemporal Mosaic Self-Patterning of Pluripotent Stem Cells.

Methods Mol Biol 2021 ;2258:105-116

Gladstone Institute of Cardiovascular Disease, Gladstone Institutes, San Francisco, CA, USA.

Pluripotent stem cells (PSCs) possess the ability to self-organize into complex tissue-like structures; however, the genetic mechanisms and multicellular dynamics that direct such patterning are difficult to control. Here, we pair live imaging with controlled induction of gene knockdown by CRISPR interference (CRISPRi) to generate changes within subpopulations of human PSCs, allowing for control over organization and analysis of emergent behaviors. Specifically, we use forced aggregation of mixtures of cells with and without an inducible CRISPRi system to knockdown molecular regulators of tissue symmetry. We then track the resulting multicellular organization through fluorescence live imaging concurrent with the induction of knockdown. Overall, this technique allows for controlled initiation of symmetry breaking by CRISPRi to produce changes in cellular behavior that can be tracked over time within high-density pluripotent stem cell colonies.
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http://dx.doi.org/10.1007/978-1-0716-1174-6_8DOI Listing
March 2021

SARS-CoV-2 infection of human iPSC-derived cardiac cells predicts novel cytopathic features in hearts of COVID-19 patients.

bioRxiv 2020 Sep 12. Epub 2020 Sep 12.

Gladstone Institutes, San Francisco, CA.

Although COVID-19 causes cardiac dysfunction in up to 25% of patients, its pathogenesis remains unclear. Exposure of human iPSC-derived heart cells to SARS-CoV-2 revealed productive infection and robust transcriptomic and morphological signatures of damage, particularly in cardiomyocytes. Transcriptomic disruption of structural proteins corroborated adverse morphologic features, which included a distinct pattern of myofibrillar fragmentation and numerous iPSC-cardiomyocytes lacking nuclear DNA. Human autopsy specimens from COVID-19 patients displayed similar sarcomeric disruption, as well as cardiomyocytes without DNA staining. These striking cytopathic features provide new insights into SARS-CoV-2 induced cardiac damage, offer a platform for discovery of potential therapeutics, and raise serious concerns about the long-term consequences of COVID-19.
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http://dx.doi.org/10.1101/2020.08.25.265561DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7491510PMC
September 2020

Microfluidic perfusion modulates growth and motor neuron differentiation of stem cell aggregates.

Analyst 2020 Jul 9;145(14):4815-4826. Epub 2020 Jun 9.

School of Chemical & Biomolecular Engineering, Georgia Institute of Technology, Atlanta, GA, USA.

Microfluidic technologies provide many advantages for studying differentiation of three-dimensional (3D) stem cell aggregates, including the ability to control the culture microenvironment, isolate individual aggregates for longitudinal tracking, and perform imaging-based assays. However, applying microfluidics to studying mechanisms of stem cell differentiation requires an understanding of how microfluidic culture conditions impact cell phenotypes. Conventional cell culture techniques cannot directly be applied to the microscale, as microscale culture varies from macroscale culture in multiple aspects. Therefore, the objective of this work was to explore key parameters in microfluidic culture of 3D stem cell aggregates and to understand how these parameters influence stem cell behavior and differentiation. These studies were done in the context of differentiation of embryonic stem cells (ESCs) to motor neurons (MNs). We assessed how media exchange frequency modulates the biochemical microenvironment, including availability of exogenous factors (e.g. nutrients, small molecule additives) and cell-secreted molecules, and thereby impacts differentiation. The results of these studies provide guidance on how key characteristics of 3D cell cultures can be considered when designing microfluidic culture parameters. We demonstrate that discontinuous perfusion is effective at supporting stem cell aggregate growth. We find that there is a balance between the frequency of media exchange, which is needed to ensure that cells are not nutrient-limited, and the need to allow accumulation of cell-secreted factors to promote differentiation. Finally, we show how microfluidic device geometries can influence transport of biomolecules and potentially promote asymmetric spatial differentiation. These findings are instructive for future work in designing devices and experiments for culture of cell aggregates.
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http://dx.doi.org/10.1039/d0an00491jDOI Listing
July 2020

Single-Cell Determination of Cardiac Microtissue Structure and Function Using Light Sheet Microscopy.

Tissue Eng Part C Methods 2020 04 3;26(4):207-215. Epub 2020 Apr 3.

Gladstone Institutes, San Francisco, California.

Native cardiac tissue is composed of heterogeneous cell populations that work cooperatively for proper tissue function; thus, engineered tissue models have moved toward incorporating multiple cardiac cell types in an effort to recapitulate native multicellular composition and organization. Cardiac tissue models composed of stem cell-derived cardiomyocytes (CMs) require inclusion of non-myocytes to promote stable tissue formation, yet the specific contributions of the supporting non-myocyte population on the parenchymal CMs and cardiac microtissues have to be fully dissected. This gap can be partly attributed to limitations in technologies able to accurately study the individual cellular structure and function that comprise intact three-dimensional (3D) tissues. The ability to interrogate the cell-cell interactions in 3D tissue constructs has been restricted by conventional optical imaging techniques that fail to adequately penetrate multicellular microtissues with sufficient spatial resolution. Light sheet fluorescence microscopy (LSFM) overcomes these constraints to enable single-cell resolution structural and functional imaging of intact cardiac microtissues. Multicellular spatial distribution analysis of heterotypic cardiac cell populations revealed that CMs and cardiac fibroblasts were randomly distributed throughout 3D microtissues. Furthermore, calcium imaging of live cardiac microtissues enabled single-cell detection of CM calcium activity, which showed that functional heterogeneity correlated with spatial location within the tissues. This study demonstrates that LSFM can be utilized to determine single-cell spatial and functional interactions of multiple cell types within intact 3D engineered microtissues, thereby facilitating the determination of structure-function relationships at both tissue-level and single-cell resolution. Impact statement The ability to achieve single-cell resolution by advanced three-dimensional light imaging techniques enables exquisite new investigation of multicellular analyses in native and engineered tissues. In this study, light sheet fluorescence microscopy was used to define structure-function relationships of distinct cell types in engineered cardiac microtissues by determining heterotypic cell distributions and interactions throughout the tissues as well as by assessing regional differences in calcium handing functional properties at the individual cardiomyocyte level.
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http://dx.doi.org/10.1089/ten.TEC.2020.0020DOI Listing
April 2020

Heparin-mediated delivery of bone morphogenetic protein-2 improves spatial localization of bone regeneration.

Sci Adv 2020 01 3;6(1):eaay1240. Epub 2020 Jan 3.

The Parker H. Petit Institute for Bioengineering and Bioscience, Georgia Institute of Technology, Atlanta, GA 30322, USA.

Supraphysiologic doses of bone morphogenetic protein-2 (BMP-2) are used clinically to promote bone formation in fracture nonunions, large bone defects, and spinal fusion. However, abnormal bone formation (i.e., heterotopic ossification) caused by rapid BMP-2 release from conventional collagen sponge scaffolds is a serious complication. We leveraged the strong affinity interactions between heparin microparticles (HMPs) and BMP-2 to improve protein delivery to bone defects. We first developed a computational model to investigate BMP-2-HMP interactions and demonstrated improved in vivo BMP-2 retention using HMPs. We then evaluated BMP-2-loaded HMPs as a treatment strategy for healing critically sized femoral defects in a rat model that displays heterotopic ossification with clinical BMP-2 doses (0.12 mg/kg body weight). HMPs increased BMP-2 retention in vivo, improving spatial localization of bone formation in large bone defects and reducing heterotopic ossification. Thus, HMPs provide a promising opportunity to improve the safety profile of scaffold-based BMP-2 delivery.
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http://dx.doi.org/10.1126/sciadv.aay1240DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6941907PMC
January 2020

Automated Design of Pluripotent Stem Cell Self-Organization.

Cell Syst 2019 11 20;9(5):483-495.e10. Epub 2019 Nov 20.

Gladstone Institute of Cardiovascular Disease, Gladstone Institutes, San Francisco, CA, USA; Department of Bioengineering and Therapeutic Sciences, University of California, San Francisco, San Francisco, CA, USA. Electronic address:

Human pluripotent stem cells (hPSCs) have the intrinsic ability to self-organize into complex multicellular organoids that recapitulate many aspects of tissue development. However, robustly directing morphogenesis of hPSC-derived organoids requires novel approaches to accurately control self-directed pattern formation. Here, we combined genetic engineering with computational modeling, machine learning, and mathematical pattern optimization to create a data-driven approach to control hPSC self-organization by knock down of genes previously shown to affect stem cell colony organization, CDH1 and ROCK1. Computational replication of the in vitro system in silico using an extended cellular Potts model enabled machine learning-driven optimization of parameters that yielded emergence of desired patterns. Furthermore, in vitro the predicted experimental parameters quantitatively recapitulated the in silico patterns. These results demonstrate that morphogenic dynamics can be accurately predicted through model-driven exploration of hPSC behaviors via machine learning, thereby enabling spatial control of multicellular patterning to engineer human organoids and tissues. A record of this paper's Transparent Peer Review process is included in the Supplemental Information.
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http://dx.doi.org/10.1016/j.cels.2019.10.008DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7089762PMC
November 2019

Author Correction: V2a interneuron differentiation from mouse and human pluripotent stem cells.

Nat Protoc 2020 Jan;15(1):181

Gladstone Institutes, San Francisco, CA, USA.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.
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http://dx.doi.org/10.1038/s41596-019-0266-zDOI Listing
January 2020

V2a interneuron differentiation from mouse and human pluripotent stem cells.

Nat Protoc 2019 11 18;14(11):3033-3058. Epub 2019 Oct 18.

Gladstone Institutes, San Francisco, CA, USA.

V2a interneurons are located in the hindbrain and spinal cord, where they provide rhythmic input to major motor control centers. Many of the phenotypic properties and functions of excitatory V2a interneurons have yet to be fully defined. Definition of these properties could lead to novel regenerative therapies for traumatic injuries and drug targets for chronic degenerative diseases. Here we describe how to produce V2a interneurons from mouse and human pluripotent stem cells (PSCs), as well as strategies to characterize and mature the cells for further analysis. The described protocols are based on a sequence of small-molecule treatments that induce differentiation of PSCs into V2a interneurons. We also include a detailed description of how to phenotypically characterize, mature, and freeze the cells. The mouse and human protocols are similar in regard to the sequence of small molecules used but differ slightly in the concentrations and durations necessary for induction. With the protocols described, scientists can expect to obtain V2a interneurons with purities of ~75% (mouse) in 7 d and ~50% (human) in 20 d.
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http://dx.doi.org/10.1038/s41596-019-0203-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7243683PMC
November 2019

Perspective: The promise of multi-cellular engineered living systems.

APL Bioeng 2018 Dec 11;2(4):040901. Epub 2018 Oct 11.

Boston University, Boston, Massachusetts 02215, USA.

Recent technological breakthroughs in our ability to derive and differentiate induced pluripotent stem cells, organoid biology, organ-on-chip assays, and 3-D bioprinting have all contributed to a heightened interest in the design, assembly, and manufacture of living systems with a broad range of potential uses. This white paper summarizes the state of the emerging field of "multi-cellular engineered living systems," which are composed of interacting cell populations. Recent accomplishments are described, focusing on current and potential applications, as well as barriers to future advances, and the outlook for longer term benefits and potential ethical issues that need to be considered.
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http://dx.doi.org/10.1063/1.5038337DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6481725PMC
December 2018

A rapid method for determining protein diffusion through hydrogels for regenerative medicine applications.

APL Bioeng 2018 Jun 12;2(2):026110. Epub 2018 Jun 12.

Wallace H. Coulter Department of Biomedical Engineering, Georgia Institute of Technology and Emory University, 313 Ferst Drive NW, Atlanta, Georgia 30332, USA.

Hydrogels present versatile platforms for the encapsulation and delivery of proteins and cells for regenerative medicine applications. However, differences in hydrogel cross-linking density, polymer weight content, and affinity for proteins all contribute to diverse diffusion rates of proteins through hydrogel networks. Here, we describe a simple method to accurately measure protein diffusion through hydrogels, within a few hours and without the use of large amounts of protein. We tracked the diffusion of several proteins of varying molecular weights along the axial direction of capillary tubes filled with alginate, collagen, or poly(ethylene glycol) hydrogels. The rate of protein diffusion decreased with increasing molecular weight. A computational model of protein diffusion through capillary tubes was also created to predict and verify experimental protein diffusion coefficients. This capillary tube-based method of measuring protein diffusion represents a simple strategy to interrogate protein diffusion through natural and synthetic hydrogels and aid in the design of better biomaterial-based delivery vehicles that can effectively modulate protein release.
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http://dx.doi.org/10.1063/1.4999925DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6324205PMC
June 2018

Phenotypic Variation Between Stromal Cells Differentially Impacts Engineered Cardiac Tissue Function.

Tissue Eng Part A 2019 05;25(9-10):773-785

1 Gladstone Institute of Cardiovascular Disease, San Francisco, California.

Impact Statement: Understanding the relationship between parenchymal and supporting cell populations is paramount to recapitulate the multicellular complexity of native tissues. Incorporation of stromal cells is widely recognized to be necessary for the stable formation of stem cell-derived cardiac tissues; yet, the types of stromal cells used have varied widely. This study systematically characterized several stromal populations and found that stromal phenotype and morphology was highly variable depending on cell source and exerted differential impacts on cardiac tissue function and induced pluripotent stem cell-cardiomyocyte phenotype. Therefore, the choice of supporting stromal population can differentially impact the phenotypic or functional performance of engineered cardiac tissues.
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http://dx.doi.org/10.1089/ten.TEA.2018.0362DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6535958PMC
May 2019

Comparable Decellularization of Fetal and Adult Cardiac Tissue Explants as 3D-like Platforms for In Vitro Studies.

J Vis Exp 2019 03 21(145). Epub 2019 Mar 21.

i3S - Instituto de Investigação e Inovação em Saúde, Universidade do Porto; INEB - Instituto de Engenharia Biomédica, Universidade do Porto; Instituto de Ciências Biomédicas Abel Salazar (ICBAS), Universidade do Porto;

Current knowledge of extracellular matrix (ECM)-cell communication translates to large two-dimensional (2D) in vitro culture studies where ECM components are presented as a surface coating. These culture systems constitute a simplification of the complex nature of the tissue ECM that encompasses biochemical composition, structure, and mechanical properties. To better emulate the ECM-cell communication shaping the cardiac microenvironment, we developed a protocol that allows for the decellularization of the whole fetal heart and adult left ventricle tissue explants simultaneously for comparative studies. The protocol combines the use of a hypotonic buffer, a detergent of anionic surfactant properties, and DNase treatment without any requirement for specialized skills or equipment. The application of the same decellularization strategy across tissue samples from subjects of various age is an alternative approach to perform comparative studies. The present protocol allows the identification of unique structural differences across fetal and adult cardiac ECM mesh and biological cellular responses. Furthermore, the herein methodology demonstrates a broader application being successfully applied in other tissues and species with minor adjustments, such as in human intestine biopsies and mouse lung.
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http://dx.doi.org/10.3791/56924DOI Listing
March 2019

Author Correction: Dynamic intercellular transport modulates the spatial patterning of differentiation during early neural commitment.

Nat Commun 2018 11 16;9(1):4901. Epub 2018 Nov 16.

The Wallace H. Coulter Department of Biomedical Engineering, Georgia Institute of Technology and Emory University, Atlanta, GA, 30332, USA.

In the original version of this Article, an incorrect DOI number was provided in the Code Availability statement regarding the deposition of the computational model. The correct DOI is 10.5281/zenodo.1413539. This error has been corrected in both the PDF and HTML versions of the Article.
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http://dx.doi.org/10.1038/s41467-018-07442-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6240069PMC
November 2018

Spatiotemporal mosaic self-patterning of pluripotent stem cells using CRISPR interference.

Elife 2018 10 9;7. Epub 2018 Oct 9.

Gladstone Institute of Cardiovascular Disease, San Francisco, United States.

Morphogenesis involves interactions of asymmetric cell populations to form complex multicellular patterns and structures comprised of distinct cell types. However, current methods to model morphogenic events lack control over cell-type co-emergence and offer little capability to selectively perturb specific cell subpopulations. Our system interrogates cell-cell interactions and multicellular organization within human induced pluripotent stem cell (hiPSC) colonies. We examined effects of induced mosaic knockdown of molecular regulators of cortical tension (ROCK1) and cell-cell adhesion (CDH1) with CRISPR interference. Mosaic knockdown of ROCK1 or CDH1 resulted in differential patterning within hiPSC colonies due to cellular self-organization, while retaining an epithelial pluripotent phenotype. Knockdown induction stimulates a transient wave of differential gene expression within the mixed populations that stabilized in coordination with observed self-organization. Mosaic patterning enables genetic interrogation of emergent multicellular properties, which can facilitate better understanding of the molecular pathways that regulate symmetry-breaking during morphogenesis.
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http://dx.doi.org/10.7554/eLife.36045DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6177255PMC
October 2018

Dynamic intercellular transport modulates the spatial patterning of differentiation during early neural commitment.

Nat Commun 2018 10 5;9(1):4111. Epub 2018 Oct 5.

The Wallace H. Coulter Department of Biomedical Engineering, Georgia Institute of Technology and Emory University, Atlanta, GA, 30332, USA.

The initiation of heterogeneity within a population of phenotypically identical progenitors is a critical event for the onset of morphogenesis and differentiation patterning. Gap junction communication within multicellular systems produces complex networks of intercellular connectivity that result in heterogeneous distributions of intracellular signaling molecules. In this study, we investigate emergent systems-level behavior of the intercellular network within embryonic stem cell (ESC) populations and corresponding spatial organization during early neural differentiation. An agent-based model incorporates experimentally-determined parameters to yield complex transport networks for delivery of pro-differentiation cues between neighboring cells, reproducing the morphogenic trajectories during retinoic acid-accelerated mouse ESC differentiation. Furthermore, the model correctly predicts the delayed differentiation and preserved spatial features of the morphogenic trajectory that occurs in response to intercellular perturbation. These findings suggest an integral role of gap junction communication in the temporal coordination of emergent patterning during early differentiation and neural commitment of pluripotent stem cells.
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http://dx.doi.org/10.1038/s41467-018-06693-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6173785PMC
October 2018

Biophysical subsets of embryonic stem cells display distinct phenotypic and morphological signatures.

PLoS One 2018 8;13(3):e0192631. Epub 2018 Mar 8.

The G. W. Woodruff School of Mechanical Engineering, Georgia Institute of Technology, Atlanta, GA, United States of America.

The highly proliferative and pluripotent characteristics of embryonic stem cells engender great promise for tissue engineering and regenerative medicine, but the rapid identification and isolation of target cell phenotypes remains challenging. Therefore, the objectives of this study were to characterize cell mechanics as a function of differentiation and to employ differences in cell stiffness to select population subsets with distinct mechanical, morphological, and biological properties. Biomechanical analysis with atomic force microscopy revealed that embryonic stem cells stiffened within one day of differentiation induced by leukemia inhibitory factor removal, with a lagging but pronounced change from spherical to spindle-shaped cell morphology. A microfluidic device was then employed to sort a differentially labeled mixture of pluripotent and differentiating cells based on stiffness, resulting in pluripotent cell enrichment in the soft device outlet. Furthermore, sorting an unlabeled population of partially differentiated cells produced a subset of "soft" cells that was enriched for the pluripotent phenotype, as assessed by post-sort characterization of cell mechanics, morphology, and gene expression. The results of this study indicate that intrinsic cell mechanical properties might serve as a basis for efficient, high-throughput, and label-free isolation of pluripotent stem cells, which will facilitate a greater biological understanding of pluripotency and advance the potential of pluripotent stem cell differentiated progeny as cell sources for tissue engineering and regenerative medicine.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0192631PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5843178PMC
June 2018

Microparticle-mediated sequestration of cell-secreted proteins to modulate chondrocytic differentiation.

Acta Biomater 2018 03 30;68:125-136. Epub 2017 Dec 30.

W.H. Coulter Department of Biomedical Engineering, Georgia Institute of Technology and Emory University, 313 Ferst Drive, Atlanta, GA 30332, USA; Petit Institute for Bioengineering and Bioscience, Georgia Institute of Technology, 315 Ferst Drive, Atlanta, GA 30332, USA. Electronic address:

Protein delivery is often used in tissue engineering applications to control differentiation processes, but is limited by protein instability and cost. An alternative approach is to control the cellular microenvironment through biomaterial-mediated sequestration of cell-secreted proteins important to differentiation. Thus, we utilized heparin-based microparticles to modulate cellular differentiation via protein sequestration in an in vitro model system of endochondral ossification. Heparin and poly(ethylene-glycol) (PEG; a low-binding material control)-based microparticles were incorporated into ATDC5 cell spheroids or incubated with ATDC5 cells in transwell culture. Reduced differentiation was observed in the heparin microparticle group as compared to PEG and no microparticle-containing groups. To determine if observed changes were due to sequestration of cell-secreted protein, the proteins sequestered by heparin microparticles were analyzed using SDS-PAGE and mass spectrometry. It was found that heparin microparticles bound insulin-like growth factor binding proteins (IGFBP)-3 and 5. When incubated with a small-molecule inhibitor of IGFBPs, NBI 31772, a similar delay in differentiation of ATDC5 cells was observed. These results indicate that heparin microparticles modulated chondrocytic differentiation in this system via sequestration of cell-secreted protein, a technique that could be beneficial in the future as a means to control cellular differentiation processes.

Statement Of Significance: In this work, we present a proof-of-principle set of experiments in which heparin-based microparticles are shown to modulate cellular differentiation through binding of cell-secreted protein. Unlike existing systems that rely on expensive protein with limited half-lives to elicit changes in cellular behavior, this technique focuses on temporal modulation of cell-generated proteins. This technique also provides a biomaterials-based method that can be used to further identify sequestered proteins of interest. Thus, this work indicates that glycosaminoglycan-based biomaterial approaches could be used as substitutes or additions to traditional methods for modulating and identifying the cell-secreted proteins involved in directing cellular behavior.
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http://dx.doi.org/10.1016/j.actbio.2017.12.038DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5803405PMC
March 2018

Functionalization of microparticles with mineral coatings enhances non-viral transfection of primary human cells.

Sci Rep 2017 10 27;7(1):14211. Epub 2017 Oct 27.

Department of Biomedical Engineering-University of Wisconsin-Madison, Madison, WI, USA.

Gene delivery to primary human cells is a technology of critical interest to both life science research and therapeutic applications. However, poor efficiencies in gene transfer and undesirable safety profiles remain key limitations in advancing this technology. Here, we describe a materials-based approach whereby application of a bioresorbable mineral coating improves microparticle-based transfection of plasmid DNA lipoplexes in several primary human cell types. In the presence of these mineral-coated microparticles (MCMs), we observed up to 4-fold increases in transfection efficiency with simultaneous reductions in cytotoxicity. We identified mechanisms by which MCMs improve transfection, as well as coating compositions that improve transfection in three-dimensional cell constructs. The approach afforded efficient transfection in primary human fibroblasts as well as mesenchymal and embryonic stem cells for both two- and three-dimensional transfection strategies. This MCM-based transfection is an advancement in gene delivery technology, as it represents a non-viral approach that enables highly efficient, localized transfection and allows for transfection of three-dimensional cell constructs.
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http://dx.doi.org/10.1038/s41598-017-14153-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5660152PMC
October 2017

Tridimensional configurations of human mesenchymal stem/stromal cells to enhance cell paracrine potential towards wound healing processes.

J Biotechnol 2017 Nov 28;262:28-39. Epub 2017 Sep 28.

Department of Bioengineering and IBB-Institute for Bioengineering and Biosciences, Instituto Superior Técnico, Universidade de Lisboa, Lisboa, Portugal; The Discoveries Centre for Regenerative and Precision Medicine, Lisbon Campus, Instituto Superior Técnico, Universidade de Lisboa, Av. Rovisco Pais, 1049-001 Lisboa, Portugal. Electronic address:

This study proposes to use alginate encapsulation as a strategy to assess the paracrine activity of 3D- and 2D-cultured human bone marrow mesenchymal stem/stromal cells (BM MSC) in the setting of wound repair and regeneration processes. A side-by-side comparison of MSC culture in three different 3D configurations (spheroids, encapsulated spheroids and encapsulated single cells) versus 2D monolayer cell culture is presented. The results reveal enhanced resistance to oxidative stress and paracrine potential of 3D spheroid-organized BM MSC. MSC spheroids (148±2μm diameter) encapsulated in alginate microbeads evidence increased angiogenic and chemotactic potential relatively to encapsulated single cells, as supported by higher secreted levels of angiogenic factors and by functional assays showing the capability of encapsulated MSC to promote formation of tubelike structures and migration of fibroblasts into a wounded area. In addition, a higher expression of the anti-inflammatory factor tumor necrosis factor alpha-induced protein 6 (TSG-6) was demonstrated by RT-PCR for encapsulated and non-encapsulated spheroids. Culture of spheroids within an alginate matrix maintains low aggregation levels below 5% and favors resistance to oxidative stress. These are important factors towards the establishment of more standardized and controlled systems, crucial to explore the paracrine effects of 3D-cultured MSC in therapeutic settings.
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http://dx.doi.org/10.1016/j.jbiotec.2017.09.020DOI Listing
November 2017

Enhanced in vivo retention of low dose BMP-2 via heparin microparticle delivery does not accelerate bone healing in a critically sized femoral defect.

Acta Biomater 2017 09 20;59:21-32. Epub 2017 Jun 20.

The Parker H. Petit Institute for Bioengineering and Bioscience, Georgia Institute of Technology, Atlanta, GA, United States; The George W. Woodruff School of Mechanical Engineering, Georgia Institute of Technology, Atlanta, GA, United States. Electronic address:

Bone morphogenetic protein-2 (BMP-2) is an osteoinductive growth factor used clinically to induce bone regeneration and fusion. Some complications associated with BMP-2 treatment have been attributed to rapid release of BMP-2 from conventional collagen scaffolds, motivating the development of tunable sustained-release strategies. We incorporated BMP-2-binding heparin microparticles (HMPs) into a hydrogel scaffold to improve spatiotemporal control of BMP-2 delivery to large bone defects. HMPs pre-loaded with BMP-2 were mixed into alginate hydrogels and compared to hydrogels containing BMP-2 alone. BMP-2 release from scaffolds in vitro, BMP-2 retention within injury sites in vivo, and bone regeneration in a critically sized femoral defect were evaluated. Compared to hydrogel delivery alone, BMP-2-loaded HMPs reduced BMP-2 release in vitro and increased early BMP-2 retention in the bone defect. BMP-2-loaded HMPs induced bone formation at both ectopic and orthotopic sites; however, the volume of induced bone was lower for defects treated with BMP-2-loaded HMPs compared to hydrogel delivery. To better understand the effect of HMPs on BMP-2 release kinetics, a computational model was developed to predict BMP-2 release from constructs in vivo. The model suggested that HMPs limited BMP-2 release into surrounding tissues, and that changing the HMP density could modulate BMP-2 release. Taken together, these experimental and computational results suggest the importance of achieving a balance of BMP-2 retention within the bone defect and BMP-2 release into surrounding soft tissues. HMP delivery of BMP-2 may provide a method of tuning BMP-2 release in vivo that can be further investigated to improve current methods of bone regeneration.

Statement Of Significance: The development of effective biomaterials for sustained protein delivery is a crucial component of tissue engineering strategies. However, in most applications, including bone repair, the optimal balance between protein presentation in the injury site and protein release into the surrounding tissues is unknown. Herein, we introduced heparin microparticles (HMPs) into a tissue engineered construct to increase in vivo retention of bone morphogenetic protein-2 (BMP-2) and enhance healing in femoral defects. Although HMPs induced bone regeneration, no increase in bone volume was observed, leading to further experimental and computational analysis of the effect of HMP-BMP-2 interactions on protein retention and release. Ultimately, this work provides insight into designing tunable protein-material interactions and their implications for controlling BMP-2 delivery.
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http://dx.doi.org/10.1016/j.actbio.2017.06.028DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6546418PMC
September 2017

Differentiation of V2a interneurons from human pluripotent stem cells.

Proc Natl Acad Sci U S A 2017 05 24;114(19):4969-4974. Epub 2017 Apr 24.

Gladstone Institute of Cardiovascular Disease, San Francisco, CA 94158;

The spinal cord consists of multiple neuronal cell types that are critical to motor control and arise from distinct progenitor domains in the developing neural tube. Excitatory V2a interneurons in particular are an integral component of central pattern generators that control respiration and locomotion; however, the lack of a robust source of human V2a interneurons limits the ability to molecularly profile these cells and examine their therapeutic potential to treat spinal cord injury (SCI). Here, we report the directed differentiation of CHX10 V2a interneurons from human pluripotent stem cells (hPSCs). Signaling pathways (retinoic acid, sonic hedgehog, and Notch) that pattern the neural tube were sequentially perturbed to identify an optimized combination of small molecules that yielded ∼25% CHX10 cells in four hPSC lines. Differentiated cultures expressed much higher levels of V2a phenotypic markers (CHX10 and SOX14) than other neural lineage markers. Over time, CHX10 cells expressed neuronal markers [neurofilament, NeuN, and vesicular glutamate transporter 2 (VGlut2)], and cultures exhibited increased action potential frequency. Single-cell RNAseq analysis confirmed CHX10 cells within the differentiated population, which consisted primarily of neurons with some glial and neural progenitor cells. At 2 wk after transplantation into the spinal cord of mice, hPSC-derived V2a cultures survived at the site of injection, coexpressed NeuN and VGlut2, extended neurites >5 mm, and formed putative synapses with host neurons. These results provide a description of V2a interneurons differentiated from hPSCs that may be used to model central nervous system development and serve as a potential cell therapy for SCI.
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http://dx.doi.org/10.1073/pnas.1608254114DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5441696PMC
May 2017

Competitive Protein Binding Influences Heparin-Based Modulation of Spatial Growth Factor Delivery for Bone Regeneration.

Tissue Eng Part A 2017 07 24;23(13-14):683-695. Epub 2017 Mar 24.

3 The Parker H. Petit Institute for Bioengineering and Bioscience, Georgia Institute of Technology , Atlanta, Georgia .

Tissue engineering strategies involving the in vivo delivery of recombinant growth factors are often limited by the inability of biomaterials to spatially control diffusion of the delivered protein within the site of interest. The poor spatiotemporal control provided by porous collagen sponges, which are used for the clinical delivery of bone morphogenetic protein-2 (BMP-2) for bone regeneration, has necessitated the use of supraphysiological protein doses, leading to inflammation and heterotopic ossification. This study describes a novel tissue engineering strategy to spatially control rapid BMP-2 diffusion from collagen sponges in vivo by creating a high-affinity BMP-2 sink around the collagen sponge. We designed an electrospun poly-ɛ-caprolactone nanofiber mesh containing physically entrapped heparin microparticles, which have been previously demonstrated to bind and retain large amounts of BMP-2. Nanofiber meshes containing 0.05 and 0.10 mg of microparticles/cm demonstrated increased BMP-2 binding and decreased BMP-2 release in vitro compared with meshes without microparticles. However, when microparticle-containing meshes were used in vivo to limit the diffusion of BMP-2 delivered by using collagen sponges in a rat femoral defect, no differences in heterotopic ossification or biomechanical properties were observed. Further investigation revealed that, although BMP-2 binding to heparin microparticles was rapid, the presence of serum components attenuated microparticle-BMP-2 binding and increased BMP-2 release in vitro. These observations provide a plausible explanation for the results observed in vivo and suggest that competitive protein binding in vivo may hinder the ability of affinity-based biomaterials to modulate growth factor delivery.
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http://dx.doi.org/10.1089/ten.tea.2016.0507DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5549832PMC
July 2017

Enhanced Immunosuppression of T Cells by Sustained Presentation of Bioactive Interferon-γ Within Three-Dimensional Mesenchymal Stem Cell Constructs.

Stem Cells Transl Med 2017 01 8;6(1):223-237. Epub 2016 Aug 8.

Gladstone Institute of Cardiovascular Disease, San Francisco, California, USA.

The immunomodulatory activity of mesenchymal stem/stromal cells (MSCs) to suppress innate and adaptive immune responses offers a potent cell therapy for modulating inflammation and promoting tissue regeneration. However, the inflammatory cytokine milieu plays a critical role in stimulating MSC immunomodulatory activity. In particular, interferon-γ (IFN-γ)-induced expression of indoleamine 2,3-dioxygenase (IDO) is primarily responsible for MSC suppression of T-cell proliferation and activation. Although pretreatment with IFN-γ is commonly used to prime MSCs for immunomodulatory activity prior to transplantation, the transient effects of pretreatment may limit the potential of MSCs to potently modulate immune responses. Therefore, the objective of this study was to investigate whether microparticle-mediated presentation of bioactive IFN-γ within three-dimensional spheroidal MSC aggregates could precisely regulate and induce sustained immunomodulatory activity. Delivery of IFN-γ via heparin-microparticles within MSC aggregates induced sustained IDO expression during 1 week of culture, whereas IDO expression by IFN-γ-pretreated MSC spheroids rapidly decreased during 2 days. Furthermore, sustained IDO expression induced by IFN-γ-loaded microparticles resulted in an increased and sustained suppression of T-cell activation and proliferation in MSC cocultures with CD3/CD28-activated peripheral blood mononuclear cells. The increased suppression of T cells by MSC spheroids containing IFN-γ-loaded microparticles was dependent on induction of IDO and supported by affecting monocyte secretion from pro- to anti-inflammatory cytokines. Altogether, microparticle delivery of IFN-γ within MSC spheroids provides a potent means of enhancing and sustaining immunomodulatory activity to control MSC immunomodulation after transplantation and thereby improve the efficacy of MSC-based therapies aimed at treating inflammatory and immune diseases. Stem Cells Translational Medicine 2017;6:223-237.
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http://dx.doi.org/10.5966/sctm.2016-0044DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5442746PMC
January 2017

Hydrolysis and Sulfation Pattern Effects on Release of Bioactive Bone Morphogenetic Protein-2 from Heparin-Based Microparticles.

J Mater Chem B 2015 Oct 28;3(40):8001-8009. Epub 2015 Aug 28.

W.H. Coulter Department of Biomedical Engineering, Georgia Institute of Technology and Emory University, 313 Ferst Drive, Atlanta, GA 30032; Petit Institute for Bioengineering and Bioscience, Georgia Institute of Technology, 315 Ferst Drive, Atlanta, GA 30332.

Glycosaminoglycans (GAGs) such as heparin are promising materials for growth factor delivery due to their ability to efficiently bind positively charged growth factors including bone morphogenetic protein-2 (BMP-2) through their negatively charged sulfate groups. Therefore, the goal of this study was to examine BMP-2 release from heparin-based microparticles (MPs) after first, incorporating a hydrolytically degradable crosslinker and varying heparin content within MPs to alter MP degradation and second, altering the sulfation pattern of heparin within MPs to vary BMP-2 binding and release. Using varied MP formulations, it was found that the time course of MP degradation for 1 wt% heparin MPs was ~4 days slower than 10 wt% heparin MPs, indicating that MP degradation was dependent on heparin content. After incubating 100 ng BMP-2 with 0.1 mg MPs, most MP formulations loaded BMP-2 with ~50% efficiency and significantly more BMP-2 release (60% of loaded BMP-2) was observed from more sulfated heparin MPs (MPs with ~100% and 80% of native sulfation). Similarly, BMP-2 bioactivity in more sulfated heparin MP groups was at least four-fold higher than soluble BMP-2 and less sulfated heparin MP groups, as determined by an established C2C12 cell alkaline phosphatase (ALP) assay. Ultimately, the two most sulfated 10 wt% heparin MP formulations were able to efficiently load and release BMP-2 while enhancing BMP-2 bioactivity, making them promising candidates for future growth factor delivery applications.
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http://dx.doi.org/10.1039/C5TB00933BDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5077163PMC
October 2015

Design Principles for Engineering of Tissues from Human Pluripotent Stem Cells.

Curr Stem Cell Rep 2016 Mar 27;2(1):43-51. Epub 2016 Jan 27.

The Gladstone Institute of Cardiovascular Disease, San Francisco, CA; Department of Bioengineering and Therapeutic Sciences, University of California San Francisco, San Francisco, CA.

Recent advances in human pluripotent stem cell (hPSC) technologies have enabled the engineering of human tissue constructs for developmental studies, disease modeling, and drug screening platforms. tissue formation can be generally described at three levels of cellular organization. Multicellular hPSC constructs are initially formed either with polymeric scaffold materials or simply via self-assembly, adhesive mechanisms. Heterotypic interactions within hPSC tissue constructs can be achieved by physically mixing independently differentiated cell populations or coaxed to simultaneously co-emerge from a common population of undifferentiated cells. Higher order tissue architecture can be engineered by imposing external spatial constraints, such as molds and scaffolds, or depend upon cell-driven organization that exploits endogenous innate developmental mechanisms. The multicellular, heterogeneous, and highly organized structure of hPSC constructs ultimately dictates the resulting form and function of engineered human tissue models.
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http://dx.doi.org/10.1007/s40778-016-0030-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4910633PMC
March 2016