Measuring dynamic cell–material interactions and remodeling during 3D human mesenchymal stem cell migration in hydrogels

Biomaterials that mimic aspects of the extracellular matrix by presenting a 3D microenvironment that cells can locally degrade and remodel are finding increased applications as wound-healing matrices, tissue engineering scaffolds, and even substrates for stem cell expansion. In vivo, cells do not simply reside in a static microenvironment, but instead, they dynamically reengineer their surroundings. For example, cells secrete proteases that degrade extracellular components, attach to the matrix through adhesive sites, and can exert traction forces on the local matrix, causing its spatial reorganization. Although biomaterials scaffolds provide initially well-defined microenvironments for 3D culture of cells, less is known about the changes that occur over time, especially local matrix remodeling that can play an integral role in directing cell behavior. Here, we use microrheology as a quantitative tool to characterize dynamic cellular remodeling of peptide-functionalized poly(ethylene glycol) (PEG) hydrogels that degrade in response to cell-secreted matrix metalloproteinases (MMPs). This technique allows measurement of spatial changes in material properties during migration of encapsulated cells and has a sensitivity that identifies regions where cells simply adhere to the matrix, as well as the extent of local cell remodeling of the material through MMP-mediated degradation. Collectively, these microrheological measurements provide insight into microscopic, cellular manipulation of the pericellular region that gives rise to macroscopic tracks created in scaffolds by migrating cells. This quantitative and predictable information should benefit the design of improved biomaterial scaffolds for medically relevant applications.

Download Full-text

A novel method for sensor-based quantification of single/multi-cellular traction dynamics and remodeling in 3D matrices

10.1101/2020.09.24.311647 ◽

2020 ◽

Author(s):

Bashar Emon ◽

Zhengwei Li ◽

Md Saddam Hossain Joy ◽

Umnia Doha ◽

Farhad Kosari ◽

...

Keyword(s):

Self Assembly ◽

Single Cells ◽

Human Colon ◽

Matrix Remodeling ◽

Tissue Stiffness ◽

Force Dynamics ◽

Cell Functions ◽

The Matrix ◽

Cancer Tumor

AbstractCells in vivo generate mechanical forces (traction) on surrounding 3D extra cellular matrix (ECM) and cells. Such traction and biochemical cues may remodel the matrix, e.g. increase stiffness, which in turn influences cell functions and forces. This dynamic reciprocity mediates development and tumorigenesis. Currently, there is no method available to directly quantify single cell traction and matrix remodeling in 3D. Here, we introduce a method to fulfil this long-standing need. We developed a high-resolution microfabricated sensor which hosts a 3D cell-ECM tissue formed by self-assembly. It measures cell forces and tissue-stiffness and can apply mechanical stimulation to the tissue. We measured single and multicellular force dynamics of fibroblasts (3T3), human colon (FET) and lung (A549) cancer cells and cancer associated fibroblasts (CAF05) with 1 nN resolution. Single cells show significant force fluctuations in 3D. FET/CAF co-culture system, mimicking cancer tumor microenvironment, increased tissue stiffness by 3 times within 24 hours.

Download Full-text

Characterization of cornea-specific bioink: high transparency, improved in vivo safety

Journal of Tissue Engineering ◽

10.1177/2041731418823382 ◽

2019 ◽

Vol 10 ◽

pp. 204173141882338 ◽

Cited By ~ 24

Author(s):

Hyeonji Kim ◽

Moon-Nyeo Park ◽

Jisoo Kim ◽

Jinah Jang ◽

Hong-Kyun Kim ◽

...

Keyword(s):

Corneal Transplantation ◽

Three Dimensional ◽

3D Culture ◽

Differentiation Potential ◽

Encapsulated Cells ◽

Tissue Integration ◽

Decellularized Extracellular Matrix ◽

Artificial Corneas ◽

Corneal Regeneration

Corneal transplantation is a typical surgical procedure for severe corneal diseases. However, the waiting time for a donor cornea has gradually increased due to a decrease in supply caused by an aging population and increased cases of laser-based surgeries. Artificial corneas were developed to meet the increase in demand; however, these approaches have suffered from material deterioration resulted by the limited tissue integration. Here, we introduce a cornea-derived decellularized extracellular matrix (Co-dECM) as a bioink for corneal regeneration. The developed Co-dECM bioink had similar quantitative measurement results for collagen and GAGs compared with that of the native cornea and also had the proper transparency for vision. The differentiation potential of human turbinate-derived mesenchymal stem cells (hTMSCs) to a keratocyte lineage was only observed in the Co-dECM group. Moreover, the developed bioink did not have any cytotoxic effect on encapsulated cells for three-dimensional (3D) culture and has great biocompatibility evident by the xeno-implantation of the Co-dECM gel into mice and rabbits for two and one month, respectively. An in vivo safety similar to clinical-grade collagen was seen with the Co-dECM, which helped to maintain the keratocyte-specific characteristics in vivo, compared with collagen. Taken together, the Co-dECM bioink has the potential to be used in various types of corneal diseases based on its corneal-specific ability and design flexibility through 3D cell printing technology.

Download Full-text

Regulation of JAM2 Expression in the Lungs of Streptozotocin-Induced Diabetic Mice and Human Pluripotent Stem Cell-Derived Alveolar Organoids

Biomedicines ◽

10.3390/biomedicines8090346 ◽

2020 ◽

Vol 8 (9) ◽

pp. 346 ◽

Cited By ~ 2

Author(s):

Roya Rasaei ◽

Eunbi Kim ◽

Ji-Young Kim ◽

Sunghun Na ◽

Jung-Hyun Kim ◽

...

Keyword(s):

Stem Cell ◽

Pluripotent Stem Cell ◽

Causative Factor ◽

Human Mesenchymal Stem Cell ◽

Therapeutic Effects ◽

Diabetic Mice ◽

Human Pluripotent Stem Cell ◽

Potential Indicator

Hyperglycemia is a causative factor in the pathogenesis of respiratory diseases, known to induce fibrosis and inflammation in the lung. However, little attention has been paid to genes related to hyperglycemic-induced lung alterations and stem cell applications for therapeutic use. In this study, our microarray data revealed significantly increased levels of junctional adhesion molecule 2 (JAM2) in the high glucose (HG)-induced transcriptional profile in human perivascular cells (hPVCs). The elevated level of JAM2 in HG-treated hPVCs was transcriptionally and epigenetically reversible when HG treatment was removed. We further investigated the expression of JAM2 using in vivo and in vitro hyperglycemic models. Our results showed significant upregulation of JAM2 in the lungs of streptozotocin (STZ)-induced diabetic mice, which was greatly suppressed by the administration of conditioned medium obtained from human mesenchymal stem cell cultures. Furthermore, JAM2 was found to be significantly upregulated in human pluripotent stem cell-derived multicellular alveolar organoids by exposure to HG. Our results suggest that JAM2 may play an important role in STZ-induced lung alterations and could be a potential indicator for predicting the therapeutic effects of stem cells and drugs in diabetic lung complications.

Download Full-text

Combinatorial morphogenetic and mechanical cues to mimic bone development for defect repair

10.1101/561837 ◽

2019 ◽

Author(s):

S. Herberg ◽

A. M. McDermott ◽

P. N. Dang ◽

D. S. Alt ◽

R. Tang ◽

...

Keyword(s):

Stem Cell ◽

Bone Formation ◽

Mechanical Loading ◽

Endochondral Ossification ◽

Bone Development ◽

Human Mesenchymal Stem Cell ◽

Bone Defects ◽

Mechanical Environment ◽

Tgf Β1

AbstractEndochondral ossification during long bone development and natural fracture healing initiates by mesenchymal cell condensation and is directed by local morphogen signals and mechanical cues. Here, we aimed to mimic these developmental conditions for regeneration of large bone defects. We hypothesized that engineered human mesenchymal stem cell (hMSC) condensations with in situ presentation of transforming growth factor-β1 (TGF-β1) and/or bone morphogenetic protein-2 (BMP-2) from encapsulated microparticles would promote endochondral regeneration of critical-sized rat femoral bone defects in a manner dependent on the in vivo mechanical environment. Mesenchymal condensations induced bone formation dependent on morphogen presentation, with dual BMP-2 + TGF-β1 fully restoring mechanical bone function by week 12. In vivo ambulatory mechanical loading, initiated at week 4 by delayed unlocking of compliant fixation plates, significantly enhanced the bone formation rate in the four weeks after load initiation in the dual morphogen group. In vitro, local presentation of either BMP-2 alone or BMP-2 + TGF-β1 initiated endochondral lineage commitment of mesenchymal condensations, inducing both chondrogenic and osteogenic gene expression through SMAD3 and SMAD5 signaling. In vivo, however, endochondral cartilage formation was evident only in the BMP-2 + TGF-β1 group and was enhanced by mechanical loading. The degree of bone formation was comparable to BMP-2 soaked on collagen but without the ectopic bone formation that limits the clinical efficacy of BMP-2/collagen. In contrast, mechanical loading had no effect on autograft-mediated repair. Together, this study demonstrates a biomimetic template for recapitulating developmental morphogenic and mechanical cues in vivo for tissue engineering.One Sentence SummaryMimicking aspects of the cellular, biochemical, and mechanical environment during early limb development, chondrogenically-primed human mesenchymal stem cell condensations promoted functional healing of critical-sized femoral defects via endochondral ossification, and healing rate and extent was a function of the in vivo mechanical environment.

Download Full-text

Encapsulation and recovery of murine hematopoietic stem and progenitor cells in a thiol-crosslinked maleimide-functionalized gelatin hydrogel

10.1101/2021.03.22.436491 ◽

2021 ◽

Author(s):

Aidan E Gilchrist ◽

Julio F. Serrano ◽

Mai T. Ngo ◽

Zona Hrnjak ◽

Sanha Kim ◽

...

Keyword(s):

Stem Cells ◽

Stem Cell ◽

Cell Fate ◽

Click Reaction ◽

Uv Light ◽

3D Culture ◽

Bone Marrow Niche ◽

Hematopoietic Stem ◽

Cell Fate Decisions

Biomaterial platforms are an integral part of stem cell biomanufacturing protocols. The collective biophysical, biochemical, and cellular cues of the stem cell niche microenvironment play an important role in regulating stem cell fate decisions. Three-dimensional (3D) culture of stem cells within biomaterials provides a route to present biophysical and biochemical stimuli such as cell-matrix interactions and cell-cell interactions via secreted biomolecules. Herein, we describe a maleimide-functionalized gelatin (GelMAL) hydrogel that can be crosslinked via thiol-Michael addition click reaction for the encapsulation of sensitive stem cell populations. The maleimide functional units along the gelatin backbone enables gelation via the addition of a dithiol crosslinker without requiring external stimuli (e.g., UV light, photoinitiator), reducing reactive oxide species generation. Additionally, the versatility of crosslinker selection enables easy insertion of thiol-containing bioactive or bioinert motifs. Hematopoietic stem cells (HSCs) and mesenchymal stem cells (MSCs) were encapsulated in GelMAL, with mechanical properties tuned to mimic the in vivo bone marrow niche. We report insertion of a cleavable peptide crosslinker that can be degraded by the proteolytic action of SortaseA, a mammalian-inert enzyme. Notably, SortaseA exposure preserves stem cell surface markers, an essential metric of hematopoietic activity used in immunophenotyping. This novel GelMAL system enables a route to producing artificial stem cell niches with tunable biophysical properties with intrinsic cell-interaction motifs and orthogonal addition of bioactive crosslinks.

Download Full-text

Defining the transcriptional signature of esophageal-to-skin lineage conversion

10.1101/2021.02.19.431899 ◽

2021 ◽

Author(s):

Maria T. Bejar ◽

Paula Jimenez-Gomez ◽

Ilias Moutsopoulos ◽

Bartomeu Colom ◽

Seungmin Han ◽

...

Keyword(s):

Stem Cell ◽

Epithelial Cells ◽

Cell Fate ◽

Cell Biology ◽

3D Culture ◽

New Paradigm ◽

Transcriptional Signature ◽

3D Culture System ◽

Cell Fate Conversion

AbstractThe ability of epithelial cells to rewire their cell fate program beyond their physiological repertoire has become a new paradigm in stem cell biology. This plasticity leaves behind the concept of strict stem cell hierarchies, opening up new exciting questions about its limits and underlying regulation. Here we developed a heterotypic 3D culture system to study the mechanisms modulating changes in the identity of adult esophageal epithelial cells. We demonstrate that, when exposed to the foreign stroma of adult skin, esophageal cells transition towards hair follicle identity and architecture. Heterotypic transplantation experiments recapitulated this cell fate conversion processin vivo. Single-cell RNA sequencing and histological analysis, capturing the temporality of this process, reveal that most esophageal cells switching towards skin identity remain in an intermediate state marked by a transient regenerative profile and a particularly strong hypoxic signature. Inhibition of HIF1a establishes the central role of this pathway in regulating epithelial cell plasticity, driving cells away from their transition state in favor of cell fate conversion.

Download Full-text

3D Stem Cell Culture

Cells ◽

10.3390/cells9102178 ◽

2020 ◽

Vol 9 (10) ◽

pp. 2178

Author(s):

Joni H. Ylostalo

Keyword(s):

Stem Cells ◽

Stem Cell ◽

Cell Biology ◽

3D Culture ◽

Basic Research ◽

The Body ◽

Culture Methods ◽

Culture Techniques ◽

3D Cultures

Much interest has been directed towards stem cells, both in basic and translational research, to understand basic stem cell biology and to develop new therapies for many disorders. In general, stem cells can be cultured with relative ease, however, most common culture methods for stem cells employ 2D techniques using plastic. These cultures do not well represent the stem cell niches in the body, which are delicate microenvironments composed of not only stem cells, but also supporting stromal cells, extracellular matrix, and growth factors. Therefore, researchers and clinicians have been seeking optimal stem cell preparations for basic research and clinical applications, and these might be attainable through 3D culture of stem cells. The 3D cultures recapitulate the in vivo cell-to-cell and cell-to-matrix interactions more effectively, and the cells in 3D cultures exhibit many unique and desirable characteristics. The culture of stem cells in 3D may employ various matrices or scaffolds, in addition to the cells, to support the complex structures. The goal of this Special Issue is to bring together recent research on 3D cultures of various stem cells to increase the basic understanding of stem cells and culture techniques, and also highlight stem cell preparations for possible novel therapeutic applications.

Download Full-text

Elucidating The In Vivo Phenotype And Niche Of The Resident Human Mesenchymal Stem Cell In An Adult Lung

10.1164/ajrccm-conference.2010.181.1_meetingabstracts.a5272 ◽

2010 ◽

Author(s):

Linda Badri ◽

Natalie Walker ◽

Takashi Ohtsuka ◽

Zhuo Wang ◽

Mario Delmar ◽

...

Keyword(s):

Stem Cell ◽

Mesenchymal Stem Cell ◽

Human Mesenchymal Stem Cell ◽

Adult Lung

Download Full-text

A novel method for sensor-based quantification of single/multicellular force dynamics and stiffening in 3D matrices

Science Advances ◽

10.1126/sciadv.abf2629 ◽

2021 ◽

Vol 7 (15) ◽

pp. eabf2629

Author(s):

Bashar Emon ◽

Zhengwei Li ◽

Md Saddam H. Joy ◽

Umnia Doha ◽

Farhad Kosari ◽

...

Keyword(s):

Self Assembly ◽

Single Cells ◽

Human Colon ◽

Matrix Remodeling ◽

Tissue Stiffness ◽

Force Dynamics ◽

Cell Functions ◽

The Matrix ◽

Cancer Tumor

Cells in vivo generate mechanical traction on the surrounding 3D extracellular matrix (ECM) and neighboring cells. Such traction and biochemical cues may remodel the matrix, e.g., increase stiffness, which, in turn, influences cell functions and forces. This dynamic reciprocity mediates development and tumorigenesis. Currently, there is no method available to directly quantify single-cell forces and matrix remodeling in 3D. Here, we introduce a method to fulfill this long-standing need. We developed a high-resolution microfabricated sensor that hosts a 3D cell-ECM tissue formed by self-assembly. This sensor measures cell forces and tissue stiffness and can apply mechanical stimulation to the tissue. We measured single and multicellular force dynamics of fibroblasts (3T3), human colon (FET) and lung (A549) cancer cells, and cancer-associated fibroblasts (CAF05) with 1-nN resolution. Single cells show notable force fluctuations in 3D. FET/CAF coculture system, mimicking cancer tumor microenvironment, increased tissue stiffness by three times within 24 hours.

Download Full-text