This protocol focuses on utilizing the inherent ability of stem cells to take cue from their surrounding extracellular matrix and be induced to differentiate into multiple phenotypes. This methods manuscript extends our description and characterization of a model utilizing a bilayered hydrogel, composed of PEG-fibrin and collagen, to simultaneously co-differentiate adipose-derived stem cells1.
Natural polymers over the years have gained more importance because of their host biocompatibility and ability to interact with cells in vitro and in vivo. An area of research that holds promise in regenerative medicine is the combinatorial use of novel biomaterials and stem cells. A fundamental strategy in the field of tissue engineering is the use of three-dimensional scaffold (e.g., decellularized extracellular matrix, hydrogels, micro/nano particles) for directing cell function. This technology has evolved from the discovery that cells need a substrate upon which they can adhere, proliferate, and express their differentiated cellular phenotype and function 2-3. More recently, it has also been determined that cells not only use these substrates for adherence, but also interact and take cues from the matrix substrate (e.g., extracellular matrix, ECM)4. Therefore, the cells and scaffolds have a reciprocal connection that serves to control tissue development, organization, and ultimate function. Adipose-derived stem cells (ASCs) are mesenchymal, non-hematopoetic stem cells present in adipose tissue that can exhibit multi-lineage differentiation and serve as a readily available source of cells (i.e. pre-vascular endothelia and pericytes). Our hypothesis is that adipose-derived stem cells can be directed toward differing phenotypes simultaneously by simply co-culturing them in bilayered matrices1. Our laboratory is focused on dermal wound healing. To this end, we created a single composite matrix from the natural biomaterials, fibrin, collagen, and chitosan that can mimic the characteristics and functions of a dermal-specific wound healing ECM environment.
1. Isolating Adipose-Derived Stem Cells (ASCs) 1, 5
Note: All procedures were performed at room temperature unless otherwise noted.
2. Preparing Chitosan Microspheres (CSMs)
Note: All procedures were performed at room temperature unless otherwise noted.
3. Determining the Number of Free Amino Groups in CSMs
Note: All procedures were performed at room temperature unless otherwise noted.
4. Loading ASC in CSM
Note: All procedures were performed at room temperature unless otherwise noted.
5. Determining the Percentage of ASC Loading and Cell Viability in CSMs
Note: All procedures were performed at room temperature unless otherwise noted.
6. Preparation and Characterization of ASC-CSM Embedded in PEG-fibrin Gels
Note: All procedures were performed at room temperature unless otherwise noted.
7. Preparation and Characterization of ASC-CSM Embedded in Collagen Gels
Note: All procedures were performed at room temperature unless otherwise noted.
8. Development of Bilayered PEG-fibrin-(ASC-CSM)-Collagen Gel Constructs
Note: All procedures were performed at room temperature unless otherwise noted.
9. Making Stock Solutions
Note: All procedures were performed at room temperature unless otherwise noted.
10. Representative Results
The overall goal of the technique presented here is to demonstrate the potential of simultaneous matrix-driven differentiation of ASC into multiple phenotypes using CSM as a delivery vehicle. We demonstrate an in vitro strategy to deliver stem cells from CSMs into a bilayered collagen-PEG-fibrin scaffold. Characterization of ASC embedded within this scaffold revealed that ASC-loaded CSMs can be “sandwiched” in between a layer of collagen and PEG-fibrin simultaneously and differentially take cue from both extracellular environments to thrive under their new conditions. We first characterized the ability for the model system to maintain cell viability and migratory capacities. Collagen supported the ability of ASCs to maintain their “stemness,” as was demonstrated by their expression of Stro-1 and their fibroblast-like morphology (Figure 2D and 2F). In contrast, PEG-fibrin induced the ASCs to differentiate toward a vascular phenotype, as is demonstrated by their tube-like structure morphology, their endothelial cell-specific expression of von Willebrand factor (Figure 2E and 2G), and pericyte-specific expression of NG2 and platelet-derived growth factor receptor beta (PDGFRβ) (data not shown). Furthermore, these observed phenotypes appeared to occur early in culture and were maintained over 11 days, as is demonstrated in Figure 3.
Tables and Figures
Benefits of Bilayer Construct:
Figure 1. Schematic depicting the overall goal and process of the technique. 1) Adipose-derived stem cells (ASCs) are loaded onto chitosan microspheres. 2) Collagen is then poured into a 6-well insert, the pH adjusted to fibrillate the collagen, and the insert placed into a 6-well plate chamber. The ASC-loaded CSM spheres are then layered over the collagen. 3) The PEGylated fibrinogen is then poured over the collagen (ASC-CSM) and gelled by the addition of thrombin. 4) The final bilayer construct can then be removed from the culture insert and used for in vitro or in vivo analysis.
Figure 2. Characterization of ASC cultured within collagen and PEG-fibrin 3D matrices. A) Phase-contrast photomicrograph of isolated ASC passaged and maintained using routine 2-dimensional cell culture techniques. Photomicrographs B, D, and F depict ASC-CSM cultured within a 3-dimensional collagen gel; whereas C, E, and G show ASC-CSM cultured within a 3-dimensional PEG-fibrin gel, both at day 12. In B and C), ASCs are shown migrating away from the CSM sphere in both scaffold types. ASCs appear to have a flattened, spindle-like morphology in collagen (B), while maintaining their expression of the stem cell marker Stro-1 (D; arrow). When cultured in PEG-fibrin ASCs exhibit more tube-like structures and are induced to express such vascular cell markers as von Willebrand Factor (E). Transmission electron microscopy depicts the typical morphology demonstrated by ASCs within each scaffold. ASCs in collagen gel appear to have smaller filopodia (fl) extending from the body of the cell (F), whereas ASCs typically formed lumenal (labeled L) structures (G; arrow).
Figure 3. Morphological analysis of ASC-CSMs between bilayers of collagen and PEG-fibrin gels. ASC-CSMs were “sandwiched” between collagen and PEG-fibrin gels and maintained in culture for 11 days. The left column depicts ASCs migrating and proliferating within the collagen matrix and appear to take on a spindle-like morphology. The right column depicts ASCs migrating away from the CSMs and forming tube-like structures throughout the PEG-fibrin gel.
ASCs are well-known for their ease of isolation and ability to differentiate toward various cell types. With the techniques described in this manuscript, we are able to exploit the plasticity of ASCs by exposing these cells to multiple biomatrices simultaneously. As cells migrate away from their CSM base and enter their surrounding extracellular environment, the cells take cue from the scaffold and can either maintain “stemness” (collagen) or be induced to differentiate toward vascular- and vascular-supportive cell types (fibrin). Since our lab is interested in skin and soft-tissue wound healing, we strategically implemented a bilayer of collagen and fibrin since collagen supports dermal and epidermal regeneration by the host, whereas fibrin naturally induces vascular network formation 9. Furthermore, it is now understood that cross-talk occurs between proliferating skin keratinocytes, fibroblast, and the underlying vascular network; and this complex mechanism is highly critical for proper wound healing 10. Without an underlying vascular network, tissue-engineered skin grafts have difficulty inosculating with host tissue. Therefore, our overall hypothesis is that collagen and fibrin, when used as a bilayer in combination with ASCs, will decrease wound healing times by improving one or more stages of blood vessel, dermal, and re-epithelialization processes.
Fibrin is a versatile biopolymer formed after thrombin-mediated cleavage of fibrinopeptide A derived from monomeric fibrinogen. Fibrin has been used clinically as a hemostatic agent (approved by the U.S. Food and Drug Administration) and as a sealant in a variety of clinical applications, including procedures such as soft-tissue dissection. Fibrin hydrogels from commercially purified allogeneic fibrinogen and thrombin have been used widely in the last decade in a variety of tissue-engineering applications. However, some major disadvantages in using a fibrin hydrogel can be 1) the potential for the scaffold to shrink, 2) low mechanical stiffness, and 3) rapid degradation before proper formation of the tissue-engineered structures. To overcome these problems, fibrin needs to be modified before use to serve as a better three-dimensional tissue-engineering scaffold. One such approach is copolymerizing the fibrin with polyethylene glycol (PEG-Fibrin). Our preliminary work has demonstrated several unique features of PEG-fibrin that make it advantageous in wound healing over other hydrogel dressings, including unmodified fibrin. PEGylated-fibrin exhibits unique features of both synthetic hydrogels and natural materials. Specifically, the presence of PEG provides a highly hydrated (>90% water) moist environment for managing exudates. Second, the presence of fibrin confers biodegradability to the material; however, prior results show that PEGylated fibrin is significantly more stable in vitro than unPEGylated fibrin11. Third, the inherent biologic activity of fibrin encourages healing by stimulating tissue and blood vessel in-growth.
The second component of our bilayer is collagen. Collagen is a natural biomaterial ubiquitously expressed in mammals and serves as a direct site of attachment for various types of cells. Different tissues express different types of collagen (type 1-type29), depending on the type of functional need of the tissue. Other inherent properties of collagen that make it highly attractive in our model are that 1) it is non-inflammatory, 2) both whole and degraded components of collagen are biocompatible, 3) it is a highly porous hydrogel, 4) it supports cell infiltration and migration, 5) it maintains multipotency of stem cells, 6) it is tunable by modulating formulation, and 7) it readily inosculates with host tissue. For our studies in skin and soft-tissue regeneration, a bilayer composite consisting of collagen is a natural choice since it is highly expressed throughout the skin, including deep dermal regions, and can be used as an immunohistochemistry marker to assess the depth of a wound.
In this protocol, we demonstrate a technique to simultaneously maintain “stemness” of ASCs while differentiating ASCs toward various vascular cell types. For the study of skin and its underlying soft tissue, collagen and PEG-fibrin are directly applicable. However, other bioscaffold materials can be implemented to study their explored unique abilities to induce ASC into other cell types. The technique implemented here helps to highlight the importance of the extracellular matrix in controlling stem cell phenotypes and lends credence to the exploration of layered composites for skin regeneration.
Summary
Critical Steps
Limitations
Possible Modifications
Troubleshooting
The authors have nothing to disclose.
S.N. was supported by a Postdoctoral Fellowship Grant from the Pittsburgh Tissue Engineering Initiative. D.O.Z. is supported by a grant awarded from The Geneva Foundation.
Name of the reagent/equipment | Company | Catalogue number | Comments |
Hanks Balanced Salt Solution (HBSS) | Gibco | 14175 | Consumable |
Fetal Bovine Serum | Hyclone | SH30071.03 | Consumable |
Collagenase Type II | Sigma-Aldrich | C6685 | Consumable |
70-μm Nylon Mesh Filter | BD Biosciences | 352350 | Consumable |
100-μm Nylon Mesh Filter | BD Biosciences | 352360 | Consumable |
MesenPRO Growth Medium System | Invitrogen | 12746-012 | Consumable |
L-Glutamine | Gibco | 25030 | Consumable |
CaCl2.2H2O | Sigma | C8106 | Consumable |
T75 Tissue Culture Flask | BD Biosciences | 137787 | Consumable |
Chitosan | Sigma-Aldrich | 448869 | Consumable |
Acetic Acid | Sigma-Aldrich | 320099 | Consumable |
N-Octanol | Acros Organics | 150630025 | Consumable |
Sorbitan-Mono-Oleate | Sigma-Aldrich | S6760 | Consumable |
Potassium Hydroxide | Sigma-Aldrich | P1767 | Consumable |
Acetone | Fisher Scientific | L-4859 | Consumable |
Ethanol | Sigma-Aldrich | 270741 | Consumable |
Trinitro Benzenesulfonic Acid | Sigma-Aldrich | P2297 | Consumable |
Hydrochloric Acid | Sigma-Aldrich | 320331 | Consumable |
Ethyl Ether | Sigma-Aldrich | 472-484 | Consumable |
8-μm Tissue Culture Plate Inserts | BD Biosciences | 353097 | Consumable |
1.5-ml Microcentrifuge Tubes | Fisher | 05-408-129 | Consumable |
MTT Reagent | Invitrogen | M6494 | Consumable |
Dimethyl Sulfoxide | Sigma-Aldrich | D8779 | Consumable |
Qtracker Cell Labeling Kit(Q Tracker 655) | Molecular probes | Q2502PMP | Consumable |
Type 1 Collagen | Travigen | 3447-020-01 | Consumable |
Sodium Hydroxide | Sigma-Aldrich | S8045 | Consumable |
12-Well Tissue Culture Plates | BD Biosciences | 353043 | Consumable |
Fibrinogen | Sigma | F3879 | Consumable |
Thrombin | Sigma | T6884 | Consumable |
Benztriazole Derivative of Polyethylene | Sunbio | DE-034GS | Consumable |
Tris Buffer Tablet (pH 7.6) | Sigma | T5030 | Consumable |
Centrifuge | Eppendorf | 5417R | Equipment |
Orbital Shaker | New Brunswick Scienctific | C24 | Equipment |
Humidified Incubator with Air-5% CO2 | Thermo Scientific | Model 370 | Equipment |
Overhead Stirrer | IKA | Visc6000 | Equipment |
Magnetic Stirrer | Corning | PC-210 | Equipment |
Vacuum Desiccator | – | – | Equipment |
Particle Size Analyzer | Malvern | STP2000 Spraytec | Equipment |
Water Bath | Fisher Scientific | Isotemp210 | Equipment |
Spectrophotometer | Beckman | Beckman Coulter DU 800UV/Visible Spectrophotometer | Equipment |
Vortex | Diagger | 3030a | Equipment |
Microplate Reader | Molecular Devices | SpectraMax M2 | Equipment |
Light/Fluorescence Microscope | Olympus | IX71 | Equipment |
Confocal Microscope | Olympus | FV-500 Laser Scanning Confocal Microscope | Equipment |
Scanning Electron Microscope | Carl Zeiss MicroImaging | Leo 435 VP | Equipment |
Transmission Electron Microscope | JEOL | JEOL 1230 | Equipment |