Here, we describe a methodology to deliver human cord blood-derived endothelial colony-forming cells (ECFCs) and bone marrow-derived mesenchymal stem cells (MSCs), embedded in a collagen/fibronectin gel, subcutaneously into immunodeficient mice. This cell/gel combination generates a human vascular network that connects with the mouse vasculature.
The future of tissue engineering and cell-based therapies for tissue regeneration will likely rely on our ability to generate functional vascular networks in vivo. In this regard, the search for experimental models to build blood vessel networks in vivo is of utmost importance 1. The feasibility of bioengineering microvascular networks in vivo was first shown using human tissue-derived mature endothelial cells (ECs) 2-4; however, such autologous endothelial cells present problems for wide clinical use, because they are difficult to obtain in sufficient quantities and require harvesting from existing vasculature. These limitations have instigated the search for other sources of ECs. The identification of endothelial colony-forming cells (ECFCs) in blood presented an opportunity to non-invasively obtain ECs 5-7. We and other authors have shown that adult and cord blood-derived ECFCs have the capacity to form functional vascular networks in vivo 7-11. Importantly, these studies have also shown that to obtain stable and durable vascular networks, ECFCs require co-implantation with perivascular cells. The assay we describe here illustrates this concept: we show how human cord blood-derived ECFCs can be combined with bone marrow-derived mesenchymal stem cells (MSCs) as a single cell suspension in a collagen/fibronectin/fibrinogen gel to form a functional human vascular network within 7 days after implantation into an immunodeficient mouse. The presence of human ECFC-lined lumens containing host erythrocytes can be seen throughout the implants indicating not only the formation (de novo) of a vascular network, but also the development of functional anastomoses with the host circulatory system. This murine model of bioengineered human vascular network is ideally suited for studies on the cellular and molecular mechanisms of human vascular network formation and for the development of strategies to vascularize engineered tissues.
1. Preparation
2. Culture of Human Cord Blood-Derived Endothelial Colony-Forming Cells (ECFCs)
This protocol assumes frozen vials of ECFC are available in the laboratory before this experiment. ECFC can be isolated from the mononuclear cell fraction of either umbilical cord blood or adult peripheral blood as previously described 7.
Repeat this procedure for subsequent passages. Keep track of the passage number as the cell population is expanded. ECFCs will be used between passages 4-8.
3. Culture of Human Bone Marrow-Derived Mesenchymal Stem Cells (MSCs)
This protocol assumes frozen vials of human MSC are available in the laboratory before this experiment. MSC can be isolated from bone marrow aspirates as previously described 11.
Repeat this procedure for subsequent passages. Keep track of the passage number as the cell population is expanded. MSCs will be used between passages 4-8.
4. Resuspension of Cells in Collagen/Fibronectin/Fibrinogen Solution (Day 0)
Prior to the experiment, make sure there are enough ECFCs and MSCs in culture; 0.8×106 ECFCs and 1.2×106 MSCs will be required for each implant and mouse.
5. Injection into Immunodeficient Nude Mouse (Day 0)
All animal experiments will be carried out with 6-week old athymic nude (nu/nu) mice.
6. Harvesting (Day 7)
7. Evaluation: Histology (H&E) and Immunohistochemistry (hCD31)
8. Representative Results
Figure 1. Typical appearance of ECFC and MSC cultures. Phase contrast micrographs displaying the typical appearance of ECFCs and MSCs in culture. (A) Confluent monolayer of cord blood-derived ECFCs displaying the characteristic cobble-stone morphology of endothelial cells. (B) Human bone marrow-derived MSCs displaying a spindle shape morphology. Scaler bars, 200 um.
Figure 2. Appearance of explanted plugs at day 7. Human cord blood-derived ECFCs and bone marrow-derived MSCs were embedded in collagen/fibronectin/fibrin gel and implanted subcutaneously into nude mice as described in the text. (A) After 7 days, once the mouse has been euthanized, cut open the skin near the area of the injection and expose the cell/gel plug by flipping the skin. (B) Appearance of the plug surgically removed from the mouse and prior to formalin fixation. The red color of the implant is an indication of vascularization.
Figure 3. Histological identification of vascular network in explanted plugs. Hematoxilin and Eosin (H&E) stained sections taken from the middle part of the explanted plugs. (A) Low magnification (10x) micrograph displaying the implant (marked by a yellow dashed line) in the context of surrounding host tissues (i.e., adipose tissue and skeletal muscle). (B) High magnification (40x) micrograph displaying multiple microvessels (yellow arrowhead pointing at some of them) inside the plug; microvessels can be identified as lumenal structures containing red blood cells.
Figure 4. Immunohistochemical identification of human lumens. Immunohistochemically stained sections taken from the middle part of the explanted plugs. Staining was carried out using a monoclonal mouse anti-human CD31 (hCD31) antibody from DakoCytomation (Clone JC70A; cat. # M0823) at a 1:100 dilution; cell nuclei were counterstained with hematoxilin. (A) Low magnification (10x) micrograph displaying the implant (delineated by a black dashed line) in the context of surrounding host tissues. Human specific, CD31-positive microvessels are stained in brown (peroxidase staining). (B) High magnification (40x) micrograph displaying multiple human microvessels (black arrowhead pointing at some hCD31-positive lumens) inside the plug.
This is an experimental model of bioengineering human vascular networks. The main characteristics of this model are: 1) microvessels are formed from human cells isolated from post-natal tissues (i.e., blood and bone marrow); 2) microvessels are formed in an adult animal; and 3) microvessels do not arise from pre-existing (host) vessels but instead they are formed, de novo, from single cells suspended in a suitable gel.
Angiogenesis plays an important role in this assay because connections to the murine vasculature are needed to achieve red blood cell-filled vessels, one of the functional read-out in this assay. In addition, host myeloid cells are recruited to the implant in the early days post transplantation, and they are necessary for the formation of the new vascular bed 12.
This experimental model offers a versatile, quantifiable, and relatively simple model system to study post-natal formation of human vascular networks in vivo. Additionally, this assay is simple to perform and it does not require an incision or surgical procedure. The model could be used to study the vasculogenic potential of different sources of human endothelial and perivascular cells. The model could be used to screen for anti- and/or pro-vasculogenic compounds. Finally, the model can be used to study the role(s) of specific genes in the formation and function of a vascular network composed of human endothelium.
The authors have nothing to disclose.
This work was partially supported by NIH grant K99EB009096-01A1 to J.M.-M.
Name of the reagent | Company | Catalogue number | Comments (optional) |
---|---|---|---|
Endothelial Basal Medium, EBM-2 | Lonza | CC-3156 | |
EGM-2 SingleQuot supplements | Lonza | CC-3162 | |
Glutamine-penicillin-streptomycin solution, 100x GPS | Mediatech, Inc. | 30-009-CI | |
Mesenchymal Stem Cell Growth Medium BulletKit, MSCBM/MSCGM | Lonza | PT-3001 | |
High glucose Dulbecco’s Modified Eagle Medium, 1x DMEM | Invitrogen | 11995-073 | |
MEM Non-essential amino acid solution, 100x NEAA | Invitrogen | 11140-050 | |
High glucose Dulbecco’s Modified Eagle, powdered DMEM | Invitrogen | 12100-046 | |
HEPES Buffer solution | Invitrogen | 15630-080 | |
Cultrex Bovine Collagen I solution | Trevigen | 3442-050-01 | |
Cultrex Human Fibronectin | Trevigen | 3420-001-01 | |
Fibrinogen from bovine plasma | Sigma-Aldrich | F8630-1G | |
Gelatin | Fisher | DF0143-17-9 | |
Dulbecco’s phosphate buffered saline, PBS | Invitrogen | 14190-250 | |
Trypsin-EDTA solution, 1x | Invitrogen | 25300-054 | |
Isoflurane, liquid for inhalation | Buxter Healthcare Corporation | NDC 10019-360-40 | |
Thrombin from bovine plasma | Sigma-Aldrich | T4648-1KU | |
Formalin Solution, Neutral Buffered | Sigma-Aldrich | HT501128 | |
Monoclonal Mouse Anti-Human CD31 Antibody | DakoCytomation | M0823 | Clone JC70A |