Aninvitromodelsystemforstudyingtheeffectsofhypoxiaonstemcell-derivedendothelialcells

Rebecca BrodyGerecht Lab, Johns Hopkins University Whiting School of Engineering

The Ingenuity Project | Baltimore Polytechnic Institute | Baltimore, MD

The cardiovascular system is a network of blood vessels that moves oxygen and nutrients throughout the body aided by the heart. Because this system is so vital, when it does not function correctly, it results in many severe diseases, collectively referred to as cardiovascular disease. Current therapies require the use of grafts, which do not have the capability to fully recover damaged vasculature and require the degradation of previously healthy areas of vasculature. With an improved understanding of the vascular system and better regenerative techniques, an alternative to revascularization surgery is possible. A potential alternative is the use of vascular cells derived from human induced pluripotent stem cells (hiPSCs). hiPSCs are human somatic cells that have been reprogrammed into pluripotent stem cells, which can then be differentiated into cells of the vascular system, such as endothelial cells (ECs)(see below). This provides a more renewable source of ECs.

The phenotype of hiPSC derived ECs has been verified through immunohistochemistry techniques. However, there has been little research on the behavior of these cells in a 3D in vitro environment, and no research on hiPSC’s response to hypoxia. Hypoxia, low oxygen in the tissues, occurs naturally in the body (see image below). Therefore, it is critical that cell behavior be observed in low oxygen environments. Here, we present an in vitro model system to study the effects of hypoxia on vascular cells from different sources, including hiPSC-derived ECs.

To determine the effect of hypoxia on hiPSC-derived endothelial cells.

Objective

Introduction

Materials and Methods

Results and Interpretation

Conclusionss

Part 1: Compare oxygen consumption by hiPSC-derived ECs and human umbilical vein endothelial cells (HUVECs, physiologic ECs) in low and high oxygen environments.Part 2: Determine the effect of low oxygen levels on the network formation of hiPSC-derived endothelial cells using confocal imaging.

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Collagen gels were used to create a three-dimensional environment that replicates the environment in the body. The gels were made using the collagen I protein, an abundant protein in the extracellular matrix. This allowed for the formation of vascular networks within the gel.

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of the gel without consuming oxygen. The levels of oxygen in a collagen gel are determined by diffusion of oxygen into the gel and cell consumption of oxygen. The levels of oxygen in the gel were also manipulated by increasing the thickness of the gel. The 2.1 mm gel produced a lower oxygen environment, while the 1.7 mm gel was higher in oxygen.

Figure 1: Comparison of the oxygen concentration in collagen gels of two thicknesses with iPSC-derived ECs or HUVECs. Representative graphs of: A- HUVECs in a 1.7 mm gel, B- HUVECs in a 2.1 mm gel, C- iPSC-derived ECs in a 1.7 mm gel, D-iPSC-derived ECs in a 2.1 mm gel. The two cell types behave similarly in the 1.7 mm gels (A,C), but in the 2.1 mm gel the HUVECs (B) consumed oxygen at a higher rate than the iPSC-derived ECs(D), reaching hypoxia (see arrow). This may suggest that the iPSC-derived ECs have adapted to the low oxygen conditions and are consuming less oxygen.

Future Work

AcknowledgementsI would like to thank Dr. Sharon Gerecht and Bria Macklin, my mentors from Johns Hopkins University; Mrs. Lisa Fridman, my research teacher; and the Ingenuity Project for their assistance and guidance in this project.

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A BFigure 3: Quantification of network characteristics of hiPSC-derived ECs in 1.7 and 2.1 mm collagen gels. Average tube length (A) and average tube volume (B) were determined using Imaris image analysis software. The average tube length and volume were greater in the 2.1 mm gel. Data are the average of four images for the 1.7 mm gels and three images for the 2.1 mm gel. Statistical significance was assessed by t-test. * p<0.05

HUVECs and iPSC-derived endothelial cells display different behaviors depending on the amount of dissolved oxygen in the collagen gels. HUVECs and iPSC-derived ECs consume oxygen similarly in higher oxygen environments (1.7 mm gel), but iPSC-derived ECs consume oxygen at a lower rate in a low oxygen environment (2.1 mm gel). When looking at network characteristics, iPSC-derived ECs form networks that are longer and wider in low oxygen environments (2.1 mm gel) compared with high oxygen environments.

We hypothesize that the hiPSC-ECs generate more effective networks in low oxygen conditions because these cells have an embryonic-like EC phenotype. ECs of the developing embryo are exposed to oxygen levels as low as 2%, so they must be able to adapt to low oxygen environments and form effective networks. To test this we will:• Compare network characteristics of HUVECs and hiPSC-ECs in low oxygen environments • Use the platform to test ECs derived from multiple stem cell lines and multiple mature EC lines• Study oxygen mediated signaling pathways

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Figure 4: Number of branch points was also considered when comparing the network formation in 1.7 and 2.1 mm gels. A branch point is the location where a new vessel sprouts off of a previously existing vessel, as shown by the arrows in A. The number of branch points was quantified in the 1.7 and 2.1 mm gels (B). The number of branch points was similar in the two conditions, which suggests that the complexity of networks is similar.

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Figure 2: Representative images of hiPSC-derived ECs in 3D gels: top view of 1.7 mm (A) and 2.1 mm (B) collagen gels and the side view of the 1.7 mm (C) and 2.1 mm (D) gels. Endothelial cells are labeled with VE-cadherin (green) and nuclei are labeled with DAPI (blue). Vascular formation is evident in both gels; however, qualitatively, the networks in the 2.1 mm (B,D) gel appear longer, wider, and more extensive. Vascular networks have formed throughout both gels. In the 2.1 mm gel (D), more network formation is present towards the bottom of the gel, where the oxygen levels are lower.

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The two cell types used in this experiment were hiPSCs-derived ECs (defined above) and HUVECs. HUVECs are mature endothelial cells harvested from a patient and grown in culture. Two million cells of each cell type were seeded into collagen gels to assess oxygen consumption and network growth.

AbstractHuman induced pluripotent stem cell-derived endothelial cells (hiPSC-ECs) represent a promising avenue to repair and replace blood vessels damaged by injury or disease. This cell type could be used to create patient specific vascular grafts in the lab, which could then be used for clinical treatments. However, little is known about how hiPSC-ECs function in comparison to physiologic endothelial cells. This includes the response of iPSC-ECs to hypoxic conditions, which are known to stimulate changes to the vasculature in vivo. We investigated these unknowns using an in vitro model system. This system uses a synthetic, three-dimensional environment, which employs the collagen I protein to mimic the physiological conditions in which blood vessels can form. It was found that hiPSC-ECs do in fact behave differently than physiologic endothelial cells, depending on the oxygen concentration of their environment. In low oxygen environments, the hiPSC-ECs consumed less oxygen than the physiologic stem cells. In addition, the networks formed by iPSC-ECs in low oxygen environments were longer and thicker. These results suggest a fundamental difference between hiPSC-ECs and physiologic ECs, but shows that hiPSC-ECs can still form effective vascular networks in low oxygen conditions.

Applications to BiotechnologyThis research has applications to the field of biotechnology. The information obtained in this experiment helps to advance the field of in vitro blood vessel models. In the future, it may be possible to create functional blood vessels in vitro, which could then be used in clinical treatments. Beyond this, the blood vessels could be created using hiPSC-ECs that have been created from cells taken directly from a patient. This personalized treatment would greatly reduce the risk of rejection if the blood vessels were grafted into a patient. In addition to personalized blood vessel grafts, we have shown that it is possible to induce lower oxygen environments by increasing the thickness of collagen gels. The low oxygen environments have a variety of applications, including disease modeling. This is especially relevant for cancer research, as tumors often create a more hypoxic environment in the body. The collagen gels could be used to replicate this disease state and test drugs or other clinical treatments.