human derived tissues 3D vascular networks for

Human iPS- human derived tissues 3D vascular networks for patient-specific drug screening
Monica Moya,1 Christina Tu,1 Luis F. Alonzo,1 Leslie Lock,1 and
Steven C. George 1
1.University of California, Irvine, Irvine, CA, USA
Introduction
Developing dynamic 3D vasculature-fed patient-specific
in vitro human microtissues has the potential to provide
whole new opportunities for drug discovery and toxicity
screening. In order to create patient-specific vascular
network models, our work will expand on our previous
work in developing microfluidic devices that can support
a metabolically active stroma with culture medium
perfused human capillaries.1 In this study we aim to
develop vessel networks derived from human induced
pluripotent stem cell-derived endothelial cells (iPS-EC).
Using iPS-derived human cells provide the potential for
patient-specific drug screening or “personalized
medicine”.
Materials and Methods
Endothelial cells were derived from the human iPS cell
line WTC11 (courtesy of Dr. Bruce Conklin) using an
unpublished method provided by Dr. James Thompson
(University of Wisconsin). The resulting differentiated
population was purified using magnetic bead sorting for
CD31, an endothelial cell surface marker. Human IPSECs were then grown in fully supplemented EGM-2
(Lonza) media prior to loading into a polydimethsiloxane
microfluidic device. The device consists of 2 fluid filled
microfluidic channels on either side of a 12 mm-sized
diamond shaped tissue microchambers. Human iPS-ECs
(2.5x106 cells/ml) or cord blood endothelial colony
forming cell-derived endothelial cells (ECFC-ECs) were
co-seeded into the central chamber with normal human
lung fibroblast (NHLFs, 5x106 cells/ml) in a fibrin matrix.
Formation of vessels was encouraged by both mechanical
(pressure gradients, insterstitial flow) and chemical
stimuli (hypoxia and nutrient deprivation). Formed
network was visualized using fluorescent imaging of
CD31 stained tissues.
Results
Cells remained viable under flow conditions in the
microfluidic device through 10 days of culture. Vessel
assembly and lumen formation were noted within a week
of culture. By day 10 human iPS-ECs supported by
stromal cells formed vessel networks whose area
encompassed 16 ± 0.03% of the chamber. (Fig 1)
Compared to control devices using primary ECFC-ECs in
place of hiPS-ECs (Fig 2), vessel networks formed by
hiPS-ECs encompassed less area of the chamber (ECFCEC vessel area: 25±0.07 %). Vessels formed by hiPS-
ECs were also noted to be slightly larger in diameter (d=
19 ± 5µm) compared to ECFC-EC controls (d = 12 ±
4µm).
200 µm
Fig. 1. Fluorescent microscopy of CD31 (green) stained
vessel network at day 10 (a) depicts the ability of iPS-ECs
to form lumenized (b) vessel structures under constant
flow conditions.
Fig. 2. Controls
devices using
ECFC-ECs form
robust networks
in microfluidic
device by day 10
Discussion and Conclusions
Our system is amendable to create vessel networks from
human induced pluripotent endothelial cells. The potential
to re-create complex multi-cellular micro-organs
completely from the same genetic source represents a
significant advance in patient-targeted therapy and
increased efficacy. Ongoing current work is focused on
introducing additional cells including cardiomyoctes
derived from the same hiPS source.
References
1. Moya, ML., Hsu, YH., Hughes CW., Lee A., George
SC., Tissue Engineering, Part C. In Press
Acknowledgments
This work was supported by the NIH UH2-TR00481,
NHLBI F32HL105055 (MLM) and NIH/NCI
F3110765866 (LFA)
Authors have nothing to disclose.