Bioengineered human vascular networks transplanted into secondary

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Bioengineered human vascular networks transplanted into secondary mice reconnect with

the host vasculature and re-establish perfusion.

Short title: Reconnectability of transplanted human vessels

Kyu-Tae Kang,1,* Patrick Allen,1,2,* Joyce Bischoff1

1Vascular Biology Program and Department of Surgery, Children's Hospital Boston, Harvard

Medical School, MA; 2Department of Biomedical Engineering, Boston University, MA

* K.T.K. and P.A. contributed equally to this study.

Corresponding Author: Dr. Joyce Bischoff

Children’s Hospital Boston

Harvard Medical School

Boston, MA 02115

Email: [email protected]

Tel (617) 919-2192

Fax (617) 730-0231

Scientific category: Vascular Biology

Blood First Edition Paper, prepublished online October 28, 2011; DOI 10.1182/blood-2011-08-375188

Copyright © 2011 American Society of Hematology

For personal use only.on April 9, 2019. by guest www.bloodjournal.orgFrom

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Abstract

The ability to form anastomoses with the host circulation is essential for vascular networks

incorporated within cell-seeded bioengineered tissues. Here, we tested whether and how rapidly

human endothelial colony forming cell (ECFC)/mesenchymal progenitor cell (MPC)-derived

bioengineered vessels, originally perfused in one mouse, could become re-perfused in a

secondary mouse. Using in vivo labeling with a systemically injected mixture of human- and

murine-specific lectins, we demonstrate that ECFC/MPC-blood vessels reconnect and are

perfused at day 3 after transplantation. Furthermore, we quantified the longitudinal change in

perfusion volume in the same implants before and after transplantation using contrast-enhanced

micro-ultrasonic imaging. Perfusion was restored at day 3 after transplantation and increased

with time, suggesting an important new feature of ECFC/MPC-blood vessels: the bioengineered

vessels can reconnect with the vasculature when transplanted to a new site. This feature extends

the potential applications of this postnatal progenitor cell-based technology for transplantable

large tissue-engineered constructs.




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Introduction

Bioengineering has emerged as a promising approach to replace dysfunctional tissues and

organs. In addition to integrity and function, bioengineered constructs must have a vascular

supply for gas exchange, nutrient delivery, and waste product elimination. Bioengineered

constructs in clinical use to date are limited to thin and/or avascular tissues such as skin1,2 and

cartilage,2,3 wherein diffusion of oxygen and nutrients is sufficient.

Significant progress has been made towards building pre-assembled networks using human

endothelial cells from sources including umbilical vein,4 embryonic stem cells,5 bone marrow,6

blood,7 and adipose tissue.8 However, few studies have focused on the ability of pre-assembled

networks to connect with the host vasculature. In 1975, Folkman and colleagues studied the fate

of endogenous blood vessels in tumor, adult and embryonic tissues implanted on the

chorioallantoic membrane of avian embryos.9 Tumor vessels regressed but new vessels from the

chorioallantois grew in (i.e. tumor angiogenesis). Adult tissue vessels regressed and were not

replaced. In contrast, vessels in the embryonic tissue remained intact and formed anastomoses

with the avian vessels. This ability to reconnect with new vasculatures would be highly desirable

in bioengineered vascular networks. Therefore, we set out to determine if human bioengineered

blood vessels are akin to the embryonic vessels. We and others showed that human ECFC

(called EPC in our previous papers) combined with perivascular cell precursors such as MPC

form functional blood vessels when coimplanted into immunodeficient mice.7,10-13 We used this

approach to design a transplantation model to test whether human ECFC/MPC-vessels, formed

in a donor mouse, can be disconnected and then reconnected with vasculature in a secondary

recipient mouse.




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To detect functional anastomoses between bioengineered and host vessels, we implemented

two new techniques: (1) in vivo labeling by tail vein injection of a mixture of human- and

murine-specific fluorescently-conjugated lectins, and (2) contrast-enhanced micro-ultrasonic

imaging to visualize and quantify perfusion over time in individual mice. Herein, we use these

techniques to demonstrate the ability of transplanted bioengineered human vessels to

disconnect/reconnect with the recipient murine vasculature and rapidly reestablish perfusion.




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Methods

2×106 cells suspended in 200 μL Matrigel at ratio of 2:3 (ECFC:MPC) were injected

subcutaneously into each severe combined immunodeficiency (SCID) mouse.11 Matrigel

implants were harvested at days 1, 3, 5 and 7 for microvessel density analysis, or transplanted

into secondary mice at day 7. Perfused human and murine vessels were identified by tail-vein

injection of a mixture of Rhodamine (red)-conjugated Ulex europaeus agglutinin I (UEA-I) and

FITC (green)-conjugated Griffonia simplifolia isolectin B4 (GS-B4). Micro-ultrasonic imaging

was used to measure perfused vascular volume of implants longitudinally. Human endothelial

cells were isolated from discarded non-identified umbilical cord blood or newborn foreskin

obtained under an IRB approved protocol. Animal experiments were conducted under a protocol

approved by the IACUC at Children’s Hospital Boston in an AAALAC-approved facility.

Detailed methods are in the Supplement.




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Results and Discussion

Detection of perfused human and murine vessels in vivo

To determine whether ECFC/MPC-vascular networks can rapidly connect to the host

vasculature in a new implantation site, we transplanted ECFC/MPC-Matrigel implants from

donor to recipient mice (Figure 1A). Perfused vessels before and after transplantation were

identified by tail-vein injection of rhodamine-conjugated UEA-I, a lectin specific for human

endothelium, and FITC-conjugated GS-IB4, a lectin specific for murine endothelium. Three

distinct patterns were observed: UEA-I-positive human vessels, GS-IB4-positive murine vessels,

and UEA-I/GS-IB4-double positive chimeric vessels (Figure 1B). This illustrates several

advantages of in vivo labeling by tail-vein injection of the lectin mixture: (1) identifying

functional perfused vessels in contrast to dead-end tubular structures, (2) distinguishing human

from murine vessels, and (3) visualizing the site of anastomosis. Point (1) is essential to exclude

vessels retained within implants after transplantation but not re-perfused.

Time course of vasculogenesis/anastomosis

Before transplantation, GS-IB4-positive murine vessels were observed at the periphery of

implants at day 3, while UEA-I-positive human vessels were observed from day 5 (Figure 1C-D).

This suggests that infiltration of angiogenic host vessels into the implant was followed by

anastomosis with nascent human vessels formed from ECFC/MPC. MPC are known to release

VEGF-A,11 providing a possible angiogenic stimulus to the host vessels. It is unclear at present

whether the injected human cells form complete vessels which anastomose with infiltrating host

vessels, or whether the human cells individually cooperate with and contribute to the angiogenic

elongation of the host vasculature. In vitro studies have shown formation of tubular structures by




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endothelial cells alone13 or when co-cultured with perivascular cells.12 Therefore, we speculate

that human ECFC and MPC form nascent vessels which then form anastomoses with infiltrating

murine vessels.

Bioengineered human blood vessels reconnect after transplantation

To test whether bioengineered human blood vessels can be disconnected from the donor

vasculature and reconnect in secondary recipient mice, we performed in vivo lectin-labeling to

detect perfused vessels and quantify microvessel density (MVD). UEA-I-positive human vessels

were observed from day 3 after transplantation, 2 days prior to the appearance of UEA-I-positive

human vessels before transplantation (Figure 1C-D). This indicates that the pre-built vascular

network facilitated anastomosis. At post-transplantation day 7, perfused MVD was similar to

pre-transplantation day 5 (98±8 vs. 99±19 vessels/mm2). Perfused human MVD at post-

transplantation day 7 (51±11 vessels/mm2) reached 77% of the level observed on pre-

transplantation day 7 (67±8 vessels/mm2; red bar graph, Figure 1D). Human CD31+ vessel

lumens were preserved during the transplantation step (Supplemental Figure S2B), suggesting

bioengineered vessels retain their luminal structure until reconnected in the recipient.

Two possibilities should be considered. One is direct reconnection in which human vessels

reconnect with recipient vessels. The other possibility is that murine vessel segments in the

primary transplants are responsible for reconnection with recipient murine vessels. To address

this, GFP-transgenic SCID mice were used as recipient mice; tail vein injection of UEA-I only

was performed at day 7 after transplantation. We observed GFP/UEA-I-positive chimeric

vessels (Figure 1E), suggesting that ECFC/MPC-human vessels can reconnect directly with

recipient vessels after transplantation.




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Perfused vascular volume in implants

To gain further insight into the extent of perfusion before and after transplantation, we

monitored perfusion at days 3 and 5 before transplantation, and days 3, 5, and 7 after

transplantation in the same implants using a contrast-enhanced ultrasonic imaging system tuned

to detect a microbubble contrast agent introduced to the animal’s systemic circulation. This

technique enabled us to quantify, by three-dimensional (3D) analysis, the proportion of the

whole implant which was perfused by the vasculature.

Figure 2A shows representative raw data images from a low-perfusion implant (post-

transplantation day 3, captured from Supplemental Video 1), and Figure 2B shows ultrasound

signal intensity over time, with contrast agent injected at 10 sec. The lack of orange pixels

within the construct (red outline, Figure 2A) and no signal increase upon injection of contrast

agent (Figure 2B) indicate very little perfusion. In contrast, representative images, graph, and

video from post-transplantation day 7 (Figure 2C-D, and Supplemental Video 2) showed a

dramatic ultrasound signal increase, indicating extensive perfusion.

Perfused vascular volume (%; Figure 2E) increased gradually before transplantation.

Transplantation interrupted construct perfusion, but by day 3 perfusion returned to 13±2 %.

Perfusion was restored to 22±2 % on day 5, which was equivalent to that measured on pre-

transplantation day 5 (17±1 %). On post-transplantation day 7, perfused vascular volume was

28±1 %. For comparison, renal vascular volume in these animals was 50±2 %. At every time

point, the experimental implants were significantly more perfused than the Matrigel-alone

negative control group.




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As shown in Figure 1D, MVD values were similar between pre-transplantation day 5 and

post-transplantation day 7, while perfused vascular volume (Figure 2E) was increased at the

latter time point. This may be due to the fact that MVD is based on two-dimensional (2D)

histological sectioning, while perfused vascular volume is a 3D ultrasound measurement of the

whole construct. Furthermore, MVD describes the number of vessels per sectional area, and is

not expected to be proportional to the volume of blood transmitted by those vessels. The two

metrics are complementary, with lectin-based MVD valued for allowing the measurement of

human and murine vessels, while perfused vascular volume is valuable as a more faithful

measurement of blood-delivery function.

Conclusion

Our goal is to develop a technology for generating vascularized tissue-engineered

constructs. In the present study, we aimed to test whether ECFC/MPC-bioengineered human

vessels originally perfused at one site could become re-perfused in a secondary site. We show,

by in vivo staining and ultrasonic imaging analyses, that ECFC/MPC rapidly formed

anastomoses and increased MVD and perfused vascular volume over 7 days in donor mice.

After transplantation, perfusion was re-established by day 3. Chen and colleagues demonstrated

that a 7 day in vitro pre-assembly step, in which ECFC-derived endothelial cells and fibroblasts

were co-incubated in a fibrin gel, accelerated anastomosis with host vasculature after

implantation.7 In contrast, the present study shows that pre-built perfused human vessels are

transplantable from one in vivo site to another. Perfusion of transplanted human blood vessels,

first detected at day 3 and increasing with time to a physiologic range, demonstrates an important

new feature of ECFC/MPC-bioengineered blood vessels – the ability to be transplanted and




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reconnect to the vasculature in a new site and rapidly re-establish perfusion. This feature extends

the potential applications of this postnatal progenitor cell-based technology for transplantable

tissue-engineered constructs.




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Acknowledgements

This study was supported by 5R01HL94262 (J.B.). K.T.K. designed and performed all

experiments, interpreted data, and wrote the manuscript. P.A. developed and performed micro-

ultrasonic imaging experiments, and wrote the manuscript. J.B. conceived the research plan,

contributed to interpretation of data and assisted with writing the manuscript.

Disclosure of Conflicts of Interest

The authors have no conflicts of interest to disclose.




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References

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8. Nor JE, Peters MC, Christensen JB, et al. Engineering and characterization of functional

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10. Au P, Daheron LM, Duda DG, et al. Differential in vivo potential of endothelial

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long-lasting vessels. Blood. 2008;111(3):1302-1305.

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Figure Legends

Figure 1. Microvessel density analysis before and after transplantation. Matrigel implants

were harvested at multiple time points before and after transplantation. (A) Diagram of

transplantation model. (B-E) Perfused human and murine vessels were identified by tail-vein

injection of a mixture of Rhodamine (red)-conjugated UEA-I and FITC (green)-conjugated GS-

IB4. (B) Representative confocal images of three distinct patterns of lectin-labeled vessels in the

implants at pre-transplantation day 7 (white arrowheads: human, murine, and chimeric vessels).

(C) Representative confocal images of lectin-labeled vessels in the implants at multiple time

points. White dotted line in the day 3 panel of pre-transplantation indicates the border of implant.

(D) Graph of microvessel density (MVD) before and after transplantation (n=3-8; means ±

S.E.M.). ∗ significant difference (P≤0.050) between groups for total MVD. † significant

difference (P≤0.050) between groups for human MVD. (E) Representative confocal images of

GFP/UEA-I(Rhodamine)-positive vessels in the implants at day 7 after transplantation into GFP-

transgenic mice. White dotted line indicates the border of implant. Scale bars represent 100 μm.

Figure 2. Perfused vascular volume before and after transplantation. ECFC/MPC/Matrigel

suspensions were injected into a SCID mouse to form vascular networks in vivo, and the degree

of perfusion in each implant was monitored at days 3 and 5 before transplantation, and days 3, 5,

and 7 after transplantation. 2D images were recorded as video (supplemental Video 1-2) and

analyzed. (A) Representative captured 2D image from low-perfused implant (post-

transplantation day 3). (B) The plot of nonlinear ultrasound signal intensity (expressed in

arbitrary units) integrated over the region of interest of the image plane of Figure 2A (Contrast




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agent was injected at approximately 10 sec). (C) Representative captured 2D image from high-

perfused implants (post-transplantation day 7). (D) Nonlinear ultrasound signal intensity as

described in B, integrated over the region of interest of image plane of Figure 2C. (E) Perfused

vascular volume was measured by collecting a stack of 2D images from the anterior to posterior

ends of the implant (supplemental Video 3-4). The difference in the percent of voxels returning

an ultrasound signal before and after contrast agent injection is perfused vascular volume (%),

the proportion of implant volume occupied by functional, perfused vessels. ∗ significant

difference (P≤0.050) between groups (n=8 for each time point; n=5 for Matrigel control).




doi:10.1182/blood-2011-08-375188Prepublished online October 28, 2011;

Kyu-Tae Kang, Patrick Allen and Joyce Bischoff reconnect with the host vasculature and re-establish perfusionBioengineered human vascular networks transplanted into secondary mice

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