Building new Flesh and Blood!

LIFELINES: The arteries and veins of the human heart (shown here injected with dyed gelatin) are critical to the organ’s function, with blood flow delivering life-giving nutrients to the cardiac muscles while removing toxic waste. In the quest to engineer transplantable organs, researchers must consider how to build comparable blood-vessel networks.© SPL/SCIENCE SOURCE

Thousands of unfortunate patients are badly in need of a replacement organ. As of April 2014, more than 122,000 such people in the United States were on the waiting list maintained by the national Organ Procurement and Transplant Network. But fewer than 30,000 of those patients will actually receive the transplant surgery they need this year. That’s because living or deceased donors constitute the only source of new organs, one that for years has not been able to keep pace with demand.

As the number of patients with severe, irreversible organ damage continues to rise, this gap will only widen. To fill the need, researchers are exploring whether they can build functional organs from scratch. Since the 1980s and 1990s, scientists and surgeons have used cells obtained from individual patients to successfully engineer and transplant human tissues, such as cartilage or skin, emboldening scientists to take tissue engineering to the next level. Building complex organs such as the heart, kidney, pancreas, or liver, however, has proven more difficult.

Even if an entire organ can be constructed, its survival in the body depends on its access to oxygen and nutrients.

A key obstacle to whole-organ engineering has been the lack of adequate numbers of functional cells needed to construct an organ. While engineering cartilage or skin has traditionally involved adult cells, such as epidermal skin cells or cartilage-forming chondrocytes, that can be easily expanded in a laboratory, many cell types required to build transplantable organs don’t grow well in culture. In the case of the heart, for example, adult human cardiomyocytes obtained through a biopsy only survive for a few hours in vitro and do not divide. Fortunately, advances in stem-cell technologies in the last decade have taken large strides toward supplying researchers with the cells they need to build more complex tissues. (See “The All-Important Pluripotent Stem Cell” below.)

As is often the case in science, however, as soon as one challenge is overcome, another presents itself. In the case of organ engineering, one major obstacle keeping researchers from crafting functioning organs is the inability to ensure adequate blood supply to the nascent organ. Even if an entire organ can be constructed using all the appropriate cell types, its survival in the body depends on its access to oxygen and nutrients. Thin layers of tissue such as cartilage can get by with the simple diffusion of these life-giving compounds across tissue boundaries and do not require the construction of blood vessels to survive once implanted in a body. But more complex engineered tissues and organs require functional blood vessels to deliver oxygen and nutrients and to remove waste products.

Building blood vessels

Tissue engineers working to construct skin grafts realized more than a decade ago that the grafts would survive much better if they were fitted with prefabricated blood vessels built out of human endothelial cells.¹ While engineered human skin–like tissues lacking such vessels took two weeks to connect to the circulatory system when grafted into mice, tissues containing preformed microvascular channels and networks connected within four days.²Tissue engineering pioneers Bob Langer of MIT and Shulamit Levenberg of Technion – Israel Institute of Technology used a similar approach to implant engineered human muscle tissue in mice, demonstrating that the addition of blood-vessel endothelial cells doubled the amount of surviving muscle tissue.³

Ensuring adequate blood supply is even more important for organ engineering. Neurons in the brain, cardiomyocytes in the heart, and hepatocytes in the liver are surrounded by complex networks of small capillaries that are essential to the functioning of these organs. Chuck Murry’s group at the University of Washington elegantly demonstrated the importance of providing blood vessels for engineered cardiac tissue when they generated patches of human embryonic stem cell–derived heart tissue. The tissue patches could contract just like human heart tissue, but the survival of the engineered tissue following transplantation into rats was 10 times higher when the research team included blood-vessel endothelial cells.⁴

But engineering functional blood vessel networks is not an easy task. Researchers must understand the mechanisms that drive the formation of blood vessels in order to guarantee consistent results and optimal survival of engineered tissues and organs. How do endothelial cells self-organize into functional networks? Do the cells require external cues to form stable vessels? How do they interact with neighboring cells to ensure expedient microvessel formation?

In 2008, vascular biologist Joyce Bischoff of Harvard University and the Children’s Hospital Boston found that combining mesenchymal stem cells (MSCs) from human bone marrow with human endothelial cells prompted the formation of robust vascular networks. When the different cell types were placed in a gel, they self-organized into tube-like structures, resembling a network of small blood vessels. After the gels were implanted into mice, the vascular structures connected to the established blood vessels of the host, and mouse blood coursed through the engineered human vessels for up to four weeks.⁵ In some ways, this was a bit surprising, because the added stem cells were not really functioning in a typical stem-cell capacity. They were not differentiating into endothelial cells, nor were they being converted into the cell types that MSCs normally give rise to, such as bone, cartilage, or fat. Instead, they were somehow acting as “builders” to help organize the “building blocks”—the endothelial cells—into a functional network.

That same year, Bruno Péault at the Children’s Hospital of Pittsburgh provided further insight into how these bone-marrow MSCs might be contributing to blood vessel formation.⁶ Through a careful analysis of MSCs throughout the body, Péault’s group showed that in addition to serving as stem cells, many MSC populations could act as pericytes, a cell type that provides growth factors to endothelial cells and increases the stability of microvessels.

How the MSCs/pericytes were able to stabilize blood-vessel networks was not clear. But it soon became apparent that some batches of MSCs were extremely effective when it came to building functional blood vessels, while others were not. As reliability and consistency are the hallmarks of successful engineering, it is now imperative for scientists to fully understand the intricate mechanisms of vascular-network formation.

Multifunctional stem cells

BLOOD BROTHERS: Pericytes are often found in association with small blood vessels, shown below from left to right on a capillary, on an arterial capillary, and alongside circular smooth muscle cells on a terminal arteriole. These cells provide growth factors that help stabilize the vessel walls.© SCIENCE SOURCEDESIGNER BLOOD VESSELS: Real blood vessels growing in culture (green) are shown on the left, while a computer simulation of blood-vessel growth (red) is shown on the right. Such simulations could help researchers develop new treatments that provide better blood supply to skin grafts, implanted ligaments, tendons, and other tissues.JEFF WEISS AND LOWELL EDGAR, UNIVERSITY OF UTAHPARALLEL LINES: Sensory nerves (green) align with arteries (red) in the developing mouse skin.MUKOUYAMA ET AL., CELL, 109:693-705, 2002. REPRINTED WITH PERMISSION FROM ELSEVIERTo determine which of the hundreds of factors released by MSCs enable the building and organization of endothelial cells into functional networks, my laboratory took inspiration from the work of Anne Eichmann at France’s National Institute of Health and Medical Research (INSERM). Eichmann determined that during human development, nerves and blood vessels form alongside each other, and specific molecules released by neurons can guide not only their own axon formation, but also the formation of blood vessels.⁷ When I heard of Eichmann’s work at a Keystone scientific conference in 2006, I wondered whether MSCs were behaving like neurons, building endothelial networks by releasing guidance molecules that mirrored those identified during neurovascular development.

To pursue this question, Jonathan Paul, a postdoctoral fellow in my laboratory, constructed 3-D patches of collagen containing human MSCs and endothelial cells as a surrogate for vascularized tissue. Our goal was to observe the formation of the 3-D blood-vessel network while measuring the expression of various genes and proteins. We hoped to identify one or more guidance molecules released by the MSCs and their receptors on the endothelial cells. If successful, we would suppress either the guidance molecules or the receptors and test whether this would abolish network formation.

Around the same time, I met with the University of Washington’s Murry, who happened to also have a postdoc, Kareen Coulombe, working on a similar question. The Murry lab had obtained two distinct MSC cell lines: one that barely formed any blood vessels when combined with endothelial cells and one that built highly robust blood-vessel networks. Coulombe, now an assistant professor at Brown University, performed a broad gene-expression analysis, screening for thousands of genes to identify the molecular signature of the MSCs that formed blood vessels.

VESSELS IN VITRO:When human endothelial cells and mesenchymal stem cells (MSCs) are placed in a gel, they self-organized into tube-like structures, resembling a network of small blood vessels. Block the release of Slit3 from the MSCs or the expression of Robo4 on the endothelial cells, and the cells fail to organize into functional blood vessels. Rather, the MSCs and the endothelial cells form clumps of cells.
See full infographic: JPG | PDF© LUCY READING-IKKANDADespite the very different approaches of our laboratories, our findings, which we published together, were surprisingly similar.⁸ Of all the neurovascular guidance molecules we analyzed, we found one ligand-receptor pair that fit the bill: MSCs released a neurovascular guidance molecule, Slit3, which bound to the endothelial cell-surface receptor Robo4. When we suppressed the release of Slit3 from the MSCs or the expression of Robo4 on the endothelial cells, the ability of the MSC-endothelial tissue patch to organize into functional human blood vessels was severely compromised. (Seeillustration.) Murry’s laboratory had also identified the Slit3-Robo4 pathway as one of the top candidates that distinguished the blood vessel–forming MSCs from those that did not support blood-vessel formation. Despite the fact that MSCs were not neurons, they were activating a pathway to organize endothelial cells into vascular networks that are typically utilized by neurons, guiding both their own growth and that of their associated vasculature.

Real-time imaging of the engineered patches allowed us to observe the MSCs gradually spreading out over a few days as endothelial cells followed suit, moving alongside MSCs that seemed to have constructed a stable cellular scaffold. When the Slit3 protein was suppressed, the MSCs and the endothelial cells formed clumps of cells sitting on top of each other instead of fanning out to generate a true network. Slit3 is known to act as a repellant factor in neural and vascular contexts, which means that it can keep cells at a distance and prevent them from coming too close. Our experiments showed that MSCs were similarly using this molecule to prevent an overcrowding of blood-vessel endothelial cells in one location.

Of course, Slit3 is unlikely to be the only factor that enables MSCs to organize endothelial cells into functional networks. MSCs release a number of attractant molecules, which signal neighboring cells to come closer. Much of the research on promoting the growth of blood vessels has traditionally focused on attractant molecules, such as vascular endothelial growth factor (VEGF), because they enable endothelial cells to migrate and line up together to form tiny branches of emerging blood vessels. However, if cells only produce attractant molecules without repellant molecules as a counterbalance, it is impossible to form a stable network. This may explain why attempts to increase the formation of blood vessels using a single gene or factor such as VEGF have failed, whereas providing cells such as MSCs that produce multiple factors has proven more successful. A complex interplay of attractant and repellant factors is likely necessary to both form and stabilize a functional network of blood vessels.

The future of organ engineering

Recent work suggests that the interaction between MSCs/pericytes and endothelial cells may also apply to blood-vessel cells derived from human induced pluripotent stem cells (iPSCs). Sharon Gerecht of Johns Hopkins University has shown that human iPSCs can generate both endothelial cells and pericytes, and that combining such iPSC-derived cells creates robust vessels similar to those we created by combining adult endothelial cells and adult MSCs/pericytes.⁹ Because iPSCs can be derived from individual patients and tailored to their specific needs while minimizing the risk of immune rejection, this approach may help equip made-to-order iPSC-derived organs with the iPSC-derived blood vessels they need to survive.

Last year, Rakesh Jain of Harvard University and Massachusetts General Hospital provided the first evidence that engineering robust blood vessels could also work using iPSCs derived from patients. The team generated endothelial cells as well as MSC/pericyte-like cells from iPSCs of patients with type 1 diabetes—who often require organ transplants following irreversible damage to their kidneys by high sugar levels and who would also benefit from engineered pancreases to manufacture insulin. Jain’s team implanted the cells into mice and found that they were able to construct robust vascular networks.¹⁰ If this approach holds true in larger animals and subsequent human studies, patient-derived iPSCs could indeed provide the necessary blood vessels required for the engineering of the pancreas, kidneys, or other organs. There is the concern, however, that tissues and organs formed using iPSCs derived from individual patients may be affected by their illnesses.

In an ideal scenario, one would obtain a blood or skin sample from a patient in need of a transplant­able organ, generate iPSCs, and expand them to create all desired cell types of the organ, including the essential blood vessel cells.

The fact that distinct MSC cell lines have very different capacities to promote blood-vessel networks is an important reminder that stem cells, like all cells in a tissue, are highly heterogeneous. Not all MSCs within our bone marrow are equally suited to double as pericyte “builders.” There may also be significant person-to-person variability in terms of MSC potency, likely due to a combination of varying genetic, epigenetic, and environmental factors. As a result, one may have to derive many distinct iPSC lines from a single patient and test the potency of each to identify the cells that are most likely to support an engineered organ. Researchers could also consider using factors to suppress or activate target genes within these cells prior to initiating the construction of blood vessels.

Regardless of the approach taken, understanding and controlling this variability will be essential to standardize vascular and tissue engineering. In an ideal scenario, one would obtain a blood or skin sample from a patient in need of a transplantable organ, generate iPSCs, and expand them to create all desired cell types of the organ, including the essential blood vessel cells. These cells could then be used to seed appropriate scaffolds and direct the formation of cellular and vascular networks to build functional, vascularized organs or parts of a functional organ, called organoids.

This may sound like science fiction, but there are plenty of reasons to be hopeful. Last year, Takanori Takebe and colleagues at Yokohama City University in Japan engineered human iPSC-derived liver organoids by converting the iPSCs into liver cells and combining them with MSCs and endothelial cells.¹¹ The vascularized liver organoids not only survived following transplantation into mice, they rescued the recipient mice whose own livers had been severely damaged. Whether such organoids would also survive long-term in larger animals or humans is not yet known, but this study is one of the best examples of how the potential for organ engineering can be successfully coupled with the formation of blood vessels.

The engineering of organs, tissues, and their constituent blood vessels has advanced rapidly in recent years, but the journey is far from over. Although we do not yet have the ability to build fully functional organs such as hearts, livers, or kidneys from scratch, we now understand more of the underlying mechanisms, and are inching toward the construction of fully vascularized organs ready for transplantation.

Jalees Rehman is an associate professor of medicine and pharmacology at the University of Illinois at Chicago. His laboratory studies the role of metabolism and oxygen landscapes as regulators of stem-cell differentiation as well as the mechanisms underlying vascular tissue engineering and regeneration. His scientific blog The Next Regeneration is part of the Scilogs blog network.

References

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2. P.L. Tremblay et al., “Inosculation of tissue-engineered capillaries with the host’s vasculature in a reconstructed skin transplanted on mice,” Am J Transplant, 5:1002-10, 2005.
3. S. Levenberg et al., “Engineering vascularized skeletal muscle tissue,” Nat Biotechnol, 23:879-84, 2005.
4. K.R. Stevens et al., “Physiological function and transplantation of scaffold-free and vascularized human cardiac muscle tissue,” PNAS, 106:16568-73, 2009.
5. J.M. Melero-Martin et al., “Engineering robust and functional vascular networks in vivo with human adult and cord blood-derived progenitor cells,” Circ Res, 103:194-202, 2008.
6. M. Crisan et al., “A perivascular origin for mesenchymal stem cells in multiple human organs,”Cell Stem Cell, 3:301-13, 2008.
7. R.H. Adams, A. Eichmann, “Axon guidance molecules in vascular patterning,” Cold Spring Harb Perspect Biol, 2:a001875, 2010.
8. J.D. Paul et al., “SLIT3-ROBO4 activation promotes vascular network formation in human engineered tissue and angiogenesis in vivo,” J Mol Cell Cardiol, 64:124-31, 2013.
9. S. Kusuma et al., “Self-organized vascular networks from human pluripotent stem cells in a synthetic matrix,” PNAS, 110:12601-06, 2013.
10. R. Samuel et al., “Generation of functionally competent and durable engineered blood vessels from human induced pluripotent stem cells,” PNAS, 110:12774-79, 2013.
11. T. Takebe et al., “Vascularized and functional human liver from an iPSC-derived organ bud transplant,” Nature, 499:481-84, 2013.