The greatest challenge in tissue engineering turned out not be the production of functioning, complex tissue structures – how to convince cells to form these intricate arrangements. Researchers are rapidly establishing the diverse set of recipes needed to produce close analogs of real tissues, including cartilage, intestines, skin, heart muscle, kidney, liver, and more. All require quite different strategies to convince the various types of cells involved to take the right actions, but the best of the results so far are close enough to the real thing to function properly when transplanted. In perhaps the most compelling of recent examples, researchers built artificial ovaries that, once implanted, enabled mice to reproduce in the normal, natural fashion. Overall, progress in building functional tissue from a patient’s own cells is in fact going very well.
What is the greatest challenge, then? It is to be found in blood vessels. Not in the production of large vessels such as arteries, as a number of research groups have succeeded in producing structures that work just as well as naturally grown arteries, but in recreating the networks of tiny capillaries that thread every cubic millimeter of naturally grown tissue. Without this capillary network, researchers are limited to the production of thin sheets and tiny organoid masses of engineered tissue – thin enough for nutrients to perfuse while the tissue is growing in a bioreactor, and thin enough for blood vessels to grow into the tissue to support it when it is transplanted. Any larger and the inner cells would starve. These thin tissues can be enough for some therapeutic uses, but if the end goal is the production of new patient-matched organs to order, then the blood vessel challenge must be solved.
Decellularization is one possible strategy, but this still requires the limited resource of donor organs; it works around the inability to generate capillary networks by using an existing set. Nonetheless, the goal for most of the tissue engineering community is to develop the means to build new capillary networks along with new tissues as they are grown. A variety of approaches are in various stages of development, such as the 3-D printing of fine-detail scaffolds that guide the creation of every last capillary, but the research community is still in search of a low-cost, efficient, reliably methodology. Today I thought I’d point out a good example of the sort of incremental research results that are representative of this line of work, the research community inching forward towards a better state of the art, one small step at a time:
Researchers have shown how to use a combination of human endothelial cells and mesenchymal stem cells to initiate a process called tubulogenesis that is crucial to the formation of blood-transporting capillaries. The work is an important step with fragile endothelial cells (ECs) made from “induced pluripotent stem cells,” or iPSCs, a type of cell that can potentially be made from the cells of any human patient. Because iPSCs can be patient-specific, researchers hope to find ways of using them to generate tissues and replacement organs that can be transplanted without risk of rejection by a patient’s immune system. But the fragility of endothelial cells during laboratory growth has limited the utilization of this critical cell type, which is found in all vasculature.
While tissue engineers have found dozens of ways to coax stems cells into forming specific kinds of cells and tissues, they still cannot grow tissues with vasculature – capillaries and the larger blood vessels that can supply the tissues with life-giving blood. Without vascularization, tissues more than a few millimeters in thickness will die due to lack of nutrients, so finding a way to grow tissues with blood vessels is one of the most sought-after advances in the field. “Ultimately, we’d like to 3-D print with living cells, a process known as 3-D bioprinting, to create fully vascularized tissues for therapeutic applications. To get there, we have to better understand the mechanical and physiological aspects of new blood-vessel formation and the ways that bioprinting impacts those processes. We are using 3-D bioprinting to build tissues with large vessels that we can connect to pumps, and are integrating that strategy with these iPS-ECs to help us form the smallest capillaries to better nourish the new tissue.”
In the process of tubulogenesis – the first step to making capillaries – endothelial cells undergo a series of changes. First, they form small, empty chambers called vacuoles, and then they connect with neighboring cells, linking the vacuoles together to form endothelial-lined tubes that can eventually become capillaries. The researchers investigated whether commercially available endothelial cells grown from iPSCs had tubulogenic potential. The test examined this potential in two types of semisolid gels – fibrin and gelatin methacrylate (GelMA). Finally, the researchers also investigated whether a second type of stem cell, human mesenchymal stem cells, could improve the likelihood of tubulogenesis. In fibrin, the team found robust tubule formation, as expected. They also found that endothelial cells had a more difficult time forming viable tubules in GelMA, but over several months and dozens of experiments the team developed a workflow to produce robust tubulogenesis in GelMA. This involved adding mesenchymal stem cells, another type of adult human stem cell that had previously been shown to stabilize the formation of tubules.
Vascularization is critical for the maintenance of multicellular life as blood vessels provide oxygen and nutrients to tissues while also removing waste. Beyond the diffusion limit for oxygen transport, cells in tissue cannot survive or maintain normal function. The formation of vascular networks in vitro and in vivo have applications in treating ischemic disease, elucidating vascular mechanisms, screening drug efficacy, and engineering functional tissues for regenerative medicine. Toward the creation of vascular networks, methods of creating both structurally and functionally mature and complex vasculature must be identified to address the challenge of fabricating clinically-relevant tissue models and engineered tissues.
One of the several strategies toward vascularization is to create new vessel networks through cell-mediated morphologic processes similar to those seen during embryonic development. De novo vascular formation, or vasculogenesis, occurs through assembly of populations of endothelial cells into a rudimentary capillary plexus. Subsequent steps in vivo ensure vascular stabilization and maturity by generating extracellular matrix (ECM) and recruiting supporting mural cells. Further cell-cell and cell-matrix interactions encourage or antagonize vessel sprouting (angiogenesis), branching, remodeling, and pruning.
Many groups have cultured endothelial cells in vitro to generate spontaneously self-assembled vascular networks. While some groups have modeled vasculogenesis in natural matrices such as collagen and fibrin, others have demonstrated capillary networks in more synthetic environments such as GelMA and polyethylene glycol (PEG)-based hydrogels. In addition to challenges associated with the biomaterial environment, a critical aspect for cell-based clinical translation is the selection of cell sources. Human umbilical vein endothelial cells (HUVECs) have been commonly used for studying vascular morphogenesis, and supporting mural cells also have a history of various cell sources such as smooth muscle cells, but these cell types suffer from poor batch-to-batch uniformity as well as difficulties with scale-up and in some cases poor translation potential. More recent work has demonstrated the ability of human bone marrow derived mesenchymal stem cells (hMSCs), which are multipotent even well into adulthood, to serve in a supporting mural role for endothelial cells.
Here the potential to derive functional endothelial cells from induced pluripotent stem cells offers high batch uniformity, standardization, scale-up, and the potential for personalization. Commercially available iPS-ECs have been previously shown to form connected capillary-like networks in 2D and 3D studies with Matrigel. We sought to further validate these findings in additional matrix formulations and with live genetic reporters to provide additional tools for cell tracking and characterization. Here, we explored a cell-based strategy to form tubule networks using clinically relevant cell sources and biomaterials. We utilize iPS-ECs in monoculture or in co-culture with hMSCs in natural fibrin and semi-synthetic GelMA environments, track the progression of vasculogenesis, and quantify the network character of nascent tubules. Our results further supports the hypothesis that tubulogenesis is driven by intracellular vacuole formation and intercellular vacuole fusion. By using cell-based strategies for vascularization we allow biology to dictate the microvascular organization and exploit cellular interactions between iPSECs and hMSCs to stabilize networks when the extracellular environment cannot.