Embryonic stem (ES) cell technology holds promise to facilitate regeneration of damaged tissues and reconstitute entire organs with identical genetic make-up as the recipient, and thus, with no opportunity for rejection. Substantial and successful tissue regeneration, however, requires integration of different cell types and adequate vascularization. Yet insufficient effort has been placed to develop adaptable technologies to induce, promote and maintain neovascular growth and maturation concurrently with cell/tissue differentiation in a three-dimensional (3D) manner. Our long-term goal is to develop new adaptive strategies for the generation of functional and sustainable vascularized tissues which can quickly integrate into living systems. It is our prediction that the ex-vivo induction of tissue / organoids requires the integrated and concurrent growth of vascular cells, parenchyma, and stroma at equivalent developmental stages. Furthermore, physical forces imposed by a 3D environment that include microflow patterns are also critical for the adequate differentiation of functional vasculature. This prediction has been formulated on the basis of our preliminary data on human cells in which we induced the formation of functional vascular channels that are fully integrated within differentiated liver parenchyma. Our data revealed that mechanical forces from shear fluid (namely, culture media) flow are critical to the development of stable endothelial networks that can, upon transplantation, connect with the host vasculature. These findings warrant further investigations of this approach to improve on the specific composition of the vascular and parenchymal progenitor cells, enhance the imposed flow dynamics and explore the long-term integration of the tissue organoids into hosts. The hypothesis for the proposed research is that the inclusion of an optimally-controlled flow environment during differentiation and morphogenesis will result in a sustainable 3D tissue with adequate vascularization (i.e., a directed artery-capillary-vein network) which can ultimately be integrated with the systemic circulation. To this end, we will pursue the following specific aims: 1) To improve the organization of stable vascular networks in engineered liver - vascular cell organoids. 2) To determine the microfluidic flow conditions needed for optimal 3D tissue growth. 3) To test the functional properties and longevity of liver explants in vitro, and upon transplantation. The research proposed is innovative because for the first time vascular growth ex vivo will be addressed throughout the entire process from individual cells to tissue formation and organoid generation in 3D and under flow conditions. We also bring novel noninvasive, quantitative tools to study vascular growth in real time. The proposed research is significant because the information gathered from these experiments is poised to provide crucial feedback to investigators in the area of bioengineering and regenerative medicine, while clarifying the basic rules that govern vascular morphogenesis in the context of hepatoparenchyma- stroma organization.
The proposed research is relevant to public health since the discovery of new and effective technologies for generating tissue in three dimensions is ultimately expected to have an important positive impact by providing possible replacement organs for patients suffering from a variety of diseases, including a number of emergencies and life-threatening conditions. This is relevant to NIH's mission because important advances in sustainable tissue transplantation, which requires the presence of a hierarchical vascular supply that is fully and quickly integrated with the host vasculature, is expected to lead to functional organoids. It is also expected that what is learned about liver organoid generation will be equally applicable to other organs that require the complete integration of functional vascular supplies to the host.
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