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Developmental Biology - Genetics

Miniature Kidney Organoids Come of Age

Method for growing kidney organoids under flow enhances their vascularization and maturation, increasing their potential for drug testing and regenerative medicine...


In recent years, researchers have created mini-organs known as organoids in the culture dish that contain many of the cell types and complex microarchitectures found in human organs, such as the kidney, liver, intestine, and even the brain. However, most organoids grown in vitro lack the vasculature required to provide oxygen and nutrients, remove metabolic waste, and facilitate communication between different cell types that drives their maturation into truly functional tissue building blocks.
For kidney organoids, lack of in vitro vascularization prevents research from emulating key kidney functions in vitro, including blood filtration, reabsorption, and urine production. Creating robustly vascularized kidney organoids can enable better modeling of kidney diseases, enhance renal drug toxicity testing and, ultimately, lead to new building blocks for renal replacement therapies.

Now, a research team at the Wyss Institute for Biologically Inspired Engineering, the Harvard Paulson School of Engineering and Applied Sciences (SEAS), Brigham and Women's Hospital, and the Harvard Stem Cell Institute led by Jennifer Lewis and Ryuji Morizane has developed a powerful new approach as part of the Institute's new 3D Organ Engineering Initiative. By exposing stem cell-derived organoids to fluidic shear stress, they were able to significantly expand organoid-derived vascular networks, and improve maturation of kidney compartments in comparison to previous static culture methods. The work is published in Nature Methods.

In 2015, Ryuji Morizane and Joseph Bonventre developed a method that enabled them to derive 3D kidney organoids from human pluripotent stem cells. "While our organoids and those generated in other laboratories contained large numbers of well-organized nephrons and primitive blood vessels, they still lacked pervasive vascular compartments with perfusable lumens," said co-corresponding author Morizane, M.D., Ph.D., Assistant Professor at Brigham and Women's Hospital and Harvard Medical School (HMS), and a member of the Harvard Stem Cell Institute.

More recently, researchers around the world have matured kidney organoids by implanting them into animals where they can connect to the host's vasculature in vivo. "For the first time, our study demonstrates that by exposing growing organoids to fluid flow, a mechanical cue known to play an important role for tissue development in the body, we can greatly enhance their vascularization and maturation in vitro," said Morizane.

To accomplish this feat, the team used expertise from the Lewis lab that has pioneered strategies to create vascularized human tissues, including 3D kidney-on-chip models, using 3D bioprinting that can be perfused and sustained for long durations.
Based on these findings, they hypothesized that fluid flow could also promote the formation of blood vessels from precursor endothelial cells found in growing kidney organoids.

"We determined the right combination of underlying extracellular matrix, media additives, and fluidic shear stress under which human stem-cell derived organoids would flourish when grown in our 3D-printed millifluidic chips," said Kimberly Homan, Ph.D., who with Navin Gupta, M.D., is a first author on the study. Gupta added that "the vascular networks form close to the epithelial structures that build the glomerular and tubular compartments, and in turn promote epithelial maturation. This integrated process works really like a two-way street." Homan is a Research Associate in Lewis' group at the Wyss Institute and SEAS, and Gupta is a Clinical Research Fellow working on Morizane's team at the Brigham.
The vessels growing on the 3D-printed chips form an interconnected network with open lumens, which can be perfused with fluids as confirmed by directly imaging fluorescent beads moving freely through them. "We were excited to see that these vascularized glomerular and tubular structures develop through some of the same stages that nephrons experience during normal kidney development in vivo."

Kimberly A. Homan PhD, Wyss Institute for Biologically Inspired Engineering, Harvard University, Cambridge, MA, USA.

Abstract
Kidney organoids derived from human pluripotent stem cells have glomerular- and tubular-like compartments that are largely avascular and immature in static culture. Here we report an in vitro method for culturing kidney organoids under flow on millifluidic chips, which expands their endogenous pool of endothelial progenitor cells and generates vascular networks with perfusable lumens surrounded by mural cells. We found that vascularized kidney organoids cultured under flow had more mature podocyte and tubular compartments with enhanced cellular polarity and adult gene expression compared with that in static controls. Glomerular vascular development progressed through intermediate stages akin to those involved in the embryonic mammalian kidney’s formation of capillary loops abutting foot processes. The association of vessels with these compartments was reduced after disruption of the endogenous VEGF gradient. The ability to induce substantial vascularization and morphological maturation of kidney organoids in vitro under flow opens new avenues for studies of kidney development, disease, and regeneration.

Authors
Kimberly A. Homan, Navin Gupta, Katharina T. Kroll, David B. Kolesky, Mark Skylar-Scott, Tomoya Miyoshi, Donald Mau, M. Todd Valerius, Thomas Ferrante, Joseph V. Bonventre, Jennifer A. Lewis and Ryuji Morizane.

These authors contributed equally: Kimberly A. Homan, Navin Gupta.
These authors jointly supervised this work: Jennifer A. Lewis, Ryuji Morizane.

Acknowledgements
The authors thank P. Galichon for flow cytometry analyses; Y. Yoda and K. Susa for cell culture and immunocytochemistry; S. Jain at The Washington University Kidney Translational Research Center (KTRC; St. Louis, MO, USA) for providing the BJFF hiPSC line; A. Moisan, C. Chen, and S. Uzel for insightful discussions; J. Weaver, B. Roman-Manso, N. Zhou, and M. Ericsson for imaging assistance; and L. Sanders for videography. This study was supported by the US National Institutes of Health (NIH; T32 fellowship training grant DK007527 to N.G.; Subaward U01DK107350 to M.T.V.; R37 grant DK039773 to J.V.B.; UG3 grant TR002155 to J.V.B., M.T.V., J.A.L., and R.M.; grant P30 DK079333 (the BJFF line) supporting The Washington University KTRC), the Harvard Stem Cell Institute (interdisciplinary grant to N.G.; seed grant to R.M. and J.A.L.), Brigham and Women’s Hospital (Research Excellence Award to N.G. and R.M.; Faculty Career Development Award to R.M.), the NIDDK Diabetic Complications Consortium (DiaComp, https://www.diacomp.org; grant DK076169 to R.M.), the NIH (Re)Building a Kidney Consortium (U01DK107350 to K.A.H. and J.A.L.), the Office of Naval Research Vannevar Bush Faculty Fellowship program (award no. N000141612823 to M.S.-S. and J.A.L.), and the Wyss Institute for Biologically Inspired Engineering (D.B.K., K.T.K., D.M., and J.A.L.). J.A.L. thanks the GETTYLAB and S. Lindenfeld for their generous donations in support of this research. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.

Contributions
K.A.H., N.G., J.A.L., R.M., D.B.K., and J.V.B. conceived the project. K.A.H., N.G., and K.T.K. designed the research, and R.M. and J.A.L. supervised the research. K.A.H., N.G., K.T.K., R.M., and D.B.K. designed, performed, and analyzed all experiments. M.T.V. provided critical insights into embryonic development, cell sources, and mouse embryonic kidneys. M.S.-S. designed and built the silicone millifluidic chips, interfacing with perfusion pumps, and analyzed the fluid flow profiles on chip. D.M. and T.M. sourced and validated antibodies, optimized staining protocols, and provided invaluable cell culture analysis and support. T.F. developed methodology for quantifying vascular and tubule features in confocal imaging stacks. All authors contributed to manuscript writing.

Competing interests
J.V.B. and R.M. are co-inventors on patents (PCT/US16/52350) on organoid technologies that are assigned to Partners Healthcare. J.V.B. or his family has received income for consulting from companies interested in biomarkers: Sekisui, Millennium, Johnson & Johnson, and Novartis. J.V.B. is a co-founder of, is a consultant to, and owns equity in Goldfinch Bio. K.A.H. is a co-founder and chairwoman of NanoHybrids Inc. J.A.L. is a co-founder of and owns equity in Voxel8 Inc.

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Feb 18, 2018   Fetal Timeline   Maternal Timeline   News  




Culturing kidney organoids under fluid flow causes endogenous endothelial progenitor cells
to create more mature vascular networks which pervade the whole organoid and interact
with epithelial compartments. Image: Wyss Institute at Harvard University.


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