Newly developed method to fabricate perfusable collecting ducts of the human kidney opens the door to disease modeling, drug testing, and organ engineering
By Benjamin Boettner
(BOSTON) — The human kidney filters about a cup of blood every minute, removing waste, excess fluid, and toxins from it, while also regulating blood pressure, balancing important electrolytes, activating Vitamin D, and helping the body produce red blood cells.
This broad range of functions is achieved in part via the kidney’s complex organization. In its outer region (cortex), more than a million microscopic units, known as “nephrons,” filter blood, reabsorb necessary nutrients, and secrete waste in the form of urine. To direct urine produced by this enormous number of blood-filtering units to a single ureter, the kidney establishes a highly branched 3-dimensional tree-like system of “collecting ducts” (CDs) during its development. In addition to directing urine flow to the ureter and ultimately out of the kidney, collecting ducts reabsorb water, which the body needs to retain, and maintain the body’s balance of salts and acidity at healthy levels.
Finding ways to recreate this system of CDs is the focus of researchers and bioengineers who are interested in understanding how CD defects cause certain kidney diseases, underdeveloped kidneys, or even the complete absence of a kidney. Importantly, being able to fabricate the kidney’s plumbing system from the bottom up would be a giant step toward tissue replacement therapies for many patients waiting for a kidney donation – alone in the U.S., 90,000 patients are on the kidney transplant waiting list. However, rebuilding this highly branched fluid-transporting ductal system is a formidable challenge and not possible yet.
Now, a team of bioengineers at the Wyss Institute at Harvard University and Harvard John A. Paulson School of Engineering and Applied Sciences (SEAS) led by Wyss Core Faculty member Jennifer Lewis, Sc.D., developed a platform that combines kidney-specific stem cell differentiation and organoid culture technology with bioengineering approaches to address this challenge. After creating an extracellular matrix (ECM) that can be 3D bioprinted and supports the differentiation of human induced pluripotent stem cells (hiPSCs) into complex CD organoids, the researchers engineered renal CDs at two different scales to mimic the tubular CD network and the drainage outlet. Upon bioprinting extensive tubular networks adjacent to larger, perfusable tubular CD structures, the two types of orthogonally-fabricated CD structures formed interconnections, demonstrating a practical way to build an integrated, tissue-scale CDnetwork. The findings are published in Cell Biomaterials.
“Our newly advanced platform enables us to create perfusable CD tubules for multiple applications, including drug discovery and disease modeling,” said Lewis, “and ultimately, biofabrication of whole organs for therapeutic use with integrated nephron units and collecting duct networks. Lewis is also the Hansjörg Wyss Professor of Biologically Inspired Engineering at SEAS and a leader of the Wyss Institute’s 3D Organ Engineering Initiative.
Our newly advanced platform enables us to create perfusable CD tubules for multiple applications, including drug discovery and disease modeling and, ultimately, biofabrication of whole organs for therapeutic use with integrated nephron units and collecting duct networks.
Reverse engineered kidney development
During normal kidney development, the so-called “ureteric bud” (UB) functions as a seed for the entire CD network. As a tiny tube that grows and splits, it creates all urine-collecting channels while also instructing other cells in the kidney’s cortex to differentiate into the blood-filtering nephrons. Emulating this tubular organization poses a particular challenge to bioengineers who aim at recreating the kidney’s CD network in the lab.
To replicate renal development, methods have been developed that allow researchers to differentiate human hiPSCs into kidney “organoids,” aggregates of cells that exhibit many organizational features of the kidney, including nephrons and CD-like tubular structures. However, organoids don’t have an inlet and outlet and their CDs are poorly formed and remain relatively unorganized, thus falling short of a functional CD system.
By using organoid biology in combination with different tissue engineering approaches, we succeeded in building renal tubular networks lined by CD cells that connect with a central outlet.
“To create a new inroad into this problem, we essentially cracked open kidney organoids and leveraged their potential to develop tubular structures. By using organoid biology in combination with different tissue engineering approaches, we succeeded in building renal tubular networks lined by CD cells that connect with a central outlet,” said co-first author Kayla Wolf, Ph.D., who, with the study’s other first author Ronald van Gaal, Ph.D., spearheaded the project in Lewis’ group as a postdoctoral fellow. She is now an Assistant Professor at Cornell University’s Meinig School of Biomedical Engineering in Ithaca, NY.
As a prerequisite for fabricating longer and more complex tubular networks from the bottom up, the team first developed and optimized a matrix made of collagen and tiny fragments of natural basement membrane components – in normal human tissues, cells produce matrix to hold tissue together and maintain its function. In the engineering of CD tissues, the extracellular matrix enabled the bioprinting process and supported the growth and differentiation of UB and UB progenitor cells placed into it.
To fabricate a larger central UB channel that could be perfused with fluid in a fluidic device, the researchers first created a hollow biomaterial channel using a “templating” method that allowed them to cast the engineered matrix in a fluid state around an elongated pin. Upon solidification of the matrix, the pin was removed, leaving behind a hollow cylindrical channel into which organoid-derived UB cells were seeded. After about a week, the UB cells had self-assembled into a hollow cylinder lined by a confluent monolayers of cells. Interestingly, Wolf and her co-workers found that, at many places, the walls of this tube started to bud into the surrounding matrix, mimicking the early stages of kidney development.
In pursuit of larger tubular CD networks, the team created smaller diameter tubes by directly bioprinting hiPSC-derived UB progenitor cells into the matrix in filamentous patterns. Over a period of days to weeks of cell culture, the filaments assembled into branched networks of tiny tubes.
From network formation to CD maturation
Importantly, when printed adjacent to a larger-diameter central channel, these tubular networks connected to the central channel via multiple lumen-to-lumen connections. “Having established a network of tubular structures connected with a larger-scale central channel, we wanted to see if it was possible to mature UB tubules to a CD-like state,” said Wolf. “Not only was this possible, but our platform revealed that perfusion during this process enhanced CD-like maturation.”
As a next step, Lewis’ team will test whether the engineered CD network can integrate with nephron-rich features. In the meantime, these CD models are of interest for understanding diseases, such as polycystic kidney disease, that primarily originate in the CD.
“To have found a promising way to engineer the collecting duct network of the human kidney, using stem cells and innovative tissue engineering methods, is a tremendous feat toward tackling the national and global transplant crisis, in addition to offering a new approach to drug testing and disease modeling in the near-term,” said Wyss Founding Director Donald Ingber, M.D., Ph.D., who is also the Judah Folkman Professor of Vascular Biology at Harvard Medical School and Boston Children’s Hospital, and the Hansjörg Wyss Professor of Biologically Inspired Engineering at Harvard John A. Paulson School of Engineering and Applied Sciences.
Other authors on the study are Sebastien Uzel, Jonathan Rubins, Aline Klaus, Amelie Printz, Pooja Nair, Katharina Kroll, Paul Stankey, and Lisa Satlin. The study was funded by the Wellcome LEAP Human Organ Physiological Engineering (HOPE) program, NIH Re(Building) a Kidney Consortium (under award NIH UC2DK126023), an NIH F32 Ruth L. Kirschstein national postdoctoral research award to Kayla Wolf, and a Rubicon grant from the Dutch Research Council to Ronald van Gaal.