Rebuilding Kidneys

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PI: Alonzo D. Cook
Co-PI’s: Beverly L. Roeder, Jonathan J. Wisco, Paul R. Reynolds

Introduction

The motivation for this work was the tremendous need for replacement kidneys. End-stage renal disease (ESRD) affects over 500,000 patients in the U.S. and costs Medicare $30 Billion annually for dialysis and transplant procedures. Our overall objective was to engineer kidneys from decellularized porcine extracellular matrix (DPECM) scaffolds and cell cultures grown from human progenitor cells. The primary reason for using human cells was to develop non-immunogenic kidneys that can be used to treat the more than 100,000 chronic kidney disease (CKD) patients awaiting a transplant, thus obviating the need for post-transplant immunosuppressive therapy and the associated risks of acute rejection, graft dysfunction, developing an infection or malignancy, or graft failure of a transplanted donor kidney.

We hypothesized that pluripotent cells would proliferate and differentiate upon reintroduction into the DPECM, would locate in the proper position within the DPECM, and completely recellularize the DPECM. The specific aims for this project were:

Specific Aim 1: Attach pluripotent human cells to DPECM.

Specific Aim 2: Optimize a recellularization process for whole porcine kidneys.

We were able to make tremendous progress towards these goals and submitted 6 publications for peer review (5 accepted, 1 pending review). We substituted Madin-Darby Canine Kidney (MDCK) epithelial cells for the human pluripotent cells in most of the work as a representative type of kidney cell that has been shown to perform equally with human cells. We ultimately demonstrated that DPECM is compatible with mouse, canine and human pluripotent stem cells. We harvested over 100 kidneys from mature swine, decellularized them using an automated system, characterized the DPECM extensively, decontaminated samples, and recellularized them using pressure and vacuum.

Academic Objectives

We were able to decellularize porcine kidneys and sterilize them for recellularization with mouse, canine and pluripotent human cells. We found that the pluripotent human cells were able to attach, proliferate and migrate within the DPECM. We were able to build the appropriate bioreactors, and are now working on developing the optimal culture conditions for 3D culture of the whole kidneys. Six manuscripts were submitted as a direct result of this work, and 2 others for the related heart project. Eight presentations were given at Conferences (see lists below). In future studies we expect that the bioengineered kidneys will be able to filter human blood and produce urine without clotting. The results of this ongoing project will guide the development of a plan to implant bioengineered kidneys in appropriate animal models. Ultimately this research will also be able to be translated into human clinical studies.

Publications on the Kidney Project

  1. Poornejad N., Schaumann L.B., Buckmiller E.M., Momtahan N., Gassman J.R., Ma H.H., Roeder B.L., Reynolds P.R., Cook A.D. The impact of decellularization agents on renal tissue extracellular matrix. J Biomater Appl. 31(4): 521-533 (2016).
  2. Poornejad, N., Schaumann, L.B., Buckmiller, E.M., Roeder, B.L., Cook, A.D., Current cell-based strategies for whole kidney regeneration. Tissue Engineering: Part B:Reviews 22(5): 358-370 (2016)
  3. Poornejad, N., Momtahan, N., Salehi, A.S.M., Scott, D.R., Fronk, C.A., Roeder, B.L., Reynolds, P.R., Bundy, B.C., Cook, A.D., Efficient decellularization of whole porcine kidneys improves reseeded cell behavior. Biomedical Materials 11(2): 025003 (2016)
  4. Poornejad, N., Nielson, J., Morris, R., Gassman, J., Reynolds, P.R., Roeder, B.L, Cook, A.D., Comparison of four decontamination treatments on porcine renal dECM structure, composition, and support of human RCTE cells. Journal of Biomaterials Applications 30(8):1154-1167 (2016).
  5. Poornejad N, Frost TS, Scott DR, Elton BB, Reynolds PR, Roeder BL, Cook AD, Freezing/thawing without cryoprotectant damages native but not decellularized porcine renal tissue, Organogenesis 11(1): 30-45 (2015).
  6. Poornejad N, Buckmiller EM, Schaumann LB, Wang H, Wisco J, Roeder BL, Reynolds PR, Cook AD. Re-epithelialization of whole porcine kidneys with renal epithelial cells. J Biomater Appl submitted Nov 2016, in review BMM-101595 (2016).

Related Publications on the Heart Project

  1. Momtahan, N., Panahi, T., Poornejad, N., Stewart, M.G., Vance, B.R., Struk, J.A., Castleton, A.A., Roeder, B.L., Sukavaneshvar, S., Cook, A.D., Using hemolysis as a novel method for assessment of cytotoxicity and blood compatibility of decellularized heart tissues. ASAIO 52(3):340-8 (2016).
  2. Momtahan, N., Poornejad, N. Struk, J.A., Castleton, A.A., Herrod, B.J., Vance, B.R., Eatough, J.P., Roeder, B.L., Reynolds, P.R., Cook, A.D., Automation of pressure control improves whole porcine heart decellularization, Tissue Engineering: Part C 21(11): 1148-1161 (2015)

Presentations on the Kidney Project

  1. Poornejad N, Fronk CA, Kirkham W, Holden G, Wisco JJ, Roeder BL, Cook AD. 2014. Porcine kidney decellularization using novel method of tonic cycle to improve cell-ECM interaction. American Institute of Chemical Engineers (AIChE) Annual Meeting, November 19, Atlanta Marriott Marquis, Poster Session: Engineering Fundamentals in Life Science, Atlanta, GA, Oral presentation, abstract 383878 (2014)
  2. Poornejad N, Fronk CA, House M, Kirkham W, Holden G, Nielsen J, Wisco JJ, Roeder BL, Cook AD, Determining the integrity of decellularized porcine kidney scaffolds. Tissue Engineering Part A. 20(S1): Poster Abstract P-144, page S-63 TERMIS, Washington, DC (2014)
  3. Nafiseh Poornejad, Robert M. Fuller, Evan M. Buckmiller, Blake J. Cannon, Beverly L. Roeder, Alonzo D. Cook, Recellularization of whole porcine kidneys
    with human epithelial and endothelial cells. Tissue Engineering Part A. 21(S1): Poster Abstract P-689, page S-158, TERMIS, Boston, MA (2015)
  4. Poornejad, N., Buckmiller, E.M., Lundwall, J.J., Ma, H.H., Roeder, B.L., Cook, A.D., Recellularization of whole porcine kidneys with human epithelial and endothelial cells. American Institute of Chemical Engineers (AIChE) Annual Meeting, Nov 9, Tissue Engineering and Regenerative Medicine Session, Salt Lake City, UT, Oral presentation, Abstract 171f (2015)
  5. Poornejad, N., Cannon, B.J., Baker, S.J., Roeder, B.L., Cook, A.D., Optimized decellularization and sterilization method of whole porcine renal scaffold. American Institute of Chemical Engineers (AIChE) Annual Meeting, Nov 11, Biomaterial Scaffolds for Tissue Engineering Session, Salt Lake City, UT, Oral presentation, Abstract 511a (2015)
  6. Pitt, W.G., Poornejad, N., Schaumann, L., Neuberger, T., Chambers, S., Roeder, B.L., Cook, A.D., The impact of decellularization agents on renal tissue extracellular matrix. American Institute of Chemical Engineers (AIChE) Annual Meeting, Nov 15, Biomaterial Scaffolds for Tissue Engineering Session I, San Francisco, CA, Oral presentation, Abstract 277e (2016)
  7. Poornejad, N., Wisco, J.J., Roeder, B.L., Cook, A.D., Non-invasive structural investigation of renal scaffold by Magnetic Resonance Imaging (MRI). American Institute of Chemical Engineers (AIChE) Annual Meeting, Nov 15, Biomaterial Scaffolds for Tissue Engineering Session II, San Francisco, CA, Oral presentation, Abstract 395e (2016)
  8. N. Poornejad, B. L. Roeder, A. D. Cook, The Impact of Decellularization Agents on Renal Tissue Extracellular Matrix, Tissue Engineering Part A. 22(S1): Abstract 168, page S-46, iPoster, TERMIS, San Diego, CA (2016)

Mentoring Environment

The graduate student responsible for this project was Nafiseh Poornejad, and she has finished her work in the Chemical Engineering department at BYU and has obtained full time employment at Johnson & Johnson. Forty undergraduates from 10 different majors worked on the project (see table below). Eight undergraduate students were authors on the 6 publications focused on the kidney project, and another 6 undergraduates were authors on the 2 related heart papers. Dr. Cook (Chemical Engineering) supervised the entire project. Dr. Jonathan Wisco (Physiology and Developmental Biology), Dr. Beverly Roeder (Biology), and Dr. Paul Reynolds (Physiology and Developmental Biology) contributed in their respective areas of expertise as advisors. Dr. Wisco advised the project on the cellular processes of regeneration, and the use of histology and MRI to analyze the results. Dr. Roeder, a veterinarian with training in comparative physiology, provided oversight in porcine kidney harvest and perfusion for decellularization, and gave guidance on the processes and equipment needed to conduct this research. Dr. Reynolds advised on tissue culture and histology techniques. The undergraduates also prepared posters for the BYU Biomedical Engineering Conference and the Utah Conference for Undergraduate Research each year, providing them an opportunity to present to their peers. Most of the students were enrolled in ChEn 498R for multiple semesters, gaining course credit toward graduation requirements. Many of the undergraduates have continued their studies in graduate school or are attending medical school.

Experiences/Successes in Mentoring

This project addressed mentoring activities from three directions: faculty mentoring a graduate student, faculty mentoring undergraduates, and a graduate student mentoring undergraduates. We followed the principles of mentoring: devoting sufficient time, involving everyone in all aspects of the project, providing individual responsibility to the students, and doing so in a spiritually uplifting way. The activities for mentoring of a graduate student included training to conduct independent research, leading a project team and preparing for a career in academia. The activities for mentoring undergraduates included designing and conducting scientific experiments, preparing written reports, and giving presentations. The faculty members and the graduate student were all involved in mentoring the undergraduates.

Dr. Cook met weekly with Nafiseh on Fridays to discuss her project, and the entire kidney team met once a week for one hour on Tuesdays with Dr. Cook and Nafiseh. In the team meetings, we began with prayer and then discussed the progress of the project and any issues that may have arisen since the last meeting. We addressed safety concerns, planned the next week’s experiments and discussed the philosophy of the project as well as the details. Each person on the team was given assignments to report on for the next meeting.

Nafiseh joined the kidney project as a team member and was reporting to an undergraduate team leader for about 6 months. It was apparent from the beginning that she possessed tremendous talent and was able to work hard and think critically. She quickly emerged as the team leader after some coaching and personal one-on-one mentoring. She then led the kidney project for over 2 years and the team produced enough data to submit 6 journal articles on the kidney project (5 have been published, 1 is submitted). Nafiseh also collaborated with the heart research team and contributed to 2 additional articles that have been published. Nafiseh became a skilled project leader and learned to understand the way in which people can be motivated to accomplish tasks. She was given opportunities to work with many students and learn their skills and attributes and was able to guide them to complete the studies that were required to be completed. Nafiseh was already a very good technical thinker and researcher, but needed additional guidance on writing since she was a foreign student. She initially struggled with the language differences, but made tremendous progress. I worked directly with her to develop her skills in writing and presenting.

Almost all of the undergraduate students in my lab were returned missionaries and had experience leading teams, or participated in YM and YW as leaders. The same principles they learned through those experiences were applied in research: plan your work and execute your plan. The students were eager to identify opportunities to contribute and I was able to direct them to the literature and help them understand the next steps that needed to take place in the research project. It was an enjoyable experience to work with such eager students.

Budget

The MEG Award of $20,000 was used as follows: