Engineering a Bioartificial                               A paper was published in Nature                                 ...
nuclei were detected using a DAPI stain and       nutrient medium. The idea is that these cellsnone were found [3].       ...
implanted into the patient and the patient’s             To characterize the contribution ofblood will be monitored for wa...
the correct locations within the kidney           allogeneic donor is available, a xenogenicconstruct and sequential perfu...
Pluripotency." Stem Cells and Development       of the National Academy of Science 89.1217.3 (2008): 519-34. Web.         ...
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Engineering a bioartificial kidney utilizing a decellularized matrix


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Engineering a bioartificial kidney utilizing a decellularized matrix

  1. 1. Engineering a Bioartificial A paper was published in Nature Medicine magazine that claimed to obtain a Kidney Utilizing a functional bioartificial heart by Decellularized Matrix recellularization of a decellularized cadaveric heart [3]. Although the experimentChristoph Neyer, Larry Liu, Regine Labog, was conducted on murine and primarily on Seyed Bozorgi heart, it was claimed that the concept of the experiment may be applied to a human heartIntroduction and other organs. Thus by mimicking the procedure of decellularizing a cadaveric 26 million Americans have chronic kidney with no recorded medical history ofkidney disease with progression of disease dysfunction and recellularizing the obtainedleading to kidney failure need for transplant extra-cellular matrix (ECM) with theto sustain life, and the number of new patients own cells, a functional bioartificialpatients with kidney failure has averaged kidney may be obtained.more than 90,000 annually [1]. Chronickidney disease is a progressive loss of renal Background on human kidney physiologyfunction which may cause: blood wastes tobuild to high levels and develop Kidneys regulate body fluid volumecomplications like high blood pressure, and composition. As an endocrine organ,anemia (low blood count), weak bones, poor kidneys synthesize renin to regulate bloodnutritional health and nerve damage [1]. pressure, erythropoietin (the main factor forChronic kidney disease (CDK) may be red blood formation in bone marrow), andcaused by diabetes, high blood pressure and active vitamin D (enhances calciumother disorders [1]. Current treatments for absorption). Nephrons, as the functional unitkidneys failure are dialysis and of kidneys, filtrate through glomerulartransplantation. Dialysis does not cure capillaries, reabsorb mostly throughkidney disease, costs a lot and patients will proximal tubule and excrete the body fluidneed to have dialysis treatments for their through collecting duct.whole life unless they are able to get akidney transplant. Kidney transplants may Design Ideas and Methodscome from living donors or a cadaver, andrequire tissue typing and blood type Decellularization of the Kidneycompatibility to reduce the risk of immunerejection of the transplanted kidney. Once a donor kidney has beenFurthermore, the treatment requires patients obtained the kidney will then beto wait for a match and take anti- rejection decellularized using detergents. This can bemedications, which are expensive and cause accomplished by first perfusing the matrixmore susceptibility to diseases. To reduce with sodium doceyl sulfate (SDS), an ionicthe risk of post- transplantation immune detergent commonly used inrejection and provide patients relief from decellularization that acts as a surfactant,waiting for a tissue match, bone marrow lysing the cells. The matrix is then perfusedstem cells, constructing a bioartificial kidney with Triton X-100, a non-ionic detergent.with the patients own renal cells will be a This process has been shown to remove overpotential treatment. 96% of all DNA traces and leaves no detectable SDS residue. In the past intact
  2. 2. nuclei were detected using a DAPI stain and nutrient medium. The idea is that these cellsnone were found [3]. will localize to the Bowmans capsule because of it unique matrix composition.Cell Source Next, mesangial cells will be added to the perfusion solution. These cells will begin to The next step in the process is to line the blood vessels present in the kidney.obtain kidney cells to seed on the Lastly, endothelial cells will be added to linematrix. Bone marrow-derived stem cells the interior of the blood vessels. The kidney(BMSCs) have been reported to differentiate construct will then be cultured in ainto renal cells, specifically mesangial cells, bioreactor designed to mimic renalendothelial cells, podocytes, and tubular physiology.cells in the kidney [8]. Mesangial cells linethe vessels of the kidney, podocytes are Culturing of Bioartificial Kidney infound in the Bowmans capsule, and tubular Bioreactorcells are present in the renal tubule, whichconnects the Bowmans capsule and the While the exact design of thecollecting duct. Using dual-wavelength flow bioreactor is beyond the scope of thiscytometric analysis [9], which is based on project, the bioreactor will need to perform athe differential ability of cells to efflux few key functions. First, it must supplyHoechst 33342 (a fluorescent DNA-binding oxygen and nutrients to the cells in thedye), enriched populations of BMSCs can be kidney. Second, it must provide an artificialobtained from adult murine bone marrow. outlet for waste products removed by theDifferentiation of the BMSCs into kidney. Lastly, it can recycle the isolatedmesangial cells is accomplished by seeding waste products back into the blood-like fluidthe BMSCs on a collagen I matrix. Non- being pumped through the blood vessels ofadherent cells were then transferred to a type the kidney to supply the kidney with wasteIV collagen matrix and subjected to a to remove. This set up also allows for thedifferentiation medium containing retinoic easy measurement of waste removalacid and platelet-derived growth factor-BB efficiency. Once the kidney exhibits(PDGF-BB) for 24h [8]. Some of the physiological waste removal capabilities, itresultant cells changed to a stellate shape can be transplanted into the patient. Theand expressed Thy1 and desmin, perfusion medium will contain nephrogeniccharacteristic of mesangial cells. growth factors such as a combination of retinoic acid, Activin-A, and Bmp7 in orderReseeding of the Decellularized kidney to promote kidney growth and function [5]. The time frame for culturing the bioartificial Our unique approach is in the kidney within the bioreactor is unknownsequential perfusion of these different cell without actually doing the experiment, buttypes into the decellularized kidney matrix. most likely after a period of weeks toFirst, tubular cells will be perfused in a months the efficacy of the efficacy of theretrograde fashion through the collecting kidney construct could be tested by addingduct of the kidney. This will result in tubular waste to the blood-like solution beingcells lining the collecting duct and renal pumped through the construct andtubule. Podocytes will then be perfused measuring the clearance. If the construct isthrough the blood vessel system of the able to provide sufficient clearance, it can bedecellularized kidney, using an oxygenated
  3. 3. implanted into the patient and the patient’s To characterize the contribution ofblood will be monitored for waste levels. bone marrow cells to the turnover rate of renal cells, male BMSCs can be transplantedProduct Characterization and Validation into a female murine kidney and tested for Y-chromosome kidney cells.In Vitro In Vivo The BMSCs were immunostainedwith CD34, CD133, c-Kit, and GATA-4 to To assess kidney function,make sure they were bone marrow derived immunofluorescence will be used tostem cells. After the BMSCs are determine whether the decellularized kidneydifferentiated in a tissue culture, the renal is composed of differentiated renal cells.cells can be isolated using immunoselection Glomerular flow rate in and out of thewith monoclonal antibodies (mABs) such as kidney within the bioreactor can beST.12, which intercalated and principal cells measured by injecting inulin into thein the renal collecting duct react to or by bioreactor. Inulin is neither reabsorbed orconjugating renal cells with cell markers and secreted by the kidney after glomerularseparating with FACS [12]. filtration so its rate of excretion is directly Cells with slow cycling time can be proportional to the rate of filtration of waterdistinguished by retention of a nucleotide and solutes across the glomerular filter. Thelabel such as bromodeoxyuridine (BrdU), normal kidney functions with a glomeruralwhich is incorporated into the DNA of cells flow rate of 90mL/min/1.73m2.during DNA synthesis [7]. If, afteradministration of a pulse of BrdU the cells Renal Blood Flow tests could also beare monitored for long periods of chase, done by determining the level of creatinine,only the slowly cycling cells retain a BUA (Blood Uric Acid), and BUN (Bloodconcentration of label sufficiently high to Urea Nitrogen) in the blood, which isallow their staining and thus adult organ- normally above levels during kidneyspecific stem cells are often called “label- disfunction.retaining cells” Discussion Flow cytometry of the acutelydispersed cells from the papillae of 8 rats Key Techniques and Improvementsrevealed that 3% of the cells were positivefor CD45, indicating that a small fraction of The most important techniquethe isolated cells were blood cells. However, introduced in our project is the sequentialless than 0.5% of the total cells were perfusion of the different cells types.positive for CD34, CD44, or c-Kit, Perfusion is done by pumping mediumindicating that if hematopoietic stem cells or through the decellularized blood vessels ofbone marrow mesenchymal stem cells were the kidney or the collecting ducts. Theisolated with the renal papillary cells, their importance of this method is that it controlscontribution to the population of the BrdU- where cells end up, but eliminates the needretaining cells (about 40% of the total cells) for a tedious manual seeding process. Sincewas small the matrix content and geometry is preserved during decellularization, the differentiated cells are likely to localize to
  4. 4. the correct locations within the kidney allogeneic donor is available, a xenogenicconstruct and sequential perfusion further kidney could theoretically be used.controls where the cells end up. If all the However, this brings with it issues ofcells were perfused at the same time, the immunogenic matrix components. There areendothelial cells, which will tend to line the some important drawbacks to consider ininterior of the blood vessels, will prevent the this case. First, the decellularized kidneyother cells from migrating further into the with implanted stem cells need to bekidney matrix. cultured in a bioreactor. However, such a bioreactor that can grow an entire kidneyFeasibility has not yet been invented and its parameters are complicated. Second, during the kidney The feasibility of this project can be growth inside the bioreactor, it will beseparated into three parts. First, it is difficult to observe and monitor celldefinitely feasible to decellularize the growth/allocation, taking into the accountkidney, as the decellularization of the that there are going to be different cell typesmurine heart was successfully conducted and layers interacting at same time. Therecently in literature. Second, it is also third drawback as well as a major challengefeasible to generate the renal cells from bone is the time thats required to complete themarrow-derived stem cells (BM-dSC) and entire process, which includes finding theother forms of stem cells. However, the part best kidney match, decellularize the selectedthats not quite feasible is the perfusion of kidney, implant the stem cells, grow kidneythe bioartificial kidney because there might inside bioreactor, implantation and potentialbe various variables not known and post-implantation treatments.parameters not quantified.Alternatives Works Cited BMSCs were chosen because of the 1. "Chronic Kidney Disease (CKD)."large number of cells that will be required to National Kidney Foundation. Web. 20 Apr.reseed the kidney matrix, there are not likely be enough native renal papillary cells < in the patient, especially if kidney ndex.cfm>.function is compromised. 2. Takahashi, K., and S. Yamanaka.Advantages and Drawbacks "Induction of Pluripotent Stem Cells from Mouse Embryonic and Adult Fibroblast The advantage of a bioartificial Cultures by Defined Factors." Cell 126.4kidney is that it only takes the decellularized (2006): 652-55. PubMed. Web. 2 May 2010.matrix from a donor kidney. Since thekidney does not need to be functional, it can 3. Ott, HC. "Perfusion-decellularizedcome from a live or cadaveric human donor. Matrix: Using Natures Platform to EngineerAn allogeneic donor is preferred to in order a Bioartificial Heart." Nature Medicine 14.2to avoid immunogenic matrix components. (2008): 213-21. Web.Allogeneic kidneys are also most similar insize and shape to the failing kidney, which 4. Peiffer, Isabelle. "Use of Xenofreeimproves the ability of the host to accept and Matrices and Molecularly-Defined Media tointegrate the bioartificial kidney. If no Control Human Embryonic Stem Cell
  5. 5. Pluripotency." Stem Cells and Development of the National Academy of Science 89.1217.3 (2008): 519-34. Web. (1992): 5487-491. Web.5. Kim, Doyeob, and Gregory Dressler."Nephrogenic Factors PromoteDifferentiation of Mouse Embryonic StemCells into Renal Epithelia." Journal of theAmerican Society of Nephrology 16 (2005):3527-534. Web.6. Yu, Junying. "Human Induced PluripotentStem Cells Free of Vector and TransgeneSequences." Science 324.5928 (2009): 797-801. Web.7. Oliver, Juan, and Omar Maarouf. "TheRenal Papilla Is a Niche for Adult KidneyStem Cells." Journal of ClinicalInvestigation 114.6 (2004): 795-804. Web.8. Imai, Enyu, and Takahito Ito. "Can BoneMarrow Differentiate into Renal Cells?"Pediatric Nephrology 17.10 (2004): 790-94.Web.9. Metsuyanim S, Harari-Steinberg O,Buzhor E, Omer D, Pode-Shakked N, et al.2009 Expression of Stem Cell Markers inthe Human Fetal Kidney. PLoS ONE 4(8):e6709. doi:10.1371/journal.pone.000670910. Duffield, Jeremy. "Restoration ofTubular Epithelial Cells during Repair of thePost ischemic Kidney Occurs Independentlyof Bone Marrow-derived Stem Cells."Journal of Clinical Investigation 115.7(2005): 1743-755. Web.11. Photograph. Acute Renal Failure. MedIndia. Web. 1 May 2010.<>.12. Fejes-Toth, G. "Differentiation of RenalBeta-intercalated Cells to Alpha-intercalatedand Principal Cells in Culture." Proceedings