Cell Transplantation (Methods in Bioengineering)
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Islet Transplantation from Living Donors 5. Discussion and Commentary 5. Younger Versus Older Pigs 6. Small Equipment and Nondisposable Items 6. Harvesting the Pancreas 6. Cleaning and Cannulating Pancreas 6. Polysucrose Purification of Tissue 6. Counting Islet Equivalents 6. Assessment of Islet Quality 6. Discussion and Commentary 6. Induction of Models Suitable for the Study Objectives 7. Controls Required for the Experiments Contents note continued: Materials and Methods 7.
Spinal Cord Section Model 7. Spinal Contusion Injury Model 7. Data Acquisition, Anticipated Results, and Interpretation 7. Function Assessment Methods 7. Discussion and Commentary 7. Preparation for Cell Suspension 8. Stereotaxic Intracerebral Cell Transplantation Rat 8. Encapsulated Cell Production 8.
Preparation of Cell Suspension 8. Making of Encapsulated Cells 8. Data Acquisition, Anticipated Results, and Interpretation 8. Discussion and Commentary 8. Conclusions Contents note continued: Preparation of Pullulan-Spermine 9. Preparation of Plasmid DNA 9. Data Acquisition, Anticipated Results, and Interpretation 9. Discussion and Commentary 9.
Western Blot Analysis Periodic Acid-Schiff Staining Discussion and Commentary Hepatocyte Differentiation and Hepatic Regeneration Data Acquisition, Anticipated Results, and Interpretation Placenta Embryological Development Placenta Tissue Structure Placenta Immunological Properties Human Epithelial Cells from Amnion Human Mesenchymal Stromal Cells from Amnion Human Mesenchymal Stromal Cells from Chorion Preparation of Solutions for Isolation and Culture Preparation for Cell Isolation Release Amniotic Epithelial Cells Release Amniotic Mesenchymal Cells Contents note continued: Placenta-Isolated Cell Characterization Detection of Transplanted Cells Anticipated Results and Discussion Cell Culture Material Notes Includes bibliographical references and index.
Other Form Print version Methods in Bioengineering. View online Borrow Buy Freely available Show 0 more links With access conditions Red Deer College Access at http: Other links Artech House Ebooks at http: Set up My libraries How do I set up "My libraries"? These 2 locations in All: John and Alison Kearney Library. Open to the public ; QH May not be open to the public Held. The kidney is derived from IM and arises from the ureteric bud and MM following precisely timed interactions between multiple signals [ 22 ].
High-throughput chemical screening and low-molecular-weight chemical compounds have recently been used in directed differentiation for kidney formation. They proposed that small chemical compounds were less expensive and more consistent than growth factors and might therefore be more suitable for generating IM cells [ 18 , 23 ]. This study demonstrated the feasibility of monitoring the nephrogenic-differentiation capacity of hiPSCs and provided a new strategy for investigating the efficiency and specificity of methods of achieving renal differentiation of hiPSCs. Additionally, in vitro differentiation of PSCs generated cells with a UB-committed IM fate with the potential to assemble spontaneously into complex, chimeric three-dimensional 3D structures upon coculture with murine embryonic kidney cells [ 19 ].
However, the differentiation efficiency was poor, though the reasons for this were unclear.
The strategy of direct differentiation from PSCs has made significant advances in the past few years [ 20 , 21 ]. The developmental origin of the kidney is well known. The protocol up to this point was similar in both groups [ 20 , 21 ], but the subsequent protocols used to induce IM differentiation from posterior primitive streak differed.
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The differentiated hESCs formed renal vesicles combined with dissociated embryonic mouse kidney cells in the study by Takasato et al. In contrast, Taguchi et al. Briefly, nephron progenitor cells of the MM could be derived from posteriorly located IM. Brachyury, encoded by the T gene, is a representative marker of the primitive streak and posterior nascent mesoderm [ 24 ]. The competence of the IM differed both temporally E8.
They finally verified the temporal kinetics of gene expression at each step of the induction process. Wnt signals are important for posteriorization [ 25 , 27 ]. Indeed, a high concentration of CHIR a Wnt agonist was used in combination with BMP4 to maintain the posterior nascent mesoderm in the posteriorization phase [ 21 ]. They also established stepwise protocols for the differentiation of both mouse ESCs and human iPSCs into metanephric nephron progenitors, thus enabling kidney generation using multiple stage-specific growth factors [ 21 ].
Bioengineering strategies to generate vascularized soft tissue grafts with sustained shape.
Coculture of embryoid bodies, containing nephron progenitors, with mouse embryonic spinal cord, a well-established inducer of kidney tubulogenesis, resulted in the formation of 3D tubular structures expressing markers characteristic of renal tubules and glomeruli [ 21 ]. Although the authors were unable to confirm urinary production or other kidney functions, this direct differentiation method for kidney regeneration appears to be the most complete and reliable study published to date.
Generating precise renal progenitor cells is essential for the development of a whole kidney de novo. The differentiation of induced IM cells into precise renal progenitor cells would allow a complete 3D kidney structure to be constructed from PSCs. However, the means of successfully regenerating a functional vascular system between the regenerated kidney and the recipient remain unknown.
Additionally, the in vivo functioning of a regenerated kidney remains unclear. However, further advances in developmental biology and bioengineering may resolve these issues and allow whole kidney regeneration. Injection of PSCs into blastocysts, the initial embryonic stage after fertilization, synchronizes the development of two line cells, and the combined blastocyst generates a chimeric body.
In the first report of this method, normal ESCs were injected into blastocysts of recombination-activating gene 2-deficient mice, which have no mature B or T lymphocytes, to generate somatic chimeras with ESC-derived mature B and T cells [ 28 ]. A recent study to generate a functional organ using blastocyst complementation by Kobayashi et al. The mouse and rat PSC-derived pancreas produced insulin, and the transplantation of PSC-derived pancreas islets improved hyperglycemia in a diabetic rodent model [ 37 ].
This study indicated that PSC-derived cellular progeny could occupy and develop in a vacant developmental niche. Furthermore, these results also demonstrated that interspecific blastocyst complementation could be used to generate organs derived from donor PSCs in vivo using a xenogeneic environment [ 36 , 37 ].
This blastocyst complementation system has already been applied to whole kidney reconstruction [ 36 ]. Nondeficient murine iPSCs were injected into blastocysts from kidney-deficient mice lacking the SAL-like 1 protein essential for kidney development, and the neonatal mice had kidneys derived almost entirely from injected iPSCs [ 36 ]. However, the vascular and nervous systems were not constructed from cells of iPSC origin, and the kidney was therefore not completely complemented.
Immunohistochemical analysis of the regenerated kidney indicated that the renal vascular system, including renal segmental, lobar, interlobar, arcuate, and interlobular arterioles, was a chimeric structure originating from both host cells and donor iPSCs [ 36 ]. Precise urinary analysis was not carried out and whether or not filtrated and reabsorbed urine was produced is unclear.
Moreover, injection of rat iPSCs into kidney-deficient mouse blastocysts failed to generate rat kidneys in mice. This result implies that the key molecules in mice involved in interactions between the mesenchyme and UB do not cross-react with those in rats. The generation of xenogeneic organs using interspecific blastocysts thus requires a host animal strain lacking all renal lineages [ 36 ].
The most important problem associated with blastocyst complementation is the ethical issue. It is impossible to exclude the possibility of generating interspecific chimeras containing brain derived from injected PSCs. Although it is difficult to establish a xenogeneic blastocyst complementation system that overcomes the xenogeneic barrier, this strategy appears to be one of the most promising methods for kidney regeneration. The clusters from dissociated S3-segment cells were induced by the hanging-drop method in 3D culture [ 44 ], while 2D culture conditions were unable to reconstruct kidney-like structures.
Surprisingly, the reconstructed kidney-like structures included all the kidney substructures, including glomeruli, proximal tubules, the loop of Henle, distal tubules, and the collecting ducts, but not the vasculature. They assumed that these cells were similar to metanephric mesenchymal cells, based on marker protein expression. However, the clusters can differentiate into collecting-duct-like cells or mesangial-like cells, which are not thought to be derived from MM [ 44 ].
The opportunity of stem cell bioengineering.
In this regard, the question of whether adult kidney stem cells can differentiate into lineages other than UB or MM remains to be answered. The kidney-like structures were not vascularized and did not produce urine. However, adult kidney stem cells remain poorly understood. These reports raise the possibility that adult stem cells may represent a safer clinical source than PSCs. Native kidney extracellular matrix ECM has been reported to provide a scaffold for cell seeding and a niche for stem cells to differentiate into whole organs [ 48 ].
The ECM plays a crucial role in kidney development and repair [ 48 — 52 ]. ECM molecules and their receptors influence organogenesis and repair by providing a scaffold for the spatial organization of cells, by secreting and storing growth factors and cytokines, and by regulating signal transduction [ 48 — 53 ]. ECM scaffolds from whole human-cadaveric and animal organs can be generated by detergent-based decellularization [ 1 , 54 ]. This strategy was used by Ott et al.
A whole-heart scaffold with intact 3D geometry and vasculature was prepared by coronary perfusion with detergents into the cadaveric heart.
The opportunity of stem cell bioengineering.
The rat heart was then seeded with neonatal cardiac cells or rat aortic endothelial cells, which subsequently induced the formation of contractile myocardium that performed stroke work [ 55 ]. Decellularized cadaveric scaffolds have also been used in several other organ systems, including the liver [ 56 ], respiratory tract [ 57 ], nerves [ 58 ], tendons [ 59 ], valves [ 60 ], bladder [ 61 ], and mammary gland [ 62 ].
Furthermore, some studies have used decellularization-recellularization technology for kidney regeneration. Many animals have been used for decellularization studies, including rats [ 63 ], rhesus monkeys [ 64 ], and pigs [ 65 ]. However, regenerated kidneys produced by this method did not have sufficient renal function to produce urine and Epo.
Notably, they generated 3D acellular renal scaffolds by perfusion decellularization of cadaveric rat, pig, and human kidneys. Endothelial and epithelial cells were repopulated by perfusion, leading to the formation of viable tissues for renal construction. However, the mechanism whereby the infused cells differentiate and are orchestrated into nephrons with vasculature to produce urine remains unclear. Decellularized cadaveric scaffolds are associated with the problem of massive thrombi, despite strong anticoagulation prophylaxis. Although this strategy still has many obstacles, it demonstrates the impact of regenerative medicine on organ transplantation and its potential as a solution for the shortage of donor organs.
The developing field of tissue engineering is an extension of cell therapy, in which biological and engineering science techniques are combined to create structures and devices to replace lost tissue or organ functions [ 67 , 68 ]. The development of bioartificial kidneys BAKs represents the intersection between regenerative medicine and renal replacement therapy [ 52 ]. A renal tubule assist device RAD containing living renal proximal tubule cells has been successfully engineered, and it demonstrated differentiated absorptive, metabolic, and endocrine functions similar to normal kidneys in animal experiments in vitro and ex vivo [ 69 ].
Briefly, renal proximal tubule segments were harvested from kidneys, and renal tubule progenitor cells were selected and expanded [ 70 ]. The tubule progenitor cells were grown in culture dishes with culture medium containing specific additives [ 68 ]. A RAD with high-flux hemofiltration cartridges containing polysulfone hollow fibers coated with pronectin-L was used as a scaffold device [ 68 ]. Renal proximal tubule cells were then seeded into the hollow fibers and the seeded cartridge was connected to a bioreactor perfusion system, in which the extracapillary space was filled with culture medium and the intracapillary space was perfused with medium.
The cell cartridges were used at least 14 days after seeding.
The RAD units included confluent monolayers of renal proximal tubule cells with characteristics including microvilli, tight junctional complexes, and endocytic vesicles demonstrated by transmission electron microscopy [ 68 ]. The tissue-engineered bioartificial RAD performed differential reabsorption and secretory transport because of the specific active transporters present in the proximal tubule cells in vivo. However, these transport functions were less efficient than those in native proximal tubules [ 68 ]. The same group reported that the RAD was able to maintain viability in a uremic environment in uremic dogs with acute renal failure when placed in series with a conventional hemofilter and an extracorporeal blood circuit [ 71 ].
Furthermore, they also performed clinical trials with BAKs [ 72 — 74 ]. The combination of regenerative medicine and bioengineering thus offers promise for the regeneration of whole kidneys. We attempted to regenerate a functional, transplantable whole kidney able to produce urine and renal hormones, such as Epo, using a xenoembryo and human mesenchymal stem cells. The embryonic metanephros, which is the mammalian renal anlagen, is thought to represent a potential source for the regeneration of functional whole kidneys [ 75 — 83 ].