Review on Embryonic Stem Cells seeded into kidney scaffolds to regenerate entire organ as well as Uretic Bud
Abstract In today’s time, the growing number of patients suffering from renal failures,the lack of available organs for transplantation,and the inadequacies and expense of dialysis has become a huge issue.The scarcity of transplant allografts for diseased organs has prompted efforts at tissue regeneration using seeded scaffolds, an approach hampered by the enormity of cell types and complex architectures. Embryonic Stem cells(ESC) have the advantage of self-replication and the potential to differentiate into multiple somatic cell types. With the correct multitude of signals, including matrix proteins, soluble or immobilized growth factors, cytokines, cell-to-cell interactions, and mechanical forces tissue regeneration and proliferation can be achieved. Organ-derived intact ECMs capture the histoarchitecture that best represents the in vivo conditions and are in theory best able to facilitate tissue regeneration. Primitive precursor cells populated and proliferated within the glomerular, vascular, and tubular structures along with necessary extracellular matrix(ECM) signals, can help in reconstituting a complete organ. After much success in regeneration of the kidney, the next challenge is to engineer renal tissue with a ureter, which would be needed for engineered kidneys to be clinically useful in transplants. Engineering kidney by seeding ESC in Kidney Scaffold Kidney has a highly complicated complicated microarchitecture composed of basement membrance of diverse protein composition that supports atleast 26 different types of cells in close apposition. Step 1: Organ Decellularization The kidney harvesting process was refined to consistently minimize vasoconstriction and clotting, and the resultant organs were rinsed and securely attached to arterial and ureteral cannulae Cells from rat kidneys were removed using sequential gentle detergent, osmotic, and enzymatic steps. The protocol using the SDS ionic detergent successfully produced acellular kidneys that were nearly transparent, furthermore light microscopy showed more complete disruption of intact cells and removal of their debris, yet with preservation of the underlying ghost-likeconnective tissue pattern throughout the cortex and medulla. In addition, immunohistochemical staining showed a completely contiguous network of laminin and collagenIV. Collagen IV and laminin are primary constituents of kidney basement membrane, and both are involved in cellular viability, migration, and differentiation.
Distribution of GFP labelled murine ES cells delivered into the renal artery of decellularized kidney scaffold
Step 3: Evidence for ECM induced differentiation and lumen formation Cell–cell and matrix-cell signaling is very essential in regeneration of a complete internal organ. Evidences of such intricate signaling leading to cell differentiation can be tested using immunohistochemical markers. Ksp-cadherin, a cell adhesion protein normally expressed in distal nephron tubular cells at later developmental stages, is thought to be consistent with development toward a renal lineage. Positive immunoreactivity for Ksp-cadherin in cells in the cortex and RT- PCR results showing expression of Ksp- cadherin are strong evidences of ECM-induced differentiation. Interestingly, the primitive ES cell cytology features (high nuclear/cytoplasmic ratio) were progressively lost with incubations over prolonged periods, permitting thick section culture consistent with apoptosis of the cells within the central core-like regions of the larger vascular and tubular structures, with greater preservation of cells lining the scaffold basement membrane, thus showing lumen formation. Differentiation of a Contractile, Ureter-Like Tissue from Embryonic Stem Cell giving rise to Uretic Bud It is now possible to produce, from embryonic or induced pluripotent stem cells, kidney organoids that represent timmature kidneys and display some physiologic functions yet without the ureters they can’t be transplanted. With the help of a sequence of growth factors and drugs mouse embryonic stem cells can be induced to differentiate into ureteric bud tissue. The isolated engineered ureteric buds differentiated from embryonic cstem cells (eUB) in three-dimensional culture can be then grafted into ex fetu mouse kidney rudiments.
eUB branches and induces nephrogenesis in renal metanephric mesenchyma
Harvested kidney
Translucent acellular kidney
Conclusion:
Immunohistochemical staining showing intact kidney membrane.
basement
Step 2: ES cell seeding and proliferation Intra-arterial ES cell seeding of the acellular whole organ scaffold showed that the pattern of distribution of the injected cells was into vascular structures and their associated glomeruli. Incubation was performed in two different media, static as well as pulsatile growth media. Incubation in the pulsatile medium showed better development as it could simulate the mechanical forces delivered by the native circulatory system. Over the ensuing incubation periods, the cells then migrated into the contiguous vascular networks The identity of the cells as being of donor (murine) origin was confirmed by anti-green fluorescent protein (GFP) staining.
The approach of using stem cells for renal regeneration is a very complex process when associated with the ECM signaling pathways. ECM signal directed regeneration helps in producing a more organized and differentiated organ, closely resembling all the intricacies of the internal organs. The challenge lies in making this ES cell generated kidney transplantable. Associating it with a ureter bud transplant, can help in developing a fully intact and functional kidney. This approach shows promise for regeneration of whole organs, as well as serving as a model for elucidating the cell signaling mechanisms that could be of use in other approaches to regenerative medicine. References: •
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Edward A. Ross, Matthew J. Williams,Takashi Hamazaki, Naohiro Terada, William L. Clapp, Christopher Adin,Gary W. Ellison,Marda Jorgensen,and Christopher D. Batich. Embryonic Stem Cells Proliferate and Differentiate when Seeded into Kidney Scaffolds. J Am Soc Nephrol 20: 2338–2347, 2009 May Sallam, Anwar A. Palakkan, Christopher G. Mills ,Julia Tarnick, Mona Elhendawi, Lorna Marson,and Jamie A. Davies. Differentiation of a Contractile, Ureter-Like Tissue, from Embryonic Stem Cell–Derived Ureteric Bud and Ex Fetu Mesenchyme.