Generation of Tubular Superstructures by Piling of Renal Stem/Progenitor Cells
Will W. Minuth, Ph.D., Lucia Denk, Tec., and Hayo Castrop, Ph.D.
Tissue Engineering: Part C, Methods. Mar 2008; 14(1): 3-13.
Summary
With hundreds of thousands of Americans suffering from various stages of renal failure, there is a great deal of interest in potential mechanisms of kidney regeneration. Under appropriate perfusive conditions, rabbit renal stem and progenitor cells have been coaxed, in vitro, into basic tubule formations that exhibit protein markers and structural characteristics representative of natural renal tubules. Whether they sufficiently exhibit the functionalities of said tubules remains to be investigated, but researchers hope this development of techniques that result in relatively large scale, coordinated production of tubule superstructures makes tissue engineering a likely player in future treatments for kidney failure.
In this experiment, researchers stripped the fibrous lining from harvested newborn rabbit kidneys and sandwiched this embryonic renal tissue in between thin sheets of polyester fleece. These arrangements were then enclosed in tissue holders and exposed to constant horizontal flow of fresh media.
Image 1: Cross-sectional illustration and scaled view of the culture subunits.
A solution of IMDM with an antibiotic-antimycotic cocktail and aldosterone (for promotion of tubule growth) was perfused through the tissue culture continuously for 13 days at 1mL/hr and 37°C. The developed tubules were fluorescently labeled with Soybean Agglutinin for analysis under microscope as whole-mount specimens and as cryosections, as well as labeled with fluorescent antibodies for detection of various other protein markers.
Ultimately, the cultured tubular structures exhibited cells arranged directionally, a visible inner channel (lumen), and ECM proteins consistent with an epithelial basal lamina—each of which are characteristic of natural renal tubules. The tubules also showed reaction with numerous markers for proteins, such as Na/K-ATPase and Fibrillin-1, that were not found in untreated embryonic cells—suggesting significant differentiation. While the exact mechanisms for growth of these tubules from single cells remains unknown, it is clear that the combination of mechanical and molecular conditions in this perfusion setup produces some promising results.
The straightforward preparation of these arrays of tubules sets forth a number of further investigations into this technique. Concerned with scalability of production, the researchers of this study performed subsequent trials in which they piled (stacked like bricks) and paved (laid like tile) several of these cell culture ‘sandwiches’ in the same chamber. The amount and degree of cell differentiation in these chambers was nearly identical to that in the single-culture chambers, and these tubules displayed the same characteristic morphologies and protein markers.
Image 2: Top: Cross-sectional views of developed tubules, exhibiting a basal lamina (asterisk) and marked lumen (arrow). Middle and bottom: Illustrations of piled and paved arrays for generation of renal tubule superstructures and the resultant perfusion of growth media.
Significance:
Hundreds of thousands of Americans in rely on multi-weekly dialysis treatment to either supplement or supplant the function of their diseased kidneys. Dialysis and transplantation may remain the treatments of choice for End Stage Renal Disease for years to come, but patients suffering from acute and less-than-complete kidney failure might be able to benefit from the implantation of these tubule superstructures to aid in kidney function. Further research will need to address the viability of these grown tubules and analyze their metabolic activity, investigate the feasibility and logistics of implantation, and ultimately locate a source of cells and confirm a technique that will produce results appropriate for humans. With kidneys in high demand and low supply in the organ transplant market, any techniques that produce results to a similar end could improve length and quality of life for thousands of people across the globe.
4 comments:
It looks like a very interesting and promising alternative to dialysis. However, since "the exact mechanisms for growth of these tubules from single cells remains unknown," how the authors can be sure that growth of these tubules under the above conditions and implantation them in vivo on a bigger scale than rabbit will not produce incapability/rejection and promise stability of the tubules?
It does seem as if the treatment options outlined here would at least require the original progenitor cells come from the recipient of the implant - your concerns are ones raised in the paper as part of a 'future outlook.' I think the point of this published research was to pave the way for much more extensive characterization of the cultures, and with more study it's hoped solutions to or shortcuts around these issues will be discovered.
re-posting to correct a typo.
Generating the tubules is interesting and likely pertinent in renal regeneration. Yet, it seems that a vast amount of challenges stand in the way of effective renal regeneration therapy.
I'm wondering whether the tubules are morphologically similar along its entire length, with a uniform distribution of markers, or if these tubules exhibit different morphologies similarly to tubules in vivo (proximal convoluted, distal tubule, etc.).
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