Directed differentiation of human induced pluripotent stem cells into mature kidney podocytes and establishment of a Glomerulus Chip
Musah S., Dimitrakakis N., Camacho D.M., Church G.M., Ingber D.E.
Nature Protocols 2018
With the use of laminin 511 coating, the authors have developed a detailed protocol for the directed differentiation of human iPS cells into mature, post-mitotic kidney glomerular podocytes with high (>90%) efficiency within 26 d and under chemically defined conditions, without genetic manipulations or subpopulation selection. They also describe how these iPS cell-derived podocytes may be induced to form within a microfluidic organ-on-a-chip culture device to build a human kidney Glomerulus Chip that mimics the structure and function of the kidney glomerular capillary wall in vitro.
Pajęcka K., Nygaard Nielsen M., Krarup Hansen T., Williams J.M.
Experimental Cell Research, 2017
Primary glomerular endothelial cells (GEnCs) is an important tool for studying glomerulosclerotic mechanisms and in drug development. Primary GEnCs are commonly cultured either on gelatin or plasma-derived fibronectin-coated surfaces, yet neither of the substrates is a major component of the healthy mature GBM. Here, the authors set out to establish a simple, reproducible model of quiescent hGEnC monolayer in vitro by determining the best commercially available ECM substrate- recombinant human laminin (rhLN)- 521, -511, -111, fibronectin (FN), collagen type IV and collagen I - based Attachment Factor. All ECM matrices except recombinant human laminin 111 (rhLN111) supported comparable cell proliferation. Laminin-521, laminin-511, and FN were best at supporting hGEnC attachment and spreading. Culturing hGEnCs on rhLN521, rhLN511 or fibronectin resulted in a physiologically relevant barrier to 70 kDa dextrans which was 82 % tighter than that formed on collagen type IV. Furthermore, only hGEnCs cultured on rhLN521 or rhLN511 showed plasma-membrane localized zonula occludens-1 and vascular endothelial cadherin indicative of proper tight and adherens junctions. The authors' recommendations based on the results are that hGEnCs is best cultured on the mature glomerular basement membrane laminin - rhLN521 – which, as the only commercially available ECM, promotes all of the characteristics of the quiescent hGEnC monolayer: cobblestone morphology, well-defined adherens junctions, and physiological perm-selectivity.
St John P.L. and Abrahamson D.R.
Kidney International, 2001
During the glomerular basement membrane assembly, laminin-111 is initially expressed in vascular clefts of comma- ann S-shaped bodies and is eventually replaced by laminin-521 which persists into maturation. Post-fixation immunoelectron microscopy of developing mouse and 2-3 days old mice kidney was performed and showed Intracellular labeling for laminin-111 and laminin-521 in developing glomerular endothelial cells and podocytes. In early capillary loop stage, GBMs laminin-111 was absent but the expression of a5 was strong (developmental switch). The results show that both endothelial cells and podocytes synthesize laminin but the highest level of laminin synthesis by endothelial cells.
Miner J.H., Cunningham J., Sanes J.R.
J Cell Biol., 1998
Here, we show that the laminin α5 chain is required during embryogenesis. The α5 chain is present in virtually all BLs of early somite stage embryos and then becomes restricted to specific BLs as development proceeds. BLs that lose α5 retain or acquire other α chains. Embryos lacking laminin α5 die late in embryogenesis. They exhibit multiple developmental defects, including failure of anterior neural tube closure. In addition to exencephaly, syndactyly, and placentopathy, several defects were seen in internal organs of Lama5 −/− embryos, including small or absent kidneys. Some of these defects were variable in severity and penetrance, but all can be associated with BLs that normally contain laminin α5.
Kikkawa Y., Virtanen I., Miner J.H.
J Cell Biol., 2003
In developing glomeruli, laminin alpha-5 (laminin-521 and -511) replaces laminin alpha-1 (laminin-111) in the glomerular basement membrane (GBM) at the capillary loop stage, a transition required for glomerulogenesis. By the capillary loop stage, laminin-111 is eliminated and at maturity, only laminin-521 is detected in the GBM where it plays a crucial role in maintaining glomerular capillary loop structures. To investigate domain-specific functions of laminin alpha5 during glomerulogenesis, the authors produced transgenic mice that express chimeric laminin composed of laminin alpha-5 domains fused to the human laminin alpha-1 globular (G) domain (Mr51). When bred onto the Lama5 -/- background, Mr51 supported GBM formation, preventing the breakdown that normally occurs in Lama5 -/- glomeruli. In addition, podocytes exhibited their typical arrangement in a single cell layer epithelium adjacent to the GBM, but the convolution of glomerular capillaries did not occur. Instead, capillaries were distended and exhibited a ballooned appearance, a phenotype similar to that observed in the total absence of mesangial cells, suggesting that the G domain of laminin alpha-5 is essential for mesangial adhesion. Finally, in vitro studies showed that integrin alpha3beta1 and the Lutheran glycoprotein mediate adhesion of mesangial cells to laminin alpha-5. These results elucidate a mechanism whereby mesangial cells organize the glomerular capillaries by adhering to laminin alpha-5 in the GBM.
Sebinger D., Ofenbauer A., Gruber P., Malik S., Werner C.
Here, the authors, explore the role of the extracellular matrix (ECM) constituents on renal structure formation during renal organogenesis. By culture of microdissected E11.5 kidney rudiments, the show that specific ECM components, including collagen I and laminin, supported nephronal and tubular structure formation of the developing organ. Culture on laminin-111 promoted development as shown by more bud tips and more nephrons.
Ekblom P., Klein G., Ekblom M., Sorokin L.
Am J Kidney Dis., 1991
Epithelial cells have a polarized morphology, with distinct basal, lateral, and apical cell surfaces. It would be of considerable interest to know how the polarized morphology develops during embryogenesis. Both the tubular and glomerular epithelial cells of the kidney develop from mesenchymal stem cells during embryogenesis. Unique conversion of nonpolar cells to polarized epithelial cells thus occurs in the embryonic kidney. This conversion also occurs in vitro if the mesenchymal cells are properly induced. Organ cultures of mesenchymal cells from the mouse embryonic kidney have therefore been much used to study the development of epithelial cell polarity. We have used this model system to study the role of basement membrane glycoproteins in development. The results obtained suggest that laminins are particularly important for epithelial cell development. There are many different types of laminins. Developing kidney tubule cells synthesize that promote development by interacting with specific integrin receptors on the cell surface. The mesenchymal stem cells also produce laminin.
Miner J.H., Patton B.L., Lentz S.I., Gilbert D.J., Snider W.D., Jenkins N.A., Copeland N.G., Sane J.R.
The Journal of Cell Biology, 1997
Using a panel of nucleotide and antibody probes, we surveyed the expression of a 1-5 in murine tissues. All five chains were expressed in both embryos and adults, but each was distributed in a distinct pattern at both RNA and protein levels. Overall, a 4 and a 5 exhibited the broadest patterns of expression, while the expression of a1 was the most restricted. Detailed analysis of developing kidney revealed that some individual basal laminae, including those of the tubule and glomerulus, changed in laminin chain composition as they matured. During glomerulogenesis there are developmental transitions in laminin trimer deposition. Initially the GBM contains laminin-111, laminin-411 and laminin-511. By the capillary loop stage during GMB maturation, laminin-111 is eliminated from the GBM and the beta 2 chain begins to accumulate while the beta 1 chain is down-regulated.
Dufek B. Meehan D.T., Delimont D., Cheung L., Gratton M.A., Phillips G., Song W., Liu S., Cosgrove D.
International Society of Nephrology, 2016
Normally, α5 laminins are highly expressed in the glomerular basement membranes (GBM). In this article, the authors show that the mesangial filopodia in the GBM are depositing mesangial matrix proteins, including laminin 211, which activates focal adhesion kinase in glomerular podocytes, resulting in the activation of genes encoding proinflammatory cytokines and metalloproteinases through a nuclear factor kB–dependent signaling pathway, which drive the progression of glomerulonephritis. The authors test whether endothelial cell-derived endothelin-1 is up-regulated in Alport glomeruli and further elevated by hypertension. Endothelin A receptor activation on mesangial cells is a key event in the initiation of Alport glomerular disease in this model.
Abrass C.K., Hansen K.M., Patton B.L.
Matrix Pathobiology, 2010
Laminin-8/9 is synthesized by rat glomerular mesangial cells and is required for PDGF-induced mesangial cell migration. Here the authors show that Lama4-/- mice have progressive glomerular and tubulointerstitial fibrosis. These mice have a significant increase in the expression of platelet-derived growth factor (PDGF)-BB, PDGF-DD, and PDGF receptor beta in association with immature glomerular and peritubular capillaries. In addition, mesangial cell exposure to alpha4-containing laminins results in the down-regulation of PDGF receptor mRNA and protein, suggesting a direct effect of LN411/LN421 on vessel maturation. These data suggest that the failure of laminin alpha4-mediated down-regulation of PDGF activity contributes to the progressive renal lesions in this animal model.
Exp Cell Res. 2012
Like all basement membranes, glomerular basement membrane (GBM) consists mainly of laminin, type IV collagen, nidogen, and heparan sulfate proteoglycan. However, the GBM is unusually thick and contains particular members of these general protein families, including laminin-521, collagen α3α4α5(IV), and agrin. Knockout studies in mice and genetic findings in humans show that the laminin and type IV collagen components are particularly important for GBM structure and function, as laminin or collagen IV gene mutations cause filtration defects and renal disease of varying severities, depending on the nature of the mutations.
Yuosif L., Russo J.D., Sorokin L.
Cell Adh Migr., 2013
α4 and α5 chain laminins are the predominant isoforms in the basal lamina of vascular endothelial cells. While the α4 chain is expressed ubiquitously throughout different developmental stages, the prominent expression of α5 chain appears postnatally and its distribution varies with vessel type.