End stage kidney disease affects hundreds of thousands of patients in the United States. The therapy of choice is kidney replacement, but availability of organs is limited, and alternative sources of tissue are needed. Generation of new kidney tissue in the laboratory has been made possible through pluripotent cell reprogramming and directed differentiation. In current procedures, aggregates of cells known as organoids are grown either submerged or at the air‐liquid interface. These studies have demonstrated that kidney tissue can be generated from pluripotent stem cells, but they also identify limitations. The first is that perfusion of cell aggregates is limited, restricting the size to which they can be grown. The second is that aggregates lack the structural integrity required for convenient engraftment and suturing or adhesion to regions of kidney injury. In this study, we evaluated the capacity of silk to serve as a support for the growth and differentiation of kidney tissue from primary cells and from human induced pluripotent stem cells. We find that cells can differentiate to epithelia characteristic of the developing kidney on this material and that these structures are maintained following engraftment under the capsule of the adult kidney. Blood vessel investment can be promoted by the addition of vascular endothelial growth factor to the scaffold, but the proliferation of stromal cells within the graft presents a challenge, which will require some readjustment of cell growth and differentiation conditions. In summary, we find that silk can be used to support growth of stem cell derived kidney tissue.
Atherosclerosis is the most common cause of heart disease and stroke. The use of animal models has advanced our understanding of the molecular signaling that contributes to atherosclerosis. Further understanding of this degenerative process in humans will require human tissue. Plaque removed during endarterectomy procedures to relieve arterial obstructions is usually discarded, but can be an important source of diseased cells. Resected tissue from carotid and femoral endarterectomy procedures were compared with carotid arteries from donors with no known cardiovascular disease. Vascular smooth muscle cells (SMC) contribute to plaque formation and may determine susceptibility to rupture. Notch signaling is implicated in the progression of atherosclerosis, and plays a receptor-specific regulatory role in SMC. We defined protein localization of Notch2 and Notch3 within medial and plaque SMC using immunostaining, and compared Notch2 and Notch3 levels in total plaques with whole normal arteries using immunoblot. We successfully derived SMC populations from multiple endarterectomy specimens for molecular analysis. To better define the protein signature of diseased SMC, we utilized sequential window acquisition of all theoretical spectra (SWATH) proteomic analysis to compare normal carotid artery SMC with endarterectomy-derived SMC. Similarities in protein profile and differentiation markers validated the SMC identity of our explants. We identified a subset of differentially expressed proteins that are candidates as functional markers of diseased SMC. To understand how Notch signaling may affect diseased SMC, we performed Jagged1 stimulation of primary cultures. In populations that displayed significant growth, Jagged1 signaling through Notch2 suppressed proliferation; cultures with low growth potential were non-responsive to Jagged1. In addition, Jagged1 did not promote contractile smooth muscle actin nor have a significant effect on the mature differentiated phenotype. Thus, SMC derived from atherosclerotic lesions show distinct proteomic profiles and have altered Notch signaling in response to Jagged1 as a differentiation stimulus, compared with normal SMC.
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