Pluripotent stem cell-derived kidney organoids hold great promise as a potential auxiliary transplant tissue for individuals with end-stage renal disease and as a platform for studying kidney... Show morePluripotent stem cell-derived kidney organoids hold great promise as a potential auxiliary transplant tissue for individuals with end-stage renal disease and as a platform for studying kidney diseases and drug discovery. To establish accurate models, it is crucial to thoroughly characterize the morphological features and maturation stages of the cellular components within these organoids. Nephrons, the functional units of the kidney, possess distinct morphological structures that directly correlate with their specific functions. High spatial resolution imaging emerges as a powerful technique for capturing ultrastructural details that may go unnoticed with other methods such as immunofluorescent imaging and scRNA sequencing. In our study, we have applied software capable of seamlessly stitching virtual slides generated from electron microscopy, resulting in high-definition overviews of tissue slides. With this technology, we can comprehensively characterize the development and maturation of kidney organoids when transplanted under the renal capsule of mice. These organoids exhibit advanced ultrastructural developments upon transplantation, including the formation of the filtration barrier in the renal corpuscle, the presence of microvilli in the proximal tubule, and various types of cell sub-segmentation in the connecting tubule similarly to those seen in the adult kidney. Such ultrastructural characterization provides invaluable insights into the structural development and functional morphology of nephron segments within kidney organoids and how to advance them by interventions such as a transplantation. Show less
Wiersma, L.E.; Avramut, M.C.; Koster, A.J.; Berg, C.W. van den; Rabelink, T.J. 2023
Pluripotent stem cell-derived kidney organoids hold great promise as a potential auxiliary transplant tissue for individuals with end-stage renal disease and as a platform for studying kidney... Show morePluripotent stem cell-derived kidney organoids hold great promise as a potential auxiliary transplant tissue for individuals with end-stage renal disease and as a platform for studying kidney diseases and drug discovery. To establish accurate models, it is crucial to thoroughly characterize the morphological features and maturation stages of the cellular components within these organoids. Nephrons, the functional units of the kidney, possess distinct morphological structures that directly correlate with their specific functions. High spatial resolution imaging emerges as a powerful technique for capturing ultrastructural details that may go unnoticed with other methods such as immunofluorescent imaging and scRNA sequencing. In our study, we have applied software capable of seamlessly stitching virtual slides generated from electron microscopy, resulting in high-definition overviews of tissue slides. With this technology, we can comprehensively characterize the development and maturation of kidney organoids when transplanted under the renal capsule of mice. These organoids exhibit advanced ultrastructural developments upon transplantation, including the formation of the filtration barrier in the renal corpuscle, the presence of microvilli in the proximal tubule, and various types of cell sub-segmentation in the connecting tubule similarly to those seen in the adult kidney. Such ultrastructural characterization provides invaluable insights into the structural development and functional morphology of nephron segments within kidney organoids and how to advance them by interventions such as a transplantation. Research Highlights High-resolution imaging is crucial to determine morphological maturation of hiPSC-derived kidney organoids. Upon transplantation, refined ultrastructural development of nephron segments was observed, such as the development of the glomerular filtration barrier. Show less
Gaykema, L.H.; Nieuwland, R.Y. van; Lievers, E.; Moerkerk, W.B.J.; Klerk, J.A. de; Dumas, S.J.; ... ; Rabelink, T.J. 2023
Immune evasive induced pluripotent stem cell (iPSC)-derived kidney organoids, known as “stealth” organoids, hold promise for clinical transplantation. To address immune rejection, we investigated... Show moreImmune evasive induced pluripotent stem cell (iPSC)-derived kidney organoids, known as “stealth” organoids, hold promise for clinical transplantation. To address immune rejection, we investigated the impact of genetically modifying human leukocyte antigen (HLA) class I in kidney organoids prior to transplantation. By using CRISPR-Cas9, we successfully knocked out beta-2-microglobulin (B2M), resulting in iPSCs devoid of HLA class I surface expression. In vitro, the B2M knockout protected kidney organoids derived from these iPSCs against T-cell rejection. To assess in vivo protection, unmodified (control) and B2M–/– kidney organoids were transplanted into humanized mice engrafted with human peripheral blood mononuclear cells (PBMCs). Successful engraftment of human PBMCs was confirmed, and after 4 weeks, we observed no discernible difference in the infiltration rate, proliferation, or cytotoxicity of CD4+ and CD8+ T cells between control and B2M–/– organoids. Both groups of organoids showed compromised tissue integrity, displaying tubulitis and loss of tubule integrity. Notably, while B2M–/– organoids failed to express HLA class I on their cell surface, there was preexisting expression of HLA class II in both control and B2M–/– organoids transplanted into mice with human PBMCs. HLA class II expression was not limited to antigen-presenting cells but also evident in epithelial cells of the kidney organoid, posing an additional immunological challenge to its transplantation. Consequently, we conclude that B2M knockout alone is insufficient to protect iPSC-derived kidney organoids from T-cell-mediated immune rejection. Additionally, our findings suggest that modulating HLA class II signaling will be necessary to prevent rejection following transplantation. Show less
Heart and kidney communicate with one another in an interdependent relationship and they influence each other's behavior reciprocally, as pathological changes in one organ can damage the other.... Show moreHeart and kidney communicate with one another in an interdependent relationship and they influence each other's behavior reciprocally, as pathological changes in one organ can damage the other. Although independent human in vitro models for heart and kidney exist, they do not capture their dynamic crosstalk. We have developed a microfluidic system which can be used to study heart and kidney interaction in vitro. Cardiac microtissues (cMTs) and kidney organoids (kOs) derived from human induced pluripotent stem cells (hiPSCs) were generated and loaded into two separated communicating chambers of a perfusion chip. Static culture conditions were compared with dynamic culture under unidirectional flow. Tissue viability was maintained for minimally 72 h under both conditions, as indicated by the presence of sarcomeric structures coupled with beating activity in cMTs and the presence of nephron structures and albumin uptake in kOs. We concluded that this system enables the study of human cardiac and kidney organoid interaction in vitro while controlling parameters like fluidic flow speed and direction. Together, this "cardiorenal-unit" provides a new in vitro model to study the cardiorenal axis and it may be further developed to investigate diseases involving both two organs and their potential treatments. Show less
The balance between safety and efficacy of T cell therapies re-mains challenging and T cell mediated toxicities have occurred. The stringent selection of tumor-specific targets and careful se... Show moreThe balance between safety and efficacy of T cell therapies re-mains challenging and T cell mediated toxicities have occurred. The stringent selection of tumor-specific targets and careful se-lection of tumor-specific T cells using T cell toxicity screenings are essential. In vitro screening options against vital organs or specialized cell subsets would be preferably included in preclin-ical pipelines, but options remain limited. Here, we set up preclinical models with human induced pluripotent stem cell (hiPSC)-derived cardiomyocytes, epicardial cells, and kid-ney organoids to investigate toxicity risks of tumor-specific T cells more thoroughly. CD8+T cells reactive against PRAME, HA-1H, CD20, or WT1, currently used or planned to be used in phase I/II clinical studies, were included. Using these hiPSC-derived preclinical models, we demonstrated that WT1-specific T cells caused on-target toxicity that correlated with target gene expression. Multiple measures of T cell reac-tivity demonstrated this toxicity on the level of T cells and hiPSC-derived target cells. In addition, phenotypic analysis illustrated interaction and crosstalk between infiltrated T cells and kidney organoids. In summary, we demonstrated the benefit of hiPSC-derived models in determining toxicity risks of tumor-specific T cells. Furthermore, our data empha-sizes the additional value of other measures of T cell reactivity on top of the commonly used cytokine levels. Show less
Essen, M.F. van; Peereboom, E.T.M.; Schlagwein, N.; Gijlswijk-Janssen, D.J. van; Nelemans, T.; Joeloemsingh, J.V.; ... ; Kooten, C. van 2023
Factor H is a pivotal complement regulatory protein that is preferentially produced by the liver and circulates in high concentrations in serum. There has been an increasing interest in the... Show moreFactor H is a pivotal complement regulatory protein that is preferentially produced by the liver and circulates in high concentrations in serum. There has been an increasing interest in the extrahepatic production of comple-ment factors, including by cells of the immune system, since this contributes to non-canonical functions of local complement activation and regulation. Here we investigated the production and regulation of factor H and its splice variant factor H-like protein 1 (FHL-1) by human myeloid cells. As validation, we confirmed the pre -dominant presence of intact factor H in serum, despite a strong but comparable mRNA expression of CFH and FHL1 in liver. Comparable levels of CFH and FHL1 were also observed in renal tissue, although a dominant staining for FHL-1 was shown within the proximal tubules. Human in vitro generated pro-and anti-inflammatory macrophages both expressed and produced factor H/FHL-1, but this was strongest in pro-inflammatory macro-phages. Production was not affected by LPS activation, but was increased upon stimulation with IFN-gamma or CD40L. Importantly, in both macrophage subsets mRNA expression of FHL1 was significantly higher than CFH. More -over, production of FHL-1 protein could be confirmed using precipitation and immunoblotting of culture su-pernatants. These data identify macrophages as producers of factor H and FHL-1, thereby potentially contributing to local complement regulation at sites of inflammation. Show less
Gaykema, L.H.; Nieuwland, R.Y. van; Dekkers, M.C.; Essen, M.F. van; Heidt, S.; Zaldumbide, A.; ... ; Kooten, C. van 2023
End stage renal disease is an increasing problem worldwide driven by aging of the population and increased prevalence of metabolic disorders and cardiovascular disease. Currently, kidney... Show moreEnd stage renal disease is an increasing problem worldwide driven by aging of the population and increased prevalence of metabolic disorders and cardiovascular disease. Currently, kidney transplantation is the only curative option, but donor organ shortages greatly limit its application. Regenerative medicine has the potential to solve the shortage by using stem cells to grow the desired tissues, like kidney tissue. Immune rejection poses a great threat towards the implementation of stem cell derived tissues and various strategies have been explored to limit the immune response towards these tissues. However, these studies are limited by targeting mainly T cell mediated immune rejection while the rejection process also involves innate and humoral immunity. In this study we investigate whether inhibition of the complement system in human induced pluripotent stem cells (iPSC) could provide protection from such immune injury. To this end we created knock-in iPSC lines of the membrane bound complement inhibitor CD55 to create a transplant-specific protection towards complement activation. CD55 inhibits the central driver of the complement cascade, C3 convertase, and we show that overexpression is able to decrease complement activation on both iPSCs as well as differentiated kidney organoids upon stimulation with anti-HLA antibodies to mimic the mechanism of humoral rejection. Show less
Understanding dynamic metabolic changes in complex biological samples often overlooks heterogeneity in cell composition. Wang et al. combine mass spectrometry imaging, isotope tracing, and... Show moreUnderstanding dynamic metabolic changes in complex biological samples often overlooks heterogeneity in cell composition. Wang et al. combine mass spectrometry imaging, isotope tracing, and multiplexed immunofluorescence microscopy to study metabolic dynamics in the kidney upon ischemia-reperfusion.A common drawback of metabolic analyses of complex biological samples is the inability to consider cell-to-cell heterogeneity in the context of an organ or tissue. To overcome this limitation, we present an advanced high-spatial-resolution metabolomics approach using matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI) combined with isotope tracing. This method allows mapping of cell-type-specific dynamic changes in central carbon metabolism in the context of a complex heterogeneous tissue architecture, such as the kidney. Combined with multiplexed immunofluorescence staining, this method can detect metabolic changes and nutrient partitioning in targeted cell types, as demonstrated in a bilateral renal ischemia-reperfusion injury (bIRI) experimental model. Our approach enables us to identify region-specific metabolic perturbations associated with the lesion and throughout recovery, including unexpected metabolic anomalies in cells with an apparently normal phenotype in the recovery phase. These findings may be relevant to an understanding of the homeostatic capacity of the kidney microenvironment. In sum, this method allows us to achieve resolution at the single-cell level in situ and hence to interpret cell-type-specific metabolic dynamics in the context of structure and metabolism of neighboring cells. Show less
Koning, M.; Dumas, S.J.; Avramut, M.C.; Koning, R.I.; Meta, E.; Lievers, E.; ... ; Rabelink, T.J. 2022
Human induced pluripotent stem cell-derived kidney organoids have potential for disease modeling and to be developed into clinically transplantable auxiliary tissue. However, they lack a functional... Show moreHuman induced pluripotent stem cell-derived kidney organoids have potential for disease modeling and to be developed into clinically transplantable auxiliary tissue. However, they lack a functional vasculature, and the sparse endogenous endothelial cells (ECs) are lost upon prolonged culture in vitro, limiting maturation and applicability. Here, we use intracoelomic transplantation in chicken embryos followed by single-cell RNA sequencing and advanced imaging platforms to induce and study vasculogenesis in kidney organoids. We show expansion of human organoid-derived ECs that reorganize into perfused capillaries and form a chimeric vascular network with host-derived blood vessels. Ligand-receptor analysis infers extensive potential interactions of human ECs with perivascular cells upon transplantation, enabling vessel wall stabilization. Perfused glomeruli display maturation and morphogenesis to capillary loop stage. Our findings demonstrate the beneficial effect of vascularization on not only epithelial cell types, but also the mesenchymal compartment, inducing the expansion of ' on target ' perivascular stromal cells, which in turn are required for further maturation and stabilization of the neo-vasculature. The here described vasculogenic capacity of kidney organoids will have to be deployed to achieve meaningful glomerular maturation and kidney morphogenesis in vitro. Show less
Gabbin, B.; Meraviglia, V.; Mummery, C.L.; Rabelink, T.J.; Meer, B.J. van; Berg, C.W. van den; Bellin, M. 2022
Heart and kidney diseases cause high morbidity and mortality. Heart and kidneys have vital functions in the human body and, interestingly, reciprocally influence each other's behavior: pathological... Show moreHeart and kidney diseases cause high morbidity and mortality. Heart and kidneys have vital functions in the human body and, interestingly, reciprocally influence each other's behavior: pathological changes in one organ can damage the other. Cardiorenal syndrome (CRS) is a group of disorders in which there is combined dysfunction of both heart and kidney, but its underlying biological mechanisms are not fully understood. This is because complex, multifactorial, and dynamic mechanisms are likely involved. Effective treatments are currently unavailable, but this may be resolved if more was known about how the disease develops and progresses. To date, CRS has actually only been modeled in mice and rats in vivo. Even though these models can capture cardiorenal interaction, they are difficult to manipulate and control. Moreover, interspecies differences may limit extrapolation to patients. The questions we address here are what would it take to model CRS in vitro and how far are we? There are already multiple independent in vitro (human) models of heart and kidney, but none have so far captured their dynamic organ-organ crosstalk. Advanced in vitro human models can provide an insight in disease mechanisms and offer a platform for therapy development. CRS represents an exemplary disease illustrating the need to develop more complex models to study organ-organ interaction in-a-dish. Human induced pluripotent stem cells in combination with microfluidic chips are one powerful tool with potential to recapitulate the characteristics of CRS in vitro. In this review, we provide an overview of the existing in vivo and in vitro models to study CRS, their limitations and new perspectives on how heart-kidney physiological and pathological interaction could be investigated in vitro for future applications. Show less
Wiersma, L.E.; Avramut, M.C.; Lievers, E.; Rabelink, T.J.; Berg, C.W. van den 2022
Background: The generation of human induced pluripotent stem cells (hiPSCs) has opened a world of opportunities for stem cell-based therapies in regenerative medicine. Currently, several human... Show moreBackground: The generation of human induced pluripotent stem cells (hiPSCs) has opened a world of opportunities for stem cell-based therapies in regenerative medicine. Currently, several human kidney organoid protocols are available that generate organoids containing kidney structures. However, these kidney organoids are relatively small ranging up to 0.13 cm(2) and therefore contain a small number of nephrons compared to an adult kidney, thus defying the exploration of future use for therapy. Method: We have developed a scalable, easily accessible, and reproducible protocol to increase the size of the organoid up to a nephron sheet of 2.5 cm(2) up to a maximum of 12.6 cm(2) containing a magnitude of nephrons. Results: Confocal microscopy showed that the subunits of the nephrons remain evenly distributed throughout the entire sheet and that these tissue sheets can attain similar to 30,000-40,000 glomerular structures. Upon transplantation in immunodeficient mice, such nephron sheets became vascularized and matured. They also show reuptake of injected low-molecular mass dextran molecules in the tubular structures, indicative of glomerular filtration. Furthermore, we developed a protocol for the cryopreservation of intermediate mesoderm cells during the differentiation and demonstrate that these cells can be successfully thawed and recovered to create such tissue sheets. Conclusion: The scalability of the procedures, and the ability to cryopreserve the cells during differentiation are important steps forward in the translation of these differentiation protocols to future clinical applications such as transplantable auxiliary kidney tissue. Show less
Differentiated kidney organoids from induced pluripotent stem cells hold promise as a treatment for patients with kidney diseases. Before these organoids can be translated to the clinic,... Show moreDifferentiated kidney organoids from induced pluripotent stem cells hold promise as a treatment for patients with kidney diseases. Before these organoids can be translated to the clinic, shortcomings regarding their cellular and extracellular compositions, and their developmental plateau need to be overcome. We performed a proteomic analysis on kidney organoids cultured for a prolonged culture time and we found a specific change in the extracellular matrix composition with increased expression of types 1a1, 2 and 6a1 collagen. Such an excessive accumulation of specific collagen types is a hallmark of renal fibrosis that causes a life-threatening pathological condition by compromising key functions of the human kidney. Here we hypothesized the need for a threedimensional environment to grow the kidney organoids, which could better mimic the in vivo surroundings of the developing kidney than standard culture on an air-liquid interface. Encapsulating organoids for four days in a soft, thiol-ene cross-linked alginate hydrogel resulted in decreased type 1a1 collagen expression. Furthermore, the encapsulation did not result in any changes of organoid structural morphology. Using a biomaterial to modulate collagen expression allows for a prolonged kidney organoid culture in vitro and a reduction of abnormal type 1a1 collagen expression bringing kidney organoids closer to clinical application. Show less
Tiemeier, G.L.; Koning, R. de; Wang, G.Q.; Kostidis, S.; Rietjens, R.G.J.; Sol, W.M.P.J.; ... ; Rabelink, T.J. 2020
Differentiation of human-induced pluripotent stem cells (hiPSCs) into vascular endothelium is of great importance to tissue engineering, disease modeling, and use in regenerative medicine. Although... Show moreDifferentiation of human-induced pluripotent stem cells (hiPSCs) into vascular endothelium is of great importance to tissue engineering, disease modeling, and use in regenerative medicine. Although differentiation of hiPSCs into endothelial-like cells (hiPSC-derived endothelial cells [hiPSC-ECs]) has been demonstrated before, controversy exists as to what extent these cells faithfully reflect mature endothelium. To address this issue, we investigate hiPSC-ECs maturation by their ability to express von Willebrand factor (VWF) and formation of Weibel-Palade bodies (WPBs). Using multiple hiPSCs lines, hiPSC-ECs failed to form proper VWF and WPBs, essential for angiogenesis, primary and secondary homeostasis. Lowering the increased intracellular pH (pHi) of hiPSC-ECs with acetic acid did result in the formation of elongated WPBs. Nuclear magnetic resonance data showed that the higher pHi in hiPSC-ECs occurred in association with decreased intracellular lactate concentrations. This was explained by decreased glycolytic flux toward pyruvate and lactate in hiPSC-ECs. In addition, decreased expression of monocarboxylate transporter member 1, a member of the solute carrier family (SLC16A1), which regulates lactate and H+ uptake, contributed to the high pHi of hiPSC-EC. Mechanistically, pro-VWF dimers require the lower pH environment of the trans-Golgi network for maturation and tubulation. These data show that while hiPSC-ECs may share many features with mature EC, they are characterized by metabolic immaturity hampering proper EC function. Show less
Berg, C.W. van den; Koudijs, A.; Ritsma, L.; Rabelink, T.J. 2020
Significance StatementThe ability to differentiate human induced pluripotent stem cells to kidney organoids in vitro holds promise for disease modeling, drug discovery, and clinical application.... Show moreSignificance StatementThe ability to differentiate human induced pluripotent stem cells to kidney organoids in vitro holds promise for disease modeling, drug discovery, and clinical application. The authors differentiated such cells to kidney tissue comprising glomerular, proximal, and distal tubular structures. Earlier research demonstrated that these structures become vascularized upon transplantation in mice and show advanced maturation. To investigate whether human induced pluripotent stem cell?derived kidney organoids can also become functional in vivo, they applied high-resolution intravital multiphoton imaging through a titanium imaging window. They demonstrated in vivo glomerular filtration and size-selective glomerular barrier function in the transplanted organoids. This technique can be instrumental for further developing stem cell?derived organoids toward clinical applications. BackgroundThe utility of kidney organoids in regenerative medicine will rely on the functionality of the glomerular and tubular structures in these tissues. Recent studies have demonstrated the vascularization and subsequent maturation of human pluripotent stem cell?derived kidney organoids after renal subcapsular transplantation. This raises the question of whether the glomeruli also become functional upon transplantation.MethodsWe transplanted kidney organoids under the renal capsule of the left kidney in immunodeficient mice followed by the implantation of a titanium imaging window on top of the kidney organoid. To assess glomerular function in the transplanted human pluripotent stem cell?derived kidney tissue 1, 2, and 3 weeks after transplantation, we applied high-resolution intravital multiphoton imaging through the imaging window during intravenous infusion of fluorescently labeled low and high molecular mass dextran molecules or albumin.ResultsAfter vascularization, glomerular structures in the organoid displayed dextran and albumin size selectivity across their glomerular filtration barrier. We also observed evidence of proximal tubular dextran reuptake.ConclusionsOur results demonstrate that human pluripotent stem cell?derived glomeruli can develop an appropriate barrier function and discriminate between molecules of varying size. These characteristics together with tubular presence of low molecular mass dextran provide clear evidence of functional filtration. This approach to visualizing glomerular filtration function will be instrumental for translation of organoid technology for clinical applications as well as for disease modeling. Show less
Berg, C.W. van den; Koudijs, A.; Ritsma, L.; Rabelink, T.J. 2020
Background The utility of kidney organoids in regenerative medicine will rely on the functionality of the glomerular and tubular structures in these tissues. Recent studies have demonstrated the... Show moreBackground The utility of kidney organoids in regenerative medicine will rely on the functionality of the glomerular and tubular structures in these tissues. Recent studies have demonstrated the vascularization and subsequent maturation of human pluripotent stem cell–derived kidney organoids after renal subcapsular transplantation. This raises the question of whether the glomeruli also become functional upon transplantation.Methods We transplanted kidney organoids under the renal capsule of the left kidney in immunodeficient mice followed by the implantation of a titanium imaging window on top of the kidney organoid. To assess glomerular function in the transplanted human pluripotent stem cell–derived kidney tissue 1, 2, and 3 weeks after transplantation, we applied high-resolution intravital multiphoton imaging through the imaging window during intravenous infusion of fluorescently labeled low and high molecular mass dextran molecules or albumin.Results After vascularization, glomerular structures in the organoid displayed dextran and albumin size selectivity across their glomerular filtration barrier. We also observed evidence of proximal tubular dextran reuptake.Conclusions Our results demonstrate that human pluripotent stem cell–derived glomeruli can develop an appropriate barrier function and discriminate between molecules of varying size. These characteristics together with tubular presence of low molecular mass dextran provide clear evidence of functional filtration. This approach to visualizing glomerular filtration function will be instrumental for translation of organoid technology for clinical applications as well as for disease modeling. Show less
Koning, M.; Berg, C.W. van den; Rabelink, T.J. 2019
Kidney organoids can be generated from human pluripotent stem cells (PSCs) using protocols that resemble the embryonic development of the kidney. The renal structures thus generated offer great... Show moreKidney organoids can be generated from human pluripotent stem cells (PSCs) using protocols that resemble the embryonic development of the kidney. The renal structures thus generated offer great potential for disease modeling, drug screening, and possibly future therapeutic application. At the same time, use of these PSC-derived organoids is hampered by lack of maturation and off-target differentiation. Here, we review the main protocols for the generation of kidney organoids from human-induced PSCs, discussing their advantages and limitations. In particular, we will focus on the vascularization of the kidney organoids, which appears to be one of the critical factors to achieve maturation and functionality of the organoids. Show less
Human induced pluripotent stem cells (hiPSCs) are used to study organogenesis and model disease as well as being developed for regenerative medicine. Endothelial cells are among the many cell types... Show moreHuman induced pluripotent stem cells (hiPSCs) are used to study organogenesis and model disease as well as being developed for regenerative medicine. Endothelial cells are among the many cell types differentiated from hiPSCs, but their maturation and stabilization fall short of that in adult endothelium. We examined whether shear stress alone or in combination with pericyte co-culture would induce flow alignment and maturation of hiPSC-derived endothelial cells (hiPSC-ECs) but found no effects comparable with those in primary microvascular ECs. In addition, hiPSC-ECs lacked a luminal glycocalyx, critical for vasculature homeostasis, shear stress sensing, and signaling. We noted, however, that hiPSC-ECs have dysfunctional mitochondrial permeability transition pores, resulting in reduced mitochondrial function and increased reactive oxygen species. Closure of these pores by cyclosporine A improved EC mitochondrial function but also restored the glycocalyx such that alignment to flow took place. These results indicated that mitochondrial maturation is required for proper hiPSC-EC functionality. Show less
Human induced pluripotent stem cells (hiPSCs) are used to study organogenesis and model disease as well as being developed for regenerative medicine. Endothelial cells are among the many cell types... Show moreHuman induced pluripotent stem cells (hiPSCs) are used to study organogenesis and model disease as well as being developed for regenerative medicine. Endothelial cells are among the many cell types differentiated from hiPSCs, but their maturation and stabilization fall short of that in adult endothelium. We examined whether shear stress alone or in combination with pericyte co-culture would induce flow alignment and maturation of hiPSC-derived endothelial cells (hiPSC-ECs) but found no effects comparable with those in primary microvascular ECs. In addition, hiPSC-ECs lacked a luminal glycocalyx, critical for vasculaturehomeostasis, shear stress sensing, and signaling. We noted, however, that hiPSC-ECs have dysfunctional mitochondrial permeability transition pores, resulting in reduced mitochondrial function and increased reactive oxygen species. Closure of these pores by cyclosporine A improved EC mitochondrial function but also restored the glycocalyx such that alignment to flow took place. These results indicated that mitochondrial maturation is required for proper hiPSC-EC functionality. Show less
Leuning, D.G.; Witjas, F.M.R.; Maanaoui, M.; Graaf, A.M.A. de; Lievers, E.; Geuens, T.; ... ; Rabelink, T.J. 2019
The bioengineering of a replacement kidney has been proposed as an approach to address the growing shortage of donor kidneys for the treatment of chronic kidney disease. One approach being... Show moreThe bioengineering of a replacement kidney has been proposed as an approach to address the growing shortage of donor kidneys for the treatment of chronic kidney disease. One approach being investigated is the recellularization of kidney scaffolds. In this study, we present several key advances toward successful re-endothelialization of whole kidney matrix scaffolds from both rodents and humans. Based on the presence of preserved glycosoaminoglycans within the decelullarized kidney scaffold, we show improved localization of delivered endothelial cells after preloading of the vascular matrix with vascular endothelial growth factor and angiopoietin 1. Using a novel simultaneous arteriovenous delivery system, we report the complete re-endothelialization of the kidney vasculature, including the glomerular and peritubular capillaries, using human inducible pluripotent stem cell - derived endothelial cells. Using this source of endothelial cells, it was possible to generate sufficient endothelial cells to recellularize an entire human kidney scaffold, achieving efficient cell delivery, adherence, and endothelial cell proliferation and survival. Moreover, human re-endothelialized scaffold could, in contrast to the non-re-endothelialized human scaffold, be fully perfused with whole blood. These major advances move the field closer to a human bioengineered kidney. Show less
Witjas, F.M.R.; Berg, B.M. van den; Berg, C.W. van den; Engelse, M.A.; Rabelink, T.J. 2019
All tissues are surrounded by a mixture of noncellular matrix components, that not only provide physical and mechanical support to cells, but also mediate biochemical signaling between cells. The... Show moreAll tissues are surrounded by a mixture of noncellular matrix components, that not only provide physical and mechanical support to cells, but also mediate biochemical signaling between cells. The extracellular matrix (ECM) of endothelial cells, also known as the perivascular matrix, forms an organ specific vascular niche that orchestrates mechano-, growth factor, and angiocrine signaling required for tissue homeostasis and organ repair. This concise review describes how this perivascular ECM functions as a signaling platform and how this knowledge can impact the field of regenerative medicine, for example, when designing artificial matrices or using decellularized scaffolds from organs. Stem Cells Translational Medicine 2019;8:375-382 Show less
