Biology subjects

LaPointe, V.

Publications and source records attributed to LaPointe, V..

6 recordsLinked to original sources

bioRxiv2023

Ultrastructural comparison of human kidney organoids and human fetal kidneys reveals features of hyperglycemic culture

Induced pluripotent stem cell (iPSC)-derived kidney organoids have the potential for a large variety of applications. However, they do not persist long in culture, for which reasons are still unclear. Furthermore, their morphological maturation, an essential feature for kidney function, has not been sufficiently assessed. Kidney organoids are transcriptionally much alike end-of-first-trimester fetal kidneys and present many of the same cell types. From large transmission electron microscopy tilescans of specific regions of interest, we compared the ultrastructures of iPSC-derived kidney organoids at various timepoints to human fetal kidneys of the first trimester. Unlike healthy fetal kidneys, large glycogen deposits developed over time in all organoid cell types, but particularly in podocytes and in chondrocytes, one of the off-target populations that contaminate the culture. Deeper investigation showed that glycogen synthase kinase 3b (GSK3{beta}) levels and activation were diminished over time, correlated with the accumulation of glycogen. Activated YAP was strongly expressed and large lipid droplets accumulated over time in proximal tubules. Accordingly, EGFR signaling increased significantly over time. Mitochondria in glomeruli and tubules contained few or no cristae, indicating mitochondrial damage. Together these features are known for hyperglycemic cultures and diabetic nephropathy. Measuring the glucose concentration in the kidney organoid culture medium showed a concentration of 2.94 g/mL, which is considered an elevated, pre-diabetic-like concentration in vitro. In summary, our ultrastructural assessment of iPSC-derived kidney organoids using an age-matched fetal human reference allowed the evaluation of cellular morphology, and we identified intracellular features that can inform the cellular state, which is particularly important while physiological testing of organoids is limited. Translational StatementKidney organoids hold promise as a future treatment for patients with end-stage kidney disease. The engineering of kidney organoids with correct and healthy morphology in vitro is therefore essential, to guarantee functionality after transplantation. The present study provided deeper insights into the structural organization and ultrastructure of cells in kidney organoids compared to age-matched human fetal kidneys. Accordingly, we found several features in the regular kidney organoid culture, which are known to occur in hyperglycemic cultures and diabetic nephropathy, indicating that the current medium composition may be inducing pathological cellular phenotypes. This study therefore creates a better understanding of current limitations in the kidney organoid culture, increases knowledge of their function and cellular organization, and sets the foundation for further research to create advanced organoids.

bioengineering↗

bioRxiv2023

Structural development of the human fetal kidney: new stages and cellular dynamics in nephrogenesis

Research on the ultrastructural development of the kidney is limited, and research on rodent kidneys prevails. Yet, large differences between rodent and human nephrogenesis exist and therefore translation between species is not desirable. At the same time, there is an increasing demand for human research, in addition to assessing the potential of novel therapies such as renal organoids. We therefore generated an interactive atlas of large transmission electron microscopy tile scans of first trimester human kidneys, specifically Carnegie stage 20 until post-conceptional week 12. Analysis identified key ultrastructural features of proximal and distal progenitor cells such as cell shape, microvilli and luminal budding in the renal vesicle. Regarding glomerular development, we identified a new W-shaped body stage and three distinct sub-stages of the well-known capillary loop stage. Chromatin organization, nuclear shape and location were used to describe tubule cell identity and maturity, indicating a specific order of tubular maturation. The greatest congruence with adult tissue was seen in proximal tubules and the least in distal tubules. Finally, cytoplasmic glycogen depositions in collecting duct cells, which are absent in adult tissue, were found to be an early feature distinguishing distal tubules from collecting ducts as well as differentiating cortical from medullary collecting ducts. The findings of this research provide new fundamental insights for researchers who aim to understand and recreate kidney development.

developmental biology↗

bioRxiv2023

A Single-Cell RNA-Seq Analysis Unravels The Heterogeniety Of Primary Cultured Human Corneal Endothelial Cells

The primary culture of donor-derived human corneal endothelial cells (CECs) is a promising cell therapy. It confers the potential to treat multiple patients from a single donor, alleviating the global donor shortage. Nevertheless, this approach has limitations preventing its adoption, particularly culture protocols allow limited expansion of CECs and there is a lack of clear parameters to identify therapy-grade CECs. To address this limitation, a better understanding of the molecular changes arising from the primary culture of CECs is required. Using single- cell RNA sequencing on primary cultured CECs, we identify their variable transcriptomic fingerprint at the single cell level, provide a pseudo temporal reconstruction of the changes arising from primary culture, and suggest markers to assess the quality of primary CEC cultures. This research depicts a deep transcriptomic understanding of the cellular heterogeneity arising from the primary expansion of CECs and sets the basis for further improvement of culture protocols and therapies.

cell biology↗

bioRxiv2022

COMPUTATIONAL EVIDENCE FOR MULTI-LAYER CROSSTALK BETWEEN THE CADHERIN-11 AND PDGFR PATHWAYS

Various cell surface receptors play an important role in the differentiation and self-renewal of human mesenchymal stem cells (hMSCs). One example of such receptors are the cadherins, which maintain cell-cell adhesion and mechanically couple cells together. Recently, cadherin-11, which is a member of the type II classical cadherin family, has been shown to be involved in the fate commitment of hMSCs. Interestingly, cadherin-11 has no known intrinsic signaling activity and is thought to affect cell behavior via interactions with other cell surface receptors. Members of the platelet-derived growth factor receptor (PDGFR) family are hypothesized to be one of the interaction partners of cadherin-11. Experiments confirmed that PDGFR- binding to extracellular cadherin-11 regions increases the PDGFR- activity, whereas the interaction between PDGFR-{beta} and cadherin-11 suppresses the activity of the growth factor receptor. Cadherin-11 knockdown experiments also decreased cell proliferation. These interactions between cadherin-11 and PDGFRs indicate a crosstalk between these receptors and their downstream signaling activities but the nature of this crosstalk is not entirely known. In this study, we used a computational model to represent the experimentally proven interactions between cadherin-11 and the two PDGFRs and we inspected whether the crosstalk also exists downstream of the signaling initiated by the two receptor families. The computational framework allowed us to monitor the relative activity levels of each protein in the network. We performed model simulations to mimic the conditions of previous cadherin-11 knockdown experiments and to predict the effect of crosstalk on cell proliferation. Overall, our predictions suggest the existence of another layer of crosstalk, namely between {beta}-catenin (downstream to cadherin-11) and an ERK inhibitor protein (e.g. DUSP1), different than the crosstalk at the receptor level between cadherin-11 and PDGFR- and -{beta}. By investigating the multi-level crosstalk between cadherin and PDGFRs computationally, this study contributes to an improved understanding of the effect of cell surface receptors on hMSCs proliferation.

systems biology↗

bioRxiv2021

Enhanced microvasculature formation and patterning in iPSC-derived kidney organoids cultured in physiological hypoxia

Functional kidney organoids have the potential to be used in implantable kidney grafts for patients with end-stage kidney disease, because they have been shown to self-organize from induced pluripotent stem cells into most important renal structures. To date, however, long-term kidney organoid culture has not succeeded, as nephrons lose their phenotype after approximately 25 days. Furthermore, the renal structures remain immature with diminishing endothelial networks with low connectivity and limited organoid invasion. We hypothesized that introducing long-term culture at physiological hypoxia, rather than the normally applied non-physiological, hyperoxic 21% O2, could initiate angiogenesis, lead to enhanced growth factor expression and improve the endothelial patterning. We therefore cultured the kidney organoids at 7% O2 instead of 21% O2 for up to 25 days and evaluated nephrogenesis, VEGF-A expression and vascularization. Whole mount imaging revealed a homogenous morphology of the endothelial network with enhanced sprouting and interconnectivity when the kidney organoids were cultured in hypoxia. Three-dimensional quantification confirmed that the hypoxic culture led to an increased average vessel length, likely due to the observed upregulation of proangiogenic VEGF-A189 mRNA and downregulation of the antiangiogenic protein VEGF-A165b measured in hypoxia. This research indicates the importance of optimization of oxygen availability in organoid systems and the potential of hypoxic culture conditions in improving the vascularization of organoids. Significance statementCulturing kidney organoids in a hypoxic environment improved patterning of the endothelial network and improved vascularization. These findings may help improve the quality of kidney organoids, and could eventually improve the kidney graft for transplantation in patients with end-stage kidney disease. Furthermore, the organoids will be more suitable for drug testing and in developmental biology. The findings might also be translatable to other organoid models containing endothelial cells. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=199 SRC="FIGDIR/small/473849v1_ufig1.gif" ALT="Figure 1"> View larger version (61K): org.highwire.dtl.DTLVardef@d5cde7org.highwire.dtl.DTLVardef@c0ff46org.highwire.dtl.DTLVardef@b76191org.highwire.dtl.DTLVardef@166823d_HPS_FORMAT_FIGEXP M_FIG C_FIG

cell biology↗

bioRxiv2021

The response of three-dimensional pancreatic alpha and beta cell co-cultures to oxidative stress

The pancreatic islets of Langerhans have low endogenous antioxidant levels and are thus especially sensitive to oxidative stress, which is known to influence cell survival and behaviour. As bioengineered islets are gaining interest for therapeutic purposes, it is important to understand how their composition can be optimized to diminish oxidative stress. We investigated how the ratio of the two main islet cell types (alpha and beta cells) and their culture in three-dimensional aggregates could protect against oxidative stress. Monolayer and aggregate cultures were established by seeding the alphaTC1 (alpha) and INS1E (beta) cell lines in varying ratios, and hydrogen peroxide was applied to induce oxidative stress. Viability, oxidative stress, and the level of the antioxidant glutathione were measured. Both aggregation and an increasing prevalence of INS1E cells in the co-cultures conferred greater resistance to cell death induced by oxidative stress. Increasing the prevalence of INS1E cells also decreased the number of alphaTC1 cells experiencing oxidative stress in the monolayer culture. In 3D aggregates, culturing the alphaTC1 and INS1E cells in a ratio of 50:50 prevented oxidative stress in both cell types. Together, the results of this study lead to new insight into how modulating the composition and dimensionality of a co-culture can influence the oxidative stress levels experienced by the cells.

cell biology↗