3D Bioprinting of Vascularized, Convoluted Renal Proximal Tubules
We aim to build a 3D proximal tubule model that contains confluent epithelium, convoluted geometry, and controlled, physiologically relevant perfusion through an open lumen that is embedded within engineered ECM. Using this model, we will study the effect of proximal tubule geometry on epithelial cell structure and function to determine the optimal curvature, degree of convolution, and tubule diameter. Ultimately, we plan to create vascularized, convoluted proximal tubule models and study vectorial transport through the interstitium (ECM) between the endothelial and epithelial tubules.
Adult Progenitors in Kidney Tubulointerstitium
We are interested in the capacities of adult kidney progenitor cells to model the kidney interstitium. Our project focuses on two such populations, a resident mesenchymal stem cell population characterized by expression of Gli1, and dedifferentiated tubular epithelial cells defined by expression of Havcr-1. We will isolate these cell types, define their differentiation capacity in vitro, and coculture them in collaboration with other RBK investigators to model the kidney interstitium in 3D.
Application of Progenitor Niche Signals to Ex Vivo Nephrogenesis
In this multi-PI project we aim to understand how the three major cell types of the developing kidney can be integrated on a scaffold to reproduce key features of the kidney. Summaries of the 4 focus areas within the project are provided below. These lines of investigation are being pursued in parallel, with the 4 participating laboratories acting as a single integrated research group with the unified goal of developing engraftable laboratory-grown kidney tissue.
Focus on: Stromal Biology
Optimal kidney functions requires complex patterning of both the nephrons and blood vessels. We study how the distinct interstitial microenvironments that exist within the embryonic and adult kidney affect the development and function of this organ. Our ultimate goal is to define the stromal signals that promote the growth of 3-D, physiologically responsive nephrons with integrated vasculature in biological scaffolds.
Focus on: Vascular Biology
Nascent blood vessels develop in a coordinated manner with kidney nephrons. We aim to establish a molecular signature for endothelial cells (ECs) in the developing kidney, from nephron stem cell generation to nephron tubule differentiation, throughout embryonic development and into adulthood. Preliminary observations reveal distinct heterogeneity in EC gene expression in the developing kidney; however its functional impact is unknown. We will define when and where blood vessels appear during nephron formation, distinguishing vasculogenic versus angiogenic events. We will test necessity and sufficiency of endothelial signals on nephron progenitor self-renewal versus differentiation. We will also determine whether specific, regionally expressed factors play functional roles in either helping to sustain progenitors or trigger NPC expansion versus differentiation.
Focus on: Biomaterials
We focus on the design and study of 3D scaffolds to support kidney cell and tissue needs. Silk protein scaffolds provide our framework due to the versatility and utility towards cell and tissue goals, including porous features for transport, tunable mechanical properties, support for long term tissue growth, controlled/slow degradation and an absence of cell-specific epitopes for signaling. We will use silk biomaterial scaffold systems to provide structure and, with the addition of appropriate ECM components, signaling cues for nephrogenesis via cells from our project collaborators (Leif, Tom, Ondine). The goal is to utilize these scaffolds to engineer modular, compartmentalized systems that support and guide nephron progenitor cell (NPC) maintenance and differentiation, and cellular signaling between stromal cells, cells of the vasculature, and NPCs.
Focus on: Nephron Progenitor Biology
We study the population dynamics of the nephron progenitor cell population with the aim of identifying the sub-populations that are best suited to new tissue formation. This in-depth study of the micro anatomy of the nephron progenitor cell niche involves understanding proliferation, adhesion, and signaling properties of the various nephron progenitor sub populations, defining their growth properties on biological scaffolds, and characterizing the influence of stromal and vascular cells on them.
Building Extracellular Matrix Scaffolds to Induce Nephron Development
Our goal is to develop nephron segments using 3D scaffolds as templates for pluripotent stem cells. This project uses extracellular matrix (ECM) to define scaffold-specific elements that drive cell differentiation. We use a high-throughput system of ECM scaffolds to screen conditions favoring differentiation. In this process we identify the requisite matrix-bound elements for differentiation and test the role of ECM remodeling by secondary cell types. Together, this system serves as a model for nephron development within a full-scale kidney scaffold and establishes the requirements for stem cell differentiation within a perfusion bioreactor system. The result of our systematic investigation is to decode critical factors involved in nephron reconstitution, important next steps in tubule repair and renal tissue regeneration.
Cellular Diversity in Human Nephrogenesis
The functional unit of the mammalian kidney is the nephron. Though there is a general appreciation for when and how different cell types of the nephron form from studies in the mouse, there is little understanding of these processes in the human kidney, and no rigorous accounting for the full range of mature cell types that underpin kidney function in either mouse or man. An understanding of the diversity of nephron cell types is essential for the goals of (Re)Building A Kidney Consortium where generating functional kidney structures is the consortium target. Further, individual cell types are likely targets for distinct disease features; as an example, mutations in genes producing podocyte specific gene products result in glomerular nephritis. We will combine two complementary strategies, MARIS (Method for Analyzing RNA following Intracellular Sorting) and Drop-seq, to obtain detailed comparative transcriptional profiles of mouse and human nephron progenitors and nephrons to address the question of cellular diversity.
Cytokines and Extracellular Matrix
Morphogenetic signals from the cell’s microenvironment play important roles in stem and progenitor cell differentiation and in multicellular morphogenesis, being driven by extracellular matrix (ECM) proteins and cytokines. We focus on how signaling from cytokines is modulated by the presence of and interactions with ECM proteins and how this modulates morphogenesis. This interaction leads to synergistic signaling between adhesion receptors and cytokine receptors. We develop molecular engineering approaches to create recombinant cytokine variants that bind the ECM with unusually high affinity, to enhance their translational potential. Recombinant ECM proteins can be built into biomaterials, or they can be assembled into a gel network to be used as a biomaterial matrix themselves. Our goal is to use these matrix and cytokine engineering approaches to support the multicellular morphogenesis that occurs between nephron progenitor cells, the developing microvasculature, and other stromal cells.
Developing Pro-regenerative Drug Therapies for Acute Kidney Injury
Acute kidney injury (AKI) is a major health problem and there are currently no effective treatments. We have identified a novel class of compounds (PTBA) that reduce AKI injury when administered days after the initiating injury. However, how PTBA prevents kidney injury remains unclear. Our goal is to use human kidney organoids generated from induced pluripotent stem cells (iPSCs) as a tool to understand the pro-regenerative mechanism-of-action of PTBA analogs during kidney injury and repair, and to further validate the kidney organoid system as a pre-clinical drug screening platform.
Differentiation of Fluorescent Reporter Human iPS Cell Lines for Monitoring Renal Cell Lineages
Our objective is to aid RBK studies of in vitro cell populations by generating kidney cell type specific human pluripotent stem cell (hPSC) reporter lines. Such cell lines will enable both the optimization of differentiation protocols tailored to achieve specific cell types, and comparisons to in vivo human kidney cell types that will advance our understanding of in vitro tissue development and maintenance. We will use CRISPR/Cas9 gene editing to create GFP knock-in cell lines, targeting genes expressed in discrete cell types and according to consortium needs. These lines will be designed to label nephron and stromal progenitors, podocytes, and proximal and distal tubules. Additionally, cell type transition points offer glimpses into the factors that drive differentiation. We will create three dual-label reporter lines to illuminate transitions between progenitor and advanced cell types including nephron progenitors, fibrotic interstitium, and the juxtaglomerular apparatus.
Differentiation of Human Pluripotent Stem Cells into Kidney Cell Lineages
Kidney function is based on the intricate interplay of a range of highly specialized cells. Thus, any approach in rebuilding the kidney depends on the generation of the right cell types in sufficient amounts and purity. This project will address this critical need. We will use state-of-the-art technology and a Quality-by-Design approach to guide a multistep differentiation process of human pluripotent stem cells obtaining pure populations of functional kidney cells. We will then characterize those cells for their ability to assemble into kidney-like structures and perform some of the critical functions executed by a healthy kidney.
Engineering Human Collecting System Reporter Pluripotent Stem Cell Lines
We propose to generate modified human pluripotent stem cell (hPSC) reporter lines with a genetically encoded activity sensor that tags key cell types important in development and function of the urinary collecting system. We will leverage an existing WTC11 hiPSC line that harbors GCaMP6 as an activity sensor thereby enhancing the functional utility of these lines by superimposing physiology onto development during or after a desired cell type or kidney organoid is formed. Multi-labeled reporter hiPSC cell lines of the collecting system in this proposal will enable RBK members to incorporate this essential component of the kidneys in efforts to optimize nephron formation, patterning and organization in scaffolds with the goal of making a kidney that maintains homeostasis and successfully expels urine.
Generating an Interconnected Kidney Tubule Architecture
To restore kidney function, filtering nephrons must connect to a tubule network to pass fluid. How cells rearrange and form new connections is not known. We are using the regenerating zebrafish kidney as a model to discover how tubule interconnections are made and to uncover signals that drive cell rearrangments required to "plumb" the kidney. Knowledge of these signals will be an important part of the molecular toolbox for growing new organs.
Generating kidney organoids and mature renal cell types via directed iPSC differentiation
We have previously shown that human kidney tissue can be generated from human pluripotent stem cells. Our project focuses on characterizing and optimizing this approach to improve tubular maturation and cellular function and generating reporter lines for the isolation of specific cell types. In the long term, human kidney tissue generated in this way may be used for drug screening, tissue regeneration or cell therapy.
Human Kidney Biopsy Single Cell Protocols and Analysis
Our partnership project aims to establish conditions for the transportation and storage of human kidney biopsies. These protocols will be optimized to ensure maximal cell viability and preservation of mRNA integrity. In the second phase of this project, we will compare single cell RNA sequencing technologies: FACS-seq and Drop-seq. Our goal is to determine which approach will be best adapted to the large-scale investigation of human kidney disease in large populations.
Identifying Kidney Cell Phenotype Factors Using Single Cell RNA Sequencing
The goal of the Penn project is to develop molecular biology and informatics tools to identify key regulatory factors that determine kidney cell phenotypes. Single cell resolution transcriptome data can reveal cryptic cell types as well as help identify regulatory systems related to cell phenotypes. We hypothesize that cell phenotype determining genes are often at low to moderate abundance in a cell, making them difficult to approach using current methods. The Penn group will develop novel molecular biology techniques to enrich single cell transcriptomes for low abundance genes. We will also develop informatics tools that will help identify targets for trans-differentiation experiments, leveraging single cell level measurements.
Molecular Characterization of Normal and Stem Cell Derived Kidney Cell Types
We will extend our hGUDMAP studies of the human fetal kidney to generate new resources for the RBK consortium. In a collaboration with ABCAM, we will characterize rabbit monoclonal antibodies for cross species (mouse and human) cell-type specificity in identifying key cell types of the developing kidney. We will validate MARIS (Method for Analyzing RNA following Intracellular Sorting) as a broadly applicable approach for acquiring transcriptional signatures from specific cell types in the developing kidneys. Finally, recognizing the importance of Six2 in regulating nephron progenitors, we will compare the Six2 regulatory landscape in the embryonic kidney with that of pluripotent stem cell derived Six2+ nephron progenitors generated in cell culture.
Rebuilding the glomerular filtration barrier by regenerating adult podocytes
Our project aims to rebuild kidney glomeruli by regenerating podocytes, terminally differentiated cells in the kidney glomerular filtering units that limit the passage of proteins from the blood into the urine, from two candidate resident stem/progenitor cells, cells of renin lineage (CoRL) and parietal epithelial cells (PECs). We will use an in vitro flow-directed microphysiological system (3D MPS) to determine to what degree CoRL and PECs are truly podocyte progenitors to rebuild a kidney, how their micro-environment regulates their fate, and what critical pathways are required for their reprogramming and fate to replace depleted adult podocytes.
Spatially-preserved expression analysis of kidney cells in human biopsy tissue
Kidney function, both under normal conditions and in the setting of disease, is predominantly controlled by the amount, localization and activation state of proteins in specific cells at specific sites and under specific spatial influences that in turn regulate cell differentiation, division, metabolism, morphology, membrane polarization, secretion and transport. Imaging mass cytometry (IMC) takes advantage of laser ionization and time of flight mass spectrometry to simultaneously identify up to 42 metal ion-conjugated antibodies from tissue sections at high spatial resolution with essentially no background signal. In the current proposal, human kidney samples will be used to develop protocols for high-fidelity staining with individual antibodies to identify cell types and protein activation states of interest in the kidney (SA 1) followed by staining of normal and diseased human kidney sections with pooled, metal ion-conjugated antibodies for multiplex IMC image reconstruction to simultaneously define the activation/expression states of multiple cells in the human kidney (SA 2).
Transcriptional profiling of in vivo derived human kidney tissue using single cell RNA sequencing
Human pluripotent stem cell derived organoid models, including kidney organoids, are most similar to fetal tissue in vitro. Therefore, while the goal of directed differentiation is to generate bona fide mature, functional cell types or organ-like systems, it is critical to have a comprehensive understanding of in vitro derived tissues relative to immature and mature human tissue. By extension, it is important to have high quality, reproducible and accessible in vivo human-tissue derived data sets in order to confidently benchmark in vitro studies. However, significant limitations exist for purifying specific populations of cells from rare human tissue samples to obtain cell-specific transcriptional profiles. The goal of this proposal is to provide high quality transcriptome data from the developing and adult human kidney using single cell RNA sequencing (scRNAseq), which is an unbiased approach that does not rely on specific reagents or bulk populations of cells.
The USC (Re)Building the Kidney Coordinating Center
The Coordinating Center will manage activities of the consortium, including research and collaboration opportunities, and facilitate communication of research results, data, and methods within the consortium and to the community. We are also creating a data hub to accelerate the pace of collaboration within the consortium and disseminate results to the broader research community.
Using cold active proteases for single cell dissociation
Single cell RNA-Seq provides a powerful particle biology approach for the study of cellular level heterogeneities. The technology for single cell RNA-Seq is rapidly evolving, yet several fundamental challenges remain. First, there is gene expression noise, resulting from the pulsatile, bursting nature of gene expression. Second, there is the technical noise that results from the technical challenges of transcript detection when working with picogram amounts of RNA. A third problem is the artifact changes in gene expresso that result from the process of generating single cell suspensions for analysis. During enzymatic dissociation at 37 deg the gene expression patterns of cells are changing. In this project we test a novel method for single cell dissociation that uses proteases active in the cold. These proteases are isolated from organisms that thrive in extreme cold environments. The use of such "cold active" proteases for cell dissociation is analogous to the use heat stable polymerases from thermophilic organisms for PCR. By carrying out cell dissociation at 5-10 deg instead of 37 deg the gene expression artifacts normally associated with this process could be dramatically reduced. The proposed improvement in methodology could have widespread impact on single cell RNA-Seq studies, rendering results more realistic representations of the in vivo state.