Application of Progenitor Niche Signals to Ex Vivo Nephrogenesis

Key Personnel

Leif Oxburgh (PI)
Maine Medical Center

  • Thomas Carroll (PI)
    University of Texas Southwestern
  • Ondine Cleaver (PI)
    University of Texas Southwestern
  • David Kaplan (PI)
    Tufts University

Project Description

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.

At the 56 minute mark of this video of Dr. Francis Collins' testimony for the Senate Health Committee, he refers to the Regenerative Medicine Innovation Project (RMIP) supplement to this project and work being done in kidney regeneration.

Focus on: Stromal Biology

Thomas Carroll
PI: Thomas Carroll
University of Texas Southwestern

Key Personnel: Alicia Fessler, Ashwani Kumar Gupta, Harini Ramalingam

Optimal kidney functions require 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.

  1. Spatiotemporal heterogeneity and patterning of developing renal blood vessels

    Daniel, E; Azizoglu, DB; Ryan, AR; Walji, TA; Chaney, CP; Sutton, GI; Carroll, TJ; Marciano, DK; Cleaver, O. Angiogenesis. April 2018.

    The kidney vasculature facilitates the excretion of wastes, the dissemination of hormones, and the regulation of blood chemistry. To carry out these diverse functions, the vasculature is regionalized within the kidney and along the nephron. However, when and how endothelial regionalization occurs remains unknown. Here, we examine the developing kidney vasculature to assess its 3-dimensional structure and transcriptional heterogeneity. First, we observe that endothelial cells (ECs) grow coordinately with the kidney bud as early as E10.5, and begin to show signs of speci cation by E13.5 when the rst arteries can be identi ed. We then focus on how ECs pattern and remodel with respect to the developing nephron and collecting duct epithelia. ECs circumscribe nephron progenitor populations at the distal tips of the ureteric bud (UB) tree and form stereotyped cruciform structures around each tip. Beginning at the renal vesicle (RV) stage, ECs form a continuous plexus around developing nephrons. The endothelial plexus envelops and elaborates with the maturing nephron, becoming preferentially enriched along the early distal tubule. Lastly, we perform transcriptional and immuno uorescent screens to characterize spatiotemporal heterogeneity in the kidney vasculature and identify novel regionally enriched genes. A better understanding of development of the kidney vasculature will help instruct engineering of properly vascularized ex vivo kidneys and evaluate diseased kidneys.

  2. (Re)Building a Kidney.

    Oxburgh, L; Carroll, TJ; Cleaver, O; Gossett, DR; Hoshizaki, DK; Hubbell, JA; Humphreys, BD; Jain, S; Jensen, J; Kaplan, DL; Kesselman, C; Ketchum, CJ; Little, MH; McMahon, AP; Shankland, SJ; Spence, JR; Valerius, MT; Wertheim, JA; Wessely, O; Zheng, Y; Drummond, IA. J Am Soc Nephrol. 28(5):1370–1378. May 2017.

    (Re)Building a Kidney is a National Institute of Diabetes and Digestive and Kidney Diseases-led consortium to optimize approaches for the isolation, expansion, and differentiation of appropriate kidney cell types and the integration of these cells into complex structures that replicate human kidney function. The ultimate goals of the consortium are two-fold: to develop and implement strategies for in vitro engineering of replacement kidney tissue, and to devise strategies to stimulate regeneration of nephrons in situ to restore failing kidney function. Projects within the consortium will answer fundamental questions regarding human gene expression in the developing kidney, essential signaling crosstalk between distinct cell types of the developing kidney, how to derive the many cell types of the kidney through directed differentiation of human pluripotent stem cells, which bioengineering or scaffolding strategies have the most potential for kidney tissue formation, and basic parameters of the regenerative response to injury. As these projects progress, the consortium will incorporate systematic investigations in physiologic function of in vitro and in vivo differentiated kidney tissue, strategies for engraftment in experimental animals, and development of therapeutic approaches to activate innate reparative responses.

Focus on: Vascular Biology

Ondine Cleaver
PI: Ondine Cleaver
University of Texas Southwestern

Key Personnel: Edward Daniel

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.

RELATED DATA

  1. Spatiotemporal heterogeneity and patterning of developing renal blood vessels

    Daniel, E; Azizoglu, DB; Ryan, AR; Walji, TA; Chaney, CP; Sutton, GI; Carroll, TJ; Marciano, DK; Cleaver, O. Angiogenesis. April 2018.

    The kidney vasculature facilitates the excretion of wastes, the dissemination of hormones, and the regulation of blood chemistry. To carry out these diverse functions, the vasculature is regionalized within the kidney and along the nephron. However, when and how endothelial regionalization occurs remains unknown. Here, we examine the developing kidney vasculature to assess its 3-dimensional structure and transcriptional heterogeneity. First, we observe that endothelial cells (ECs) grow coordinately with the kidney bud as early as E10.5, and begin to show signs of speci cation by E13.5 when the rst arteries can be identi ed. We then focus on how ECs pattern and remodel with respect to the developing nephron and collecting duct epithelia. ECs circumscribe nephron progenitor populations at the distal tips of the ureteric bud (UB) tree and form stereotyped cruciform structures around each tip. Beginning at the renal vesicle (RV) stage, ECs form a continuous plexus around developing nephrons. The endothelial plexus envelops and elaborates with the maturing nephron, becoming preferentially enriched along the early distal tubule. Lastly, we perform transcriptional and immuno uorescent screens to characterize spatiotemporal heterogeneity in the kidney vasculature and identify novel regionally enriched genes. A better understanding of development of the kidney vasculature will help instruct engineering of properly vascularized ex vivo kidneys and evaluate diseased kidneys.

  2. (Re)Building a Kidney.

    Oxburgh, L; Carroll, TJ; Cleaver, O; Gossett, DR; Hoshizaki, DK; Hubbell, JA; Humphreys, BD; Jain, S; Jensen, J; Kaplan, DL; Kesselman, C; Ketchum, CJ; Little, MH; McMahon, AP; Shankland, SJ; Spence, JR; Valerius, MT; Wertheim, JA; Wessely, O; Zheng, Y; Drummond, IA. J Am Soc Nephrol. 28(5):1370–1378. May 2017.

    (Re)Building a Kidney is a National Institute of Diabetes and Digestive and Kidney Diseases-led consortium to optimize approaches for the isolation, expansion, and differentiation of appropriate kidney cell types and the integration of these cells into complex structures that replicate human kidney function. The ultimate goals of the consortium are two-fold: to develop and implement strategies for in vitro engineering of replacement kidney tissue, and to devise strategies to stimulate regeneration of nephrons in situ to restore failing kidney function. Projects within the consortium will answer fundamental questions regarding human gene expression in the developing kidney, essential signaling crosstalk between distinct cell types of the developing kidney, how to derive the many cell types of the kidney through directed differentiation of human pluripotent stem cells, which bioengineering or scaffolding strategies have the most potential for kidney tissue formation, and basic parameters of the regenerative response to injury. As these projects progress, the consortium will incorporate systematic investigations in physiologic function of in vitro and in vivo differentiated kidney tissue, strategies for engraftment in experimental animals, and development of therapeutic approaches to activate innate reparative responses.

Focus on: Biomaterials

David Kaplan
PI: David Kaplan
Tufts University

Key Personnel: Shreyas Jadhav, Sophia Szymkowiak

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.

RELATED DATA

  1. Polycystin 2 regulates mitochondrial Ca(2+) signaling, bioenergetics, and dynamics through mitofusin 2.

    Kuo, Ivana Y.; Brill, Allison L.; Lemos, Fernanda O.; Jiang, Jason Y.; Falcone, Jeffrey L.; Kimmerling, Erica P.; Cai, Yiqiang; Dong, Ke; Kaplan, David L.; Wallace, Darren P.; Hofer, Aldebaran M.; Ehrlich, Barbara E. Sci Signal. 12(580). May 2019.

    Mitochondria and the endoplasmic reticulum (ER) have an intimate functional relationship due to tethering proteins that bring their membranes in close (~30 nm) apposition. One function of this interorganellar junction is to increase the efficiency of Ca(2+) transfer into mitochondria, thus stimulating mitochondrial respiration. Here, we showed that the ER cation-permeant channel polycystin 2 (PC2) functions to reduce mitochondria-ER contacts. In cell culture models, PC2 knockdown led to a 50% increase in mitofusin 2 (MFN2) expression, an outer mitochondrial membrane GTPase. Live-cell super-resolution and electron microscopy analyses revealed enhanced MFN2-dependent tethering between the ER and mitochondria in PC2 knockdown cells. PC2 knockdown also led to increased

  2. Scaffolding kidney organoids on silk.

    Gupta, Ashwani Kumar; Coburn, Jeannine M.; Davis-Knowlton, Jessica; Kimmerling, Erica; Kaplan, David L.; Oxburgh, Leif. J Tissue Eng Regen Med. 13(5):812–822. May 2019.

    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.

  3. (Re)Building a Kidney.

    Oxburgh, L; Carroll, TJ; Cleaver, O; Gossett, DR; Hoshizaki, DK; Hubbell, JA; Humphreys, BD; Jain, S; Jensen, J; Kaplan, DL; Kesselman, C; Ketchum, CJ; Little, MH; McMahon, AP; Shankland, SJ; Spence, JR; Valerius, MT; Wertheim, JA; Wessely, O; Zheng, Y; Drummond, IA. J Am Soc Nephrol. 28(5):1370–1378. May 2017.

    (Re)Building a Kidney is a National Institute of Diabetes and Digestive and Kidney Diseases-led consortium to optimize approaches for the isolation, expansion, and differentiation of appropriate kidney cell types and the integration of these cells into complex structures that replicate human kidney function. The ultimate goals of the consortium are two-fold: to develop and implement strategies for in vitro engineering of replacement kidney tissue, and to devise strategies to stimulate regeneration of nephrons in situ to restore failing kidney function. Projects within the consortium will answer fundamental questions regarding human gene expression in the developing kidney, essential signaling crosstalk between distinct cell types of the developing kidney, how to derive the many cell types of the kidney through directed differentiation of human pluripotent stem cells, which bioengineering or scaffolding strategies have the most potential for kidney tissue formation, and basic parameters of the regenerative response to injury. As these projects progress, the consortium will incorporate systematic investigations in physiologic function of in vitro and in vivo differentiated kidney tissue, strategies for engraftment in experimental animals, and development of therapeutic approaches to activate innate reparative responses.

Focus on: Nephron Progenitor Biology

Leif Oxburgh
PI: Leif Oxburgh
Maine Medical Center

Key Personnel: Prasenjit Sarkar

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.

RELATED DATA

  1. Kidney-in-a-lymph node: A novel organogenesis assay to model human renal development and test nephron progenitor cell fates.

    Francipane, Maria Giovanna; Han, Bing; Oxburgh, Leif; Sims-Lucas, Sunder; Li, Zhongwei; Lagasse, Eric. J Tissue Eng Regen Med. July 2019.

    Stem cell-derived organoids are emerging as sophisticated models for studying development and disease and as potential sources for developing organ substitutes. Unfortunately, although organoids containing renal structures have been generated from mouse and human pluripotent stem cells, there are still critical unanswered questions that are difficult to attain via in vitro systems, including whether these nonvascularized organoids have a stable and physiologically relevant phenotype or whether a suitable transplantation site for long-term in vivo studies can be identified. Even orthotopic engraftment of organoid cultures in the adult does not provide an environment conducive to vascularization and functional differentiation. Previously, we showed that the lymph node offers an alternative transplantation site where mouse metanephroi can differentiate into mature renal structures with excretory, homeostatic, and endocrine functions. Here, we show that the lymph node lends itself well as a niche to also grow human primary kidney rudiments and can additionally be viewed as a platform to interrogate emerging renal organoid cultures. Our study has a wide-ranging impact for tissue engineering approaches to rebuild functional tissues in vivo including-but not limited to-the kidney.

  2. Scaffolding kidney organoids on silk.

    Gupta, Ashwani Kumar; Coburn, Jeannine M.; Davis-Knowlton, Jessica; Kimmerling, Erica; Kaplan, David L.; Oxburgh, Leif. J Tissue Eng Regen Med. 13(5):812–822. May 2019.

    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.

  3. Long-Term Culture of Nephron Progenitor Cells Ex Vivo.

    Brown, Aaron C.; Gupta, Ashwani K.; Oxburgh, Leif. Methods Mol Biol. 1926:63–75. 2019.

    Nephrons differentiate from the cap mesenchyme of the fetal kidney. Nephron progenitor cells that populate the cap mesenchyme efficiently balance self-renewal and epithelial differentiation to enable repeated rounds of nephron formation during development. Here we describe a method to isolate and propagate these cells from the embryonic mouse kidney. Using this method, nephron progenitor cells from a single litter of mice can be propagated to hundreds of millions of cells that express appropriate markers of the undifferentiated state and retain epithelial differentiation capacity in vitro.

  4. (Re)Building a Kidney.

    Oxburgh, L; Carroll, TJ; Cleaver, O; Gossett, DR; Hoshizaki, DK; Hubbell, JA; Humphreys, BD; Jain, S; Jensen, J; Kaplan, DL; Kesselman, C; Ketchum, CJ; Little, MH; McMahon, AP; Shankland, SJ; Spence, JR; Valerius, MT; Wertheim, JA; Wessely, O; Zheng, Y; Drummond, IA. J Am Soc Nephrol. 28(5):1370–1378. May 2017.

    (Re)Building a Kidney is a National Institute of Diabetes and Digestive and Kidney Diseases-led consortium to optimize approaches for the isolation, expansion, and differentiation of appropriate kidney cell types and the integration of these cells into complex structures that replicate human kidney function. The ultimate goals of the consortium are two-fold: to develop and implement strategies for in vitro engineering of replacement kidney tissue, and to devise strategies to stimulate regeneration of nephrons in situ to restore failing kidney function. Projects within the consortium will answer fundamental questions regarding human gene expression in the developing kidney, essential signaling crosstalk between distinct cell types of the developing kidney, how to derive the many cell types of the kidney through directed differentiation of human pluripotent stem cells, which bioengineering or scaffolding strategies have the most potential for kidney tissue formation, and basic parameters of the regenerative response to injury. As these projects progress, the consortium will incorporate systematic investigations in physiologic function of in vitro and in vivo differentiated kidney tissue, strategies for engraftment in experimental animals, and development of therapeutic approaches to activate innate reparative responses.