THREE-DIMENSIONAL DIFFERENTIATION OF EPIBLAST SPHEROIDS TO KIDNEY ORGANOIDS MODELS STAGE-SPECIFIC EPITHELIAL PHYSIOLOGY, MORPHOGENESIS, AND DISEASE
Human pluripotent stem cells (hPSCs) have dual value as microphysiological laboratory models and regenerative therapeutics. hPSCs are epithelial cells, but the extent to which hPSCs and descendant epithelia can reconstitute lineage-specific functions remains poorly understood. Here the Inventors show that hPSCs in three-dimensional cultures and their differentiated descendants can functionally recapitulate tissue-specific epithelial morphogenesis, physiology, and disease.
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This application is a divisional under 35 U.S.C. § 121 of co-pending U.S. application Ser. No. 15/756,846 filed Mar. 1, 2018, which is a 35 U.S.C. § 371 National Phase Entry Application of International Application No. PCT/US2016/050271, filed Sep. 2, 2016, which designates the U.S. and which claims benefit under 35 U.S.C. § 119(e) of U.S. Provisional Application No. 62/213,740, filed Sep. 3, 2015, the contents of each of which are incorporated herein by reference in their entireties.
SEQUENCE LISTINGThe instant application contains a Sequence Listing which has been submitted electronically in ASCII format and is hereby incorporated by reference in its entirety. Said ASCII copy, created on Feb. 28, 2018, is named 2016-09-02_SequenceListing_043214-085701-PCT.txt and is 2,600 bytes in size.
FIELD OF THE INVENTIONHuman stem cells can be cultured in three-dimensional cultures recapitulate tissue-specific epithelial morphogenesis, physiology, and disease.
BACKGROUNDBoth undifferentiated stem cells and terminally differentiated somatic cells form epithelia. These can function to establish axes for differentiation in the embryo, or to perform barrier and transport roles in adult organs such as the kidney. 3D cell culture in vitro is a powerful tool for investigating epithelial morphogenesis, physiology, and disease, being readily accessible to microscopic inspection, chemical treatment, and experimental manipulation. Studies of epithelial cell lines such as Madin-Darby Canine Kidney (MOCK) cells have, for instance, revealed polarity and apoptosis pathways contributing mechanistically to lumen formation. Conventional epithelial cell lines, however, are lineage-restricted and lack genetic diversity. As a result, the 3D structures that arise are relatively simple, and it has been challenging to perform controlled comparisons of different epithelia of the same genetic background, or the same epithelia with different genetic backgrounds. Despite these limitations, interest in the cellular microenvironment and 3D culture systems has been increasing steadily, particularly for stem cell applications. There is a significant need for genetically diverse cell culture platforms that accurately reconstitute tissue-specific epithelial function, particularly in humans where species-specific toxicology and disease pathophysiology is of significant biomedical relevance.
Human pluripotent stem cells (hPSCs) are capable of extensive self-renewal and can differentiate into all somatic cell types. They therefore represent a reproducible source of diverse human epithelia for laboratory studies and regeneration. hPSCs, including both embryo-derived embryonic stem cells (ESCs) and patient-derived induced pluripotent stem cells (iPSCs), are genetically diverse, with thousands of lines in production representing specific patient populations or gene-targeted knockout mutants. Undifferentiated hPSCs are polarized epithelial cells and are typically cultured either as flat colonies in adherent monolayer cultures or as dense aggregates in 3D suspensions. These epithelial cultures are proposed to model the implantation-stage epiblast epithelium. In contrast, mouse (m)ESCs adopt a compact, clustered morphology in culture that resembles the inner cell mass (ICM), a structure which precedes the epiblast developmentally.
Recently, mESCs surrounded by MATRIGEL™ extracellular matrix (ECM) were shown to form polarized rosettes with small cavities when, suggesting the possibility of modeling amniotic cavity formation in vitro. However, because those experiments were performed with mESCs cultured under conditions that do not sustain pluripotency, it remains unclear whether the observed rosettes represent ICM, epiblast, or differentiating subtypes, which can also form spheroids. Experiments in pluripotency-sustaining media using epiblast-stage hPSCs are required to address this question and establish a human model of epiblast cavitation.
Described herein is the discovery that hPSCs in three-dimensional cultures and their differentiated descendants can functionally recapitulate tissue-specific epithelial morphogenesis, physiology, and disease. Undifferentiated hPSCs form spheroid colonies surrounding hollow, amniotic-like cavities, modeling the embryonic epiblast. A two-step protocol differentiates spheroids into convoluted, tubular organoids with developmental and structural characteristics of kidney nephrons, including proximal tubules, podocytes and endothelial cells. Kidney tubules and epiblast cavities differentially accumulate fluorescent cargoes and respond to nephrotoxic chemical injury.
SUMMARY OF THE INVENTIONDescribed herein is a method of generating human organoids, including providing a quantity of human pluripotent stem cells (hPSCs), culturing the hPSCs in a culture medium to form epiblast spheroids, and differentiating the epiblast spheroids in a differentiation medium, wherein the differentiation medium includes one or more agents capable of differentiating the epiblast spheroids into organoids. In various embodiments, the hPSCs are cultured in absence of leukemia inhibitory factor (LIF) and doxycycline prior to forming epiblast spheroids. In various embodiments, the hPSCs are cultured in the presence of Y27632 prior to forming epiblast spheroids. In various embodiments, the culture medium includes MATRIGEL™. In various embodiments, the culture medium includes collagen I. In various embodiments, culturing the hPSCs includes depositing a first layer of culture medium on a surface, placing the hPSCs on the deposited culture medium, and adding a second layer of culture medium over the hPSCs. In various embodiments, culturing the hPSCs in a culture medium includes about 1, 2 or more days. In various embodiments, the one or more agents include CHIR99021. In various embodiments, the one or more agents comprise B27. In various embodiments, differentiating the epiblast spheroids includes about 7, 8, 9, 10, 11, 12, or 13 or more days. In various embodiments, the epiblast spheroids express one or more of podocalyxin (PODXL), zonula occluden (ZO-1) and β-catenin. In various embodiments, the epiblast spheroids are cavitated. In various embodiments, the organoids are tubular. In various embodiments, the organoids are kidney organoids. In various embodiments, the organoids express one or more of podocalyxin (PODXL), zonula occluden (ZO-1), and lotus tetragonolobus lectin (LTL). For example, hPSCs can be maintained feeder-free on about 3% Reduced Growth Factor GeiTrex for at least one passage in media such as mTeSR1, or a hESC conditioned media (CM)+leukemia inhibitory factor (LIF)+dox for hLR5 iPSCs). In various embodiments, hPSCs are primed by withdrawing LIF and doxycycline. In various embodiments, withdrawal of LIF and doxycycline includes substitution with FGF2. In various embodiments, cells are plated a specific density relative to the culture surface and media volume.
Also described herein is quantity of organoids made by a method of generating human organoids, including providing a quantity of human pluripotent stem cells (hPSCs), culturing the hPSCs in a culture medium to form epiblast spheroids, and differentiating the epiblast spheroids in a differentiation medium, wherein the differentiation medium includes one or more agents capable of differentiating the epiblast spheroids into organoids. In various embodiments, the hPSCs are cultured in absence of leukemia inhibitory factor (LIF) and doxycycline prior to forming epiblast spheroids. In various embodiments, the hPSCs are cultured in the presence of Y27632 prior to forming epiblast spheroids. In various embodiments, the culture medium includes MATRIGEL™. In various embodiments, the culture medium includes collagen I. In various embodiments, culturing the hPSCs includes depositing a first layer of culture medium on a surface, placing the hPSCs on the deposited culture medium, and adding a second layer of culture medium over the hPSCs. In various embodiments, culturing the hPSCs in a culture medium includes about 1, 2 or more days. In various embodiments, the one or more agents include CHIR99021. In various embodiments, the one or more agents comprise B27. In various embodiments, differentiating the epiblast spheroids includes about 7, 8, 9, 10, 11, 12, or 13 or more days. In various embodiments, the epiblast spheroids express one or more of podocalyxin (PODXL), zonula occluden (ZO-1) and β-catenin. In various embodiments, the epiblast spheroids are cavitated. In various embodiments, the organoids are tubular. In various embodiments, the organoids are kidney organoids. In various embodiments, the organoids express one or more of podocalyxin (PODXL), zonula occluden (ZO-1), and lotus tetragonolobus lectin (LTL). In various embodiments, the hPSCs are genetically modified using genomic editing, such as CRISPR.
Further described herein is a method of generating tubular organoids, including providing a quantity of epiblast spheroid and differentiating the epiblast spheroids in a differentiation medium, wherein the differentiation medium includes one or more agents capable of differentiating the epiblast spheroids into organoids. In various embodiments, the one or more agents comprise CHIR99021. In various embodiments, the one or more agents comprise B27. In various embodiments, differentiating the epiblast spheroids includes about 7, 8, 9, 10, 11, 12, or 13 or more days. In various embodiments, the tubular organoids are kidney organoids. In various embodiments, the kidney organoids express one or more of podocalyxin (PODXL), zonula occluden (ZO-1), and lotus tetragonolobus lectin (LTL). In various embodiments, differentiating the epiblast spheroids into kidney organoids includes further culturing in a second differentiation medium including RPMI and B27. In various embodiments, differentiating the epiblast spheroids into kidney organoids includes further culturing in a second differentiation medium including CHIR99021, RPMI and B27 for about 24 hours, replacement of the second differentiation medium with a third differentiation medium including CHIR99021, RPMI, B27 and insulin and additional culturing for about 48 hours, addition of IWP2 and continued culturing for about 48 hours, and additional replacement of the third differentiation medium with the second differentiation medium.
Also described herein is a method of screening a compound for an effect on tubular organoids, including providing a quantity of tubular organoids, adding one or more compounds to the tubular organoids, determining changes to phenotype or activity of the tubular organoids, and correlating the changes with an effect of the compounds on tubular organoids, thereby screening the one or more compounds for an effect on tubular organoids.
In various embodiments, determining changes to phenotype or activity includes detecting one or more markers in the tubular organoids. In various embodiments, the one or more markers comprise kidney injury molecule (KIM-1). In various embodiments, an increase in KIM-1 expression correlates with a toxic effect of the compound. In various embodiments, the tubular organoids are kidney organoids.
All references cited herein are incorporated by reference in their entirety as though fully set forth. Unless defined otherwise, technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Allen et al., Remington: The Science and Practice of Pharmacy 22nd ed., Pharmaceutical Press (Sep. 15, 2012); Hornyak et al., Introduction to Nanoscience and Nanotechnology, CRC Press (2008); Singleton and Sainsbury, Dictionary of Microbiology and Molecular Biology 3rd ed., revised ed., J. Wiley & Sons (New York, N.Y. 2006); Smith, March's Advanced Organic Chemistry Reactions, Mechanisms and Structure 7th ed., J. Wiley & Sons (New York, N.Y. 2013); Singleton, Dictionary of DNA and Genome Technology 3rd ed., Wiley-Blackwell (Nov. 28, 2012); and Green and Sambrook, Molecular Cloning: A Laboratory Manual 4th ed., Cold Spring Harbor Laboratory Press (Cold Spring Harbor, N.Y. 2012), provide one skilled in the art with a general guide to many of the terms used in the present application. For references on how to prepare antibodies, see Greenfield, Antibodies A Laboratory Manual 2nd ed., Cold Spring Harbor Press (Cold Spring Harbor N.Y., 2013); Köhler and Milstein, Derivation of specific antibody-producing tissue culture and tumor lines by cell fusion, Eur. J. Immunol. 1976 Jul. 6(7):511-9; Queen and Selick, Humanized immunoglobulins, U.S. Pat. No. 5,585,089 (1996 December); and Riechmann et al., Reshaping human antibodies for therapy, Nature 1988 Mar. 24, 332(6162):323-7.
One skilled in the art will recognize many methods and materials similar or equivalent to those described herein, which could be used in the practice of the present invention. Indeed, the present invention is in no way limited to the methods and materials described. For purposes of the present invention, the following terms are defined below.
As used in the description herein and throughout the claims that follow, the meaning of “a,” “an,” and “the” includes plural reference unless the context clearly dictates otherwise. Also, as used in the description herein, the meaning of “in” includes “in” and “on” unless the context clearly dictates otherwise.
As described, human pluripotent stem cells (hPSCs) have dual value as microphysiological laboratory models and regenerative therapeutics. hPSCs are epithelial cells, but the extent to which hPSCs and descendant epithelia can reconstitute lineage-specific functions remains poorly understood. Here the Inventors show that hPSCs in three-dimensional cultures and their differentiated descendants can functionally recapitulate tissue-specific epithelial morphogenesis, physiology, and disease.
Undifferentiated hPSCs form spheroid colonies surrounding hollow, amniotic-like cavities, modeling the embryonic epiblast. A two-step protocol differentiates spheroids into convoluted, tubular organoids with developmental and structural characteristics of kidney nephrons, including proximal tubules, podocytes and endothelial cells. Kidney tubules and epiblast cavities differentially accumulate fluorescent cargoes and respond to nephrotoxic chemical injury.
CRISPR/Cas9 knockout of podocalyxin or polycystic kidney disease genes produces disease relevant, tissue-specific phenotypes in kidney organoids, which are distinct from effects in epiblast spheroids. The Inventors' findings establish a reproducible and versatile three-dimensional framework for human microphysiology, disease modeling, and regenerative medicine applications.
The kidney is an organ of major interest to the field of regenerative medicine. Kidney epithelia are highly specialized and dysfunction of specific cell types can result in a variety of clinical disorders. For instance, polycystic kidney disease (PKD) features cystic expansion from tubular epithelial cells, whereas glomerulopathies involve injury to the podocyte epithelium through which blood plasma is filtered into the tubules. In proof-of-principle for using hPSCs to model kidney disease, the Inventors have described a ciliary phenotype in undifferentiated iPSCs and descendant epithelial cells from PKD patients. Recently, hPSCs have been directed to differentiate in vitro into tubular epithelia expressing markers typical of kidney progenitor cells, proximal tubules and podocytes.
However, the markers used in these studies are not exclusive to the kidney, and no study to date has demonstrated a disease-relevant phenotype in hPSC-derived kidney cells. Reconstitution of kidney-specific morphogenesis, microphysiology, and injury/disease states in hPSC-derived kidney cells is therefore important to more conclusively identify these epithelia and to advance their translational application.
Here, the Inventors establish adherent, 3D growth conditions for reconstitution of two distinct epithelial structures, epiblast spheroids and kidney tubular organoids, which arise sequentially in a single continuous culture of hPSCs. Using small molecule treatments and genome edited hPSCs, the Inventors demonstrate that these structures are capable of reconstituting tissue-specific epithelial transport, toxicity responses, and disease phenotypes. The Inventors' results reveal both common and tissue-specific features in hPSCs and descendant epithelia, with relevance for functional studies of human microphysiology, pathophysiology, and regenerative medicine.
Described herein is a method of generating human organoids, including providing a quantity of human pluripotent stem cells (hPSCs), culturing the hPSCs in a culture medium to form epiblast spheroids, and differentiating the epiblast spheroids in a differentiation medium, wherein the differentiation medium includes one or more agents capable of differentiating the epiblast spheroids into organoids. In various embodiments, the hPSCs are cultured in absence of leukemia inhibitory factor (LIF) and doxycycline prior to forming epiblast spheroids. In various embodiments, the hPSCs are cultured in the presence of Y27632 prior to forming epiblast spheroids. In various embodiments, the culture medium includes MATRIGEL™. In various embodiments, the culture medium includes collagen I. In various embodiments, culturing the hPSCs includes depositing a first layer of culture medium on a surface, placing the hPSCs on the deposited culture medium, and adding a second layer of culture medium over the hPSCs. In various embodiments, culturing the hPSCs in a culture medium includes about 1, 2 or more days. In various embodiments, the one or more agents include CHIR99021. In various embodiments, the one or more agents comprise B27. In various embodiments, differentiating the epiblast spheroids includes about 7, 8, 9, 10, 11, 12, or 13 or more days. In various embodiments, the epiblast spheroids express one or more of podocalyxin (PODXL), zonula occluden (ZO-1) and β-catenin. In various embodiments, the epiblast spheroids are cavitated. In various embodiments, the organoids are tubular. In various embodiments, the organoids are kidney organoids. In various embodiments, the organoids express one or more of podocalyxin (PODXL), zonula occluden (ZO-1), and lotus tetragonolobus lectin (LTL).
For example, hPSCs can be maintained feeder-free on about 3% Reduced Growth Factor GeiTrex for at least one passage in media such as mTeSR1, or a hESC conditioned media (CM)+leukemia inhibitory factor (LIF)+dox for hLR5 iPSCs). In various embodiments, hPSCs are primed by withdrawing LIF and doxycycline. In various embodiments, withdrawal of LIF and doxycycline includes substitution with FGF2. In various embodiments, cells are plated a specific density relative to the culture surface and media volume. For example, about 30-60,000 cells/well of a 24-well plate or 4-well chamber slide pre-coated with GeiTrex in media supplemented with 10 μM Rho-kinase inhibitor Y27632. In another example, about 5-20,000 of a 96-well plate were resuspended in 75 ul of either buffered collagen I (containing 10 mM HEPES and 1×DMEM), reduced growth factor MATRIGEL™ (BD Biosciences), or a 1:1 mixture of the two, incubated for 45 minutes at 37 degrees, and then overlaid with 100 ul of media plus Y27632.
For example, 48 hours after 3-D culture in the “sandwich” layers of culture medium, hPSC epiblast spheroids are differentiated in a differentiation medium including CHIR990021, at a concentration of, for example about 12 μM CHIR, and for a period of about 36 hours. In another example for kidney cell differentiation, the differentiation medium is changed to RB (Advanced RPMI+Glutamax+B27 Supplement) and replaced every three days thereafter. In another embodiment, epiblast spheroids are differentiated in a differentiation medium including CHIR990021, at a concentration of, for example about 12 μM CHIR, and media that is RB minus insulin (RBNI) for a period of about 24 hours, RBNI for 48 hours, addition of 5 IWP2 for 48 hours, RBNI for 48 hours, and RB every three days thereafter.
Also described herein is a quantity of organoids made by a method of generating human organoids, including providing a quantity of human pluripotent stem cells (hPSCs), culturing the hPSCs in a culture medium to form epiblast spheroids, and differentiating the epiblast spheroids in a differentiation medium, wherein the differentiation medium includes one or more agents capable of differentiating the epiblast spheroids into organoids. In various embodiments, the hPSCs are cultured in absence of leukemia inhibitory factor (LIF) and doxycycline prior to forming epiblast spheroids. In various embodiments, the hPSCs are cultured in the presence of Y27632 prior to forming epiblast spheroids. In various embodiments, the culture medium includes MATRIGEL™. In various embodiments, the culture medium includes collagen I. In various embodiments, culturing the hPSCs includes depositing a first layer of culture medium on a surface, placing the hPSCs on the deposited culture medium, and adding a second layer of culture medium over the hPSCs. In various embodiments, culturing the hPSCs in a culture medium includes about 1, 2 or more days. In various embodiments, the one or more agents include CHIR99021. In various embodiments, the one or more agents comprise B27. In various embodiments, differentiating the epiblast spheroids includes about 7, 8, 9, 10, 11, 12, or 13 or more days. In various embodiments, the epiblast spheroids express one or more of podocalyxin (PODXL), zonula occluden (ZO-1) and β-catenin. In various embodiments, the epiblast spheroids are cavitated. In various embodiments, the organoids are tubular. In various embodiments, the organoids are kidney organoids. In various embodiments, the organoids express one or more of podocalyxin (PODXL), zonula occluden (ZO-1), and lotus tetragonolobus lectin (LTL). In various embodiments, the hPSCs are genetically modified using genomic editing, such as CRISPR.
Also described herein is a method of generating tubular organoids, including providing a quantity of epiblast spheroid and differentiating the epiblast spheroids in a differentiation medium, wherein the differentiation medium includes one or more agents capable of differentiating the epiblast spheroids into organoids. In various embodiments, the one or more agents comprise CHIR99021. In various embodiments, the one or more agents comprise B27. In various embodiments, differentiating the epiblast spheroids includes about 7, 8, 9, 10, 11, 12, or 13 or more days. In various embodiments, the tubular organoids are kidney organoids. In various embodiments, the kidney organoids express one or more of podocalyxin (PODXL), zonula occluden (ZO-1), and lotus tetragonolobus lectin (LTL). In various embodiments, differentiating the epiblast spheroids into kidney organoids includes further culturing in a second differentiation medium including RPMI and B27. In various embodiments, differentiating the epiblast spheroids into kidney organoids includes further culturing in a second differentiation medium including CHIR99021, RPMI and B27 for about 24 hours, replacement of the second differentiation medium with a third differentiation medium including CHIR99021, RPMI, B27 and insulin and additional culturing for about 48 hours, addition of IWP2 and continued culturing for about 48 hours, and additional replacement of the third differentiation medium with the second differentiation medium.
Also described herein is a method of screening a compound for an effect on tubular organoids, including providing a quantity of tubular organoids, adding one or more compounds to the tubular organoids, determining changes to phenotype or activity of the tubular organoids, and correlating the changes with an effect of the compounds on tubular organoids, thereby screening the one or more compounds for an effect on tubular organoids.
In various embodiments, determining changes to phenotype or activity includes detecting one or more markers in the tubular organoids. In various embodiments, the one or more markers comprise kidney injury molecule (KIM-1). In various embodiments, an increase in KIM-1 expression correlates with a toxic effect of the compound. In various embodiments, the tubular organoids are kidney organoids.
Example 1 3D CultureCell lines included H9 (WA09), BJ, HDFn, hLR5, hfib2-iPS4, and hfib2-iPS5 (human) and J1, R1, and v6 (mouse). Cells were maintained feeder-free on 3% Reduced Growth Factor GeiTrex (Life Technologies) for at least one passage in media (mTeSR1 for hPSCs; N2/827 supplement+2i in for mESCs; hESC conditioned media (CM)+leukemia inhibitory factor (LIF)+dox for hLR5 iPSCs) and dissociated with Accutase® or TrypLE™. LD-iPSCs were derived from native hLR5 iPSCs by withdrawing LIF and doxycycline and substituting with FGF2. For thin gel sandwich colonies, cells were plated at 60,000 (primed) or 30,000 (native) cells/well of a 24-well plate or 4-well chamber slide pre-coated with GeiTrex in media supplemented with 10 μM Rho-kinase inhibitor Y27632 (StemGent). The following day the media was replaced with 500 μL 1.5 GeiTrex in mTeSR1. Media was changed after 24 hours. For thick gel cultures, 20,000 (epiblast-stage) or 6,000 (naïve) cells/well of a 96-well plate were resuspended in 75 ul of either buffered collagen I (containing 10 mM HEPES and 1×DMEM), reduced growth factor MATRIGEL™ (BD Biosciences), or a 1:1 mixture of the two, incubated for 45 minutes at 37 degrees, and then overlaid with 100 ul of media plus Y27632. For serial passaging in thin gels, colonies with lumens in 3D cultures were dissociated 72 hours after plating, replated at a density of 300,000 cells/well of a 6 well plate, and cultured for 72 hours in either 2D or 3D conditions before dissociation, cell counting, and replating. For suspension, 20,000 dissociated hPSCs were plated in mTeSR1 media in one well of a low-adherence 6-well plate. For all cells, media was changed daily.
Example 2 Tubular Organoid Differentiation60,000-120,000 H9 hPSCs were plated, sufficient to produce scattered, isolated spheroid colonies. 48 hours after sandwiching, hPSC spheroids were treated with 12 μM CHIR for 36 hours, then changed to RB (Advanced RPMI+Glutamax+B27 Supplement) and replaced every three days thereafter. Alternatively, spheroids were treated with 12 μM CHIR in RB minus insulin (RBNI) for 24 hours, RBNI for 48 hours, 5 μM IWP2 for 48 hours, RBNI for 48 hours, and RB every three days thereafter, as described for 2D cardiomyocyte differentiation. For 2D kidney differentiation, cells were plated overnight and then treated with 8 μM CHIR in APEL™ media (StemCell Technologies) for 48-72 hours, 30 ng/ml FGF2+1 μg/ml heparin in APEL™ media for 96 hours, and subsequently cultured in APEL™ media for 10-15 days. For stochastic differentiation, hPSCs in 2D or 3D cultures were treated with 10% fetal bovine serum (FBS) in DMEM+P/S and observed for 19 days. Immunofluorescence and Electron Microscopy
To fix while preserving 3D architecture, an equal volume of 8% paraformaldehyde was added to the culture media (4% final concentration) for 15 minutes at room temperature. After fixing, samples were washed in PBS, blocked in 5% donkey serum (Millipore)/0.3% Triton-X-100/PBS, incubated overnight in 3% bovine serum albumin/PBS with primary antibodies, washed, incubated with Alexa-Fluor secondary antibodies (Invitrogen), washed, and stained with DAPI or mounted in Vectashield™ H-1000. Primary antibodies included OCT4 (sc-5279; Santa Cruz), NANOG (RCAB0004PF; Cosmobio), brachyury (sc-17745; Santa Cruz), TRA-1-60 (MAB4360, Millipore), TRA-1-81 (MAB4381; Millipore), acetylated o-tubulin (051M4770; Sigma), ZO-1 (339100; Invitrogen), podocalyxin (AF1658 and AF1556; R&D), CDX2 (−88, Biogenex), AQP1 (AB2219; Millipore), WT-1 (sc-192; Santa Cruz), LHX1 (Developmental Studies Hybridoma Bank), mPODXL (AF1556; R&D), hPODXL (AF1658, R&D), HNA (MAB1281, Millipore), LTL (FL-1321, Vector Labs), SYNPO (sc-21537; Santa Cruz), CD31 (555444; BD), crumbs 3 (HPA013835, Sigma), Na,K-ATPase (ab7671, Abeam), and cleaved caspase-3 (MAB835; R&D). Fluorescence images were captured using a Nikon epifluorescence 90-1 (upright), Eclipse Ti (inverted), or confocal C1 microscopes. For electron microscopy, structures were scraped from the plate after 5 minutes of fixation, pelleted at 300 g for four minutes, and the pellet was gently released by pipetting into cacodylate buffer containing 4% formaldehyde and 2% glutaraldehyde, postfixed with osmium tetroxide, dehydrated in serial ethanols, and embedded in epoxy resin. Semi-thin sections were cut at 1 mm and stained with toluidine blue to identify tubular structures with apparent lumens by light microscopic examination. Ultrathin sections (75 nm) were cut, mounted on 200 mesh copper grids, counterstained with uranyl acetate and lead citrate, and examined in a JEOL JEM-1010 transmission electron microscope.
Example 3 Permeability AssaysTo test permeability, media was supplemented with 20 mM HEPES plus Lucifer Yellow™ carbohydrazide potassium salt (Invitrogen, 38 μM) and Rhodamine-8 isothiocyanate dextran (Sigma, 0.5 and imaged by confocal microscopy. For microinjection, 5 rhodamine-conjugated dextran solution in mTeSR1 was diluted 1:1 with Phenol Red Solution (0.5%, Sigma) for visualization. 2 nl was microinjected via a pulled glass capillary microneedle on a Nanoject-2 micromanipulator, and monitored in real-time by wide-field epifluorescence. For TEER, 50,000 hPSCs were plated on 24-well transwell plates (Corning) pre-coated with dilute MATRIGEL™. The media was gently exchanged for 10 days until cells were completely confluent. TEER was measured using an EVOM 2™ device (World Precision Instruments).
Example 4 KIM-1 InductionOrganoids in identically-plated wells of a 24-well plate were treated with increasing concentrations of gentamicin and cisplatin for 36-48 hours, fixed, and processed for immunofluorescence with KIM-1 antibodies AKG7.9 (Bonventre laboratory) or 1400 (Biogen). Immunofluorescence for KIM-1 was observed at moderate, sub-toxic doses which did not induce gross tubular disintegration.
Example 5 RNA Interference16 hours after plating, hPSCs were transfected with Dharmacon Smartpool™ siRNAs directed against PODXL, OCT4, or scrambled control in mTeSR1 without antibiotics. Ten hours later, the media was changed and the cells were either cultured in 2D or sandwiched for 3D culture.
Example 6 Cas9/CRISPR MutagenesisConstructs encoding green fluorescent protein (GFP)-tagged Cas9 (Addgene 44719) and a guide RNA (Addgene 64711) targeting the second exon of PODXL (GCTACACCTTCACAAGCCCGGGG) [SEQ ID NO: 1], the first exon of PKD2 (GCGTGGAGCCGCGATAACCCCGG) [SEQ ID NO: 2], or the thirty-sixth exon of PKD1 (GTGGGTGCGAGCTTCCCCCCGGG) [SEQ ID NO: 3] were transiently transfected into H9 hESCs, and GFP expressing cells were isolated by flow cytometric sorting, clonally expanded, and screened for clones with biallelic loss-of-function indels. −200,000 sorted hESCs were plated per well of a 6-well plate in hESC-conditioned mTeSR1 plus Y27632. Media was replaced the following morning without Y27632 and cells were clonally expanded and the PODXL gRNA region was amplified by PCR. Chromatogram sequences were analyzed manually and mutations were confirmed by immunoblot and immunofluorescence.
Example 7 Transcriptome ProfilinghPSCs plated in 2D or 3D were prepared side-by-side using the RNEasy™ Mini Kit (Qiagen). Samples were QC'd on the Agilent Bioanalyzer™ to check for high integrity samples. Qualifying samples were then prepped using the TruSeq™ stranded mRNA library kit (Illumina). Sequencing was performed on an Illumina NextSeq500 75×75 paired end high output run. Samples were aligned to hg19 reference sequence using Tophat2 and differential expression calculated using Cuffdiff.
Example 8 RT-PCRRNA was prepared on days 2, 10, 14, and 21 after plating during the differentiation time course using the RNeasy Mini Kit (Qiagen). RNA from all time points was reverse transcribed side-by-side using the M-MLV Reverse Transcription System (Promega). Quantitative RT-PCR reactions were run in duplicate using cDNA (diluted 1:10), 300 nM primers, and iQ SYBR Green™ Supermix (Bio-Rad) with the iQ5 Multi-Color Real-Time PCR Detection System (Bio-Rad), using P-actin as the housekeeping gene.
Example 9 Quantification and Statistical AnalysisFor fluorescence intensity quantifications, images were taken in a single imaging session and at identical exposures and processed identically. The number of cavitated colonies (ellipsoid with lumen) versus flat colonies (non-ellipsoid or without a lumen) was scored manually in phase contrast images of living cells, in which lumens were discerned more easily than in fixed samples. For apoptosis, cleaved caspase-3 expression was scored manually, confirmed by nuclear condensation, and divided by the total number of nuclei per wide-field epifluorescent image. For ZO-1 areas, individual colonies or subregions expressing ZO-1 were traced manually and surface areas calculate using NIS Elements (Nikon). For each colony, the summed ZO-1 expressing area was expressed as a percentage of the total surface area and then averaged. To quantify intensities, line scans of equal length were drawn through randomly selected structures imaged with identical exposures to obtain raw fluorescence values in NIS Elements software (Nikon). The averaged line scan values were plotted with error bars. For CHIR-induced differentiation, −6000 individual cells were identified in low-magnification immunofluorescence images using Cell Profiler 2.0 and fluorescence intensities were measured automatically. Statistical comparisons utilized a two-tailed I test for two samples with unequal variance (heteroscedastic). Immunoblots were quantified using the ImageJ Gel Analyzer.
Example 10 Cavity and Tubule Morphogenesis in a Continuous 3D Culture of hPSCsTo reconstitute human epithelia from undifferentiated hPSCs and their somatic descendants, the Inventors developed an adherent, 3D culture system for hPSCs that first produced hollow spheroids and subsequently tubules (
The Inventors tested hPSC spheroids for pluripotency and self-renewal, which are the key functional characteristics of undifferentiated hPSCs. Cavitated spheroids were repeatedly dissociated, replated, and sandwiched (
The Inventors hypothesized that hPSC spheroids model the epiblast epithelial mass, which forms an early amniotic cavity in human and primate implantation-stage embryos. Conversely, hPSCs resembling the more primitive ICM were predicted not to cavitate. Indeed, naive' hLR5 iPSCs, which form compact, ICM-like colonies similar to mouse (m)ESCs, did not form lumens in 3D cultures even after five days of growth, correlating with low podocalyxin expression and discontinuous ZO-1 localization (
CHIR added at the time of sandwiching inhibited spheroid formation in a dose-dependent manner, with differentiating cells undergoing EMT and radiating outwards away from their colonies of origin (
In the preceding experiment, the Inventors observed that 5˜M CHIR induced higher levels of BRY expression in 3D cultures than in 2D cultures plated side-by-side (
With the new protocol, the Inventors' yield was approximately 90 tubular aggregates per well of a 24-well plate, starting with H9 (WA09) hESCs (
The Inventors observed progression of EGAD+distal tubule to L TL+proximal tubule to capsule-like structures containing POD XL+ cells (presumptive podocytes), consistent with nephron segmentation (
Using this protocol, H9 hESCs and three different hiPSC lines produced organoids incorporating proximal tubules, endothelial cells, and podocytes, in kidney-like arrangements, with hESCs showing the highest efficiency of differentiation (
To test the barrier functions of pluripotent and descendant epithelia, the Inventors developed a real-time assay to visualize molecular diffusion kinetics into and out of lumens, using fluorescent compounds of different sizes. In epiblast spheroids, Lucifer Yellow™ (L Y, 521 Da) added to the culture media for 2-4 hours gradually accumulated within cavities, whereas rhodamine-conjugated dextran (RD, 10,000 Da) was excluded from lumens and instead accumulated in apical intercellular regions and formed a bright halo around the lumen (
The Inventors further applied this system to clarify the role of podocalyxin, an apical sialomucin proposed to regulate epithelial cell differentiation, polarity, and lumen morphogenesis in both epiblast and kidney. Podocalyxin knockout (PODXL−/−.) hPSCs were generated using the clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 genome editing system, and clones were selected by chromatogram analysis (
Podocalyxin knockdown by RNA interference (RNAi) was previously shown to disrupt tight junction organization. In PODXL−/−—hESCs, however, the junctional components ZO-1, occludin, and filamentous actin appeared properly localized, and transepithelial electrical resistance (TEER) was indistinguishable from wild-type controls (
Contrary to these findings in PODXL−/−—hESCs, a −90% knockdown of podocalyxin by RNA silencing (siPODXL) resulted in mislocalization of ZO-1 to small patches of residual podocalyxin, confining ZO-1 to −50% of its normal surface area (
The Inventors next investigated podocalyxin function in human kidney cell types. PODXL−/−—hPSCs differentiated efficiently into kidney organoids featuring both tubules (ZO-1+LTL+) and podocytes (ZO-1.SYNPO+), despite the complete loss of podocalyxin expression from these cell types (
Lastly, the Inventors investigated the potential of kidney organoids to functionally model polycystic kidney disease (PKD), which is characterized by the expansion of kidney tubules to form cysts. Biallelic, loss-of function mutations in PKD1 or PKD2 are proposed to contribute strongly to PKD cystogenesis. The Inventors therefore applied the CRISPR/Cas9 genome editing system to introduce biallelic, truncating mutations in PKD1 or PKD2 in hPSCs (
To test whether these lines might produce phenotypes relevant to PKD in the kidney lineage, the Inventors cultured organoids derived from PKD hPSCs for several weeks side-by-side with isogenic, unmodified controls. Remarkably, in the PKD hPSC cultures, the Inventors observed formation of large, translucent, cyst-like structures alongside tubular organoids (
The epithelial characteristics of hPSCs and derived somatic lineages are poorly understood. Reconstitution of epithelial physiology and morphogenesis in both undifferentiated hPSCs and their differentiated descendants is important for advancing their potential as human laboratory models and regenerative therapeutics. The described culture system and assays establish a framework for generating and functionally profiling undifferentiated hPSCs and descendant epithelia in three dimensions. hPSCs are a well-characterized, homogenous, and genetically diverse cell type that includes patient-specific, immunocompatible iPSCs. Well-functioning epithelia may therefore have applicability for regenerative medicine.
The Inventors demonstrate, for the first time, that undifferentiated, epiblast-stage hPSCs form cavitated spheroids in 3D culture, similar to rosettes recently derived from mESCs, but with expanded lumens. Importantly, spheroids form under non-differentiating conditions and remain fully pluripotent, excluding the possibility that they represent a differentiated lineage 14 Spheroid formation is furthermore restricted to the epiblast-stage, but is not characteristic of ICM-stage hPSCs or mESCs. The primate epiblast in vivo is an expanding, discoid epithelial mass that surrounds a highly dynamic early amniotic cavity.
As ethical and legal barriers restrict the study and culture of human embryos at this stage, hPSC spheroids provide an ethically acceptable and experimentally accessible 3D model for epiblast cavitation and subsequent differentiation. Mechanistically, lumen morphogenesis in epiblast spheroids occurs via apicobasal polarization, similar to MOCK cells. The extensive lineage flexibility and genetic diversity of hPSCs is a major advantage over existing epithelial cell models, enabling direct comparisons of human epithelia from diverse tissues and genetic backgrounds. Using genetically modified hPSCs, the Inventors identify podocalyxin as a key mediator of epiblast spheroid lumenogenesis, which functions independently of tight junctions in this cell type.
The Inventors' results suggest a molecular model whereby the combination of apicobasal polarization, tight junction organization, and podocalyxin expression distinguishes epiblast-stage hPSCs from ICM-stage progenitors and promotes formation of the early amniotic cavity (
However, even wild-type cells frequently form cysts in these systems. A reproducible system for PKD-specific cyst formation from tubules is therefore an important goal for the field, particularly in humans where species-specific pathophysiology and therapy is of clinical interest. The Inventors find that loss-of-function PKD mutations result in cyst formation from hPSC-derived tubular cells, which is not observed in isogenic controls. This finding suggests that PKD-specific cystogenesis from tubules is a cell-intrinsic phenomenon that can be modeled in a minimal system in vitro. As cystogenesis was observed for both PKD1 and PKD2 mutants, and was specific to the kidney organoids but not epiblast spheroids, the phenotype is both gene-specific and lineage-specific in this system. Cysts arise at relatively low frequencies, consistent with the focal appearance of cysts throughout the kidneys of PKD patients and mouse models. Further studies are required to determine the cellular basis of cystogenesis in this system and whether iPSCs from PKD patients, which have heterozygous mutations and variable genetic backgrounds, also produce cysts. As cysts are a relatively rare phenomenon, improvements in iPSC differentiation efficiencies may be required to perform such experiments. In addition to tubular cells, the hPSC system produces podocytes, which are morphologically and functionally distinct from kidney tubules. hPSC-derived podocytes form polarized domains segregating junctional components such as CRB3 and ZO-1, consistent with biochemical and microscopic analyses of podocytes in vivo. The combination of these proteins with podocalyxin, synaptopodin, and WT1 is not known to be co-expressed in any population other than kidney podocytes, nor would such cells be expected to appear alongside L TL+tubular cells in other organs. Using CRISPR knockout lines, the Inventors demonstrate that podocalyxin functions in these cells to segregate junctional complexes basolaterally, analogous to findings during rodent nephrogenesis and in Podxr1 mice
As alterations in podocalyxin expression are characteristic of human glomerular disease states, further studies of PODXL−/− podocytes may produce insights into cellular pathophysiology and treatment. The hPSC system does have limitations. For instance, the Inventors have not yet observed formation of a vascularized glomerulus from hPSC podocytes and neighboring endothelia. Dedicated studies involving fluid flow and the tissue microenvironment in vivo are required to further develop this system into fully functional nephrons, for more advanced disease modeling and therapeutic application. In conclusion, the Inventors have developed a 3D culture system that reconstitutes functional, structured epithelia representing the epiblast, kidney tubular cells, and podocytes. These pluripotent and descendant epithelia share certain key structural features, but they can nevertheless recapitulate stage-specific transport characteristics and morphogenesis mechanisms. This provides an accurate and reproducible platform in which to model human microphysiology, injury, and disease at distinct developmental stages. Genome-modified tubular organoids functionally recapitulate kidney disease phenotypes, strengthening the identification of these structures as kidney and establishing innovative cellular systems for studying human renal physiology and pathophysiology in vitro. The described methodologies are broadly applicable and adaptable to diverse tissues and genetically diverse backgrounds, and can be utilized immediately to experimentally investigate molecular pathways relevant to human epithelial diseases. In the longer term, this system may provide a useful setting in which to optimize and test the functionality of patient-derived epithelia in vitro, prior to regenerative graft administration.
Example 19 3D CultureCell lines included H9 (WA09), BJ, HDF, hLR5, hfib2-iPS4, and hfib2-iPS5 (human) and J1, R1, and v6 (mouse). Cells were maintained feeder-free on 3% Reduced Growth Factor GelTrex™ (Life Technologies) for at least one passage in media (mTeSR1 for hPSCs; N2/B27 supplement+2i in for mESCs; hESC conditioned media (CM)+leukemia inhibitory factor (LIF)+dox for hLR5 iPSCs) and dissociated with Accutase® or TrypLE™. LD-iPSCs were derived from naïve hLR5 iPSCs by withdrawing LIF and doxycycline and substituting with FGF2. For thin gel sandwich colonies, cells were plated at 60,000 (primed) or 30,000 (naive) cells/well of a 24-well plate or 4-well chamber slide pre-coated with GelTrex™ in media supplemented with 10 μM Rho-kinase inhibitor Y27632 (StemGent). The following day the media was replaced with 500 μL 1.5% GelTrex™ in mTeSR1. Media was changed after 24 hours. For thick gel cultures, 20,000 (epiblast-stage) or 6,000 (naive) cells/well of a 96-well plate were resuspended in 75 ul of either buffered collagen I (containing 10 mM HEPES and 1×DMEM), reduced growth factor MATRIGEL™ (BD Biosciences), or a 1:1 mixture of the two, incubated for 45 minutes at 37 degrees, and then overlaid with 100 ul of media plus Y27632. For serial passaging in thin gels, colonies with lumens in 3D cultures were dissociated 72 hours after plating, replated at a density of 300,000 cells/well of a 6 well plate, and cultured for 72 hours in either 2D or 3D conditions before dissociation, cell counting, and replating. For suspension, 20,000 dissociated hPSCs were plated in mTeSR1 media in one well of a low-adherence 6-well plate. For all cells, media was changed daily.
Example 20 Tubular Organoid Differentiation60,000-120,000 H9 hPSCs were plated, sufficient to produce scattered, isolated spheroid colonies. 48 hours after sandwiching, hPSC spheroids were treated with 12 μM CHIR for 36 hours, then changed to RB (Advanced RPMI+Glutamax+B27 Supplement) and replaced every three days thereafter. Alternatively, spheroids were treated with 12 μM CHIR in RB minus insulin (RBNI) for 24 hours, RBNI for 48 hours, 5 μM IWP2 for 48 hours, RBNI for 48 hours, and RB every three days thereafter, as described for 2D cardiomyocyte differentiation. For 2D kidney differentiation, cells were plated overnight and then treated with 8 μM CHIR in APEL™ media (StemCell Technologies) for 48-72 hours, 30 ng/ml FGF2+1 μg/ml heparin in APEL™ media for 96 hours, and subsequently cultured in APEL™ media for 10-15 days. For stochastic differentiation, hPSCs in 2D or 3D cultures were treated with 10% fetal bovine serum (FBS) in DMEM+P/S and observed for 19 days.
Example 21 Immunofluorescence and Electron MicroscopyTo fix while preserving 3D architecture, an equal volume of 8% paraformaldehyde was added to the culture media (4% final concentration) for 15 minutes at room temperature. After fixing, samples were washed in PBS, blocked in 5% donkey serum (Millipore)/0.3% Triton-X-100/PBS, incubated overnight in 3% bovine serum albumin/PBS with primary antibodies, washed, incubated with Alexa-Fluor secondary antibodies (Invitrogen), washed, and stained with DAPI or mounted in Vectashield H-1000. Primary antibodies included OCT4 (sc-5279; Santa Cruz), NANOG (RCAB0004PF; Cosmobio), brachyury (sc-17745; Santa Cruz), TRA-1-60 (MAB4360, Millipore), TRA-1-81 (MAB4381; Millipore), acetylated □-tubulin (051M4770; Sigma), ZO-1 (339100; Invitrogen), podocalyxin (AF1658 and AF1556; R&D), CDX2 (-88, Biogenex), AQP1 (AB2219; Millipore), WT-1 (sc-192; Santa Cruz), LHX1 (Developmental Studies Hybridoma Bank), mPODXL (AF1556; R&D), hPODXL (AF1658, R&D), HNA (MAB1281, Millipore), LTL (FL-1321, Vector Labs), SYNPO (sc-21537; Santa Cruz), CD31 (555444; BD), crumbs 3 (HPA013835, Sigma), Na,K-ATPase (ab7671, Abcam), and cleaved caspase-3 (MAB835; R&D). Fluorescence images were captured using a Nikon epifluorescence 90-I (upright), Eclipse Ti (inverted), or confocal C1 microscopes. For electron microscopy, structures were scraped from the plate after 5 minutes of fixation, pelleted at 300 g for four minutes, and the pellet was gently released by pipetting into cacodylate buffer containing 4% formaldehyde and 2% glutaraldehyde, post-fixed with osmium tetroxide, dehydrated in serial ethanols, and embedded in epoxy resin. Semi-thin sections were cut at 1 mm and stained with toluidine blue to identify tubular structures with apparent lumens by light microscopic examination. Ultrathin sections (75 nm) were cut, mounted on 200 mesh copper grids, counterstained with uranyl acetate and lead citrate, and examined in a JEOL JEM-1010 transmission electron microscope.
Example 22 Permeability AssaysTo test permeability, media was supplemented with 20 mM HEPES plus Lucifer Yellow™ carbohydrazide potassium salt (Invitrogen, 38 μM) and Rhodamine-B isothiocyanate dextran (Sigma, 0.5 μM), and imaged by confocal microscopy. For microinjection, 5 μM rhodamine-conjugated dextran solution in mTeSR1 was diluted 1:1 with Phenol Red Solution (0.5%, Sigma) for visualization. 2 nl was microinjected via a pulled glass capillary microneedle on a Nanoject-2 micromanipulator, and monitored in real-time by wide-field epifluorescence. For TEER, 50,000 hPSCs were plated on 24-well transwell plates (Corning) pre-coated with dilute MATRIGEL™. The media was gently exchanged for 10 days until cells were completely confluent. TEER was measured using an EVOM 2™ device (World Precision Instruments).
Example 23 KIM-1 InductionOrganoids in identically-plated wells of a 24-well plate were treated with increasing concentrations of gentamicin and cisplatin for 36-48 hours, fixed, and processed for immunofluorescence with KIM-1 antibodies AKG7.9 (Bonventre laboratory) or 1400 (Biogen). Immunofluorescence for KIM-1 was observed at moderate, sub-toxic doses which did not induce gross tubular disintegration.
Example 24 RNA Interference16 hours after plating, hPSCs were transfected with Dharmacon Smartpool™ siRNAs directed against PODXL, OCT4, or scrambled control in mTeSR1 without antibiotics. Ten hours later, the media was changed and the cells were either cultured in 2D or sandwiched for 3D culture.
Example 25 Cas9/CRISPR MutagenesisConstructs encoding green fluorescent protein (GFP)-tagged Cas9 (Addgene 44719) and a guide RNA (Addgene 64711) targeting the second exon of PODXL (GCTACACCTTCACAAGCCCGGGG) [SEQ ID NO: 1], the first exon of PKD2 (GCGTGGAGCCGCGATAACCCCGG) [SEQ ID NO: 2], or the thirty-sixth exon of PKD1 (GTGGGTGCGAGCTTCCCCCCGGG) [SEQ ID NO: 3] were transiently transfected into H9 hESCs, and GFP-expressing cells were isolated by flow cytometric sorting, clonally expanded, and screened for clones with biallelic loss-of-function indels. ˜200,000 sorted hESCs were plated per well of a 6-well plate in hESC-conditioned mTeSR1 plus Y27632. Media was replaced the following morning without Y27632 and cells were clonally expanded and the PODXL gRNA region was amplified by PCR. Chromatogram sequences were analyzed manually and mutations were confirmed by immunoblot and immunofluorescence.
Example 26 Transcriptome ProfilinghPSCs plated in 2D or 3D were prepared side-by-side using the RNEasy Mini Kit (Qiagen). Samples were QC'd on the Agilent Bioanalyzer to check for high integrity samples. Qualifying samples were then prepped using the TruSeq stranded mRNA library kit (Illumina). Sequencing was performed on an Illumina NextSeq500 75×75 paired end high output run. Samples were aligned to hg19 reference sequence using Tophat2 and differential expression calculated using Cuffdiff.
Example 27 RT-PCRRNA was prepared on days 2, 10, 14, and 21 after plating during the differentiation time course using the RNeasy Mini Kit (Qiagen). RNA from all time points was reverse transcribed side-by-side using the M-MLV Reverse Transcription System (Promega). Quantitative RT-PCR reactions were run in duplicate using cDNA (diluted 1:10), 300 nM primers, and iQ SYBR Green™ Supermix (Bio-Rad) with the iQ5 Multi-Color Real-Time PCR Detection System (Bio-Rad), using β-actin as the housekeeping gene.
Example 28 Quantification and Statistical AnalysisFor fluorescence intensity quantifications, images were taken in a single imaging session and at identical exposures and processed identically. The number of cavitated colonies (ellipsoid with lumen) versus flat colonies (non-ellipsoid or without a lumen) was scored manually in phase contrast images of living cells, in which lumens were discerned more easily than in fixed samples. For apoptosis, cleaved caspase-3 expression was scored manually, confirmed by nuclear condensation, and divided by the total number of nuclei per wide-field epifluorescent image. For ZO-1 areas, individual colonies or subregions expressing ZO-1 were traced manually and surface areas calculate using NIS Elements (Nikon). For each colony, the summed ZO-1 expressing area was expressed as a percentage of the total surface area and then averaged. To quantify intensities, line scans of equal length were drawn through randomly selected structures imaged with identical exposures to obtain raw fluorescence values in NIS Elements software (Nikon). The averaged line scan values were plotted with error bars. For CHIR-induced differentiation, ˜6000 individual cells were identified in low-magnification immunofluorescence images using Cell Profiler 2.0 and fluorescence intensities were measured automatically. Statistical comparisons utilized a two-tailed t test for two samples with unequal variance (heteroscedastic). Immunoblots were quantified using the ImageJ Gel Analyzer.
Example 29 hPSCs Form Cavitated Spheroids in 3D CultureTo evaluate the tissue-specific functions of undifferentiated hPSCs and descendant hPSC-KCs, the Inventors developed an adherent, 3D culture system for hPSCs that first produced epiblast spheroids and subsequently kidney tubules (
The Inventors further tested hPSC spheroids for pluripotency and self-renewal, which are the key functional characteristics of undifferentiated hPSCs. In nine serial passages, dissociated cavity-lining spheroid cells generated new cavitated spheroids after sandwiching, or alternatively flat colonies when the final passage was into monolayer conditions (
During the development of humans and many other mammals, the ICM of the early embryo differentiates into the epiblast, from which all somatic cells are derived. The Inventors hypothesized that hPSC spheroids model the epiblast epithelial mass, which forms a columnar epithelium surrounding an early amniotic cavity in human and primate implantation-stage embryos. Conversely, hPSCs resembling the more primitive ICM were predicted not to cavitate. Indeed, ‘naïve’ hLR5 iPSCs, which form compact, ICM-like colonies similar to mouse (m)ESCs, did not form lumens in 3D cultures even after five days of growth, whereas ‘primed’ hLR5-derived (LD-)iPSCs, which resemble epiblast-stage hPSCs, formed cavities efficiently in sandwich cultures (
To differentiate epiblast spheroids into descendant epithelia, the Inventors applied a directed differentiation regimen originally designed for cardiomyocyte generation from 2D cultures, involving the sequential inhibition of glycogen synthase kinase-3β (GSK3β) and Wingless-related integration site (WNT) signaling. Remarkably, rather than form cardiomyocytes, spheroid cells underwent epithelial-to-mesenchymal transition (EMT) to form a confluent monolayer that by day 10 aggregated into folds and initiated mesenchymal-to-epithelial transition (MET) into convoluted, translucent, tubular organoids (
The Inventors examined these tubular organoids for the expression of kidney markers. Lotus tetragonolobus lectin (LTL), a marker of kidney proximal tubules, reacted strongly with tubular structures and appeared enriched in tubular lumens (
Using this protocol, H9 hESCs and three different hiPSC lines produced organoids incorporating cells and structures with characteristics of proximal tubules, endothelial cells, and podocytes, in kidney-like segmental arrangements (
Tissue-specific functions or disease phenotypes have not been demonstrated in hPSC-KCs. The Inventors therefore investigated the potential of kidney organoids to upregulate kidney injury molecule-1 (KIM-1), a clinical biomarker of proximal tubule injury. When treated with the nephrotoxic drugs cis-diamminedichloroplatinum(II) (cisplatin) or gentamicin, KIM-1 immunofluorescence was detected at the luminal surface of tubules in ˜80% of organoids, and was confirmed using two different antibodies (
To test whether epiblast spheroids and kidney tubules exhibit tissue-specific barrier functions, the Inventors developed a real-time assay to visualize molecular diffusion kinetics into and out of lumens, using fluorescent compounds of different sizes. In epiblast spheroids, Lucifer Yellow™ (LY, 521 Da) added to the culture media for 2-4 hours gradually accumulated within cavities, whereas rhodamine-conjugated dextran (RD, 10,000 Da) was excluded from lumens and instead accumulated in apical intercellular regions and formed a bright halo around the lumen (
Podocalyxin is an apical sialomucin expressed highly in both epiblast and kidney podocytes. To investigate the functional role of podocalyxin in these cell types, podocalyxin knockout (PODXL−/−) hPSCs were generated using the clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 genome editing system (
Podocalyxin is proposed to regulate lumenogenesis through tight junction organization. In PODXL−/− hESCs, however, the junctional components ZO-1, occludin, and filamentous actin appeared properly localized, and transepithelial electrical resistance (TEER) was indistinguishable from wild-type controls (
The Inventors next investigated podocalyxin function in human kidney cell types. In tissue sections from adult human kidneys, podocalyxin was highly expressed in the glomeruli, but was not detected in the tubules (
To investigate podocalyxin function in human podocyte-like cells, the Inventors produced kidney organoids from PODXL−/− hPSCs. In contrast to wild-type organoids, in PODXL−/− organoids, the appearance of linear ZO1+SYNPO+ tracks was strongly reduced, and junctional markers adopted a more diffuse expression pattern (
Lastly, the Inventors investigated the potential of kidney organoids to functionally model polycystic kidney disease (PKD), which is characterized by the expansion of kidney tubules to form cysts. Biallelic, loss-of-function mutations in PKD1 or PKD2 are proposed to contribute strongly to PKD cystogenesis. The Inventors therefore applied the CRISPR/Cas9 genome editing system to introduce biallelic, truncating mutations in PKD1 or PKD2 in hPSCs (‘PKD hPSCs’). Chromatogram analyses and immunoblotting confirmed frame-shift mutations at the target site and demonstrated the absence of the corresponding full-length proteins (
To test whether these lines might produce phenotypes relevant to PKD in the kidney lineage, the Inventors cultured renal organoids derived from PKD hPSCs for several weeks side-by-side with isogenic, unmodified controls. Remarkably, in the PKD hPSC cultures, the Inventors observed formation of large, translucent, cyst-like structures alongside tubular organoids (
The epithelial characteristics of hPSCs and derived kidney cells are poorly understood. Reconstitution of epithelial physiology and morphogenesis in these cell types is important for advancing their potential as human laboratory models and regenerative therapeutics. The described culture system and assays establish a framework for generating and functionally profiling undifferentiated hPSCs and descendant hPSC-KCs in three dimensions. hPSCs are a well-characterized, homogenous, and genetically diverse cell type that includes patient-specific, immunocompatible iPSCs. Well-functioning hPSC-derived epithelia may therefore have applicability for regenerative medicine.
The Inventors demonstrate, for the first time, that undifferentiated, epiblast-stage hPSCs form cavitated spheroids in 3D culture, similar to rosettes recently derived from mESCs, but with expanded lumens. By directly comparing 2D and 3D cultures, the Inventors' studies reveal that spheroid formation at the epiblast stage can significantly affect subsequent cell fate decisions, producing tubular organoids instead of cardiomyocytes. These organoids recapitulate key characteristics of kidney development and physiology in vitro, which have been challenging to model using primary adult or embryonic kidney cells. In contrast to previous protocols for kidney directed differentiation from hPSCs, the Inventors' simple, two-step procedure of spheroid formation followed by GSK3β inhibition in growth-factor reduced MATRIGEL™ does not require exogenous supplementation with fibroblast growth factor 2 (FGF2), activin, or bone morphogenetic protein. The tubular structures are surrounded by dilute ECM in an adherent, microplate format which is experimentally accessible, scalable, and potentially high-throughput. These structures exhibit a lineage complexity that differs from conventional kidney cell lines and organoids. All the major components of the developing proximal nephron—tubular cells, endothelial cells, nephron progenitors, and podocyte-like cells—are represented within each individual organoid, in kidney-like architectures. The proximal tubules transport fluorescent cargoes in a characteristic manner, which is distinct from the pluripotent spheroid epithelia from which they derive. When injured, tubules express a clinical biomarker, KIM-1, a response that is highly characteristic of the proximal tubule in vivo but lost in de-differentiated primary cultures. This may provide a quantifiable human standard with which to predict proximal tubule nephrotoxicity, a frequent cause of failure in drug development.
As the Inventors' studies of PODXL−/− hPSCs illustrate, this advanced differentiation system can be combined with CRISPR/Cas9 genome editing to determine the function of specific genes in different human cell types, on an isogenic genetic background. The Inventors' results suggest a molecular model whereby the combination of apicobasal polarization, tight junction organization, and podocalyxin expression distinguishes epiblast-stage hPSCs from ICM-stage progenitors and promotes formation of the early amniotic cavity (
PKD is among the most common monogenic diseases and of major interest to both clinicians and cell biologists. Existing cellular systems have reported quantitative differences in the formation of simple spheroids or ‘cysts’ attributed to defects in PKD gene expression. However, even wild-type cells frequently form cysts in these systems. A reproducible system for PKD-specific cyst formation from tubules is therefore an important goal for the field, particularly in humans where species-specific pathophysiology and therapy is of clinical interest. The Inventors find that loss-of-function PKD mutations result in cyst formation from hPSC-derived tubular cells, which is not observed in isogenic controls. This finding suggests that PKD-specific cystogenesis from tubules is a cell-intrinsic phenomenon that can be modeled in a minimal system in vitro. As cystogenesis was observed for both PKD1 and PKD2 mutants, and was specific to the kidney organoids but not epiblast spheroids, the phenotype is both gene-specific and lineage-specific in this system. Further studies are required to determine the cellular basis of cystogenesis in this system and whether iPSCs from PKD patients, which have heterozygous mutations and variable genetic backgrounds, also produce cysts. As cysts are a relatively rare phenomenon, improvements in iPSC differentiation efficiencies may be required to perform such experiments.
The described hPSC system does have limitations. For instance, the Inventors have not yet observed formation of a vascularized glomerulus from hPSC podocyte-like cells and neighboring endothelia. The tubules also do not contain a full brush border. Although SIX2+ mesenchyme was observed adjacent to tubular cells, the Inventors did not observe evidence of ureteric bud markers in these tubules. Rather, the tubules have characteristics of proximal tubules derived from the SIX2+ mesenchyme, which was induced to differentiate through a non-developmental pathway. Neurons were abundant in these cultures and might possibly represent a source of inductive signals for kidney tubular differentiation in the absence of ureteric bud, similar to embryonic spinal cord. A further limitation of this hPSC-based system is the lack of widely-available fluorescent reporter lines with which to perform lineage tracing experiments. One possible solution to this problem would be to adapt this protocol for mouse EpiSCs, which are similar to hPSCs in phenotype. For instance, EpiSCs from the SIX2-TdTomato reporter mouse might be used to determine with greater certainty whether all tubular cells in the Inventors' system derive from the SIX2+ mesenchyme, using developing kidneys from this mouse as positive controls. Overall, the Inventors' findings suggest that while kidney differentiation is indeed occurring from hPSCs, this process in vitro does not fully recapitulate developmental kidney nephrogenesis. Dedicated studies involving fluid flow and the tissue microenvironment in vivo are required to further develop this system into fully functional nephrons, for more advanced disease modeling and therapeutic application.
In conclusion, the Inventors have developed a 3D culture system that reconstitutes functional, structured epithelia modeling the epiblast, kidney tubular cells, and podocyte-like cells. These pluripotent and descendant epithelia share certain key structural features, but they can nevertheless recapitulate stage-specific transport characteristics and morphogenesis mechanisms. This provides an accurate and reproducible platform in which to model human microphysiology, injury, and disease at distinct developmental stages. Genome-modified tubular organoids functionally recapitulate kidney disease phenotypes, strengthening the identification of these structures as kidney and establishing innovative cellular systems for studying human renal physiology and pathophysiology in vitro. The described methodologies are broadly applicable and adaptable to diverse tissues and genetically diverse backgrounds, and can be utilized immediately to experimentally investigate molecular pathways relevant to human epithelial diseases. In the longer term, this system may provide a useful setting in which to optimize and test the functionality of patient-derived epithelia in vitro, prior to regenerative graft administration.
The various methods and techniques described above provide a number of ways to carry out the invention. Of course, it is to be understood that not necessarily all objectives or advantages described may be achieved in accordance with any particular embodiment described herein. Thus, for example, those skilled in the art will recognize that the methods can be performed in a manner that achieves or optimizes one advantage or group of advantages as taught herein without necessarily achieving other objectives or advantages as may be taught or suggested herein. A variety of advantageous and disadvantageous alternatives are mentioned herein. It is to be understood that some preferred embodiments specifically include one, another, or several advantageous features, while others specifically exclude one, another, or several disadvantageous features, while still others specifically mitigate a present disadvantageous feature by inclusion of one, another, or several advantageous features.
Furthermore, the skilled artisan will recognize the applicability of various features from different embodiments. Similarly, the various elements, features and steps discussed above, as well as other known equivalents for each such element, feature or step, can be mixed and matched by one of ordinary skill in this art to perform methods in accordance with principles described herein. Among the various elements, features, and steps some will be specifically included and others specifically excluded in diverse embodiments.
Although the invention has been disclosed in the context of certain embodiments and examples, it will be understood by those skilled in the art that the embodiments of the invention extend beyond the specifically disclosed embodiments to other alternative embodiments and/or uses and modifications and equivalents thereof.
Many variations and alternative elements have been disclosed in embodiments of the present invention. Still further variations and alternate elements will be apparent to one of skill in the art. Among these variations, without limitation, are sources of spheroids, organoids, methods of generating, characterizing, and producing such cell products, and the particular use of the products created through the teachings of the invention. Various embodiments of the invention can specifically include or exclude any of these variations or elements.
In some embodiments, the numbers expressing quantities of ingredients, properties such as concentration, reaction conditions, and so forth, used to describe and claim certain embodiments of the invention are to be understood as being modified in some instances by the term “about.” Accordingly, in some embodiments, the numerical parameters set forth in the written description and attached claims are approximations that can vary depending upon the desired properties sought to be obtained by a particular embodiment. In some embodiments, the numerical parameters should be construed in light of the number of reported significant digits and by applying ordinary rounding techniques. Notwithstanding that the numerical ranges and parameters setting forth the broad scope of some embodiments of the invention are approximations, the numerical values set forth in the specific examples are reported as precisely as practicable. The numerical values presented in some embodiments of the invention may contain certain errors necessarily resulting from the standard deviation found in their respective testing measurements.
In some embodiments, the terms “a” and “an” and “the” and similar references used in the context of describing a particular embodiment of the invention (especially in the context of certain of the following claims) can be construed to cover both the singular and the plural. The recitation of ranges of values herein is merely intended to serve as a shorthand method of referring individually to each separate value falling within the range. Unless otherwise indicated herein, each individual value is incorporated into the specification as if it were individually recited herein. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g. “such as”) provided with respect to certain embodiments herein is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention otherwise claimed. No language in the specification should be construed as indicating any non-claimed element essential to the practice of the invention.
Groupings of alternative elements or embodiments of the invention disclosed herein are not to be construed as limitations. Each group member can be referred to and claimed individually or in any combination with other members of the group or other elements found herein. One or more members of a group can be included in, or deleted from, a group for reasons of convenience and/or patentability. When any such inclusion or deletion occurs, the specification is herein deemed to contain the group as modified thus fulfilling the written description of all Markush groups used in the appended claims.
Preferred embodiments of this invention are described herein, including the best mode known to the inventors for carrying out the invention. Variations on those preferred embodiments will become apparent to those of ordinary skill in the art upon reading the foregoing description. It is contemplated that skilled artisans can employ such variations as appropriate, and the invention can be practiced otherwise than specifically described herein. Accordingly, many embodiments of this invention include all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. Moreover, any combination of the above-described elements in all possible variations thereof is encompassed by the invention unless otherwise indicated herein or otherwise clearly contradicted by context.
Furthermore, numerous references have been made to patents and printed publications throughout this specification. Each of the above cited references and printed publications are herein individually incorporated by reference in their entirety.
In closing, it is to be understood that the embodiments of the invention disclosed herein are illustrative of the principles of the present invention. Other modifications that can be employed can be within the scope of the invention. Thus, by way of example, but not of limitation, alternative configurations of the present invention can be utilized in accordance with the teachings herein. Accordingly, embodiments of the present invention are not limited to that precisely as shown and described.
Claims
1. A method of screening a compound for an effect on tubular organoids, the method comprising:
- providing a quantity of tubular organoids;
- adding one or more compounds to the tubular organoids; and
- determining changes to phenotype or activity of the tubular organoids.
2. The method of claim 1, wherein determining changes to phenotype or activity comprises detecting and/or measuring expression levels of one or more markers in the tubular organoids.
3. The method of claim 2, wherein the one or more markers comprise kidney injury molecule (KIM-1).
4. The method of claim 3, wherein an increase in KIM-1 mRNA or KIM-1 polypeptide expression correlates with a toxic effect of the compound.
5. The method of claim 1, wherein the tubular organoids are kidney organoids.
6. The method of claim 1, wherein the tubular organoids express one or more of podocalyxin (PODXL), zonula occluden (ZO-1), and lotus tetragonolobus lectin (LTL).
7. The method of claim 1, wherein the kidney organoids are differentiated from human pluripotent stem cells (hPSCs).
8. The method of claim 1, wherein determining the phenotype or activity of tubular organoids comprises an assay selected from the group consisting of:
- (a) a KIM-1 induction assay;
- (b) a permeability assay;
- (c) an immunofluorescence assay;
- (d) an RT-PCR assay;
- (e) transcriptome profiling; and
- (f) microscopy.
9. The method of claim 1, wherein the tubular organoids are genome-modified kidney organoids.
10. The method of claim 1, wherein the tubular organoids are derived from a subject with a kidney disease.
11. The method of claim 9, wherein the tubular organoids are modified by a CRISPR/Cas9 genome editing system.
12. The method of claim 10, wherein the kidney disease is polycystic kidney disease.
13. The method of claim 1, wherein the tubular organoids are generated by the method comprising:
- (i) providing a quantity of human pluripotent stem cells (hPSCs);
- (ii) culturing the hPSCs in a first culture medium comprising a ROC kinase inhibitor for at least 24 hours and then culturing the hPSCs sandwiched between two layers of a gelatinous protein mixture secreted by Engelbreth-Holm-Swarm mouse sarcoma cells to form epiblast spheroids; wherein the first medium does not comprise exogenous fibroblast growth factor 2 (FGF2), activin or bone morphogenetic protein; and
- (iii) contacting the epiblast spheroids from step (ii) with a second culture medium comprising at least 12 μM CHIR99021 for at least 24 hours, wherein the second medium does not comprise exogenous fibroblast growth factor 2 (FGF2), activin or bone morphogenetic protein; and
- (iv) culturing the epiblast spheroids from step (iii) with a third culture medium comprising B27 for at least 48 hours, wherein the third medium does not comprise exogenous fibroblast growth factor 2 (FGF2), activin or bone morphogenetic protein, thereby differentiating the epiblast spheroids into tubular organoids.
14. A method of screening a compound for the treatment of polycystic kidney disease, the method comprising:
- providing a quantity of genome-modified kidney organoids;
- adding one or more compounds to the kidney organoids; and
- determining changes to phenotype or activity of the kidney organoids.
Type: Application
Filed: Aug 13, 2020
Publication Date: Dec 3, 2020
Applicant: THE BRIGHAM AND WOMEN'S HOSPITAL, INC. (Boston, MA)
Inventors: Benjamin S. Freedman (Boston, MA), Joseph V. Bonventre (Boston, MA)
Application Number: 16/992,362
