Patent Application
20180258404
Descried herein are methods and compositions related to production of nephron progenitor cells and kidney organoids. The techniques described herein find use in regenerative medicine applications.Human stem cells can be cultured in three-dimensional cultures recapitulate tissue-specific epithelial morphogenesis, physiology, and disease.
Chronic kidney disease affects 9-13% of the U.S. adult population and is a serious public health problem worldwide. Disease progression is marked by gradual, irreversible loss of nephrons, the individual functional units of the kidney. The ability to generate functional kidney tissue from hPSCs may allow the development of cell therapies for kidney disease as well as strategies for modeling kidney development and disease and for drug screening. Nephrons are made up of glomeruli, which filter the blood plasma into a multicomponent tubular system that reabsorbs and/or secretes solutes and water to produce urine. The many different epithelial cell types in nephrons have complicated efforts to generate them in vitro. However, studies have shown that all of these epithelial cell types except those in the collecting ducts derive from a multipotent nephron progenitor cell (NPC) population present in the metanephric cap mesenchyme during kidney development. The NPCs, which express the markers SIX2, SALL1, WT1, and PAX2, are found in humans only during kidney organogenesis, which ceases by birth and cannot be reinitiated after birth, even during repair after kidney injury. However, these NPC markers are also expressed in the more primitive mesonephric mesenchyme that derives from the anterior intermediate mesoderm (IM) and forms the transiently functional mesonephros. This suggests that careful attention to the early separation of anterior versus posterior intermediate mesoderm (IM) fate is likely to be critical for the proper induction of metanephric NPCs (
Several studies have attempted to differentiate mouse and human PSCs into cells of the kidney lineage. Published protocols to produce SIX2+ NPCs from hPSCs have several limitations. First, differentiation efficiency is too low for large-scale production of NPCs. One explanation for this may be that most protocols have not distinguished anterior from posterior IM in early steps of directed differentiation. The metanephric mesenchyme derives from cells of the primitive streak, which persist as cells of the posterior IM. In contrast the ureteric bud, the precursor to the adult kidney collecting duct system, originates in the anterior IM, a cell population incapable of giving rise to the metanephric mesenchyme. Second, existing protocols use poorly defined components, such as mouse embryonic spinal cord, which would not be suitable for clinical applications. Finally, previous studies could generate nephron rudiments, but not mature nephron segments containing the mature kidney epithelial cell types or a single contiguous nephron-like structure with characteristics of multiple nephron segments, precluding their use for modeling human kidney development, disease, and injury.
Here the Inventors describe an efficient, chemically defined system for differentiating hPSCs into multipotent NPCs capable of forming nephron-like structures. By carefully recapitulating the stages of metanephric kidney development in two-dimensional monolayer culture, the Inventors generate NPCs that co-express the critical markers SIX2, SALL1, WT1, and PAX2 with 90% efficiency within 9 days of initiation of differentiation—a substantial improvement over previous methods (
Described herein is a method for generating metanephric mesenchyme, including providing a quantity of human pluripotent stem cells (“hPSCs”), generating late primitive streak cells, inducing formation of posterior intermediate mesoderm cells, and differentiating into metanephric mesenchyme cells. In other embodiments, the human pluripotent stem cells are human embryonic stem cells (“hESCs”). In other embodiments, the human pluripotent stem cells are human induced pluripotent stem cells (“hiPSCs”). In other embodiments, generating late primitive streak cells includes culturing in CHIR99021 for about 3-5 days. In other embodiments, the method further includes addition of Noggin. In other embodiments, inducing formation of posterior intermediate mesoderm cells includes culturing in the presence of activin for about 2-4 days. In other embodiments, differentiating into metanephric mesenchyme cells includes addition of FGF9. In other embodiments, the metanephric mesenchyme lineage cells are further differentiated into nephronic progenitor cells (NPCs) by addition of CHIR99021.
In other embodiments, late primitive streak cells express one or more of: T and TBX. In other embodiments, posterior intermediate mesoderm cells express one or more of: WT1 and HOXD11. In other embodiments, metanephric mesenchyme lineages cells express one or more of: SIX2, SALL1, WT1, and PAX2. In other embodiments, NPCs express one or more of: SIX2, SALL1, WT1, PAX2, and EYA1. In other embodiments, differentiation into metanephric mesenchyme cells is at least 50% efficient. In other embodiments, differentiation into metanephric mesenchyme cells is at least 70% efficient.
Further described herein is a composition of metanephric mesenchyme cells generated by a method for generating metanephric mesenchyme, including providing a quantity of human pluripotent stem cells (“hPSCs”), generating late primitive streak cells, inducing formation of posterior intermediate mesoderm cells, and differentiating into metanephric mesenchyme cells. In other embodiments, the human pluripotent stem cells are human embryonic stem cells (“hESCs”). In other embodiments, the human pluripotent stem cells are human induced pluripotent stem cells (“hiPSCs”). In other embodiments, generating late primitive streak cells includes culturing in CHIR99021 for about 3-5 days. In other embodiments, the method further includes addition of Noggin. In other embodiments, inducing formation of posterior intermediate mesoderm cells includes culturing in the presence of activin for about 2-4 days. In other embodiments, differentiating into metanephric mesenchyme cells includes addition of FGF9. In other embodiments, the metanephric mesenchyme lineage cells are further differentiated into nephronic progenitor cells (NPCs) by addition of CHIR99021. In other embodiments, late primitive streak cells express one or more of: T and TBX. In other embodiments, posterior intermediate mesoderm cells express one or more of: WT1 and HOXD11. In other embodiments, metanephric mesenchyme lineages cells express one or more of: SIX2, SALL1, WT1, and PAX2. In other embodiments, NPCs express one or more of: SIX2, SALL1, WT1, PAX2, and EYA1. In other embodiments, differentiation into metanephric mesenchyme cells is at least 50% efficient. In other embodiments, differentiation into metanephric mesenchyme cells is at least 70% efficient. A composition of nephron progenitor cells generated by the described method.
Described herein is a method of generating kidney organoids, including providing a quantity of nephron progenitor cells (“NPCs”), and culturing the NPCs in a suspension culture for at about 11 days. In other embodiments, the method includes addition of one or more of: CHIR99021 and FGF9. In other embodiments, the kidney organoids comprise one or more cell types selected from: podocyte-like cells, proximal tubules, descending limbs of Henle, thick ascending limbs of Hendle, and distal convoluted tubules. In other embodiments, podocyte-like cells express one or more of: NPHS1+, PODXL+, and WT1+. In other embodiments, proximal tubules express one or more of: LTL+ and AQP1+. In other embodiments, descending limbs of Henle express one or more of: CDH1+ and AQP1+. In other embodiments, thick ascending limbs of Henle express one or more of CDH1+ and UMOD+. In other embodiments, distal convoluted tubules express one or more of CDH1+UMOD−. In other embodiments, NPCs are derived from human pluripotent stem cells (“hPSCs”). In other embodiments, hPSCs are derived from a patient suffering a disease mutation. In other embodiments, hPSCs have been genomically edited using CRISPR.
Also described herein is a quantity of organoids made by a method of generating kidney organoids, including providing a quantity of nephron progenitor cells (“NPCs”), and culturing the NPCs in a suspension culture for at about 11 days. In other embodiments, the method includes addition of one or more of: CHIR99021 and FGF9. In other embodiments, the kidney organoids comprise one or more cell types selected from: podocyte-like cells, proximal tubules, descending limbs of Henle, thick ascending limbs of Hendle, and distal convoluted tubules. In other embodiments, podocyte-like cells express one or more of: NPHS1+, PODXL+, and WT1+. In other embodiments, proximal tubules express one or more of: LTL+ and AQP1+. In other embodiments, descending limbs of Henle express one or more of: CDH1+ and AQP1+. In other embodiments, thick ascending limbs of Henle express one or more of CDH1+ and UMOD+. In other embodiments, distal convoluted tubules express one or more of CDH1+UMOD−. In other embodiments, NPCs are derived from human pluripotent stem cells (“hPSCs”). In other embodiments, hPSCs are derived from a patient suffering a disease mutation. In other embodiments, hPSCs have been genomically edited using CRISPR.
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 July, 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.
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.
Human pluripotent stem cells (hPSCs) are attractive sources for regenerative medicine and disease modeling in vitro. To date, however, the protocols used for the differentiation of hPSCs into specific kidney cell types with high efficiency, without the need for less well defined inducers such as embryonic spinal cords, have eluded many researchers. Significant advances have been made within the past decade that draw upon the Inventors' knowledge of kidney development to differentiate PSCs into cells of the kidney lineage. By recapitulating metanephric kidney development in vitro, the Inventors generated nephron progenitor cells (NPCs) with ˜80-90% purity within 9 days, without subpopulation selection during the directed differentiation protocol. hPSC-derived NPCs possess the developmental potential of their in vivo counterparts, forming renal vesicles that self-pattern into nephron structures. In both 2D and 3D culture, NPCs form kidney organoids containing epithelial nephron-like structures expressing markers of podocytes, proximal tubules, loops of Henle, distal tubules in organized, continuous structures that resemble the nephron in vivo.
A recent study revealed that the origin of the metanephros is distinct from the ureteric bud or pro/mesonephric lineages. They showed that the metanephros arises from the posterior intermediate mesoderm, whereas the ureteric bud and the pro/mesonephros is derived from anterior intermediate mesoderm. Therefore, the Inventors hypothesized that the specific induction of posterior intermediate mesoderm cells from hPSCs would greatly facilitate the induction of NPCs and avoid contamination with pronephric or mesonephric cells. Previous studies revealed that locations in the primitive streak define the subsequent differentiation into each segment of mesoderm i.e. paraxial, intermediate or lateral plate mesoderm. In addition, the timing of cell migration from the primitive streak defines the anterior-posterior axis in mesoderm, suggesting that the late stage of the primitive streak induces posterior mesoderm. The Inventors optimized the time of treatment with the GSK-3β inhibitor, CHIR99201 (CHIR), an inducer of the primitive streak, to induce late stage primitive streak. Additionally, the Inventors employed BMP4 inhibitors, as high BMP4 activity induces more posterior aspects of the primitive streak, which develops into lateral plate mesoderm. With this approach, the Inventors found a highly efficient protocol to induce SIX2+SALL1+WT1+PAX2+EYA1+ NPCs from both human ESCs and iPSCs with 80-90% efficiency within 9 days of differentiation. After the induction of NPCs, the Inventors transiently treated cells with CHIR (3 μM), generating multi-segmented nephron structures with characteristics of podocytes, proximal tubules, loops of Henle, and distal tubules sequenced in a self-assembled tubule in a manner that reflects normal nephron structure. Further analyses of other organoid compartments revealed CDH1+AQP2+ tubules and PDGFRI3+, endomucin+, or α-SMA+ interstitial cells in the kidney organoids (
The protocols to differentiate hPSCs into NPCs and kidney organoids provide novel platforms in vitro to study human kidney development and developmental disorders, inherited kidney diseases, kidney injury, nephrotoxicity testing, and kidney regeneration. In addition, the organoids provide systems in vitro for the study of intracellular and intercellular kidney compartmental interactions using differentiated cells. Since the protocols were derived to follow the steps of kidney development as the Inventors know them in vivo, the Inventors can induce intermediate cell populations at each step of differentiation: late mid primitive streak, posterior intermediate mesoderm, NPCs, pre-tubular aggregates, renal vesicles, and nephrons (
The maintenance of a differentiated phenotype in vitro will also allow for cellular biochemical analyses and the study of inter-compartmental interactions in ways that will likely more closely mimic the status in vivo than typical cell culture studies where the cells are generally dedifferentiated. The presence of CDH1+AQP2+ tubules and PDGFRβ+, endomucin+, or α-SMA+ interstitial cells, will permit studies of nephron-interstitial cell interactions. Ultimately, the protocol has the potential to serve as a foundation to provide organoids for kidney regenerative therapies.
In comparing the Inventors' protocol to previous published protocols to induce kidney lineage cells, there are many differences in efficiency, specificity, and simplicity. The Inventors' protocols yield NPCs, with much higher induction efficiency, from both hESCs and hiPSCs when compared to previous studies, including the Inventors' own. High induction efficiency at each step of differentiation, simultaneously, indicates high specificity of kidney induction. One study reported relatively high efficiency (˜60%) of SIX2+ cell induction with embryoid body formation, yet co-culture with mouse embryonic spinal cords is required to generate kidney epithelial cells while the Inventors' protocols use monolayer culture and chemically defined components. The Inventors were able to generate NPCs and organoids using fully defined conditions without the addition of any non-purified non-human factors, which is desirable for regenerative utility in humans. In addition, the Inventors' protocols use 96-well, round bottom, ultra-low attachment plates to generate 3D kidney organoids, which enables mass production of kidney organoids, while the other protocols to generate organoids require pelleting cells in eppendorf tubes or co-culture with mouse embryonic spinal cords. As previous studies have shown that the efficiency of the same differentiation protocol differs in separate hPSC lines, adjustments must be made to achieve similar results in varying lines. The Inventors define how to adjust the protocol for different lines of hPSCs, which further facilitates the applicability of the Inventors' differentiation protocols. The dose of growth factors can greatly influence the costs of the directed differentiation protocols. The Inventors were able to use lower doses of FGF9 than those used in the excellent protocol of Takasato et al. This has substantial financial advantages at the present time.
Other protocols result in generation of CD31+ endothelia-like cells and CDH1+GATA3+ collecting duct-like cells which on the surface can be perceived as a shortcoming of the Inventors' protocol. On the other hand the Inventors have now documented the presence of CDH1+AQP2+ tubules and PDGFRβ+, endomucin+, or α-SMA+ interstitial cells in the kidney organoids (
Described herein is a method for generating metanephric mesenchyme, including providing a quantity of human pluripotent stem cells (“hPSCs”), generating late primitive streak cells, inducing formation of posterior intermediate mesoderm cells, and differentiating into metanephric mesenchyme cells. In other embodiments, the human pluripotent stem cells are human embryonic stem cells (“hESCs”). In other embodiments, the human pluripotent stem cells are human induced pluripotent stem cells (“hiPSCs”). In other embodiments, generating late primitive streak cells includes culturing hPSCs in CHIR99021 for about 3-5 days. In other embodiments, this includes about 4 days. In various embodiments, the concentration of CHIR99021 is about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 μM. In various embodiments, the concentration of CHIR99021 is about 8-10 μM. In other embodiments, the method further includes addition of noggin In various embodiments, the concentration of noggin is about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 ng/ml. In various embodiments, the concentration of noggin is about 5 ng/ml. In other embodiments, inducing formation of posterior intermediate mesoderm cells includes culturing in the presence of activin for about 2-4 days. In other embodiments, this includes about 3 days. In various embodiments, the concentration of activin is about 5-10, 10-20-30 ng/ml. In various embodiments, the concentration of activin is about 10 ng/ml. In other embodiments, differentiating into metanephric mesenchyme cells includes addition of FGF9. In various embodiments, the concentration of FGF9 is about 5-10, 10-20-30 ng/ml. In various embodiments, the concentration of FGF9 is about 10 ng/ml. In other embodiments, the metanephric mesenchyme lineage cells are further differentiated into nephronic progenitor cells (NPCs) by addition of CHIR99021. In various embodiments, the concentration of CHIR99021 is about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 μM. In various embodiments, the concentration of CHIR99021 is about 5 μM. In other embodiments, late primitive streak cells express one or more of: T and TBX. In other embodiments, posterior intermediate mesoderm cells express one or more of: WT1 and HOXD11. In other embodiments, metanephric mesenchyme lineages cells express one or more of: SIX2, SALL1, WT1, and PAX2. In other embodiments, NPCs express one or more of: SIX2, SALL1, WT1, PAX2, and EYA1. In other embodiments, differentiation into metanephric mesenchyme cells is at least 50, 60, 70% or more efficient. In other embodiments, differentiation into metanephric mesenchyme cells is at least 70, 80, 90% or more efficient.
For example, hPSCs can be dissociated into single cells and maintained in a culture medium supplemented with the ROCK inhibitor Y27632 and optionally, FGF2 (10 ng/ml). Cells that are about 30, 40, 50, 60, or 70% confluent are then cultured in basic differentiation medium supplemented with CHIR99021 (8-10 μM) for 4 days to induce late primitive streak cells Noggin (5 ng/ml) was also used for hiPSC differentiation in addition to CHIR (10 To induce posterior intermediate mesoderm, cells were then cultured in Advanced RPMI+1X L-GlutaMAX+activin (10 ng/mL) for 3 days. For induction of nephron progenitor cells, the media was then changed to Advanced RPMI+1X L-GlutaMAX+FGF9 (10 ng/ml) for 7 days. CHIR (3 μM) can be added to the media from day 9 to 11 of differentiation to induce renal vesicles. On day 14, cells were switched to the basic differentiation medium and cultured for an additional 7 to 14 days (total of 21 to 28 days). The medium was replaced every 2 or 3 days. A variety of growth factors and small molecules were tested for differentiation. For organoid formation, hPSCs on day 9 of differentiation, which represents metanephric mesenchyme cells, arare dissociated resuspended in the basic differentiation medium supplemented with CHIR (3 μM) and FGF9 (10 ng/mL) and cultured at 37° C., 5% CO2 for 2 days. The medium is then changed to the basic differentiation medium supplemented with FGF9 10 ng/mL and cultured for 3 more days. After that, the organoids were cultured in basic differentiation medium with no additional factors for 7-21 days (a total of 21-35 days).
Further described herein is a composition of metanephric mesenchyme cells generated by a method for generating metanephric mesenchyme, including providing a quantity of human pluripotent stem cells (“hPSCs”), generating late primitive streak cells, inducing formation of posterior intermediate mesoderm cells, and differentiating into metanephric mesenchyme cells. In other embodiments, the human pluripotent stem cells are human embryonic stem cells (“hESCs”). In other embodiments, the human pluripotent stem cells are human induced pluripotent stem cells (“hiPSCs”). In other embodiments, generating late primitive streak cells includes culturing in CHIR99021 for about 3-5 days. In other embodiments, the method further includes addition of Noggin. In other embodiments, inducing formation of posterior intermediate mesoderm cells includes culturing in the presence of activin for about 2-4 days. In other embodiments, differentiating into metanephric mesenchyme cells includes addition of FGF9. In other embodiments, the metanephric mesenchyme lineage cells are further differentiated into nephronic progenitor cells (NPCs) by addition of CHIR99021. In other embodiments, late primitive streak cells express one or more of: T and TBX. In other embodiments, posterior intermediate mesoderm cells express one or more of: WT1 and HOXD11. In other embodiments, metanephric mesenchyme lineages cells express one or more of: SIX2, SALL1, WT1, and PAX2. In other embodiments, NPCs express one or more of: SIX2, SALL1, WT1, PAX2, and EYA1. In other embodiments, differentiation into metanephric mesenchyme cells is at least 50% efficient. In other embodiments, differentiation into metanephric mesenchyme cells is at least 70% efficient. A composition of nephron progenitor cells generated by the described method.
Described herein is a method of generating kidney organoids, including providing a quantity of nephron progenitor cells (“NPCs”), and culturing the NPCs in a suspension culture for at about 11 days. In other embodiments, the method includes addition of one or more of: CHIR99021 and FGF9. In other embodiments, the kidney organoids comprise one or more cell types selected from: podocyte-like cells, proximal tubules, descending limbs of Henle, thick ascending limbs of Hendle, and distal convoluted tubules. In other embodiments, podocyte-like cells express one or more of: NPHS1+, PODXL+, and WT1+. In other embodiments, proximal tubules express one or more of: LTL+ and AQP1+. In other embodiments, descending limbs of Henle express one or more of: CDH1+ and AQP1+. In other embodiments, thick ascending limbs of Henle express one or more of CDH1+ and UMOD+. In other embodiments, distal convoluted tubules express one or more of CDH1+UMOD−. In other embodiments, NPCs are derived from human pluripotent stem cells (“hPSCs”). In other embodiments, hPSCs are derived from a patient suffering a disease mutation. In other embodiments, hPSCs have been genomically edited using CRISPR.
Also described herein is a quantity of organoids made by a method of generating kidney organoids, including providing a quantity of nephron progenitor cells (“NPCs”), and culturing the NPCs in a suspension culture for at about 11 days. In other embodiments, the method includes addition of one or more of: CHIR99021 and FGF9. In other embodiments, the kidney organoids comprise one or more cell types selected from: podocyte-like cells, proximal tubules, descending limbs of Henle, thick ascending limbs of Hendle, and distal convoluted tubules. In other embodiments, podocyte-like cells express one or more of: NPHS1+, PODXL+, and WT1+. In other embodiments, proximal tubules express one or more of: LTL+ and AQP1+. In other embodiments, descending limbs of Henle express one or more of: CDH1+ and AQP1+. In other embodiments, thick ascending limbs of Henle express one or more of CDH1+ and UMOD+. In other embodiments, distal convoluted tubules express one or more of CDH1+UMOD−. In other embodiments, NPCs are derived from human pluripotent stem cells (“hPSCs”). In other embodiments, hPSCs are derived from a patient suffering a disease mutation. In other embodiments, hPSCs have been genomically edited using CRISPR. In other embodiments, NPCs are derived from hPSCs by a method for generating metanephric mesenchyme, including providing a quantity of human pluripotent stem cells (“hPSCs”), generating late primitive streak cells, inducing formation of posterior intermediate mesoderm cells, and differentiating into metanephric mesenchyme cells. In other embodiments, the human pluripotent stem cells are human embryonic stem cells (“hESCs”). In other embodiments, the human pluripotent stem cells are human induced pluripotent stem cells (“hiPSCs”). In other embodiments, generating late primitive streak cells includes culturing hPSCs in CHIR99021 for about 3-5 days. In other embodiments, this includes about 4 days. In various embodiments, the concentration of CHIR99021 is about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 μM. In various embodiments, the concentration of CHIR99021 is about 8-10 μM. In other embodiments, the method further includes addition of noggin In various embodiments, the concentration of noggin is about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 ng/ml. In various embodiments, the concentration of noggin is about 5 ng/ml. In other embodiments, inducing formation of posterior intermediate mesoderm cells includes culturing in the presence of activin for about 2-4 days. In other embodiments, this includes about 3 days. In various embodiments, the concentration of activin is about 5-10, 10-20-30 ng/ml. In various embodiments, the concentration of activin is about 10 ng/ml. In other embodiments, differentiating into metanephric mesenchyme cells includes addition of FGF9. In various embodiments, the concentration of FGF9 is about 5-10, 10-20-30 ng/ml. In various embodiments, the concentration of FGF9 is about 10 ng/ml. In other embodiments, the metanephric mesenchyme lineage cells are further differentiated into nephronic progenitor cells (NPCs) by addition of CHIR99021. In various embodiments, the concentration of CHIR99021 is about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 μM. In various embodiments, the concentration of CHIR99021 is about 5 μM. In other embodiments, late primitive streak cells express one or more of: T and TBX. In other embodiments, posterior intermediate mesoderm cells express one or more of: WT1 and HOXD11. In other embodiments, metanephric mesenchyme lineages cells express one or more of: SIX2, SALL1, WT1, and PAX2. In other embodiments, NPCs express one or more of: SIX2, SALL1, WT1, PAX2, and EYA1. In other embodiments, differentiation into metanephric mesenchyme cells is at least 50, 60, 70% or more efficient. In other embodiments, differentiation into metanephric mesenchyme cells is at least 70, 80, 90% or more efficient.
Also described herein is a method of screening a compound for an effect on kidney organoids, including providing a quantity of kidney organoids, adding one or more compounds to the kidney organoids, determining changes to phenotype or activity of the kidney organoids, and correlating the changes with an effect of the compounds on kidney organoids, thereby screening the one or more compounds for an effect on kidney organoids.
In various embodiments, determining changes to phenotype or activity includes detecting one or more markers in the tubular organoids.
H9 human ESCs (passage 45-65), and HDF-α human iPSCs (hiPSC derived from healthy fibroblasts; passage 22-42) were maintained in ReproFF2 (ReproCELL, #RCHEMD006) supplemented with FGF2 (10 ng/mL) (Peprotech, #100-18B) in 6-well tissue culture plates (Falcon, #353046) coated with 1% vol/vol LDEV-Free hESC-qualified Geltrex (Life Technologies, #A1413302) in a 37° C. incubator with 5% CO2. hPSCs were passaged using Dissociation Solution for human ES/iPS cells (ReproCELL, #RCHETP002) at a 1:3 split ratio every 7 days according to the manufacturer's protocol. H9 was purchased from WiCell. HDF-α human iPSCs was previously established in the Inventors' laboratory.
hPSCs grown on Geltrex were washed once with PBS (Life Technologies, #10010-049) and dissociated into single cells with Accutase (STEMCELL Technologies, #07920). Cells were then plated at a density of 2-2.4×104 (H9) or 1-1.4×104 (HDF, 2C) cells/cm2 onto 24-well tissue culture plates (TPP, #92024) coated with 1% Geltrex in ReproFF2 supplemented with the ROCK inhibitor Y27632 (10 μM) (TOCRIS, #1254) and FGF2 (10 ng/ml). After 72 hours, cells (50% confluent) were briefly washed in PBS and then cultured in basic differentiation medium consisting of Advanced RPMI 1640 (Life Technologies, #12633-020) and 1X L-GlutaMAX (Life Technologies, #35050-061) supplemented with CHIR99021 (8-10 μM) (TOCRIS, #4423) for 4 days to induce late primitive streak cells Noggin (5 ng/ml) was also used for hiPSC differentiation in addition to CHIR (10 μM). To induce posterior intermediate mesoderm, cells were then cultured in Advanced RPMI+1X L-GlutaMAX+activin (10 ng/mL) (R&D, #338-AC-050) for 3 days. For induction of nephron progenitor cells, the media was then changed to Advanced RPMI+1X L-GlutaMAX+FGF9 (10 ng/ml) (R&D, #273-F9-025/CF) for 7 days. CHIR (3 μM) was added to the media from day 9 to 11 of differentiation to induce renal vesicles. On day 14, cells were switched to the basic differentiation medium and cultured for an additional 7 to 14 days (total of 21 to 28 days). The medium was replaced every 2 or 3 days. A variety of growth factors and small molecules were tested for differentiation.
hPSCs on day 9 of differentiation, which represents metanephric mesenchyme cells, were dissociated with Accutase and resuspended in the basic differentiation medium supplemented with CHIR (3 μM) and FGF9 (10 ng/mL), and placed in 96-well, round bottom, ultra-low attachment plates (Corning, #7007) at 1×105 cells per well. The plates were centrifuged at 1500 rpm for 15 seconds, and the cells then cultured at 37° C., 5% CO2 for 2 days. The medium was then changed to the basic differentiation medium supplemented with FGF9 10 ng/mL and cultured for 3 more days. After that, the organoids were cultured in basic differentiation medium with no additional factors for 7-21 days (a total of 21-35 days).
3D kidney organoids were cultured in basic differentiation medium supplemented with gentamicin 5×10−4, 5×10−2, or 5 mg/mL (Sigma, #G1264) for 48 hours or cisplatin 5 or 50 μM (Sigma, #P4394) for 2, 6, 24 or 48 hours after day 21 of differentiation. Organoids were then fixed with 4% paraformaldehyde (Electron Microscopy Sciences, #RT15710) for 20 minutes for both whole-mount and frozen section immunohistochemistry.
Cell cultures were washed once with PBS and fixed in 4% paraformaldehyde for 15 minutes at room temperature (RT). Fixed cells were washed three times in PBS and incubated in blocking buffer (0.3% Triton X-100 and 5% normal donkey serum) for 1 hour at RT. The cells were then incubated with primary antibody overnight at 4° C. or for 2 hours at RT in antibody dilution buffer (0.3% Triton X-100 and 1% BSA in PBS). Cells were then washed three times in PBS and incubated with Alexa Fluor 488-, 555-, or 647-conjugated secondary antibodies (1:500) (Life Technologies) in antibody dilution buffer for 1 hour at RT. For immunostaining with biotinylated LTL (Vector Labs, #B-1325), Streptavidin/Biotin Blocking Kit (Vector Labs, #SP-2002) and Alexa Fluor 488- or 647-conjugated streptavidin (Lafe Technologies) were used according to manufacturer's instructions. Nuclei were counterstained with DAPI (Sigma, #D8417). Immunofluorescence was visualized using an inverted fluorescence microscope (Nikon Eclipse Ti). Quantification was performed using ImageJ by counting three to five representative fields per experiment at 20× magnification. The sample number of biological replicates in each experiment is shown in figure legends.
3D kidney organoids were fixed with 4% paraformaldehyde in PBS for 20 minutes at RT in a 96-well plate, then washed three times in PBS. The organoids were then incubated in blocking buffer (0.3% Triton X-100 and 5% normal donkey serum) for 1 hour at RT, then washed three times in PBS. The organoids were incubated with primary antibodies in antibody dilution buffer (0.3% Triton X-100 and 1% BSA in PBS) overnight at 4° C. The organoids were then washed with PBS three times for 1 hour each, with the third washing performed overnight at 4° C. For immunostaining with biotinylated LTL (Vector Labs, #B-1325), a Streptavidin/Biotin Blocking Kit (Vector Labs, #SP-2002) was used according to the manufacturer's protocol. The organoids were incubated with secondary antibodies in antibody dilution buffer for 1 hour at RT, then washed with PBS three times for 30 minutes each. Nuclei were counterstained with DAPI for more than 30 minutes. The organoids were then mounted with Vectashield (Vector Labs, #H-1200) and examined by confocal microscopy (Nikon Cl, Tokyo, Japan).
3D kidney organoids were fixed with 4% paraformaldehyde in PBS for 20 minutes in a 96-well plate, washed three times in PBS, then incubated with 30% sucrose (w/w) overnight at 4° C. The organoids were mounted with O.C.T compound (Fisher Scientific, #23-730-571) to make frozen blocks and were cut into 10-μm sections. The sections were washed three times in PBS for 5 minutes each, then incubated in blocking buffer (0.3% Triton X-100 and 5% normal donkey serum) for 1 hour. The sections were incubated with primary antibodies in antibody dilution buffer (0.3% Triton X-100 and 1% BSA in PBS) for 2 hours, then washed three times in PBS. The sections were incubated with secondary antibodies in antibody dilution buffer for 1 hour, then washed three times in PBS. The sections were then treated with Vectashield with DAPI. Imaging was performed with a Nikon Cl confocal microscope.
Total RNA was purified from cells using the RNAeasy MiniKit (Qiagen). 500 ng of RNA was used for reverse transcription with High-Capacity cDNA Reverse Transcription Kit (Life Technologies, #4368814) according to the manufacture's protocol. RT-PCR reactions were run in duplicate using cDNA (diluted 1:10), 300 nM forward and reverse primers, and iTAQ SYBR Green Supermix (Bio-Rad, #172-5122). Quantitative RT-PCR was performed using the iQ5 Multicolor Real-Time PCR Detection System (Bio-Rad). All samples were run with two technical replicates. β-actin was used as the housekeeping gene. Values were calculated by the delta delta CT method. Primer sequences are listed in Table 1.
Cells were dissociated using Accutase for 10 minutes, and cell clumps were removed with a 40-μm cell strainer (Corning, #352340). Cells were fixed with 2% paraformaldehyde for 15 minutes on ice and then permeabilized with 0.1% Triton for 15 minutes on ice. Cells were then blocked with PBS+5% donkey serum for 15 minutes and incubated with primary antibodies (PAX8 1:2500, LHX1 1:100, SIX2 1:1000, SALL1 1:100, WT1 1:100) for 30 minutes. After washing three times with 1% BSA in PBS, cells were incubated with secondary antibodies (Alexa Fluor 488-conjugated donkey anti-rabbit 1:5000 [Life Technologies], Cy5-conjugated donkey anti-mouse 1:2500 [Jackson ImmunoResearch] or Alexa Fluor 647-conjugated donkey anti-mouse 1:5000 [Life Technologies]) for 20 minutes on ice. Cells were then washed three times with 1% BSA in PBS. Flow cytometry was performed using MACSQuant (Miltenyi Biotec). Optimal dilution ratios of antibodies were determined using negative controls, undifferentiated H9 and human proximal tubular cell line (HKC-8) that does not express PAX8, LHX1, SIX2, SALL1, or WT1. HKC-8 was kindly provided by Dr. Lorraine Racusen (Johns Hopkins Hospital).
3D kidney organoids were fixed with 4% PFA for 20 minutes and subsequently fixed with electron microscopy (EM) fixation buffer consisting of 1.5% glutaraldehyde, 1% paraformaldehyde, 70 mM NaPO4 pH 7.2, and 3% sucrose in water overnight at 4° C. The organoids were washed three times in 0.2 M cacodylate buffer pH 7.4 for 10 minutes each and were incubated with 1% OsO4 for 1 hour on ice. The organoids were then washed three times in 0.2M cacodylate buffer pH 7.4 for 10 minutes each, dehydrated through a graded series of ethanol solutions, and embedded in Epon. 70 nm sections were cut and analyzed on a JEM-1010 (JEOL).
HKC-8 was maintained in DMEM/F12 (Life Technologies, #11320-033) supplemented with 10% fetal bovine serum (FBS) in a 37° C. incubator with 5% CO2, and was passaged every 3 or 4 days. NIH3T3-Wnt4 was maintained in DMEM (Corning, #10-013-CV) supplemented with 10% FBS in a 37° C. incubator with 5% CO2, and was passaged every 3 or 4 days. NIH3T3-Wnt4 was kindly provided by Dr. Andrew P. McMahon. A mouse ureteric bud cell line was maintained in DMEM (Corning, #10-013-CV) supplemented with 10% FBS in a 37° C. incubator with 5% CO2, and was passaged every 3 or 4 days. A mouse ureteric bud cell line was kindly provided by Dr. Jonathan Barasch. Mycoplasma contamination was tested by DAPI staining in all cell lines.
Recent efforts to direct the differentiation of PSCs into cells of the kidney lineage have focused the first step of differentiation on induction of the posterior primitive streak using a combination of growth factors that includes BMP4. The inclusion of BMP4 is justified by evidence that a gradient of Wnt3a and BMP4 patterns the anterior-posterior axis of the mouse primitive streak. However, recent developmental studies on early mesoderm patterning led us to reconsider this rationale. First, cells originating in the posterior primitive streak give rise to lateral plate mesoderm and not IM, from which the kidneys are derived (
To test this hypothesis, the Inventors treated human embryonic stem cells (hESCs; H9) with varying doses and durations of CHIR, which the Inventors and others previously showed could effectively differentiate hPSCs into T+primitive streak, solely or in combination with multiple developmental growth factors and small-molecule inhibitors of developmental signaling pathways (
To confirm the reproducibility of posterior IM induction in other hPSC lines, the Inventors next tested the combination of high-dose CHIR with activin, BMP4, FGF2, FGF8, FGF9, IDE-1, JAG1, Noggin or Y-27632 for 4 days followed by treatment with activin in HDF-α human induced pluripotent stem cells (hiPSCs). Intrinsic differences between HDF-α hiPSCs and H9 ESCs mandated slight modifications to the protocol to optimize the production of posterior IM cells. HDF-α hiPSCs required a higher dose of CHIR (10 μM) to induce T+TBX6+ primitive streak with an efficiency similar to that of H9 hESCs (
As the posterior primitive streak is induced by a BMP4 signal gradient, the Inventors hypothesized that CHIR treatment of HDF-α hiPSCs might induce endogenous BMP4 production that promotes differentiation into the posterior primitive streak and subsequently lateral plate mesoderm. The addition of low dose noggin (5 ng/mL), a BMP4 signaling antagonist, with CHIR in the first step of differentiation suppressed BMP4 to an optimal level to yield cells that expressed T and TBX6 but not FOXF1 on day 4 (
To differentiate WT1+HOXD11+ posterior IM cells into NPCs of the metanephric mesenchyme, the Inventors treated them on day 7 with varying doses of FGF9 (5-200 ng/mL), which the Inventors and others have previously shown to induce SIX2+ cells. A low dose of FGF9 (10 ng/mL) was sufficient to induce SIX2+ cells with an efficiency of 90% within 1-2 days of treatment (
Even with continuous FGF9 treatment, the Inventors observed between days 10 and 14 of differentiation that some of the NPCs spontaneously downregulated SIX2 expression and differentiated into round, polarized clusters of PAX8+LHX1+ cells reminiscent of renal vesicles (
To test whether the formation of epithelial structures could be facilitated by 3D culture conditions, on day 10, at the time of FGF9 withdrawal, the Inventors replated NPCs in ultra-low-attachment, round-bottom 96-well plates and cultured them for 1 week. NPCs formed 3D spherical aggregates, one per 96-well, in suspension culture. Whole-mount staining of aggregates at day 16 revealed the presence of LTL+tubules and clusters of NPHS1+PODXL+WT1+ cells (
Although the Inventors observed spontaneous differentiation of SIX2+ NPCs into PAX8+LHX1+ renal vesicles and early nephron epithelia in both 2D and 3D culture, the efficiency of this process was relatively low. During mouse nephrogenesis, Wnt9b, secreted by the ureteric bud, induces the metanephric cap mesenchyme to undergo a mesenchymal to epithelial transition via upregulation of Wnt4 in nephron progenitors, and the inductive Wnt signals are subsequently suppressed during formation of renal vesicles, possibly by Dkk1. This induction can be mimicked ex vivo or in vitro by the transient treatment of isolated mouse metanephric mesenchyme or FACS-sorted SIX2+ cells with the GSK-3β inhibitor BIO. The Inventors therefore sought to improve the efficiency of generating renal vesicles through transient rather than sustained activation of Wnt signaling in NPCs. Through a systematic screen of growth factors and small molecules, including CHIR, (
By day 21 of differentiation, the renal vesicles spontaneously formed elongated epithelial nephron structures without additional factors (
Next the Inventors investigated whether a 3D culture environment could promote the formation of more organized nephron structures with tubules possessing a lumen. The Inventors replated cells cultured in 2D on days 9, 11 and 14 corresponding to NPCs, pre-tubular aggregates and renal vesicles, respectively, into 3D suspension culture and applied the same protocol as with 2D (
Electron microscopy of the kidney organoids at day 21 of differentiation revealed ultrastructural features characteristic of mature renal epithelia. Structures resembling foot processes were noted on the surface of podocyte-like cells, which were encapsulated by a layer of cells reminiscent of Bowman's capsule (
The observation that no additional exogenous chemicals are required for hPSC-derived renal vesicles to form more mature nephron structures indicated that the signals for nephron formation are endogenously activated after renal vesicles are formed. As patterning of the nephron into its different segments begins at the renal vesicle stage during development, the Inventors postulated that developmental patterning could be mimicked by chemical modulation of these endogenous signals. Addition of the Notch signaling inhibitor DAPT from day 14 to 21 of differentiation resulted in a marked suppression of proximal tubule formation in both 2D and 3D culture (
Drug nephrotoxicity is an important cause of acute kidney injury in hospitalized patients. Currently there are no patient-specific models to assay nephrotoxicity in vitro. To test whether the Inventors' organoids could be used to study kidney injury and toxicity in vitro, the Inventors treated 3D hESC-derived kidney organoids after 21 days of differentiation for 48 hours with gentamicin (5 mg/mL), a commonly used antibiotic with well-established proximal tubular toxicity, or for 24 hours with cisplatin (5 an anticancer drug with proximal and distal tubular toxicity. Organoids were then fixed and immunostained for Kidney Injury Molecule-1 (KIM-1), a biomarker that is highly upregulated in the proximal tubules following acute kidney injury, together with LTL and E-cadherin to identify proximal and distal tubules, respectively.
Staining of both whole mount and frozen sections of gentamicin-treated organoids demonstrated clear KIM-1 expression at the luminal surface of LTL+tubules but not in E-cadherin+ tubules, and real-time PCR showed KIM-1 upregulation by gentamicin in a dose-dependent manner (
The Inventors describe the generation of segmentally patterned nephron structures from hPSCs by directed differentiation. The Inventors' protocol efficiently induces NPCs that spontaneously form renal vesicles in both 2D and 3D culture, which subsequently differentiate into self-organized nephron-like structures containing glomeruli, proximal tubules, loops of Henle, and distal tubules in a contiguous, ordered arrangement analogous to that of nephrons. To the Inventors' knowledge, no previous study has converted hPSCs into nephron structures with mature contiguous, ordered segments. Ref 18 generated SIX2+ cells, with an efficiency of 20%, that formed 3D aggregates containing isolated tubular structures but not continuous nephron structures with all epithelial components, and the protocol generated cells of both the metanephric mesenchyme and ureteric bud lineages, suggesting a lack of specificity. In comparison, the Inventors' method generates SIX2+SALL1+WT1+PAX2+ NPCs with 90% efficiency, and the cells spontaneously give rise to nephron structures containing all the major epithelial derivatives of the metanephric mesenchyme without detectable ureteric bud derivatives. Induction of SIX2+ cells from posterior IM in the Inventors' protocol required very low doses of FGF9 (10 ng/mL) compared to a concentration 20 times higher in ref. 18, suggesting that the WT1+HOXD11+ posterior IM cells are primed to respond to FGF9 and differentiate into NPCs. Taguchi and colleagues introduced the concept that targeting axial stem cells and posterior IM could facilitate the derivation of NPCs of the metanephric mesenchyme. Their protocol, based on an embryoid body culture system, required more intermediate steps and growth factors at each step and used mouse embryonic spinal cord to induce NPCs to undergo tubulogenesis. In addition, the persistent expression of SALL1 in the tubular structures indicated that they were still at an immature stage of nephron development. The Inventors' protocol to generate posterior IM and NPCs uses 2D monolayer culture, fewer steps, fewer chemicals and is fully chemically defined and more rapid. A key difference between the Inventors' protocol and previous ones is the Inventors' strategy to induce late-stage mid primitive streak rather than posterior primitive streak, based on developmental studies showing that the posterior primitive streak gives rise to lateral plate mesoderm rather than IM. By precisely defining the appropriate anterior-posterior position within the primitive streak (with the dose of CHIR and suppression of BMP4) and the timing of cell migration out of the primitive streak (with the duration of CHIR treatment), the Inventors could generate the correct precursor population that would give rise to NPCs. As predicted, posteriorization of the primitive streak with the addition of BMP4 causes hPSCs to differentiate into FOXF1+ lateral plate mesoderm. Finally, the Inventors show that minor modifications in the protocol optimize the efficiency of directed differentiation in both hESC and hiPSC lines. Variability in the levels of endogenous BMP4 signaling markedly affected the Inventors' ability to differentiate an hiPSC line into posterior IM, but this could be addressed by adjusting BMP4 levels with the addition of the antagonist Noggin.
As shown in previous studies of differentiating hPSCs to other lineages, the Inventors find that closely recapitulating critical developmental stages in vitro improves differentiation efficiencies and produces cells that most closely resemble their in vivo counterparts. The Inventors' hPSC-derived NPCs express all of the markers of metanephric mesenchyme and possess the intrinsic ability to spontaneously differentiate into renal vesicles and nephrons. Although the absence of ureteric bud and vascular progenitors in the Inventors' system precludes the generation of collecting ducts and glomerular capillaries, respectively, the NPC-derived renal vesicles self-organize into nephrons without these components in both 2D and 3D contexts.
The ability to generate 3D kidney organoids containing self-organized nephrons will facilitate studies of kidney development, disease and injury and of cell replacement therapies. Similar organoid systems have shown promising results for modeling the brain and gastric symptoms. The Inventors' data demonstrating that Notch inhibition suppresses proximal tubular differentiation confirms the utility of the Inventors' system for studying mechanisms of human kidney development, for which no models currently exist. Using gentamicin and cisplatin, the Inventors have also shown how the presence of the major epithelial components of the nephron in the organoids allows screening for toxic drug effects on multiple nephron segments. Given the individual variation in drug sensitivity in humans, the generation of kidney organoids from human iPSCs would enable drug testing in a patient-specific manner.
All maintenance culture experiments described here use ReproFF2 and 6-well plates coated with 1% LDEV-Free hESC-qualified Geltrex.
If differentiated cells are observed, remove those cells by aspiration. Those differentiated cells are usually located at the center of the colony when the size of each colony becomes too large. Some differentiated cells, however, sometimes exhibit fibroblast-like morphology at the periphery of the colonies. In that case, it is difficult to remove those differentiated cells; therefore, it is better to retry transition from “on feeder” to “feeder-free” from the beginning.
It is important to be careful with treatment with CTK solution which can be detrimental if exposed to cells too long. If one wait too long, colonies will be lost when subsequently washed with PBS. Hence, it is important to perform a visual check, with or without a microscope, for cell detachment after 2 min of exposure to CTK. When one notice that periphery of colonies starting to roll up, it is time to proceed to the next step. The Inventors recommend checking cells every 15 sec after 2 min of incubation with CTK solution.
The Inventors' protocols use feeder-free hPSC culture in ReproFF2 medium with lactose dehydrogenase elevating virus (LDEV)-Free hESC-qualified Geltrex-coated plates. The Inventors maintain hPSCs in 6-well plates coated with 1% LDEV-Free hESC-qualified Geltrex with ReproFF2 medium, supplemented with fibroblast growth factor 2 (FGF2), 10 ng/ml (step 1-6). If hPSCs were initially cultured on mouse embryonic fibroblast (MEF) feeders, the Inventors recommend passaging the cells at least 5 times under feeder-free conditions with ReproFF2. If it is too difficult to maintain hPSCs in ReproFF2, the Inventors recommend using StemFit Basic supplemented with FGF2 (10 ng/ml) maintenance of hPSCs in 6-well plates coated with 1% LDEV-Free hESC-qualified Geltrex (Box 1). hPSCs are passaged every 7 days whether ReproFF2 or StemFit Basic is used.
All differentiation experiments use 24-well plates coated with 1% LDEV-Free hESC-qualified Geltrex.
If the confluency is not high enough, passage the cells at 1:1 ratio until the confluency is nearly 80%. Less confluency will result in poor viability of cells and inefficient differentiation once initiated.
When one collect cells from 1 well of 6-well plates, usually one have 2˜5 million cells. If one have fewer than 1 million cells, the cell confluency in the maintenance hPSC culture was too low, which will result in poor viability of cells when differentiation is initiated.
The cells are plated for differentiation when the cells are passaged (step 7-13). The Inventors usually prepare 2 wells of 6-well plates, and use one well for differentiation and one well for continued passaging. Plating density significantly affects the differentiation efficiency, and each line of hPSCs requires adjustment. Pluripotency of hPSCs needs to be well maintained in the undifferentiated cells, and hence differentiated colonies need to be removed by aspiration. For differentiation, the cells are dissociated with Accutase for 15 min, resuspended in ReproFF2 supplemented with FGF2 10 ng/ml and Y27632 10 μM, and plated onto 24-well plates pre-coated with 1% LDEV-Free hESC-qualified Geltrex. The cells are cultured for 72 hours until the cells reach approximately 50% confluency.
The concentration of CHIR and addition of a BMP4 inhibitor depends on the cell line, the passaging number, and maintenance culture conditions. For H9, 8 μM of CHIR was best with ReproFF2 culture. For HDF, 10 μM of CHIR with 5 ng/ml noggin was best. If one use other cell lines or other culture media, adjust the protocol as follows: First, adjust the plating cell number to obtain 50% confluency when differentiation is initiated. Second, find the highest concentration of CHIR (3˜10 μM) which does not lead to cell detachment and death during 4 days of CHIR treatment. Third, test addition of a BMP4 inhibitor (noggin 5˜25 ng/ml or dorsomorphin 100˜500 nM), if the adjustment of the plating cell number and CHIR was not sufficient to induce SIX2+ cells.
This stage is important to achieve high efficiency of differentiation to NPCs. Check the morphology very carefully. If the cells still form a homogenous flat monolayer (too loose,
After 3 days of activin A treatment, the markers for posterior intermediate mesoderm, namely WT1 and HOXD11, become positive. Then, the cells are treated with FGF9, 10 ng/ml, for 2 days to induce NPCs. On day 9 of differentiation, a critical marker for NPCs, SIX2, becomes positive. SIX2 staining is very bright when the differentiation is induced appropriately (
Check the morphology of cells. If many cells form round polarized structures with lumens resembling renal vesicles (
From this nephron progenitor cell stage, the Inventors can apply the same differentiation treatment in either 2D or 3D culture. When the Inventors switch to 3D culture, the Inventors use 96-well, round bottom, ultra-low attachment plates, and plate 100,000 cells/well. Usually, the Inventors obtain 2˜3 million cells from one well of 24-well plates, which is sufficient to generate many organoids. In both 2D and 3D culture, the Inventors treat NPCs with CHIR 3 μM and FGF9 10 ng/ml for 2 days in order to induce pre-tubular aggregates (PAX8+LHX1+). Then, the Inventors switch back to FGF9, 10 ng/ml alone, and culture the cells for 3 days to differentiate them into renal vesicles (PAX8+LHX1+LAM+). After that, the Inventors use only the basic differentiation medium without any growth factors, and the cells form segmented-nephron structures within one week. The kidney organoids generated by the Inventors' protocols are stable in the basic differentiation medium for up to 3 months with feeding every 2˜3 days.
Typically, nephron structures are visible after 3˜5 days of culture after the “renal vesicle stage” in 2D culture (
There are a variety of possible applications using NPCs and kidney organoids. As an example of one of these applications, the Inventors show a nephrotoxicity assay with cisplatin, a known nephrotoxicant (
The differentiation efficiency is affected by the variability intrinsic to hPSC lines. The Inventors have clarified how to adjust the protocol for different cell lines grown initially in different culture conditions, reflecting the Inventors' experience with 2 different culture media and multiple hPSC lines. The Inventors recommend use of H9 and ReproFF2, as the simplest methods to achieve high differentiation efficiency. The Inventors believe that the the Inventors' adjustment method will enable researchers in different environments to generate NPCs and kidney organoids with different culture systems and different cell lines.
Another limitation of the Inventors' protocols is that the cells in the interstitial space of kidney organoids were not well characterized in the Inventors' original study because of lack of validated antibodies in human kidney samples and definitive morphological characteristics. Those cells were presumably derived from SIX2-negative population which accounted for 10˜20% of cells at the NPC stage, and could be collecting duct cells, pericytes, endothelial cells, smooth muscle cells, fibroblasts or others according to published studies. The Inventors' recent results showed CDH1+AQP2+ tubules (characteristic of connecting tubules/collecting ducts) and PDGFRIβ+ (characteristic of pericytes), endomucin+(characteristic of endothelial cells), or α-SMA+ (characteristic of myofibroblasts) interstitial cells in the organoids (
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 generating intermediate mesoderm, metanephric mesenchyme, nephronic progenitor cells, methods of generating intermediate mesoderm, metanephric mesenchyme, nephronic progenitor cells, 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.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/US2016/052350 | 9/16/2016 | WO | 00 |
| Number | Date | Country | |
|---|---|---|---|
| 62219704 | Sep 2015 | US |
