Patent Application
20250115872
The instant application contains a Sequence Listing that has been submitted electronically as an XML file named “29618-0376WO1_SL_ST26.xml”. The XML file, created on Jan. 31, 2023, is 25,788 bytes in size. The material in the XML file is hereby incorporated by reference in its entirety.
Generation of brown adipocytes from human pluripotent stem cells.
Understanding of human brown adipose tissue (BAT) development is limited. Current in vitro models to study human BAT are mostly based on brown adipocytes differentiated in vitro from primary cell lines and stromal vascular cells (Samuelson and Vidal-Puig, 2020). Several protocols have also been established to differentiate human pluripotent stem cells (hPSCs) into brown adipocytes using either transgenic overexpression of transcription factors (Ahfeldt et al., 2012) or treatment with growth factors and small molecules (Takeda et al., 2017). Other reported methods rely on serum-based spontaneous differentiation or treatment of hPSCs with a cytokine cocktail (Guenantin et al., 2017; Hafner et al., 2016; Nishio et al., 2012). Recently, protocols allowing the generation of brown adipocytes from hPSCs by recapitulating developmental cues have also been reported (Carobbio et al., 2021; Zhang et al., 2020). However, to date, a well characterized roadmap of brown fat lineage development is still missing. Therefore, benchmarking these protocols against the normal trajectory of brown adipocytes differentiation in vivo has not been possible.
Provided herein are methods of generating a Gata6-positive brown adipocyte precursor cell, the method comprising culturing PAX3-positive somitic progenitor cells in a medium comprising effective amounts of each of an FGF signaling pathway activator, a BMP signaling pathway activator, a TGFβ signaling pathway activator, a Wnt signaling pathway inhibitor, and a thyroid hormone receptor activator under conditions and for a time sufficient for the PAX3-positive cells to differentiate into a Gata6-positive brown adipocyte precursor cell.
Provided herein are methods wherein the PAX3-positive somatic progenitor cells are generated by a method comprising culturing a pluripotent cell, preferably an induced pluripotent stem cell (iPSC) or embryonic stem (ES) cell in a medium comprising effective amounts of each of an hepatocyte growth factor (HGF) signaling pathway activator, an insulin-like growth factor (IGF) signaling pathway activator, and an fibroblast growth factor (FGF) signaling pathway activator, a Wnt signaling pathway activator, and a bone morphogenetic protein (BMP) signaling pathway inhibitor, to generate a PAX3-positive somatic progenitor cell.
Provided herein are methods further comprising culturing the PAX3-positive somatic progenitor cell in a medium comprising an HGF signaling pathway activator and an IGF signaling pathway activator but lacking an FGF signaling pathway activator and a BMP signaling pathway inhibitor for between 8 and 23 days.
In some embodiments, the methods further comprise dissociating the PAX3-positive somatic progenitor cells after culturing the cells between 8 and 23 days. In some embodiments, the HGF signaling pathway activator comprises HGF. In some embodiments, the IGF signaling pathway activator comprises IGF-1. In some embodiments, the FGF signaling pathway activator comprises at least one of FGF-1, FGF-2, FGF-3, FGF-4, FGF-5, FGF-6, FGF-7, FGF-8, FGF-9, FGF-10, FGF-11, FGF-12, FGF-13, FGF-14, FGF-15, FGF-16, FGF-17, FGF-18, FGF-19, FGF-20, FGF-21, FGF-22, FGF-23, and combinations thereof. In some embodiments, the FGF signaling pathway activator comprises FGF-2. In some embodiments, the BMP signaling pathway inhibitor comprises at least one of LDN-193189, dorsomorphin, Noggin, Follistatin, Cerberus, and combinations thereof. In some embodiments, the BMP signaling pathway inhibitor comprises LDN-193189. In some embodiments, the BMP signaling pathway activator comprises at least one of BMP2, BMP4, BMP7, and combinations thereof. In some embodiments, the Wnt signaling pathway inhibitor comprises at least one of C59, XAV939, IWR-1, JW74, JW55, G007-LK, MSC2504877, benzimidazolone, WNT974, RK-287107, E7499, IWP-L6, GNF-1331, GNF-6231, ETC-131, IWP-29, LGK947, ETC159, ETC-1922159, and combinations thereof. In some embodiments, the Wnt signaling pathway inhibitor comprises C59. In some embodiments, the TGFβ signaling pathway activator comprises at least one of TGFβ1, TGFβ2, TGFβ3, and combinations thereof. In some embodiments, the TGFβ signaling pathway activator comprises TGFβ1. In some embodiments, the thyroid hormone receptor activator comprises at least one of triiodothyronine (T3), T4, resmetirom, eprotirome, sobetirome, Sob-AM2, VK2809, MB07344, IS25 TG68, and combinations thereof. In some embodiments, the thyroid hormone receptor activator comprises T3. In some embodiments, the HGF signaling pathway activator in some methods is different than the HGF signaling pathway activator in other parts of the same method. In some embodiments, the IGF1 signaling pathway activator in some methods is different than the IGF1 signaling pathway activator in other parts of the same method. In some embodiments, the FGF2 signaling pathway activator in some methods is different than the FGF2 signaling pathway activator in other parts of the same method. In some embodiments, dissociating the PAX3-positive somatic progenitor cells comprises applying one or more of Type IV collagenase and trypsin EDTA. In some embodiments, the methods further comprise seeding the dissociated cells at a density of 60,000-100,000/cm2. In some embodiments, the methods further comprise culturing the Gata6-positive cells in adipogenic differentiation medium to generate brown adipocytes. In some embodiments, adipogenic differentiation medium comprises Dulbecco's Modified Eagle Medium (DMEM) high glucose medium supplemented with serum-free serum replacement, 1× Insulin-Transferrin-Selenium (ITS), isobutylmethylxanthine (IBMX), 1-ascorbic acid, T3, a TGFβ inhibitor, dexamethasone, epidermal growth factor (EGF), hydrocortisone, and rosiglitazone.
Provided herein are in vitro methods of generating a UCP1-positive, Gata6-positive, and/or Pparg positive brown adipocyte organoid, the method comprising culturing a PAX3-positive somite-like structure in a medium comprising effective amounts of each of an FGF signaling pathway activator, a BMP signaling pathway activator, a TGFβ signaling pathway activator, a Wnt signaling pathway inhibitor, and triiodothyronine (T3), under conditions and for a time sufficient for the PAX3-positive somite-like structure to differentiate into a UCP1-positive, Gata6-positive, and/or Pparg positive brown adipocyte organoid.
Provided herein are methods wherein the PAX3-positive somite-like structure is generated by a method comprising culturing a pluripotent cell, preferably an induced pluripotent stem cell (iPSC) or embryonic stem (ES) cell in a medium comprising effective amounts of each of a Wnt activator and a bone morphogenetic protein (BMP) inhibitor, to generate a PAX3-positive somite-like structure.
In some embodiments, the HGF signaling pathway activator comprises HGF. In some embodiments, the IGF signaling pathway activator comprises IGF-1. In some embodiments, the FGF signaling pathway activator comprises at least one of FGF-1, FGF-2, FGF-3, FGF-4, FGF-5, FGF-6, FGF-7, FGF-8, FGF-9, FGF-10, FGF-11, FGF-12, FGF-13, FGF-14, FGF-15, FGF-16, FGF-17, FGF-18, FGF-19, FGF-20, FGF-21, FGF-22, FGF-23, and combinations thereof. In some embodiments, the FGF signaling pathway activator comprises FGF-2. In some embodiments, the BMP signaling pathway inhibitor comprises at least one of LDN-193189, dorsomorphin, Noggin, Follistatin, Cerberus, and combinations thereof. In some embodiments, the BMP signaling pathway inhibitor comprises LDN-193189. In some embodiments, the BMP signaling pathway activator comprises at least one of BMP2, BMP4, BMP7, and combinations thereof. In some embodiments, the Wnt signaling pathway inhibitor comprises at least one of C59, XAV939, IWR-1, JW74, JW55, G007-LK, MSC2504877, benzimidazolone, WNT974, RK-287107, E7499, IWP-L6, GNF-1331, GNF-6231, ETC-131, IWP-29, LGK947, ETC159, ETC-1922159, and combinations thereof. In some embodiments, the Wnt signaling pathway inhibitor comprises C59. In some embodiments, the TGFβ signaling pathway activator comprises at least one of TGFβ1, TGFβ2, TGFβ3, and combinations thereof. In some embodiments, the TGFβ signaling pathway activator comprises TGFβ1. In some embodiments, the thyroid hormone receptor activator comprises at least one of triiodothyronine (T3), T4, resmetirom, eprotirome, sobetirome, Sob-AM2, VK2809, MB07344, IS25 TG68, and combinations thereof. In some embodiments, the thyroid hormone receptor activator comprises T3. In some embodiments, the methods further comprise culturing the organoids in adipogenic differentiation medium to generate brown adipocyte organoids. In some embodiments, the adipogenic differentiation medium comprises Dulbecco's Modified Eagle Medium (DMEM) high glucose medium supplemented with serum-free serum replacement, 1× Insulin-Transferrin-Selenium (ITS), isobutylmethylxanthine (IBMX), 1-ascorbic acid, T3, a TGFβ inhibitor, dexamethasone, epidermal growth factor (EGF), hydrocortisone, and rosiglitazone.
Unless otherwise defined, all 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. Methods and materials are described herein for use in the present invention; other, suitable methods and materials known in the art can also be used. The materials, methods, and examples are illustrative only and not intended to be limiting. All publications, patent applications, patents, sequences, database entries, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control.
Other features and advantages of the invention will be apparent from the following detailed description and figures, and from the claims.
Brown Adipocytes (BAs) represent a specialized type of mammalian adipocytes able to unlink nutrient catabolism and ATP generation to dissipate energy as heat. This mechanism is physiologically important in mammals, allowing non-shivering thermogenesis to regulate body temperature in response to cold exposure. BA activity in humans shows an inverse correlation with body mass index and percentage of total body fat. Increasing brown fat activity either with drug treatment or with cell therapy approaches are considered promising approaches for the treatment of metabolic syndrome and obesity. Thus, the generation of brown adipocytes from human pluripotent stem cell has a huge potential for cell therapy and drug discovery.
Provided herein are novel differentiation methods to obtain functional BAs from human pluripotent stem cells. The methods include a treatment step with the signaling cues identified using FGF2, BMP7, TGFb1, a Wnt inhibitor and Triiodothyronin following dissociation and replating of the cultures to generate GATA6-positive adipocyte precursors. These progenitors can then be very efficiently differentiated into BA expressing the characteristic marker UCP1 after culture in adipogenic differentiation medium. The BAs differentiated under these conditions are functional, responding to adrenergic stress by lipolysis, heat production, and increased Oxygen consumption as compared to normal BAs. This protocol leads to the production of 2-dimensional cultures highly enriched in BA and BA precursors that could be used for drug screening or cell therapy.
Unless specifically defined otherwise, all technical and scientific terms used herein shall be taken to have the same meaning as commonly understood by one of ordinary skill in the art (e.g., in cell culture, molecular genetics, and biochemistry).
As used herein, the singular forms “a,” “an,” and “the” include the plural reference unless the context clearly dictates otherwise. Thus, for example, a reference to “a disease,” “a disease state”, or “a nucleic acid” is a reference to one or more such embodiments, and includes equivalents thereof known to those skilled in the art and so forth.
As used herein, the term “about” or “approximately” in the context of a numerical value or range means ±10% of the numerical value or range recited or claimed, unless the context requires a more limited range.
As used herein, “effective” when referring to an amount of a therapeutic compound refers to the quantity of the compound that is sufficient to yield a desired therapeutic response without undue adverse side effects (such as toxicity, irritation, or allergic response) commensurate with a reasonable benefit/risk ratio when used in the manner of this disclosure.
By “reference” is meant a standard or control condition.
The terms “subject,” “patient,” “individual,” and the like as used herein are not intended to be limiting and can be generally interchanged. The subject is a mammal in need of such treatment, e.g., a human with a metabolic disorder.
As used herein, a “symptom” associated with a disorder includes any clinical or laboratory manifestation associated with the disorder, and is not limited to what the subject can feel or observe.
As used herein, “treating” encompasses, e.g., inhibition, regression, or stasis of the progression of a disorder. Treating also encompasses the amelioration of a symptom or symptoms of the disorder. As used herein, “inhibition” of disease progression or a disease complication in a subject means preventing or reducing the rate, frequency, or risk of disease progression and/or disease complications in the subject. The terms “preventing” and “prevention” refer to the administration of a therapeutic protocol to a clinically asymptomatic individual who is susceptible or predisposed to a particular adverse condition, disorder, or disease, and thus relates to reducing the risk of the occurrence of symptoms and/or their underlying cause.
The transitional term “comprising,” which is synonymous with “including,” “containing,” or “characterized by,” is inclusive or open-ended and does not exclude additional, unrecited elements or method steps. By contrast, the transitional phrase “consisting of” excludes any element, step, or ingredient not specified in the claim. The transitional phrase “consisting essentially of” limits the scope of a claim to the specified materials or steps “and those that do not materially affect the basic and novel characteristic(s)” of the claimed invention.
Other features and advantages of the invention will be apparent from the following description of the preferred embodiments thereof, and from the claims. Unless otherwise defined, all 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. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable methods and materials are described below.
The present disclosure provides methods for generating BAT cells, preferably human BAT cells, from progenitor cells, e.g., paraxial mesoderm (presomitic mesoderm) (PAM) cells. PAM cells exhibit characteristics of progenitor cells of the Paraxial Mesoderm. In one embodiment, the PAM cells are characterized by the following properties:
Optionally, they may have long term self-renewal properties, e.g., they can be maintained in culture more than 6 months. The multipotency of the PAM cells can be tested in vitro, e.g., by in vitro differentiation into adipose, skeletal, dermal or muscle cell lineages using the protocols described below, and in particular in the Examples.
In some embodiments, the progenitor cells, e.g., paraxial mesoderm cells, are induced paraxial mesoderm (iPAM) cells, preferably derived from induced pluripotent stem (iPS) cells or embryonic stem (ES) cells, e.g., human pluripotent stem cells. Methods for obtaining and generating PAM cells are known in the art; see, e.g., WO2013030243 and Chal et al. 2015; Loh et al., Cell. 2016 Jul. 14; 166(2):451-67; Shelton et al., Stem Cell Reports. 2014 Sep. 9; 3(3): 516-529; Xi et al., Cell Rep. 2017 Feb. 7; 18(6): 1573-1585; Choi et al., Cell Rep. 2016 Jun. 7; 15(10):2301-12; and Hosoyama et al., Stem Cells Transl Med. 2014 May; 3(5):564-74.
In some embodiments, the methods include culturing iPS or ES cells in the presence of one a Wnt activator, e.g., a Wnt ligand, GSK3beta inhibitor, or member of the R-spondin family, or of both a Wnt activator and a BMP inhibitor, e.g., an ALK inhibitor. In some embodiments, progenitor cells isolated from a subject, e.g., autologous or primary cells, are differentiated using the described media; methods for isolating CD34+ BAT progenitor cells from the skeletal muscle of a subject are described in US PG Pub 20160303100.
Studying brown fat lineage development has proven complicated due to the difficulty of identifying intermediate stages in differentiation. Single cell sequencing techniques together with lineage tracing methods now provide powerful approaches for exploring cell states during differentiation programs. Disclosed herein is a characterization of the development of interscapular mouse brown fat using scRNAseq. The development of paraxial mesoderm-derived cell populations in the dorsal trunk at the forelimb level of mouse embryos which contains the interscapular brown fat was analyzed. This analysis identified a previously unrecognized brown adipocyte differentiation stage characterized by expression of the transcription factor Gata6. Using bioinformatics tools and lineage tracing analysis in mouse, reveal that these cells derive from a multipotent fibroblastic precursor population and give rise to Pparg-positive preadipocytes which differentiate into UCP1-positive brown adipocytes (
Our data also suggest that signaling cues known to be important for brown adipogenesis such as Wnt inhibition and BMP activation act at the Gata6-positive precursor stage. We show that Bmp4/7 and Bmpr1a/2 are enriched in the Gata6-positive progenitors. This is consistent with the well-established role of Bmps in the promotion of brown adipogenesis (Schulz et al., 2013; Tseng et al., 2008). We also observed transient expression of the Wnt inhibitors Sfrp1, 2 and 4 and Dkk2 in the Gata6-positive precursors, consistent with the well-known anti-adipogenic role of Wnt signaling (Bagchi and MacDougald, 2021; Longo et al., 2004). The thyroid hormone and the FGF/IGF1/PI3K pathways which play a role in the control of thermogenesis and brown adipose development and physiology were also activated in the Gata6 precursors (Mullur et al., 2014; Ohta and Itoh, 2014). We modified a protocol to produce highly efficient generation of functional human brown adipocytes in vitro using defined serum-free culture conditions. ScRNAseq analysis of the human cells differentiating in vitro show that they recapitulate a developmental sequence very similar to that observed in mouse in vivo. This sequence results in the production of GATA6-positive brown adipocyte precursors and then to PPARG-positive preadipocytes and finally to UCP1-positive adipocytes which exhibit functional properties characteristic of brown adipocytes.
Our analyses using the STREAM or Waddington OT pipelines suggest that brown adipocyte precursors share a common precursor with other somite-derived mesenchymal lineages such as dermis, muscle connective tissue, cartilage precursors and smooth muscle (
Gata6 (GATA binding protein 6), is a zinc finger transcription factor which plays an essential role in the development of extraembryonic tissues (Koutsourakis et al., 1999). Its null mutation in mouse leads to lethality shortly after implantation. Gata6 has also been implicated in the development of several organs including heart, lung, gut, pancreas, and skin but its expression during brown fat development has so far not been reported (Freyer et al., 2015; Morrisey et al., 1996). Gata6 was however identified in silico as a transcription factor potentially regulating the expression of brown fat specific genes such as Zic1 and TCA cycle enzymes such as citrate synthase (Cs) in the ProFat database (Cheng et al., 2018). Also, a GATA recognition motif was identified in ATACseq data of differentiating human adipocytes in vitro (Zhang et al., 2020). In cardiomyocytes, GATA-6 was shown to associate with Ppara to regulate the expression of the glucose transporter Glut4 (Yao et al., 2012). Together, these data suggest that Gata6 could play a role in the regulation of energy metabolism in differentiating brown adipocytes.
Our transcriptome analysis of the brown adipocyte lineage development in mouse identified signaling cues which allowed us to modify our paraxial mesoderm development protocol to generate brown fat from hPSCs. This also provided us with a benchmark to which the differentiating hPSCs could be compared. Our comparison of the differentiated human adipocytes generated in vitro in our protocol with those generated by two other recently published protocols (Carobbio et al., 2021; Zhang et al., 2020) suggest that the transcriptional signature of the adipocytes generated in our conditions is closer to endogenous fetal brown adipocytes. In humans, BAT activity shows an inverse correlation with body mass index and percentage of total body fat (Cypess et al., 2009; Saito et al., 2009). Moreover, the graft of ectopic BAT leads to improvement of glucose metabolism in diabetic or obese mice (Gunawardana and Piston, 2012; Stanford et al., 2013). Our work could therefore help the development of cell therapy strategies involving the graft of human brown adipocytes generated in vitro for the treatment of obesity, metabolic syndrome, and diabetes.
Methods for generating BAT and/or BA progenitor cells from source cells, e.g., embryonic stem cells (ESC) or induced pluripotent stem cells (iPSCs), as described herein can include an induction step of culturing PAX3-positive progenitors in the presence of a cocktail comprising FGF2/BMP7/TGBb1/a Wnt inhibitor/T3, preferably in adherent conditions or organoid conditions; an example is shown in
In some embodiments, Gata6-positive cells are produced by the following methods. In some embodiments, human induced pluripotent stem cells (iPSCs) are dissociated to single cells using enzymes and seeded at a density of 10,000-50,000/cm2 on cell-culture dishes in feeder-free maintenance medium supplemented with a ROCK inhibitor. In some embodiments, the cells make small pluripotent colonies and are treated with basal medium supplemented with a basal medium which includes insulin-transferrin-selenium, a GSK3 inhibitor, and a BMP inhibitor (presomitic mesoderm/PSM medium) for between 2 and 5 days. In some embodiments, over the next 3 days, the cells are cultured in a basal medium supplemented with basal medium which includes insulin-transferrin-selenium, a GSK3 inhibitor, and a BMP inhibitor, and an FGF activator. To differentiate cells into PAX3-positive somitic progenitors, in some embodiments, cells are treated with high glucose media supplemented with serum free media, non-essential amino acid supplement, synthetic basal medium, an HGF activator, an IGF activator, an FGF activator, and a BMP inhibitor for between 1-5 days. In some embodiments, the cells were further treated with a Wnt activator to differentiate into PAX3-positive somitie progenitors. In some embodiments, the PAX3-positive cells are further differentiated into PAX7 and EBF2-positive progenitors in high glucose media supplemented with serum free media, non-essential amino acid supplement, synthetic basal medium, an HGF activator, an IGF activator, for between 5-30 days. To generate brown adipocyte precursors, in some embodiments, the cells are dissociated to single cells enzymes in PBS, filtered, and seeded at a density of 30,000-150,000/cm2 on cell-culture dishes and treated with high glucose media supplemented with serum-free media, a basal medium which includes insulin-transferrin-selenium, an FGF activator, PROCN inhibitor (Wnt inhibitor), a BMP activator, a TGFβ activator, and a thyroid hormone receptor activator (together referred to as adipocyte precursor medium) for between 1-10 days. In some embodiments, after 4-6 days cells are cultured in high glucose media supplemented with serum-free medium, a basal medium which includes insulin-transferrin-selenium, a phosphodiesterase inhibitor, an antioxidant (e.g., vitamin C), a thyroid hormone receptor activator, a TGFβ inhibitor, corticosteroid, an EGF activator, a second corticosteroid, and insulin sensitizer (together referred to as adipogenic differentiation medium) for between 20-50 days, wherein the adipogenic differentiation medium is changed every 2-3 days.
In some embodiments, human induced plutipotent stem cells (iPSCs) are dissociated to single cells using Accutase and seeded at a density of 30,000-33,000/cm2 on Matrigel-coated dishes in mTESR-1 supplemented with 10 μM Y-27632. In some embodiments, the cells make small pluripotent colonies and are treated with DMEM/F12 GlutaMAX supplemented with 1% ITS, 3 μM CHIR99021, and 0.5 μM LDN-193189 (PSM medium) for 3 days. In some embodiments, over the next 3 days, the cells are cultured in DMEM/F12 GlutaMAX supplemented with 1% ITS, 3 μM CHIR99021, 0.5 μM LDN-193189, and 20 ng/ml FGF-2. To differentiate cells in PAX3-positive somitic progenitors, in some embodiments, cells are treated with Dulbecco's Modified Eagle Medium (DMEM) high glucose media supplemented with 15% KSR, 1× Non-Essential Amino Acid (NEAA) Solution, 0.01 mM bME, 10 ng/ml HGF, 2 ng/ml IGF-1, 20 ng/ml FGF-2, and 0.1 μM LDN-193189 for 2 days. In some embodiments, the PAX3-positive cells are further differentiated into PAX7 and EBF2-positive progenitors in DMEM high glucose supplemented with 15% KSR, 1×NEAA, 0.01 mM bME, 10 ng/ml HGF, and 2 ng/ml IGF-1 for between 8-23 days. To generate brown adipocyte precursors, in some embodiments, the cells are dissociated to single cells using Type IV Collagenase and Trypsine EDTA in PBS, filtered through a 30 μm filter and seeded at a density of 60,000-100,000/cm2 on Matrigel-coated dishes and treated with DMEM high glucose supplemented with 5% KSR, 1% ITS, 2 ng/ml FGF-2, 20 nM C59, 20 ng/ml BMP7, 10 ng/ml TGFb1, 2 nM T3 (Adipocyte precursor medium) for between 4-6 days. In some embodiments, after 4-6 days cells are cultured in DMEM high glucose supplemented with 5% KSR, 1% ITS, 500 μM IBMX, 25.5 g/ml L-Ascorbic acid, 2 nM T3, 5 μM TGFb inhibitor SB431542, 1 μM Dexamethasone, 10 ng/ml EGF, 4 μg/ml Hydrocortisone, 1 μM Rosiglitazone (adipogenic differentiation medium) for between 30-40 days, wherein the adipogenic differentiation medium is changed every 2-3 days.
Recombinant proteins, activators/agonists, and antagonists/inhibitors are typically supplemented each at 0.1 ng/mL to 1 mg/mL of culture medium. Small compounds/molecules activators or inhibitors are typically supplemented each at 1 nM to 1 mM. Then, cultures are changed to an adipocyte differentiation medium, and refreshed every 2-3 days. Adipogenic differentiation medium comprises a DMEM-based medium containing a serum replacement supplement as known in the art or described herein, 1× Insulin-Transferin-Selenium (ITS, Gibco) and further comprising 0.5 mM isobutylmethylxanthine (IBMX), 125 nM indomethacin, 1 nM triiodothreonine (T3), 5 mM dexamethasone, and 1 mM rosiglitazone (see also a related adipogenic cocktail described in Sharma et al, PO 2014 and US20150030662).
Provided herein are methods to produce BAs and/or BAT in 3D. In some embodiments, BAs containing organoids can be obtained from source cells, e.g., ESCs or iPSCs, e.g., human iPSCs, by a protocol that involves an induction step of culturing PAX3-positive progenitors with FGF2/BMP7/TGBb1/a Wnt inhibitor/T3 treatment described in the protocol detailed above. Briefly, in some embodiments, iPSC cultures are dissociated to single cells using enzymes and aggregated in non-adhesive plates to form 3D structures. These structures, called somitoids, are cultured in basal medium supplemented with a basal medium which includes insulin-transferrin-selenium, a GSK3 inhibitor, and a BMP inhibitor (presomitic mesoderm/PSM medium) supplemented with serum to generate PAX3-expressing somite-like structures and dermomyotomal cells. In some embodiments, the cells were further treated with a Wnt activator to generate PAX3-expressing somite-like structures. In some embodiments, somitoids are then cultured in high glucose media supplemented with with serum-free media, a basal medium which includes insulin-transferrin-selenium, an FGF activator, PROCN inhibitor (Wnt inhibitor), a BMP activator, a TGFβ activator, and a thyroid hormone receptor activator (together referred to as adipocyte precursor medium) for between 2-15 days. In some embodiments, to differentiate the Gata6-positive precursors into lipid vesicle containing BA, somitoids are cultured in the adipogenic differentiation medium (high glucose media supplemented with serum-free medium, a basal medium which includes insulin-transferrin-selenium, a phosphodiesterase inhibitor, an antioxidant (e.g., vitamin C), a thyroid hormone receptor activator, a TGFβ inhibitor, corticosteroid, an EGF activator, a second corticosteroid, and insulin sensitizer (together referred to as adipogenic differentiation medium) for between 20-40 days. This method leads to the production of tridimensional organoids highly enriched in UCP1 positive BAs. These organoids act as glucose sinks, and serve as a substrate to graft in patients for cell therapy of obesity, type II diabetes, or metabolic syndrome.
Provided herein are methods to produce BAs and/or BAT in 3D. In some embodiments, BAs containing organoids can be obtained from source cells, e.g., ESCs or iPSCs, e.g., human iPSCs, by a protocol that involves an induction step of culturing GATA6+BA progenitors with FGF2/BMP7/TGBb1/T3 treatment described in the protocol detailed above. Briefly, in some embodiments, iPSC cultures are dissociated to single cells using Accutase and aggregated in non-adhesive U-bottom plates to form 3D structures. These structures, called somitoids, are cultured in the DMEM/F12 GlutaMAX supplemented with 1% ITS, 3 μM CHIR99021, and 0.5 μM LDN-193189 (PSM medium) supplemented with 5% FCS for 11 days to generate PAX3-expressing somite-like structures and dermomyotomal cells. In some embodiments, somitoids are then cultured in DMEM high glucose supplemented with 5% KSR, 1% ITS, 2 ng/ml FGF-2, 20 nM C59, 20 ng/ml BMP4, 10 ng/ml TGFb1, 2 nM T3 (Adipocyte precursor medium) for between 4-12 days. In some embodiments, to differentiate the precursors into lipid vesicle containing BA, somitoids are cultured in the adipogenic differentiation medium (DMEM high glucose supplemented with 5% KSR, 1% ITS, 500 μM IBMX, 25.5 g/ml L-Ascorbic acid, 2 nM T3, 5 μM TGFb inhibitor SB431542, 1 μM Dexamethasone, 10 ng/ml EGF, 4 g/ml Hydrocortisone, 1 μM Rosiglitazone) for between 20-40 days. This method leads to the production of tridimensional organoids highly enriched in UCP1 positive BAs. These organoids act as glucose sinks, and serve as a substrate to graft in patients for cell therapy of obesity, type II diabetes, or metabolic syndrome.
Recombinant proteins, activators/agonists, and antagonists/inhibitors are typically supplemented each at 0.1 ng/mL to 1 mg/mL of culture medium. Small compounds/molecules activators or inhibitors are typically supplemented each at 1 nM to 1 mM. Then, cultures are changed to an adipocyte differentiation medium, and refreshed every 2-3 days. Adipogenic differentiation medium comprises a DMEM-based medium containing a serum replacement supplement as known in the art or described herein, 1× Insulin-Transferin-Selenium (ITS, Gibco) and further comprising 0.5 mM isobutylmethylxanthine (IBMX), 125 nM indomethacin, 1 nM triiodothreonine (T3), 5 mM dexamethasone, and 1 mM rosiglitazone (see also a related adipogenic cocktail described in Sharma et al, PO 2014 and US20150030662).
The present methods can include the use of agonists or activators of the canonical Wnt/beta catenin signaling pathway, characterized by a Wnt dependant inhibition of glycogen synthase kinase 3β (GSK-3β), leading to a subsequent stabilization of β-catenin, which then translocates to the nucleus to act as a transcription factor. As used herein the term “activator” denotes a molecule, e.g., antibody, protein, nucleic acid, or small molecule that enhances Wnt signaling activity. For example, for the canonical Wnt/β-catenin signaling pathway, this activity can be measured by Wnt reporter activity using established multimers of LEF/TCF binding sites reporters, and/or inhibition of GSK-3β, and/or activation of canonical Wnt target genes such as T, Tbx6, Msgn1, or Axin2.
In some embodiments, GSK-3β inhibitors activate the Wnt pathway. Inhibitors of GSK-3β are known in the art and include lithium chloride (LiCl), Purvalanol A, olomoucine, alsterpaullone, kenpaullone, SB216763 (3-(2,4-dichlorophenyl)-4-(1-methyl-1H-indol-3-yl)-1H-pyrrole-2,5-dione) and SB415286 (3-[(3-chloro-4-hydroxyphenyl)amino]-4-(2-nitrophenyl)-1H-pyrrole-2,5-dio-ne) which are maleimide derivatives, benzyl-2-methyl-1,2,4-thiadiazolidine-3,5-dione (TDZD-8), 2-thio(3-iodobenzyl)-5-(1-pyridyl)-[1,3,4]-oxadiazole (GSK3 inhibitor II), 2,4-dibenzyl-5-oxothiadiazolidine-3-thione (OTDZT), (2′Z,3′E)-6-Bromoindirubin-3′-oxime (BIO), a 4 Dibromoacetophenone (i.e., Tau Protein Kinase I (TPK I) Inhibitor), 2-Chloro-1-(4,5-dibromo-thiophen-2-yl)-ethanone, N-(4-Methoxybenzyl)-N′-(5-nitro-1,3-thiazol-2-yl)urea (AR-A014418), and indirubins (e.g., indirubin-5-sulfonamide; indirubin-5-sulfonic acid (2-hydroxyethyl)-amide indirubin-3′-monoxime; 5-iodo-indirubin-3′-monoxime; 5-fluoroindirubin; 5, 5′-dibromoindirubin; 5-nitroindirubin; 5-chloroindirubin; 5-methylindirubin, 5 bromoindirubin), 4-Benzyl-2-methyl-1,2,4-thiadiazolidine-3,5-dione (TDZD-8), 2-thio(3-iodobenzyl)-5-(1-pyridyl)-[1,3,4]-oxadiazole (GSK3 inhibitor II), 2,4-Dibenzyl-5-oxothiadiazolidine-3-thione (OTDZT), (2′Z,3′E)-6-Bromoindirubin-3′-oxime (BIO), a 4 Dibromoacetophenone (i.e., Tau Protein Kinase I (TPK I) Inhibitor), 2-Chloro-1-(4,5-dibromo-thiophen-2-yl)-ethanone, (vi) N-(4-Methoxybenzyl)-N′-(5-nitro-1,3-thiazol-2-yl)urea (AR-A014418), and H-KEAPPAPPQSpP-NH2 (L803) or its cell-permeable derivative Myr-N-GKEAPPAPPQSpP-NH2 (L803-mts). Other GSK30 inhibitors are disclosed in U.S. Pat. Nos. 6,417,185; 6,489,344; 6,608,063 and Published U.S. Applications Nos. 20160375006 2; 0040138273; 20040106574; 20040077707; 20040034037; 20030216574; and 20030130289.
Other activators of Wnt signaling include WAY-316606 (SFRP Inhibitor) Bodine et al., Bone. 2009 June; 44(6):1063-8; (hetero)arylpyrimidines (Gilbert et al., Bioorg Med Chem Lett. 2010 Jan. 1; 20(1):366-70); IQ1 (PP2A Activator) Miyabayashi et al., Proc Natl Acad Sci USA. 2007 Mar. 27; 104(13):5668-73 (2007); QS11 (ARFGAP1 Activator) Zhang et al., Proc Natl Acad Sci USA. 2007 May 1; 104(18):7444-8; SB-216763 (GSK3 Inhibitor) Coghlan et al., Chem Biol. 2000 October; 7(10):793-803 (2000); CHIR99021 (6-[[2-[[4-(2,4-Dichlorophenyl)-5-(5-methyl-1H-imidazol-2-yl)-2-pyrimidinyl]amino]ethyl]amino]-3-pyridinecarbonitrile, GSK3 Inhibitor); DCA (beta-catenin Activator) Pai et al., Mol Biol Cell. 2004 May; 15(5):2156-63; and 2-amino-4-[3,4-(methylenedioxy) benzyl-amino]-6-(3-methoxyphenyl) pyrimidine (AMBMP) Liu et al., Inflamm Res. 2016 January; 65(1):61-9. Wnt activators can be effective in the methods provided herein at a range between 0.1 micromolar and 10 micromolar. For example, the Wnt activators described herein can be used at 0.2 μM, 0.3 μM, 0.4 μM, 0.5 μM, 0.6 μM, 0.7 μM, 0.8 μM, 0.9 μM, 1.0 μM, 1.1 μM, 1.2 μM, 1.3 μM, 1.4 μM, 1.5 μM, 1.6 μM, 1.7 μM, 1.8 μM, 1.9 μM, 2.0 μM, 3 μM, 4, μM, 5 μM, 6 μM, 7 μM, 8 μM, 9 μM, 10 μM, and any concentrations in between.
Wnt ligands in natural or modified forms can also be used as Wnt activator in the form of purified or recombinant proteins. Wnt ligands comprise a large family of Wnt activators including but not limited to Wnt-1, Wnt-2, Wnt-2b, Wnt-3a, Wnt-4, Wnt-5a, Wnt-5b, Wnt-6, Wnt-7a, Wnt-7a/b, Wnt-7b, Wnt-8a, Wnt-8b, Wnt-9a, Wnt-9b, Wnt-10a, Wnt-10b, Wnt-11, Wnt-16b, Wnt-3. The R-spondins family encompassing R-spondin1, R-spondin2, R-spondin3, and R-spondin4 are potent Wnt signaling enhancer through their binding to LGR4/5 surface receptors. Natural or modified, synthetic, polypeptides fragments generated from known Wnt activator polypeptide sequence can also be potential Wnt activators. When the activator of the Wnt signaling pathway is a protein, it may be a purified protein or a recombinant protein or a synthetic protein. Alternatively, conditioned media produced from a cell line engineered to express one or several Wnt ligands can be used as a Wnt activator. Wnt signaling can also be activated by blocking negative regulators of Wnt signaling, such as Axin and APC using RNA interference.
In some embodiments, the methods disclosed herein use Wnt inhibitors. The terms “Wnt antagonist”, “Wnt inhibitor”, and “inhibitor of Wnt signaling” are used interchangeably herein to mean an agent that antagonizes, inhibits, suppresses, or negatively regulates Wnt modulation of a cell's activity. Likewise, the phrases “antagonizing Wnt signaling” and “inhibiting Wnt signaling” are used interchangeably herein to mean antagonizing, inhibiting, or otherwise negatively regulating Wnt modulation of a cell's activity. Wnt inhibitors may act anywhere along a Wnt signaling pathway to antagonize Wnt signaling. For example, Wnt inhibitors may antagonize activation of the Wnt co-receptors, for example by blocking Wnt binding; or they may antagonize the activity of a protein in a Wnt-responsive intracellular signaling cascade, e.g., the Wnt/β-catenin, Wnt/PCP or Wnt/Ca2+ signaling pathways, for example by promoting β-catenin degradation or preventing translocation of stabilized β-catenin to the nucleus. Wnt inhibitors can include small-molecule inhibitors and small proteins.
Non-limiting examples of Wnt inhibitors include members of the Dkk family (Dickkopf proteins), including Dkk-1, Dkk-2, Dkk-3, Dkk-4, Soggy-1/DkkL1. Further examples of Wnt inhibitors include secreted frizzled related proteins (sFRPs) including sFRP-1, sFRP-, sFRP-3, sFRP-4, and sFRP-5. Further examples of Wnt inhibitors include can be a porcupine inhibitor. Other examples of Wnt inhibitors include APCDD1, APCDD1L, Draxin, IWP, IWP-2, IWP-4, Ginsenoside Rh4, M2912, KY-05009, ICG-001; INCB28060/INC280/NVP-INC280, IWR-1-endo, KY02111, FH535, WIKI4, CCT251545, Salinomycin, Prodigiosin, IQ-1, KYA1797K, NCB-0846, PNU-74654, LF3, iCRT14, Adavivint, PRI-724, Triptonide, M435-1279, Resibufogenin, LGK974, C59, ETC-159, JW55, RCM-1, MSAB, Ant1.4Br/Ant 1.4C1, Niclosamide, apicularen, bafilomycin, XAV939, IWR, G007-LK, G244-LM, Lanatoside C, pyrvinium, NSC668036, 2,4-diamino-quinazoline, Quercetin, Isoquercitrin, ICG-001, PKF115-584, BC2059/Tegavivint, Shizokaol D, IGFBP-4, LMBR1L, Notum, SOST/Sclerostin, USAG1, WIF-1. Wnt inhibitors can be effective in the methods provided herein at a range between 0.1 micromolar and 10 micromolar. For example, the Wnt inhibitors described herein can be used at 0.2 μM, 0.3 μM, 0.4 μM, 0.5 μM, 0.6 μM, 0.7 μM, 0.8 μM, 0.9 μM, 1.0 μM, 1.1 μM, 1.2 μM, 1.3 μM, 1.4 μM, 1.5 μM, 1.6 μM, 1.7 μM, 1.8 μM, 1.9 μM, 2.0 μM, 3 μM, 4, μM, 5 μM, 6 μM, 7 μM, 8 μM, 9 μM, 10 μM, and any concentrations in between.
A bone morphogenetic protein (BMP) activator or agonist refers to a molecule. e.g., antibodies, protein, nucleic acids, or small molecules that enhances BMP signaling activity. Typically, a BMP signaling pathway agonist/activator binds to or directly activates components of the biological pathway in which BMP participates, such as a BMP receptor protein (e.g. BMP type I receptors ALK2 and/or ALK3) or downstream SMAD proteins). Examples of known BMP signaling activators include BMP7, BMP4, and BMP2. BMP activators can be effective in the methods provided herein at a range between 1 ng/ml and 1000 ng/ml. For example, the BMP activators described herein can be used at 1 ng/ml, 10 ng/ml, 100 ng/ml, 200 ng/ml, 300 ng/ml, 400 ng/ml, 500 ng/ml, 600 ng/ml, 700 ng/ml, 800 ng/ml, 900 ng/ml, 1000 ng/ml, and any concentrations in between. A bone morphogenetic protein (BMP) antagonist or inhibitor refers to a molecule, e.g., antibodies, proteins, nucleic acids, or small molecules, that inhibits or attenuates the biological activity of the BMP signaling pathway either by directly interacting with BMP or by acting on components of the biological pathway in which BMP participates, such as a BMP receptor protein (e.g. BMP type I receptors ALK2 and/or ALK3) or downstream SMAD proteins). Typically, a compound is deemed to be an inhibitor of the BMP signaling pathway if, after culturing cells in the presence of said compound, the level of phosphorylated Smad 1, 5 or 8 is decreased compared to cells cultured in the absence of said compound. Levels of phosphorylated Smad proteins can be measured by Western blot using antibodies specific for the phosphorylated form of said Smad proteins.
Examples include noggin, an inhibitor of the transduction activity of the BMP type I receptors ALK2 and/or ALK3, chordin, or LDN-193189 (DM3189), a dorsomorphin derivative. Noggin, chordin, follistatin, and gremlin block BMP signaling by sequestrating secreted BMP, preventing its binding to the receptor. The inhibitor of the BMP signaling pathway may be a BMP antagonist, a chemical compound that blocks BMP type I and/or type II receptors activity (BMP type I/II receptor inhibitor), an inhibitor of BMP type I and/or type II gene expression, or a molecule which inhibits any downstream step of the BMP signaling pathway. The inhibitor of BMP signaling may be a natural or a synthetic compound. When the inhibitor of the BMP signaling pathway is a protein, it may be a purified protein or a recombinant protein or a synthetic protein. Non-limiting examples of BMP inhibitors include A77-01, A83-01, LDN193189 dihydrochloride, dormorphin dihydrochloride, DMG-1, SB505124, ML347, M4K2163, K02288, DMH1, DMH2, dorsomorphin, noggin, follistatin, and cerberus.
It is well known in the art that an inhibitor of BMP type I receptors may block the BMP signaling pathway, see for example Yu et al, Nat Chem Biol. 2008.
In one non-limiting example, the inhibitor of BMP type I receptors is Dorsomorphin, a chemical compound or any derivatives thereof generated by structure-activity studies (Cuny G D et al., 2008), e.g., LDN-193189. Dorsomorphin (6-[4-(2-Piperidin-1-yl-ethoxy)phenyl]-3-pyridin-4-yl-pyrazolo[1,5-a]pyrimidine, also known as Compound C, specifically inhibits BMP type I receptors (ALK2, 3, and 6) (Yu P B et al., 2008). For additional BMP inhibitors, see WO2013030243, which is herein incorporated by reference in its entirety. BMP inhibitors can be effective in the methods provided herein at a range between 1 nM and 1000 nanomolar. For example, the BMP inhibitors described herein can be used at 1 nM, 10 nM, 100 nM, 200 nM, 300 nM, 400 nM, 500 nM, 600 nM, 700 nM, 800 nM, 900 nM, 1000 nM, and any concentrations in between.
A Hepatocyte Growth factor signaling pathway agonist/activator is a molecule e.g., antibody, protein, nucleic acid, or small molecule, that enhances HGF signaling activity. Typically, a HGF signaling pathway agonist/activator binds to or directly activates the c-Met proto-oncogene (HGFR) receptor. Examples of known HGF signaling activators include Hepatocyte Growth Factor-Scatter Factor (HGF-SF), HGF variants such as described further in U.S. Pat. Nos. 5,227,158; 5,316,921 and 5,328,837; HGFR activating antibodies such as MAb DO-24, 6E10 or 3D6 described in Pietronave et al, Am J Physiol Heart Circ Physiol. 2010 April; 298(4):H1155-65 and U.S. Ser. No. 08/884,669. Among the intracellular signaling pathways transduced by HGFR activation are MAPK, STAT3, and PI3K/Akt signaling axis. These intracellular pathways can be monitored using biochemical reporter assays and for transduction cascade activation, as known in the art. HGF signaling activity can be measured by biological assays such as mitogenic, motogenic, or morphogenic activities as a result of HGF binding to a HGF receptor. In particular, c-MET activation can lead to the disruption of cadherin-based cell-cell contacts, and promote cell motility as evidenced by a cell-scattering phenotype, which was first described with MDCK cells treated with HGF (Zhu et al., Cell Growth Differ. 1994 April; 5(4):359-66). HGF activators can be effective in the methods provided herein at a range between 1 and 1000 ng/ml. For example, the HGF activators described herein can be used at 1 ng/ml, 10 ng/ml, 100 ng/ml, 200 ng/ml, 300 ng/ml, 400 ng/ml, 500 ng/ml, 600 ng/ml, 700 ng/ml, 800 ng/ml, 900 ng/ml, 1000 ng/ml, and any concentrations in between.
An Insulin-like Growth factor signaling pathway agonist/activator is a molecule e.g., antibody, protein, nucleic acid, or small molecule that enhances IGF signaling activity. Examples of known IGF activators include IGF-1 (e.g., somatomedin C), MGF, IGF-2 and Insulin, demethylasterriquinone B-1, (DMAQ-B1; Salituro et al, Recent Prog Horm Res. 2001; 56:107-26). Typically, an IGF signaling pathway agonist/activator binds to or directly activates IGF1 receptor (IGF1Ra, b), and/or the insulin receptor (IR), and/or Insulin receptor-related receptor IR-related receptor (IRR). IGF signaling transduction results in the activation of several intracellular pathways including RAS-MAP kinase pathway, PI3K/AKT, and PI3K/mTor signaling pathways. These intracellular pathways activity can be monitored using biochemical reporter assays and transduction cascade activation, as known in the art. IGF activators can be effective in the methods provided herein at a range between 1 and 1000 ng/ml. For example, the IGF activators described herein can be used at 1 ng/ml, 10 ng/ml, 100 ng/ml, 200 ng/ml, 300 ng/ml, 400 ng/ml, 500 ng/ml, 600 ng/ml, 700 ng/ml, 800 ng/ml, 900 ng/ml, 1000 ng/ml, and any concentrations in between.
A Fibroblast Growth factor signaling pathway agonist/activator is a molecule e.g., antibody, protein, nucleic acid, or small molecule that enhances FGF signaling activity. Examples of FGF signaling pathway agonist/activator are natural or recombinant proteins ligands including the 23 identified FGF ligands, e.g., FGF-1, FGF-2, FGF-3, FGF-4, FGF-5, FGF-6, FGF-7, FGF-8, FGF-9, FGF-10, FGF-11, FGF-12, FGF-13, FGF-14, FGF-15, FGF-16, FGF-17, FGF-18, FGF-19, FGF-20, FGF-21, FGF-22, and FGF-23. FGF activators bind and/or activate one or several FGF receptors (FGFR1 to 4) leading to the activation of several signaling pathways including RAS-MAPK, PI3K-AKT, PLCγ, and STAT signaling pathways. These intracellular pathways activity can be monitored using biochemical reporter assays and transduction cascade activation, as known in the art. FGF activators can be effective in the methods provided herein at a range between 1 and 1000 ng/ml. For example, the FGF activators described herein can be used at 1 ng/ml, 10 ng/ml, 100 ng/ml, 200 ng/ml, 300 ng/ml, 400 ng/ml, 500 ng/ml, 600 ng/ml, 700 ng/ml, 800 ng/ml, 900 ng/ml, 1000 ng/ml, and any concentrations in between.
A TGFβ signaling pathway activator is a molecule, e.g., an antibody, protein, nucleic acid, or small molecule that enhances TGFβ signaling activity. Typically, TGFβ agonist/activator binds to or activates the TGF-β receptor (TGF-βR). Examples of TGFβ activators include, but are not limited to, TGFβ1, TGFβ2, and TGFβ3. TGFβ activators can be effective in the methods provided herein at a range between 1 and 1000 ng/ml. For example, the TGFβ activators described herein can be used at 1 ng/ml, 10 ng/ml, 100 ng/ml, 200 ng/ml, 300 ng/ml, 400 ng/ml, 500 ng/ml, 600 ng/ml, 700 ng/ml, 800 ng/ml, 900 ng/ml, 1000 ng/ml, and any concentrations in between.
A TGFβ signaling pathway inhibitor is a molecule, e.g., an antibody, protein, nucleic acid, or small molecule that inhibits TGFβ signaling activity. Typically, TGFβ inhibition binds to or blocks the TGF-β receptor (TGF-βR). Examples of TGFβ inhibitors include, but are not limited to, SB431542, A83-01, RepSox, SB525334, SB505124, LY364947, galunisertib, D4476, GW788388, SD208, R268712, IN1130, SM16, A77-01, and AZ12799734. TGFβ inhibitors can be effective in the methods provided herein at a range between 0.1 and 10 micromolar. For example, the TGFβ inhibitors described herein can be used at 0.1 μM, 0.2 μM, 0.3 μM, 0.4 μM, 0.5 μM, 0.6 μM, 0.7 μM, 0.8 μM, 0.9 μM, 1.0 μM, 1.1 μM, 1.2 μM, 1.3 μM, 1.4 μM, 1.5 μM, 1.6 μM, 1.7 μM, 1.8 μM, 1.9 μM, 2.0 μM, 3 μM, 4, μM, 5 μM, 6 μM, 7 μM, 8 μM, 9 μM, 10 μM, and any concentrations in between.
A thyroid hormone receptor activator is a molecule, e.g., an antibody, protein, nucleic acid, or small molecule that enhances thyroid hormone receptor signaling activity. Typically, thyroid hormone receptor agonist/activator binds to or activates the thyroid hormone receptor. Examples of thyroid hormone receptor agonist/activator activators include, but are not limited to, triiodothyronine (T3), T4, resmetirom, eprotirome, sobetirome, Sob-AM2, VK2809, MB07344, IS25 and TG68.
The Gata6-positive brown adipose precursors (BAs, or BAT) produced using methods described herein can be used in a number of different ways. For example, the cells can be used in a transplantation protocol in which BAT cells are transplanted into a subject to treat metabolic disorders such as diabetes, insulin-resistance, obesity, hypertension, an insulin resistance disorder, or hepatic steatosis. The cells can optionally be derived from induced pluripotent stem (iPS) cells or embryonic stem (ES) cells, cells obtained from the subject themselves (e.g., autologous), or can be derived from allogeneic cells. The Gata6-positive brown adipose precursor cells can be transplanted into areas where BAT is already present, e.g., a supraclavicular region, the nape of the neck, over the scapula, alongside the spinal cord, near proximal branches of the sympathetic nervous system that terminate in BAT depots, around at least one of the kidneys, the renal capsule, the liver, the skin, or elsewhere. The Gata6-positive brown adipose precursors cells can be suspended in a suitable transplant media, such as phosphate buffered saline or other salines. The cell transplant mixture can be injected via a syringe with a needle ranging from 30 to 18 gauge, with the gauge of the needle being dependent upon such factors as the overall viscosity of the adipocyte suspension, into a target location. Preferably, needles ranging from 22 to 18 gauge and 30 to 27 gauge can be used. See, e.g., US20170000827; Liu et al., Cell Res 23, 851-854 (2013); and US20170014455, which are incorporated by reference in their entireties. In some embodiments, the cells are present in a biocompatible semisolid or gel matrix, e.g., a hydrogel matrix, suitable for transplantation; for example, a hyaluronic acid-based hydrogel (see Tharp et al., Diabetes. 2015 November; 64(11):3713-24); Tharp and Stahl, Front Endocrinol (Lausanne). 2015; 6: 164; Vaicik et al., J. Mater. Chem. B, 2015, 3, 7903-7911); collagen/alginate microspheres (Yao et al., Biofabrication, 4(4): 045003 (2012)); or adipose tissue derived soluble extracellular matrix (sECM) and methylcellulose (MC) (see Kim et al., PLoS ONE 11(10): e0165265) can be used. See also Cho et al., Cell Transplat 16(4):421-434 (2007). Compositions comprising these hydrogels and the Gata6-positive brown adipose precursors cells described herein are also within the scope of the present disclosure.
In some embodiments, the methods described herein include treating a subject with the Gata6-positive brown adipose precursor cells or cells derived from Gata6-positive brown adipose precursor cells for metabolic-related indications, e.g., diabetes, insulin-resistance, obesity, hypertension, an insulin resistance disorder, or hepatic steatosis. The terms “treatment”, “treating” and the like are used herein to generally mean obtaining a desired pharmacologic and/or physiologic effect. The effect may be prophylactic in terms of completely or partially preventing a disease or symptom thereof and/or may be therapeutic in terms of a partial or complete cure for a disease and/or adverse effect attributable to the disease. “Treatment” as used herein covers any treatment of a disease in a mammal, and includes: (a) preventing the disease from occurring in a subject which may be predisposed to the disease but has not yet been diagnosed as having it; (b) inhibiting the disease, i.e., arresting its development; or (c) relieving the disease, i.e., causing regression of the disease. The therapeutic agent maybe administered before, during or after the onset of disease or injury. The treatment of ongoing disease, where the treatment stabilizes or reduces the undesirable clinical symptoms of the patient, is of particular interest. Such treatment is desirably performed prior to complete loss of function in the affected tissues. The subject therapy will desirably be administered during the symptomatic stage of the disease, and in some cases after the symptomatic stage of the disease. In some embodiments, the cells would be administered by injection, graft, infusion, implant, or any combination thereof
In addition to their use in transplantation, the Gata6-positive brown adipose precursor cells derived using the present methods can also be used, e.g., for in vitro screening of drugs to determine their effect on BAT, including thermogenic assays. Thus, included herein are methods for screening test compounds, e.g., polypeptides, polynucleotides, inorganic or organic large or small molecule test compounds, to identify agents useful in the treatment of metabolic disorders such as diabetes, insulin-resistance, obesity, hypertension, an insulin resistance disorder, or hepatic steatosis. The methods can be used to identify whether a test compound has an effect on BAT, e.g., to increase BAT numbers or activity, or to decrease BAT numbers or activity. In some embodiments, the methods used to identify whether a test compound has an effect on BAT include testing for an increase of UCP1 expression. In some embodiments, the methods used to identify whether a test compounds has an effect on BAT include testing for glycerol release. In some embodiments, the methods used to identify whether a test compounds has an effect on BAT include testing for an increase in oxygen consumption. In some embodiments, the methods used to identify whether a test compounds has an effect on BAT include testing for an increase in temperature. In some embodiments, the methods used to identify whether a test compounds has an effect on BAT include measuring metabolism and thermogenic capacity of cells including measurement of glycolysis, mitochondrial respiration, lipid synthesis, beta-oxidation and mitochondrial uncoupling. In some embodiments, the methods used to identify whether a test compounds has an effect on BAT include measuring response on RNA and protein expression of BAT specific genes.
As used herein, “small molecules” refers to small organic or inorganic molecules of molecular weight below about 3,000 Daltons. In general, small molecules useful for the invention have a molecular weight of less than 3,000 Daltons (Da). The small molecules can be, e.g., from at least about 100 Da to about 3,000 Da (e.g., between about 100 to about 3,000 Da, about 100 to about 2500 Da, about 100 to about 2,000 Da, about 100 to about 1,750 Da, about 100 to about 1,500 Da, about 100 to about 1,250 Da, about 100 to about 1,000 Da, about 100 to about 750 Da, about 100 to about 500 Da, about 200 to about 1500, about 500 to about 1000, about 300 to about 1000 Da, or about 100 to about 250 Da).
The test compounds can be, e.g., natural products or members of a combinatorial chemistry library. A set of diverse molecules should be used to cover a variety of functions such as charge, aromaticity, hydrogen bonding, flexibility, size, length of side chain, hydrophobicity, and rigidity. Combinatorial techniques suitable for synthesizing small molecules are known in the art, e.g., as exemplified by Obrecht and Villalgordo, Solid-Supported Combinatorial and Parallel Synthesis of Small-Molecular-Weight Compound Libraries, Pergamon-Elsevier Science Limited (1998), and include those such as the “split and pool” or “parallel” synthesis techniques, solid-phase and solution-phase techniques, and encoding techniques (see, for example, Czarnik, Curr. Opin. Chem. Bio. 1:60-6 (1997)). In addition, a number of small molecule libraries are commercially available. A number of suitable small molecule test compounds are listed in U.S. Pat. No. 6,503,713, incorporated herein by reference in its entirety.
Libraries screened using the methods of the present invention can comprise a variety of types of test compounds. A given library can comprise a set of structurally related or unrelated test compounds. In some embodiments, the test compounds are peptide or peptidomimetic molecules. In some embodiments, the test compounds are nucleic acids.
In some embodiments, the test compounds and libraries thereof can be obtained by systematically altering the structure of a first test compound, e.g., a first test compound that is structurally similar to a known natural binding partner of the target polypeptide, or a first small molecule identified as capable of binding the target polypeptide, e.g., using methods known in the art or the methods described herein, and correlating that structure to a resulting biological activity, e.g., a structure-activity relationship study. As one of skill in the art will appreciate, there are a variety of standard methods for creating such a structure-activity relationship. Thus, in some instances, the work may be largely empirical, and in others, the three-dimensional structure of an endogenous polypeptide or portion thereof can be used as a starting point for the rational design of a small molecule compound or compounds. For example, in one embodiment, a general library of small molecules is screened, e.g., using the methods described herein.
In some embodiments, a test compound is applied to a test sample comprising a Gata6-positive brown adipose precursor cell or tissue a brown adipose cell obtained using a method described herein, and one or more effects of the test compound is evaluated. For example, the methods can evaluate the ability of the test compound to alter expression of one or more of UCP-1, Dio2, Cidea2, FABP4, C/ebp, Cox7a1, Cox7a2, Cox8a, Zic1, Ebf2, Cd34, and/or Prdm16, and/or proliferation of BAT progenitors, and/or fat storage evaluated for example by Oil Red O staining and lipid dye, and/or BAT thermogenic activity, and/or BAT metabolic activity, as evaluated by measure of glycolysis, mitochondrial respiration, lipid synthesis, beta-oxidation and mitochondrial uncoupling.
Methods for evaluating each of these effects are known in the art. For example, ability to modulate expression of a protein can be evaluated at the gene or protein level, e.g., using quantitative PCR or immunoassay methods. In some embodiments, high throughput methods, e.g., protein or gene chips as are known in the art (see, e.g., Ch. 12, Genomics, in Griffiths et al., Eds. Modern genetic Analysis, 1999, W. H. Freeman and Company; Ekins and Chu, Trends in Biotechnology, 1999, 17:217-218; MacBeath and Schreiber, Science 2000, 289(5485):1760-1763; Simpson, Proteins and Proteomics: A Laboratory Manual, Cold Spring Harbor Laboratory Press; 2002; Hardiman, Microarrays Methods and Applications: Nuts & Bolts, DNA Press, 2003), can be used to detect an effect on the BAT. Ability to modulate signaling via the VEGFR2, atrial natriuretic peptide receptor (NPR), beta3 adrenergic receptor (03-AR), OxR1, BMPRII, Alk7 (Acvr1c), FGFR, and/or Irisin receptor signaling pathways can be evaluated, e.g., using genetically encoded reporter assays and/or using biochemical assays for known intracellular transduction pathways transduction including, p38, PKC, Pi3K, and phosphoSMADs (see, e.g., Tchivileva et al., Mol Immunol. 2009 July; 46(11-12): 2256-2266; Kumar et al., 1356(2):221-228, 1997; Moshinsky et al., J Biomol Screen. 2003 August; 8(4):447-52; Zilberberg et al., BMC Cell Biol. 2007; 8: 41; Logeart-Avramoglou et al., Anal Biochem. 2006 Feb. 1; 349(1):78-86]. In some embodiments, differentiation of iPSCs into brown adipocytes can be determined by using cells that express a reporter. For example, a UCP1-mCherry construct can be introduced immediately 5′ to the start codon of UCP1, separated by a P2A, to create a fluorescent human iPSC reporter line using CRISPR-Cas9.
Thermogenic assays can include exposing the cells to thermogenesis-inducing factors, including but not limited to a GPR120 activator (e.g., GW9508 (see Quesada-Lopez et al., Nat Commun. 2016 Nov. 17; 7:13479) or Berberine (see Zhang et al., Nat Commun. 2014 Nov. 25; 5:5493) or exposure to cold.
A test compound that has been screened by a method described herein and determined to increase expression of UCP-1, Dio2, Cidea2, FABP4, C/ebp, Ebf2, and/or Prdm16, and/or increase proliferation of BAT progenitors, and/or increase fat storage and lipolysis evaluated for example by Oil Red O staining, and/or increase BAT mitochondrial content, and/or increase BAT thermogenic activity, and/or increase BAT metabolic activity, can be considered a candidate compound for treating a metabolic disorder.
Alternatively, the methods can be used to identify compounds that decrease BAT activity, e.g., to encourage weight gain or to treat conditions associated with increased BAT activity, hypermetabolism, underweight or weight loss associated with chronic disease. A test compound that has been screened by a method described herein and determined to decrease expression of UCP-1, Dio2, Cidea2, FABP4, C/ebp, Ebf2, and/or Prdm16, and/or decrease proliferation of BAT progenitors, and/or decrease fat storage and lipolysis evaluated for example by Oil Red O staining, and/or decrease BAT mitochondrial content, and/or decrease BAT thermogenic activity, and/or decrease BAT metabolic activity, can be considered a candidate compound for treating a condition associated with increased BAT activity, hypermetabolism, underweight, or weight loss associated with chronic disease.
A candidate compound that has been screened, e.g., in an in vivo model of a disorder, e.g., a metabolic disorder such as diabetes, insulin-resistance, obesity, hypertension, an insulin resistance disorder, or hepatic steatosis, and determined to have a desirable effect on the disorder, e.g., on one or more symptoms of the disorder, can be considered a candidate therapeutic agent. Candidate therapeutic agents, once screened in a clinical setting, are therapeutic agents. Candidate compounds, candidate therapeutic agents, and therapeutic agents can be optionally optimized and/or derivatized, and formulated with physiologically acceptable excipients to form pharmaceutical compositions.
Test compounds identified as “hits” (e.g., test compounds that increase expression of UCP-1, increase proliferation of BAT, increase Oil Red O staining, or to increase expression of UCP-1, Dio2, Cidea2, FABP4, C/ebp, Ebf2, and/or Prdm16, and/or increase proliferation of BAT progenitors, and/or increase fat storage evaluated for example by Oil Red O staining, and/or increase BAT mitochondrial content, and/or increase BAT thermogenic activity, and/or increase BAT metabolic activity) in an Gata6-positive brown adipose precursor screen can be selected and systematically altered, e.g., using rational design, to optimize binding affinity, avidity, specificity, or other parameter. Such optimization can also be screened for using the methods described herein. Thus, in one embodiment, the invention includes screening a first library of compounds using a method known in the art and/or described herein, identifying one or more hits in that library, subjecting those hits to systematic structural alteration to create a second library of compounds structurally related to the hit, and screening the second library using the methods described herein.
Test compounds identified as hits can be considered candidate therapeutic compounds, useful in treating metabolic disorder such as diabetes, insulin-resistance, obesity, hypertension, an insulin resistance disorder, or hepatic steatosis. A variety of techniques useful for determining the structures of “hits” can be used in the methods described herein, e.g., NMR, mass spectrometry, gas chromatography equipped with electron capture detectors, fluorescence and absorption spectroscopy. Thus, the invention also includes compounds identified as “hits” by the methods described herein, and methods for their administration and use in the treatment, prevention, or delay of development or progression of a disorder described herein.
Test compounds identified as candidate therapeutic compounds can be further screened by administration to an animal model of metabolic disorder such as diabetes, insulin-resistance, obesity, hypertension, an insulin resistance disorder, or hepatic steatosis, as described herein. The animal can be monitored for a change in the disorder, e.g., for an improvement in a parameter of the disorder, e.g., a parameter related to clinical outcome. In some embodiments, the parameter is weight, and an improvement would be weight loss. In some embodiments, the subject is a human, e.g., a human with obesity, and the parameter is weight loss. In some embodiments, the subject is a human with, or an animal model of, a metabolic disorder such as diabetes, insulin-resistance, hypertension, an insulin resistance disorder, or hepatic steatosis, and the parameter is improved blood glucose control, improved insulin sensitivity, improved blood pressure control or improved liver enzyme levels. In this context, “improved” means returned to or near normal levels.
The invention is further described in the following examples, which do not limit the scope of the invention described in the claims.
NCRM1 (NIH CRM control iPSC line (male)) human iPSC and other cell lines were routinely cultured on Matrigel (Corning, 354263) coated culture plates (Corning, 353046) in mTeSR (Stemcell Technologies, 85850). Upon confluency, cultures were dissociated into single cells using Accutase (Corning, 25-058-C). Cells were seeded on Matrigel coated culture plates in mTeSR supplemented with 10 μM Y-27632 dihydrochloride (R&D Systems, 1254/10) at a density of 56,000/cm2 for routine maintenance of iPSC lines. On the following day, medium was replaced with mTeSR only and medium was changed every day. Cultures became confluent every fourth day and were passaged as described above. For freezing, cultures were dissociated into single cells with Accutase and frozen in NutriFreez D10 Cryopreservation Medium (Biological Industries, 01-0020-50).
iPSCs were differentiated into presomitic mesoderm as described previously (Chal et al., 2016). Briefly, confluent maintenance cultures were dissociated into single cells using Accutase (Corning, 25-058-C) and cells were seeded at a density of 30,000-33,000/cm2 on Matrigel (Corning, 354263) coated culture plates (Corning, 353046) in mTeSR (Stemcell Technologies, 85850) supplemented with 10 μM Y-27632 dihydrochloride (R&D Systems, 1254/10). Next day, the cells formed small compact colonies. On day 0 of differentiation, to induce presomitic mesoderm, cells were treated with CL medium [DMEM/F12 GlutaMAX (Thermo Fisher Scientific, 10565042)+1% Insulin-Transferrin-Selenium (Gibco, 41400045)+3 μM CHIR 99021 (R&D Systems, 4423)+0.5 μM LDN-193189 (Stemgent, 04-0074)]. Cells were treated with CL medium for 3 days and medium was refreshed every day. On day 3 of differentiation, cells were changed to CLF medium [CL medium+20 ng/ml FGF-2 (PeproTech, 450-33)]. Cells were treated with CLF medium for 3 days and medium was changed every day.
To further differentiate presomitic mesoderm cells to dermomyotomal fate, cells were changed to HIFL medium [DMEM high glucose (Thermo Fisher Scientific, 11965-118)+Penicillin/Streptomycin (Life Technologies, 15140122)+15% KnockOut™ Serum Replacement (Life Technologies, 10828-028)+NEAA (Thermo Fisher Scientific, 11140-050)+0.01 mM 2-Mercaptoethanol (Life Technologies, 21985-023)+10 ng/ml HGF (PeproTech, 315-23)+2 ng/ml IGF-1 (PeproTech, 250-19)+20 ng/ml FGF-2 (PeproTech, 450-33)+0.5 μM LDN-193189 (Stemgent, 04-0074)]. Cells were treated with HIFL for two days and medium was changed every day. To let the PAX3 expressing dermomyotomal cells into myogenic and non-myogenic lineages, cells were changed to HI medium (HIFL medium without FGF-2 and LDN-193189) and medium was changed every day.
On day 16-30 of differentiation, cells were replated to differentiate cells into brown adipocyte lineage. Cultures were dissociated into single cells with 2.5 mg/ml Collagenase, (Type IV, Thermo Fisher Scientific, 17104019) and 0.05% Trypsine EDTA (Thermo Fisher Scientific, 25200-056) in PBS (Gibco, 14190) and filtered through a 30 μm (CellTrics, 04-0042-2316) filter and seeded on Matrigel coated plates at a density of 60,000-100,000/cm2 in BCTFT medium [DMEM high glucose (Thermo Fisher Scientific, 11965-118)+Penicillin/Streptomycin (Life Technologies, 15140122)+5% KnockOut™ Serum Replacement (Life Technologies, 10828-028)+1% Insulin-Transferrin-Selenium (Gibco, 41400045)+10 ng/ml FGF-2 (PeproTech, 450-33)+10 ng/ml BMP7 (Thermo Fisher Scientific, PHC9544)+20 nM Porcn Inhibitor II, C59 (Millipore Sigma, 500496)+10 ng/ml TGFb1 (PeproTech, 100-21-10)+2 nM 3,3′,5-Triiodo-L-thyronine sodium salt (Sigma-Aldrich, T6397)]. Cells were treated with BCTFT for 4-6 days and medium was refreshed every day. Adipocyte precursors were either frozen to be used later or differentiated into adipocytes. For freezing, cultures were dissociated into single cells using 0.05% Trypsine EDTA (Thermo Fisher Scientific, 25200-056) in PBS (Gibco, 14190) and cells were frozen in NutriFreez D10 Cryopreservation Medium (Biological Industries, 01-0020-50).
To differentiate adipocyte precursors into adipocytes, cells were cultured in adipogenic medium [DMEM high glucose (Thermo Fisher Scientific, 11965-118)+Penicillin/Streptomycin (Life Technologies, 15140122)+5% KnockOut™ Serum Replacement (Life Technologies, 10828-028)+1% Insulin-Transferrin-Selenium (Gibco, 41400045)+500 μM IBMX (3-Isobutyl-1-methylxanthine, Sigma-Aldrich, 17018)+25.5 g/ml L-Ascorbic acid (Sigma-Aldrich, A4544)+2 nM T3 (3,3′,5-Triiodo-L-thyronine sodium salt, Sigma-Aldrich, T6397)+5 μM TGFb inhibitor SB431542 (Selleck Chemicals, S1067)+1 μM Dexamethasone (Sigma-Aldrich, D4902)+10 ng/ml EGF (Peprotech, AF-100-15)+4 g/ml Hydrocortisone (Sigma-Aldrich, H0888)+1[M Rosiglitazone (Sigma-Aldrich, R2408)] for 30-40 days. Medium was changed every third day.
To perform functional assays and immunofluorescence analysis, 30-40 day old replated cultures were dissociated with 2.5 mg/ml Collagenase, (Type IV, Thermo Fisher Scientific, 17104019) and 0.05% Trypsine EDTA (Thermo Fisher Scientific, 25200-056) in 1× Phosphate Buffered Saline (Gibco, 14190) and cells were seeded onto suitable plate format required for the assay.
Human fetal tissues were obtained by the University of Washington Birth Defects Research Laboratory (BDRL) under a protocol approved by the University of Washington Institutional Review Board. BAT tissues were isolated at 98 days (H28540), 115 days (H28572), 122 days (H28560), 125 days (H28626) and 135 days estimated post-conceptual age from the interscapular and scapular region. Tissues were rinsed in 1× Hanks' Balanced Salt Solution (HBSS, Thermo Fisher Scientific, 14185052) before processing. For immunofluorescence analysis, tissues were fixed with 4% Paraformaldehyde (Electron Microscopy Sciences, 15710) overnight at 4° C., washed 3 times in 1× Phosphate Buffered Saline (PBS, Sigma-Aldrich, P5493) and stored at 4° C. in PBS supplemented with 0.1% Sodium Azide (Sigma-Aldrich, 71290) until further processing. For bulk mRNA sequencing, tissues were snap frozen in liquid nitrogen and stored at −80° C. until further processing.
Pax7-iCre/+; Rosa26-mTmG/+ embryos were generated by crossing Rosa26-mTmG/mTmG (Muzumdar et al., 2007) males with Pax7-iCre/+ females in timed matings and embryos were collected at E14.5. Gata6-EGFPCreERT2 mice were generated by knocking an EGFPCreERT2 cassette into the endogenous Gata6 locus as reported previously (Donati et al., 2017). They were crossed with Rosa26-fl/STOP/fl-tdTomato mice (Madisen et al., 2010). For lineage tracing experiments, pregnant females were injected intraperitoneally with 50 lg/g of tamoxifen (Sigma) at E12.5 and embryos were collected at E15.5. For experiments other than lineage tracing, wildtype CD1 IGS mice (Charles River) were used.
UCP1 gene was targeted using the CRISPR-Cas9 system-based genome editing to generate a reporter NCRM1 iPSC line which already had been modified at PAX7 locus to introduce Venus. To target the UCP1 locus, a single guide RNA (sense strand encoding gRNA=CACCGGGTTTGCTGCCCGGCGGAC (SEQ ID NO: 1), antisense strand encoding gRNA=AAACGTCCGCCGGGCAGCAAACCC) (SEQ ID NO: 2) targeting the 5′ region of the gene was designed using the MIT Crispr Design Tool (crispr.mit.edu). The guide RNA was cloned into PSpCas9 (BB)-2A-GFP(PX458) (Addgene, 48138) following protocol from Ran et al., 2013. The final vector was sequence to ensure no mutations were generated during cloning. To generate a targeting vector for homology dependent repair, we cloned 5′ and 3′ 1 kb long homology sequence (HA) flanking a nuclear localization sequence (NLS) region from H2B gene sequence, fluorescent protein mCherry sequence and self-cleaving P2A peptide sequence (5′HA-H2B-mCherry-P2A-3′HA) in a pUC19 vector backbone using Gibson Assembly (New England Biolabs (NEB), E5510S). To mutate the PAM site for the guide RNA, the assembled targeting vector was mutated using site-directed mutagenesis using In-Fusion HD Cloning Plus (Takara, 638909). NCRM1 iPSCs were transfected with both Guide RNA vector (PSpCas9 (BB)-2A-GFP) and targeting vector (pUC19-5′HA-H2B-mCherry-P2A-3′HA) using Lipofectamine™ Stem Transfection Reagent (Thermo Fisher Scientific, STEM00001). 24-hours after transfection, cells were sorted using flow cytometry (S3 cell sorter, Biorad) for GFP positive cells and plated at low density for clonal expansion in Matrigel (Corning, #354263) coated culture plates (Corning, 353046) in mTeSR (Stemcell Technologies, 85850) supplemented with 10 μM Y-27632 dihydrochloride (R&D Systems, 1254/10) and Penicillin/Streptomycin (Life Technologies, 15140122). After appearance of small colonies, the colonies were sub-cultured and genotyped using PCR for targeted homozygous insertion of the H2B-mCherry-P2A in the UCP1 locus before the transcription start site. Positive clones were sequenced and clones with no undesired mutation were further validated using immunofluorescence and RT-qPCR. Two positive clones were differentiated into adipocytes and expression of UCP1 in mitochondria and mCherry in the nucleus was confirmed. To generate the human PAX3-Venus iPSC line using the CRISPR/Cas9 technology we followed a similar strategy. We generated a Venus knock-in allele by inserting the Venus sequence in front of the coding sequence of exon 1. Guide RNA (sense strand encoding gRNA=5′-CCGGCCAGCGTGGTCATCCTGGG-3′ (SEQ ID NO: 3), antisense strand encoding gRNA=5′-TGCCCCCAGGATGACCACGCTGG-3′ (SEQ ID NO: 4)) targeting the 5′ region of the gene was designed using the MIT Crispr Design Tool (crispr.mit.edu). The targeting plasmid (pBSKS-2A-3xNLS) was designed to contain NLS sequence, 1.5 kb of the 5′ genomic region of the PAX3 gene, 1 kb of the 3′ sequence and 2A sequence. Targeting vector, together with the Cas9 plasmid was electroporated into cells by nucleofection and clones were sub-cultured and genotyped using PCR for targeted homozygous insertion of the NLS-Venus-2A in the PAX3 locus.
Samples were harvested and RNA was extracted using NucleoSpin® RNA kit (Macherey and Nagel, 740955) following manufacturer's instructions. DNA digestion was performed on column and RNA quality and concentration was measured using Nanodrop. For reverse transcription, lug RNA was in a 20 ul reaction volume to generate cDNA using iScript™ cDNA Synthesis Kit (Bio-Rad, 1708891). Typically, cDNA was diluted 1:10 with nuclease free water. For real time quantitative PCR, 3.5 ul of cDNA, 1.5 ul of 10 μM forward and reverse primer mix (300 μM of forward and reverse primers) and 5 ul of iTaq™ Universal SYBR® Green Supermix (Bio-Rad, 172-5124) was used for 10 ul reaction volume. For each sample and gene primer set, 3 technical replicates were performed. PCR primers were designed using primer 3, typically spanning splice junctions wherever possible. Primers were validated for amplification efficiency and specificity using a standard curve and melting curves, respectively. PCRs were run on a Bio-Rad CFX384 thermocycler with the following cycling program: initial denaturation step (95° C. for 1 minute), 40 cycles of amplification and SYBR green signal detection (denaturation at 95° C. for 5 seconds, annealing/extension and plate read at 60° C. for 40 seconds), followed by final rounds of gradient annealing from 65° C. to 95° C. to generate dissociation curves. Relative gene expression was calculated using ΔΔCt method and RPL37A (ribosomal protein L37a) was used as a housekeeping gene. A list of qPCR primers is provided.
Cells were cultured on Matrigel coated plastic culture plates (Corning, #353046), glass bottom 24-well plates (Cellvis, P24-1.5H-N), μ-Dish 35 mm (Ibidi, 81156) or μ-Plate 24 Well Black (Ibidi, 82406). Cultures were washed with 1× Phosphate Buffered Saline (PBS, Sigma-Aldrich, P5493) and fixed with 400 Paraformaldehyde (Electron Microscopy Sciences, 15710) for 20 minutes at room temperature. Cultures were washed with PBS and stored in PBS supplemented with 0.2% Sodium Azide (Sigma-Aldrich, 71290) until used for immunostaining.
Mouse embryos were dissected out at different stages in Hanks' Balanced Salt Solution (HBSS) (Gibco, 14185-052). After several washes in HBBS, embryos were fixed in 4% Paraformaldehyde (Electron Microscopy Sciences, 15710) overnight at 4° C. Embryos were then washed in PBS and transferred to 15% sucrose solution (Sigma-Aldrich, 84097) in PBS overnight at 4° C. followed by incubation in 30% sucrose solution for overnight at 4° C. Embryos were then embedded in Tissue-Tek O.C.T. Compound (VWR, 25608-930) and stored at −80° C. until sectioned. 10-20 m thick sections were cut and stored at −20° C. until used for immunostaining.
Fixed cells or sections were permeabilized using 1% Triton-X (Millipore Sigma, T8787) for 10 minutes at room temperature. Samples were washed in PBS and were incubated in blocking solution [PBS supplemented with 3% donkey serum (Jackson ImmunoResearch, 017-000-121) and 0.1% Triton-X]for 1 hour at room temperature. Samples were then incubated with primary antibody diluted in blocking solution at 4° C. overnight. Next day, samples were washed 3 times with PBS for 5 minutes. Samples were then incubated with secondary antibody and Hoechst 33342 (ThermoFisher Scientific, H3570) diluted in blocking solution for 1 hour at room temperature followed by 3 washes with PBS for 5 minutes each. Cultured cell samples were stored in PBS until imaged. Mouse tissue slides were mounted using Fluoromount aqueous mounting medium (Sigma-Aldrich, F4680) and were stored at 4° C. until analyzed. Samples were imaged using EVOS FL imaging system (Thermo Fisher Scientific) or LSM780 confocal microscope (Zeiss). A list of primary and secondary antibodies is provided.
iPSC-BA cultures were washed with 1× Phosphate Buffered Saline (PBS, Sigma-Aldrich, P5493) and fixed with 4% Paraformaldehyde (Electron Microscopy Sciences, 15710) for 20 minutes at room temperature. Cell were then incubated with 0.5 mM BODIPY™ (Invitrogen™, D3922) in PBS for 20 minutes at room temperature. Cells were washed 3 times with PBS and visualized with EVOS FL imaging system (Thermo Fisher Scientific).
Agilent Seahorse XFe96 Analyzer was used to measure oxygen consumption in live iPSC-BAs and precursor cells following manufacturer's instructions. 30,000 iPSC-BAs and precursor cells were cultured on Matrigel (Corning, 354263) coated Seahorse XF96 cell culture microplates (Agilent Technologies, 101085-004). Assay medium was prepared using Seahorse XF DMEM (Agilent Technologies, 103575-100) supplemented with 1 mM pyruvate (Agilent Technologies, 103578-100), 2 mM glutamine (Agilent Technologies, 103579-100), and 10 mM glucose (Agilent Technologies, 103577-100). Basal respiration was measured 3 times in the assay medium. Cells were then treated with 1.5 μM Oligomycin (Tocris, 4110) and 3 measurement were taken. Cells were then treated with 10 μM Forskolin (Sigma-Aldrich, F6886) or DMSO (Sigma-Aldrich, D2650) and oxygen consumption rate (OCR) was measured for 70 minutes (12 measurements). To block the electron transport chain, cells were then treated with 1p M Rotenone (Sigma-Aldrich, R8875) and Antimycin A (Sigma-Aldrich, A8674) for 3 measurements. Cells were then washed with 1× Phosphate Buffered Saline (PBS, Sigma-Aldrich, P5493) and fixed with 4% Paraformaldehyde (Electron Microscopy Sciences, 15710) for 10 minutes at room temperature. Seahorse XF96 cell culture microplates were imaged for DAPI and mCherry using GE INCELL Analyzer 2200 Widefield High-Content Imager. Cells were automatically segmented using custom code in Fiji and number of DAPI and mCherry positive cells were quantified. OCR data from each well was normalized with DAPI positive cell number in each well. Four replicates were performed. Each experiment consisted of 40 technical replicates for each cell type.
iPSC-BAs and iPSC precursors were cultured on Matrigel (Corning, 354263) coated 96-well Black Clear-Bottom Plates (Corning, 3603) and were incubated in DMEM/H with 250 nM ERthermAC (Sigma, SCT057) for 30 min at 37° C. After washing with PBS, fresh 90 μl DMEM/H without phenol red was added prior to imaging. Fluorescence in stained cells were detected with a GloMax Discover Multimode Detection System (Promega) using 520 nm excitation and emission at 590 nm. Temperature inside the machine was equilibrated at 25° C. After measuring 3 points of basal fluorescence, 10 μl forskolin (final concentration 10 μM, F6886, Sigma) was added to initiate thermogenesis. 10 μl PBS was added for vehicle control group. Fluorescence was recorded every 5 min over 120 min. Results are interpreted as relative intensity (intensity was normalized to basal measurements).
iPSC-BAs were rinsed with 0.1M Phosphate buffer pH 7.5 and fixed in 2.5% glutaraldehyde (Sigma, G7651) in 0.1M phosphate buffer at 4° C., for 2 days. Samples were washed in 0.1M phosphate buffer and post-fixed in 1% osmic acid in 0.1M phosphate buffer for 1 hour at room temperature. Dehydration was performed by serial incubation in 50%, 70%, 80%, 95%, and 100% ethanol before incubation in propylene oxide for 30 minutes. Samples were impregnated by incubation in propylene oxide/araldite (1:1 v/v) for 60-90 min, in propylene oxide/araldite (1:2 v/v) for 1 hour and in 100% araldite overnight at 4° C. The samples were finally incubated in Araldite which was allowed to polymerize for 24 hours at 56° C. Ultrathin sections (85 nm) were cut with a ultracut Leica EM UC. Sections were contrasted 8 min with 2% uranyl acetate and 8 min with lead citrate (Reynolds). All pictures were taken with the Zeiss EM 900 microscope and a GATAN camera (Orius SC 1000).
iPSC-BAs were cultured in a (Corning, 354263) coated 96-well culture plate (Genesee Scientific, 25-109). Cells were serum starved for 16 hours and cultured in serum-free DMEM/F12 GlutaMAX (Thermo Fisher Scientific, 10565042) containing 0.5% BSA. Next day, cells were washed with KRB-HEPES buffer [118.5 mM NaCl, 4.75 mM KCl, 1.92 mM CaCl2), 1.19 mM KH2PO4, 1.19 mM MgSO4, 25 mM NaHCO3, 6 mM glucose and 10 mM HEPES, pH 7.4] containing 4% fatty-acid-free BSA (Bioworld, 22070017-1). Cells were treated with DMSO or 10 μM Forskolin in KRB-HEPES buffer supplemented with 4% fatty-acid-free BSA for at 37° C., 5% C02 for 4 hours. Cell culture medium was collected for glycerol measurement using the free glycerol reagent (Sigma-Aldrich, F6428) following manufacturer's instructions. A standard curve was generated using Glycerol Standard Solution (Sigma-Aldrich, G7793). Test samples, standards and water control were incubated with the Free Glycerol Reagent for 5 minutes at 37° C. and absorbance was recorded at A540 using a spectrophotometer. To normalize with total protein content, Bradford assay was performed using DC Protein Assay (Biorad, 500-0116) following manufacturers protocol.
To stain glycogen in differentiating iPSC-BAs, cells were treated with Periodic Acid-Schiff stain (Sigma, 395B). iPSC-BAs were fixed with 4% paraformaldehyde (Electron Microscopy Sciences, 15710) at room temperature for 15 minutes. Fixed cells were washed several times with distilled water and immersed in Periodic Acid Solution for 5 minutes at room temperature. Cells were then washed in distilled water and incubated with Schiff's Reagent for 15 minutes at room temperature. Cells were washed in running tap water for 5 minutes. Stained cells were visualized in brightfield using EVOS FL imaging system (Thermo Fisher Scientific).
Bulk mRNA Sequencing
UCP1-mCherry knock-in line was differentiated into brown adipocytes as described above. Cells were harvested on day +40 of differentiation and RNA was extracted using NucleoSpin® RNA kit (Macherey and Nagel, 740955) following manufacturer's instructions. DNA digestion was performed on column and RNA quality and concentration was measured using Nanodrop. Human fetal tissues were harvested as described above and RNA was extracted using NucleoSpin® RNA kit following manufacturer's instructions.
RNA library preparations and sequencing reactions were conducted at GENEWIZ, LLC. (South Plainfield, NJ, USA). RNA samples received were quantified using Qubit 2.0 Fluorometer (Life Technologies, Carlsbad, CA, USA) and RNA integrity was checked using Agilent TapeStation 4200 (Agilent Technologies, Palo Alto, CA, USA). RNA sequencing libraries were prepared using the NEBNext Ultra II RNA Library Prep Kit for Illumina using manufacturer's instructions (NEB, Ipswich, MA, USA). Briefly, mRNAs were initially enriched with Oligod(T) beads. Enriched mRNAs were fragmented for 15 minutes at 94° C. First strand and second strand cDNA were subsequently synthesized. cDNA fragments were end repaired and adenylated at 3′ends, and universal adapters were ligated to cDNA fragments, followed by index addition and library enrichment by PCR with limited cycles. The sequencing library was validated on the Agilent TapeStation (Agilent Technologies, Palo Alto, CA, USA), and quantified by using Qubit 2.0 Fluorometer (Invitrogen, Carlsbad, CA) as well as by quantitative PCR (KAPA Biosystems, Wilmington, MA, USA).
The sequencing libraries were clustered on a single lane of a flowcell. After clustering, the flowcell was loaded on the Illumina HiSeq instrument (4000 or equivalent) according to manufacturer's instructions. The samples were sequenced using a 2×150 bp Paired End (PE) configuration. Image analysis and base calling were conducted by the HiSeq Control Software (HCS). Raw sequence data (.bcl files) generated from Illumina HiSeq was converted into fastq files and de-multiplexed using Illumina's bcl2fastq 2.17 software. One mismatch was allowed for index sequence identification.
For bulk RNA-seq data analysis, we performed quality control on the sequence data (FastQ files) using FastQC (v0.11.9) and used STAR RNA-seq aligner (v2.7.3a) (Dobin et al., 2013) to map the sequenced reads to the human reference genome (GRCh38 release 101 from ENSEMBL). Mapped reads were quantified using featureCounts (v2.0.1) (Liao et al., 2014). Starting from the raw gene counts, normalization and differential expression analysis were done in the R environment (v3.6.0), using the DESeq2 package (v 1.22.2) (Love et al., 2014). Genes were defined as differentially expressed when the false discovery rate was lower than 0.05. Gene Ontology (GO) enrichment analysis was performed on the differentially expressed genes using EnrichR (Kuleshov et al., 2016), choosing the WikiPathway 2019 Human resource.
To quantify the thermogenic potential of each sample we relied on the ProFAT webtool (Cheng et al., 2018). Starting from the raw count matrix, barplot and heatmap, representing the adipose browning capacity of each sample, were generated as standard output of ProFAT.
To produce the heat-map for genes of interest (linked to muscle, pluripotency and brown adipose tissue) we used normalized read counts produced by DESeq2. To calculate up- or down-regulation, we computed the log difference of the average of the biological replicates against the baseline value (average over all conditions) for each gene.
Single cell analysis of differentiating iPSCs and mouse embryos cells was performed using inDrops as previously reported (Klein et al.).
UCP1-mCherry iPSC line was differentiated into brown adipocytes as described above. Cultures were harvested on day 20 of the primary differentiation and day 20 and 40 after replating.
The cultures were washed with PBS (Gibco, 14190) and dissociated into single cells using 2.5 mg/ml Collagenase, (Type IV, Thermo Fisher Scientific, 17104019) and 0.05% Trypsine EDTA (Thermo Fisher Scientific, 25200-056) in PBS for 5-15 minutes at 37° C. Dissociated cells were run through 70 μm cell strainer (Celltreat, 229483) followed by 30 μm cell strainer (CellTrics, 04-0042-2316), spun at 300 g for 5 minutes and resuspended in DMEM (Thermo Fisher Scientific, 11965-118) +5% fetal bovine serum (VWR, 89510-186). Cells were again spun at 300 g for 5 minutes and resuspended in DMEM+5% fetal bovine serum, this was repeated 2-3 times to remove debris. Final resuspension was made in PBS+0.1% bovine serum albumin (Gibco, 15260-037). Cell density was adjusted to 200,000 cells/ml. For day 20, 2500 cells were collected from each sample. For samples 20 day after replating and 40 day after replating, at least 3000 cells were collected from each sample from two independent differentiations.
For analyzing mouse embryonic tissues, embryos were collected on embryonic day (E) 11.5, 12.5, 13.5, 14.5 and 15.5 from wildtype CD1 IGS mice (Charles River). For each stage, back tissue dorsal to rib cage at the level of forelimb was dissected out. Neural tube/spinal cord and dorsal root ganglion were removed wherever possible. For E15.5, epidermis and underlying dermis was removed before dissociating the tissue. Dissected tissues were washed several times in Hanks' Balanced Salt Solution (HBSS, Thermo Fisher Scientific, 14170112). Tissues were transferred to microfuge tube with 200 μl of 2.5 mg/ml Collagenase, (Type IV, Thermo Fisher Scientific, 17104019) and 0.05% Trypsine EDTA (Thermo Fisher Scientific, 25200-056) in PBS (Gibco, 14190), chopped into small pieces using scissors and incubated at 37° C. for 15 minutes with intermittent shaking. Tissues were mechanically dissociated by triturating several times using wide-bore 1 ml pipette. The resulting cell suspension was mixed with HBBS+10% FBS, filtered through a 30 μm cell strainer and spun at 300 g for 5 minutes. To remove red blood cells, cell pellet was resuspended and incubated with RBC Lysis Solution (Qiagen, 158902) for 5 minutes at room temperature. Cells were washed with HBSS+10% FBS for 2-3 times to remove debris. Final suspension was made in PBS+0.1% BSA. Cell density was adjusted to 200,000/ml. For each stage, cells were collected from two littermate embryos. For E11.5-14.5 embryo, 3000 cells were encapsulated and sequenced from each embryo. For E15.5, 5000 cells were collected and sequenced from each embryo.
Single cells were encapsulated using inDrops technique as reported previously (Klein et al.). Cells were barcoded using v3 sequencing adapters and were sequenced on an Illumina NextSeq 500 using the NextSeq 75 High Output Kits using standard Illumina sequencing primers and 61 cycles for Read1, 14 cycles for Read2, 8 cycles each for IndexRead1 and IndexRead2. Sequence FASTQ files were processed according to indrops.py pipeline (available at github.com/indrops/indrops). Single cell transcriptomes were mapped to mouse (GRCm38/mm10) and human (GRCh38/hg19) reference transcriptomes. Samtools version 1.3.1, rsem version 1.3.0 and Bowtie version 1.2.2 was used with parameter -e 100.
A weighted histogram of transcript counts per cell barcode vs cell barcode abundance was used to identify transcripts originating from abundant cell barcodes. Only transcript counts originating from abundant cell barcodes were included in downstream analysis. Basic filtering parameters were used to exclude cells expressing <500 genes and genes expressed in less than 3 cells. The filtered counts were normalized by total number of counts for each biological sample. Top 1000 variable genes were identified according to Satija 2015. Cell doublets were identified using Scrublet and filtered out (PMID: 30954476). For each cell, fraction of counts due to mitochondrial genes was determined and cells with high fraction were filtered out. The cell cycle was scored as in Tirosh et al. 2016 (PMID: 27806376). Each cell was given a cell cycle score based on the expression of G2/M and S phase markers. The cells not expressing the markers from G2/M and S phase were identified to be in G0/G1 stage. Source of variation between the libraries were regressed out using bkknn batch correction function (PMID: 31400197). Single cell data were projected into a low dimensional space by principal component analysis (PCA). UMAP (McInnes et al., 2018) was used to embed the neighborhood graph. Cell clusters were identified using Leiden graph-clustering method [PMID: 30914743, Blondel et al, 2008]. Differentially expressed genes were identified by a Wilcoxon rank-sum test by comparing cells of each cluster with cells of all the other clusters.
Tracksplot were made using each genes are plotted as a filled line plot where the y are the genes processed expression values and x is each of the cells (Wolf et al., 2018). Stream: Stream pipeline was used to layout the trajectories inside each datasets. Original code and documentation: (Chen et al., 2019) Neural cells were removed from the dataset analyzed with stream as there developmental origin differ from the rest of the clusters.
Trajectory inference analysis was done using Cellrank (Lange et al., 2022). Waddington-OT kernel (Schiebinger et al., 2019) was used to infer the temporal couplings of cells from the different samples collected independently at various timepoints. Transition matrices were calculated to show the amount of mass transported from a cell type to another from a start and an end point.
Single cell RNA sequencing data from mouse E18 periaortic (pVAT) brown adipose tissue described in Angueira et al. 2021 was processed through the same processing pipeline as described above. Cell state predictions on single cell clusters from pVAT described in Angueira et al. 2021 were made using a kNN-classifier trained on our E13.5-E15.5 mouse single cell clusters (Diaz-Cuadros et al., 2020).
In mice, interscapular brown adipose tissue (BAT) develops in between the dermis, trapezius muscle, pectoralis muscle, and deep dorsal muscle bundles. To characterize the developmental trajectory of the developing brown fat, we isolated dorsal tissues at the forelimb level at E11.5, E12.5, E13.5, E14.5, and E15.5 (
Most of the somite-derived cells, except endothelial and skeletal muscle cells, lie closely together on the UMAP embedding suggesting that they have related transcriptional signatures (
This developmental trajectory of the brown fat lineage was confirmed by immuno-histochemistry of sections of the dorsal region of the developing mouse brachial trunk with markers of the adipocyte lineage. Immunofluorescence analysis of E12.5 to E15.5 embryos confirmed the expression of Ebf2 in the presumptive interscapular brown fat region but also in skeletal muscle and precartilage (
Angueira et al. (2021) recently identified progenitors that give rise to brown adipocytes in mouse periaortic (pVAT) brown adipose cells analyzed by scRNAseq at E18 and perinatal stages (Angueira et al., 2021). We reanalyzed their E18 dataset which contains 16,967 single cell transcriptomes. Following UMAP embedding and Leiden clustering, we identified 13 clusters representing the cell types described in the original study (
The transcription factor Gata6 has been previously associated with the brown adipocyte lineage (Cheng et al., 2018; Wang et al., 2014), but its developmental expression has not been characterized. In our dataset, we observed a striking restriction of Gata6 expression to the brown adipocyte and smooth muscle lineages (
To experimentally demonstrate that Gata6 is expressed by precursors which give rise to brown adipocytes, we used a Gata6-CreERT2:Rosa26-tdTomato mouse line to label Gata6-expressing cells during embryonic development (Donati et al., 2017). We induced recombination using tamoxifen injection at E12.5 and analyzed the contribution of labelled cells to brown adipose tissue at E15.5 (
To further investigate the origin of these Gata6 precursors, we used a Pax7-Cre:Rosa26-mTmG mouse line to analyze the contribution of Pax7 descendants to Gata6-positive brown fat precursors at E14.5 (Hutcheson et al., 2009). As expected, dorsal, ventral and limb skeletal muscle as well as Pparg-expressing prospective brown adipocytes precursors were identified as Pax7 descendants (
To further examine whether Gata6 expression is conserved in human brown adipogenesis, we performed immunofluorescence analysis on 98- and 135-day-old (estimated post-conceptual age) human fetal tissue from the interscapular and scapular regions. We detected expression of EBF2 and GATA6 in cells adjacent to skeletal muscle tissue (detected by MYHC antibody) under the dorsal dermis and dispersed between the deeper dorsal muscle bundles at 98 days of development (
We next decided to take advantage of our characterization of mouse brown fat development to inform the design of a differentiation protocol aiming to recapitulate this process from human pluripotent stem cells. We previously developed efficient protocols to differentiate human paraxial mesoderm and skeletal muscle from iPS cells in vitro (Chal et al., 2016; Chal et al., 2015; Diaz-Cuadros et al., 2020). As brown adipocytes originate mostly from the paraxial mesoderm and share a common precursor with skeletal muscles in the dermomyotome, we first aimed to recapitulate the development of Pax-3 positive dermomyotomal cells in vitro. To achieve this, we first treated human iPSCs with the Wnt agonist CHIR and the BMP inhibitor LDN in serum-free conditions to differentiate them to presomitic mesoderm (PSM) (
To promote the brown adipogenic fate in these cultures, we next sought to identify potential signaling pathways involved in brown adipogenesis from the mouse dataset. Mouse knock-out experiments have identified BMP activation and Wnt inhibition as important signals required for brown adipocyte development (Atit et al., 2006; Longo et al., 2004; Tseng et al., 2008). However, these studies have not precisely defined the time window when these cues are present during development. We observed expression of the BMP ligands (Bmp7, Bmp4) and of the Wnt signaling antagonists (Sfrp1, Sfrp2, Sfrp4, Dkk2) mostly at the FP stage in the mouse dataset (
Thus, to recapitulate the sequence of signaling cues to which brown adipocyte precursors are exposed during their differentiation in vivo, we dissociated the primary cultures around day 20 of differentiation to eliminate the myofibers and replated the cells in a medium containing BMP7, the Wnt antagonist C-59, TGFb1, FGF2 and Triiodothyronine (BCTFT medium) for 4-6 days (
To follow the differentiation of iPSCs into brown adipocytes, we generated a UCP1-mCherry fluorescent human iPSC reporter line using CRISPR-Cas9. In this knock-in line, an mCherry construct was introduced immediately 5′ to the start codon of UCP1, from which it was separated by a P2A peptide. Thus, the reporter is produced from the same transcript as UCP1 and targeted to the nucleus using an H2B tag (
iPSC-BAs contain abundant mitochondria (a characteristic of brown adipocytes) as revealed by immunofluorescence for mitochondrially encoded cytochrome c oxidase II (MT-C02) and electron microscopy analysis at 60 days of differentiation (
To further characterize the differentiation of iPSCs into adipocytes, we performed scRNAseq of the cultures at 40 days (d40) and 60 days (d60) of differentiation. After quality control and filtering, we analyzed 2744 and 5322 cells from d40 and d60 samples, respectively. UMAP projection followed by Leiden clustering analysis shows that at d40, most of the cells belong to a large cluster which expresses Fibroblastic markers such as PDGFRA and HIC1 (
We next merged the three time points (d20, d40, and d60) to generate a UMAP projection and performed a Leiden clustering analysis on this combined dataset (
To further establish the identity of cells differentiated in vitro, we directly compared the d60 human cultures to the mouse scRNaseq dataset. The list of expressed mouse genes was converted into their human orthologs and the two datasets were merged and analyzed together using UMAP projection followed by Leiden clustering. Human and mouse cells identified as Fibroblastic Progenitors, BAPre and Brown adipocytes were found in the same clusters in the merged dataset indicating the similarity of the human cells differentiated in vitro to the mouse brown adipocyte lineage differentiating in vivo (
The single cell dissociation required for scRNAseq disrupts the mature lipid filled brown adipocytes, which are large buoyant cells that moreover cannot be encapsulated using the microfluidics platform used for the Indrops pipeline and are thus absent from the scRNA dataset. Accordingly, we only detected ˜1% of cells expressing the UCP1 transcript in the d60 scRNAseq dataset whereas counting the UCP1-mCherry positive cells using fluorescence microscopy in d60 cultures yields an estimate of ˜40-50% positive cells. Nevertheless, our scRNAseq analysis suggests that in d60 cultures, ˜40% of the cells belong to the BAPre and brown adipocyte clusters while another 40% of the cells are in the Progenitors cluster. Therefore, an approximate 80% of the cells of the scRNAseq dataset represent different stages of the brown adipocyte lineage differentiation. Together, this suggests that the vast majority (possibly up to 80-90%) of the d60 cultures corresponds to cells at various stages of the differentiation of the brown adipocyte lineage. In conclusion, this analysis suggests that a brown adipocyte lineage developmental trajectory highly similar to that observed for mouse interscapular fat in vivo can be recapitulated in vitro from human iPS cells. This results in the production of immature brown adipocytes approximately equivalent to those of the E15.5 mouse embryo.
To molecularly characterize the iPSC-BAs which are mostly lost during dissociation in the scRNAseq analysis, we performed bulk mRNA sequencing of undifferentiated iPSCs and cultures at 60 days of differentiation. In parallel, as a reference for differentiated adipocytes, we analyzed human fetal BAT (fBAT) isolated from 115, 122 and 125-day post-conceptual age fetuses from the interscapular and scapular regions. As an outgroup, we used bulk RNAseq data from iPSC-derived skeletal muscle cultures (iPSC-SkM) (Al Tanoury et al., 2021). Brown adipocyte-specific genes such as UCP1, CIDEA, PARGC1A and DIO2 were specifically expressed in iPSC-BAs and human fetal BAT (
Next, we sought to investigate the functional potential of iPSC-BAs generated in vitro. Generally, brown adipocytes are characterized by their potential to generate heat in response to a beta-adrenergic stimulus and downstream activation of the cAMP pathway (Wang and Seale, 2016). This activation can be mimicked by treatment with forskolin which activates cAMP signaling (Kriszt et al., 2017; Wang et al., 2020; Zhang et al., 2020). To test the capacity of iPSC-BAs to respond to adrenergic stress, we used forskolin in 40 day-old replated adipogenic cultures. Cells treated with forskolin for 4 hours show increased UCP1 mRNA levels compared to vehicle-treated cells (
The process of thermogenesis is driven by the flow of protons through UCP1 in the mitochondrial inner membrane. To examine whether forskolin-induced thermogenesis in iPSC-BAs is coupled to proton leak in mitochondria, we measured oxygen consumption rate (OCR) with a Seahorse metabolic flux analyzer in 40 day-old replated cultures (
iPSC cultures were dissociated to single cells and aggregated in non-adhesive U-bottom plates to form 3D structures. The structures were cultured in DMEM/F12 GlutaMAX supplemented with 1% ITS, 3 μM CHIR99021, and 0.5 μM LDN-193189 (PSM medium) supplemented with 5% FCS for 11 days to generate PAX3-expressing somite-like structures and dermomyotomal cells. The PAX3-expressing somite-like structures and dermomyotomal cells were then cultured in DMEM high glucose supplemented with 5% KSR, 1% ITS, 2 ng/ml FGF-2, 20 nM C59, 20 ng/ml BMP4, 10 ng/ml TGFb1, 2 nM T3 (adipocyte precursor medium) between 4-12 days. To differentiate the precursors into lipid vesicle containing BA, organoids were cultured in adipogenic differentiation medium (DMEM high glucose supplemented with 5% KSR, 1% ITS, 500 μM IBMX, 25.5 g/ml L-Ascorbic acid, 2 nM T3, 5 μM TGFb inhibitor SB431542, 1 μM Dexamethasone, 10 ng/ml EGF, 4 g/ml Hydrocortisone, 1 μM Rosiglitazone) between 20-40 days. A resulting brown adipose tissue (BAT) organoid was cryosectioned and stained with phalloidin (
It is to be understood that while the invention has been described in conjunction with the detailed description thereof, the foregoing description is intended to illustrate and not limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.
This application claims the benefit of U.S. Provisional Application Ser. No. 63/305,390, filed on Feb. 1, 2022. The entire contents of the foregoing are incorporated herein by reference.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/US2023/061754 | 2/1/2023 | WO |
| Number | Date | Country | |
|---|---|---|---|
| 63305390 | Feb 2022 | US |
