Patent Application
20220298483
The present invention relates to a composition and a method for inducing chondrogenesis.
Cartilage and joint diseases are one of the most common clinical conditions affecting quality of life worldwide, due to increases in the two major risk factors of age and obesity. Current treatments for arthritic diseases are largely limited to symptom palliation using analgesics for mild cases, and immunosuppressants and/or surgery for more serious cases. However, these treatments are not disease-modifying or curative; moreover, cartilage is unable to regenerate. While more novel treatment using autologous cartilage tissue or chondrocytes are being tested, the rarity of the cell/tissue source, ex vivo culture difficulties, and low cell viability after transplantation have resulted in limited success with these methods. Thus, continued investigation into more efficacious methods of regenerating cartilage is ongoing to find curative treatments for these common and debilitating diseases.
Human multipotent mesenchymal stem cells (MSCs) are versatile somatic stem cells with immunomodulatory properties. First isolated in adult bone marrow (BM), MSCs have subsequently been found in numerous post-natal organ/tissues as well as directly differentiated from pluripotent stem cells such as human embryonic stem cells (ESCs) (Barberi et al., 2005; Yen et al., 2009) and induced pluripotent stem cells (iPSCs) (Kimbrel et al., 2014; Wang et al., 2018). MSCs readily differentiate into chondrocytes, osteoblasts, adipocytes, and fibroblasts, and their easy accessibility compared to many other cell types including chondrocytes render them ideal for use in cartilage-related diseases. However, chondrogenic differentiation efficiency is low, due to the requisite cumbersome 3-dimensional (3D) pellet culture and the relative lack of knowledge on molecular mechanisms involved in chondrogenesis, compared to osteogenesis and adipogenesis. The most common factors used to induce MSC chondrogenesis are TGFβ1 and TGFβ3, two pleomorphic growth factors, but information on mechanisms involved for either of these factors are surprisingly scarce despite decades of use (Wang et al., 2014). Moreover, TGFβ is well known to induce fibrosis, as well as ossification in cartilage (Chen et al., 2012; Kurpinski et al., 2010). It is difficult to avoid osteogenic specification with use of proteins from this family during chondrogenic differentiation.
Thus, there clearly is a need for more precision and efficiency in achieving MSC chondrogenesis for therapeutic application.
The present application describes a composition for inducing chondrogenesis comprising an Wnt antagonist and a pharmaceutically acceptable carrier.
The present application also provides a method for inducing chondrogenesis comprising administering an Wnt antagonist and a pharmaceutically acceptable carrier to a subject.
The present application further provides a method for treating a cartilage-related disease comprising administering a therapeutically effective amount of an Wnt antagonist and a pharmaceutically acceptable carrier to a subject.
The present application provides a composition for inducing chondrogenesis comprising an Wnt antagonist and a pharmaceutically acceptable carrier. The present application also provides a method for inducing chondrogenesis and a method for treating a cartilage-related disease by applying the above composition.
β-catenin/Wnt signaling, a major developmental and oncogenic pathway, is also important in skeletal related-tissue/organ development, especially for bone and cartilage tissue. We hypothesized that inhibition of the Wnt/β-catenin pathway may more precisely induce MSC chondrogenesis than TGFβ. To ascertain this, we utilized multiple sources of human MSCs—including iPSC-derived MSCs (iPSC-MSCs), ESC-derived MSCs (ESC-MSCs), and BM-MSCs—and also explored the role of AJ-β-catenin interactions in MSC chondrogenesis.
The antagonist of the Wnt signaling includes, for example, but not limited to Dickkopf (Dkk) proteins, secreted Frizzled-Related Proteins (sFRPs), and Wnt Inhibitory Factor-1 (WIF-1). In one embodiment, the Wnt antagonist can be XAV, DIF-3, iCRT3, ICG-001, IWP-2, IWP-4, Dkk, Soggy, sFRP, WIF-1, APCDD1, APCDD1L, Draxin, LMBR1L, Notum, SOST/Sclerostin, USAG1, or any combination thereof.
In the present application, the Wnt antagonist is preferably a Wnt/β-catenin antagonist. Some examples of the Wnt/β-catenin antagonist include, for example, but not limited to XAV, DIF-3 and iCRT3 In one embodiment, the Wnt/β-catenin antagonist is XAV.
In the present application, a stem cell can be further provided with the compositions or the methods. The stem cell can include totipotent stem cells, pluripotent stem cells, multipotent stem cells, and/or unipotent stem cells. The stem cell has an ability to differentiate into chondrocyte, for example, embryonic stem cell, induced pluripotent stem cell (iPSC), mesenchymal stem cell (MSC) and/or marrow stromal cell. In one preferred embodiment, the stem cell is mesenchymal stem cell (MSC). The MSC may be, for example, but not limited to iPSC-derived MSCs (iPSC-MSCs), ESC-derived MSCs (ESC-MSCs), BM-MSCs, adipose tissue-derived MSC, umbilical cord-derived MSC, placenta-derived MSC, uterus-derived MSC and dental pulp-derived MSC.
In the present application, the composition may further comprises a chondrocyte differentiation agent. The chondrocyte differentiation agent may be selected from the known agents. The chondrocyte differentiation agent may comprises, for example, but not limited to insulin growth factor (IGF1), Ruxolitinib, Tofacitinib, Baricitinib, angiopoietin-like 3 protein (ANGPTL3), oral salmon calcitonin, iNOS inhibitors, cholecalciferol, collagen hydrolyzate, bone morphogenetic protein 7 (BMP7), hyaluronic acid, a steroid, a non-steroidal anti-inflammatory agent (NSAID), TPX-100 and the like.
The present application also provides a method for inducing chondrogenesis comprising administering the composition to a subject, wherein the composition comprises an Wnt antagonist and a pharmaceutically acceptable carrier.
In one embodiment, the administration is in vitro or in vivo.
In one embodiment, the administration is local or systemic.
In some embodiments, the method can further comprise administering a stem cell having an ability to differentiate into chondrocyte to the subject. In one embodiment, the MSC can be co-cultured with the Wnt antagonist, and the mixture of the MSC and the Wnt antagonist is then administered to the subject. The subject can be a vertebrate. For example, the subject can be human, monkey, mice, rat, rabbit, pig, dog, cat and the like.
In some embodiments, the subject is the stem cell. The stem cell can be treated with the Wnt antagonist and the carrier to be induced to differentiate into chondrocyte.
The method may further comprises administering a chondrocyte differentiation agent to the subject. The chondrocyte differentiation agent can be administered prior to, simultaneously with, or after the administration of Wnt antagonist.
The present application further provides a method for treating a cartilage-related disease comprising administering a therapeutically effective amount of an Wnt antagonist and a pharmaceutically acceptable carrier to a subject.
In some embodiments, the method can further comprise administering a stem cell having an ability to differentiate into chondrocyte to the subject. The subject can be a vertebrate. For example, the subject can be human, monkey, mice, rat, rabbit, pig, dog, cat and the like.
The cartilage-related disease comprises, for example, but not limited to osteoarthritis, degenerative joint disease, osteochondritis dissecans, rheumatoid arthritis, articular joint injury, achondroplasia, and cartilage defects.
In one embodiment, the administration is in vitro or in vivo.
In one embodiment, the administration is local or systemic.
In one embodiment, the administration can be via injection, implantation, oral administration, and/or subcutaneous administration.
In the treating method of the present application, the therapeutically effective amount means the amount of the Wnt antagonist that, when administered to the subject, is sufficient to induce the chondrogenesis of the cells and/or the tissues, the formation of cartilage, and/or the treatment of cartilage-related disease. The therapeutically effective amount depends on the severity and course of the disease, previous treatment, the subject's health, weight, and response to medication, the judgment of the treating physician and the like. For example, the therapeutically effective amount of the Wnt antagonist can be 0.1 ug-100 mg per gram of the subject.
The treating method may further comprises administering a chondrocyte differentiation agent to the subject. The chondrocyte differentiation agent can be administered prior to, simultaneously with, or after the administration of Wnt antagonist.
In the present application, the pharmaceutically acceptable carrier can be one or more compatible conventional solid or liquid delivery systems as are well known in the art. Some examples of the pharmaceutically acceptable carrier are water, saline, phosphate buffer, sugars, starches, cellulose and its derivatives, powdered tragacanth, malt, gelatin, collagen, talc, stearic acids, magnesium stearate, calcium sulfate, vegetable oils, polyols, agar, alginic acids, and other suitable non-toxic substances and medicinal agents used in pharmaceutical formulations.
Materials and Methods
Cell Culture
Human iPSC-MSCs were derived from iPSCs generated from fetal endothelial cells through lentiviral transduction of OCT-4 and SOX-2 (Ho et al., 2010), and human ESC-MSCs were derived from H1 (Wisconsin Alumni Research Foundation, Madison, Wis., USA) as previously reported (Peng et al., 2016; Wang et al., 2018; Yen et al., 2009). BM-MSCs were obtained from commercial sources (Promocell, Heidelberg, Germany). All MSCs were cultured and expanded in low-glucose Dulbecco's Modified Eagle's medium (DMEM) (Gibco-Thermo Fisher Scientific, MA, USA), with 10% FBS (Hyclone-Thermo Fisher Scientific) and 100 U/ml penicillin, 100 g/ml streptomycin, and 2 mM L-glutamine (all from Gibco-Thermo Fisher Scientific) as previously described (Pittenger et al., 1999; Yen et al., 2005).
In Vitro Chondrogenic Differentiation
In vitro chondrogenic differentiation using 3D pellet culture was performed as described previously (Liu et al., 2011; Wang et al., 2018; Yen et al., 2013), 2×105 trypsinized were centrifuged 450×g for 10 minutes to form a pellet in expansion medium. After 16 hours, the medium was changed to chondrogenic basal medium (ChBM) consisting of low-glucose DMEM supplemented with 100 units/ml penicillin, 100 μg/ml streptomycin (all from Gibco-Thermo Fisher Scientific), 1% Insulin-transferrin-sodium selenite media supplement (Sigma-Aldrich, St Louis, Mo., USA), 2 mM L-glutamine (Gibco-Thermo Fisher Scientific), 10 μM L-Ascorbic acid 2-phosphate (Sigma-Aldrich), 100 nm dexamethasone (Sigma-Aldrich). Addition of TGFβ1 (10 ng/mL; R&D Systems, Minneapolis, Minn., USA), TGFβ3 (10 ng/mL; R&D Systems), XAV939 (XAV; 10 μM; Sigma-Aldrich) or CHIR99021 (CHIR; 10 μM; Sigma-Aldrich) to ChBM was performed as indicated. Cell pellets were harvested at the indicated day for analysis. Micromass culture-based chondrogenic differentiation was performed as previously reported, using 2×107 cells/ml of MSCs suspended in expansion medium and 20-μl drops spotted in the center of each well of a 24-well culture plate (Craft et al., 2015; Greco et al., 2011). After adhesion for 1.5 hour in a humidified incubator at 37° C. with 5% CO2, the medium was changed to ChBM with indicated regulators for chondrogenic induction.
Alcian Blue Staining and Quantification
Samples were washed with PBS and fixed with 4% paraformaldehyde, followed by pH 1.0 1% Alcian blue 8GX (Sigma-Aldrich) staining at room temperature overnight to detect sulfated proteoglycan matrix (Lev and Spicer, 1964). Quantification of Alcian blue staining extracted with 6M Guanidine-HCl was measured in 650 nm absorbance (Song et al., 2018).
Quantitative Real-Time PCR
Total RNA was isolated using TRI® reagent (Sigma-Aldrich), and quantified using NanoDrop spectrophotometer (Nyxor Biotech, Paris, France) (Wang et al., 2013). Reverse transcription was performed using RevertAid H Minus reverse transcriptase (Thermo Fisher Scientific), and PCR was performed on ABI PRISM 7500 system (Applied Biosystem, Foster City, Calif., USA) with SYBR Fast qPCR kit (KAPA Biosystems, Boston, Mass., USA). mRNA expression level was calculated by using the ΔΔCt method, and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) level were used as housekeeping control. The results are normalized to ChBM to represent effects of indicated modulators. The sequences of primers for each gene are listed in Table 1.
Immunofluorescent Staining
Immunofluorescent staining was performed as previously reported (Wang et al., 2013). Paraformaldehyde-fixed cell samples were permeabilized with 0.1% Triton X-100 and nonspecific binding was blocked by 5% bovine serum albumin Primary antibodies against alpha smooth muscle actin (αSMA) (Sigma-Aldrich), β-catenin (BD Biosciences, San Jose, Calif., USA) or N-cadherin (Abcam, Cambridge, UK) were stained with 1:100 dilution for 24 hours, followed by species specific secondary antibody incubation overnight. Fluorescent signals were acquired by confocal microscopy (Leica TCS SP5 II, Wetzlar, Germany) and analyzed by Image J (NIH, Maryland, USA).
Proximity Ligation Assay (PLA)
Micromass cultured iPSC-MSCs were used for PLA and performed according to the manufacturer's protocol (Sigma-Aldrich). Antibodies against N-cadherin (Abcam) and β-catenin (BD Biosciences) were used for recognition. After ligation and amplification, the PLA signals from each pair of probes in close proximity (<40 nm) were visualized by confocal microscope (Leica TCS SP5 II) and signal counts were analyzed by Image J.
In Vivo Chondrogenesis
All animal work was performed according to protocols approved by the Institutional Animal Care and Use Committee. In vivo ectopic chondrogenesis was performed as previously reported with modifications (Zhao et al., 2014). Mouse BM-MSCs were cultured as 3D pellets in ChBM alone or with addition of TGFβ3, CHIR, or XAV for 3 days, then subcutaneously transplanted to dorsal skin of wild-type C57BL/6J mice, with local injections of modulators every 3 days till the pellet sample was harvested at Day 20. Collected samples were then frozen in optimal cutting temperature (OCT) compound and sliced to 5 mm in thickness for histological staining.
Gene Expression and Pathway Analysis
Transcriptome data of human primary smooth muscle cells (GSE109859), MSCs (GSE128949 and GSE108186), chondrocytes (GSE68038), and osteoblasts (GSE121892) were obtained from public database. Datasets are merged to perform principal component analysis (PCA) using Partek® Genomics Suite (St. Louis, Mo., USA). Ranked Gene Set Enrichment Analysis (GSEA) was performed with software version 4.1.0 (Subramanian et al., 2005). Upstream Analysis were performed using IPA software (Qiagen, https://www.qiagenbioinformatics.com).
Quantification and Statistical Analysis
All data represent three replicates or more from separate experiments. Analysis of variance (ANOVA) followed by Tukey's post-hoc test was performed to evaluate significance for comparisons for multiple groups, with p<0.05 as significant. Analysis were performed using GraphPad Prism software and data are shown as mean±standard deviation (S.D.).
Results
Wnt/β-Catenin Antagonism Significantly Enhanced MSC Chondrogenesis while Agonism Resulted in the Opposite and Upregulation of the Master Osteogenic Transcription Factor Runx2
To investigate the effects of Wnt modulation on MSC chondrogenesis, we treated 3D pellet-cultured human MSCs—including iPSC-MSCs, ESC-MSCs and BM-MSCs—in complete chondrogenic induction medium (ChM) along with a small molecule Wnt/β-catenin agonist CHIR which inhibit GSK3β to disrupt the β-catenin destruction complex, or antagonist XAV, a tankyrase inhibitor which stabilizes the β-catenin destruction complex. TGFβ3 was utilized in ChM since it has been found to possess higher chondrogenic potential than TGFβ1 (Puetzer et al., 2010). We found that in ChM conditions, Wnt inhibition by XAV increased condensation of the pellets whereas agonism by CHIR decreased pellet integrity in all 3 sources of MSCs at Day 20.
Quantification of glycosaminoglycans (GAGs) showed a significant increase in the expression of this structural extracellular matrix of cartilage with Wnt/β-catenin antagonism, whereas the opposite was seen with Wnt/β-catenin agonism (
TGFβ Rapidly Increased Alpha Smooth Muscle Actin (αSMA) and RUNX2 Expression in MSCs During Chondrogenic Induction.
While TGFβ1 and TGFβ3 have long been used for MSC chondrogenesis, it is also well established that TGFβ can induce smooth muscle differentiation (Kurpinski et al., 2010) and upregulate an osteogenic program (Wu et al., 2016). By analyzing the transcriptomic data of human primary BM-MSCs and smooth muscle cells from public database (GSE128949 for human primary BM-MSCs; GSE109859 for human primary smooth muscle cells) with Ranked Gene Set Enrichment Analysis (GSEA), gene set of Signaling by TGFB Family Members was positively enriched in human BM-MSCs compared to smooth muscle cells with normalized enrichment score (NES) equals to 2.17 and the nominal p value less than 0.001 (
Wnt/β-Catenin Antagonism Alone Induced More Rapid MSC Chondrogenesis than TGFβ
Since Wnt/β-catenin antagonism strongly enhances MSC chondrogenesis and TGFβ agonism significantly induce other non-chondrogenic lineage markers during this process, we examined the possibility of replacing TGFβ completely with Wnt/β-catenin antagonism to achieve more specific MSC chondrogenic differentiation. After 20 days of chondrogenic differentiation, MSC pellets treated with either TGFβ3 or XAV formed well-condensed spheres and strong Alcian Blue staining compared to ChBM, while pellets treated with CHIR did not undergo condensation but became disintegrated at the end of the culture time period (
Similar to 3D pellet culture, micromass culture of MSCs with XAV treatment at Day 10 demonstrated better structural condensation and stronger Alcian Blue intensity compared to ChBM culture and even TGFβ3-treated culture, while CHIR treatment resulted in minimal evidence of any micromass structure or Alcian Blue staining. Quantitation of GAG production was in line with the morphologic findings, with XAV treatment resulting in the highest production of GAG (
To validate our in vitro data, we performed subcutaneous transplantation into wildtype mice of mouse BM-MSCs cultured in ChBM medium with injections of various modulators (TGFβ3, XAV, or CHIR) every three days for 20 days (
Wnt/β-Catenin Antagonism but not TGFβ Agonism During MSC Chondrogenesis Decreased Canonical Wnt/β-Catenin Transcriptional Activity Including RUNX2 Expression
The significant enhancement of MSC chondrogenesis with Wnt/β-catenin antagonism alone led us to investigate the role of the canonical Wnt/β-catenin pathway during the differentiation process. As a transcription factor, β-catenin undergoes translocation from the cytoplasm to the nucleus when activated. Using immunofluorescent staining and quantification, we found that pellet-cultured MSCs treated with XAV had the lowest nuclear β-catenin levels compared to control ChBM culture or TGFβ3 treatment, while CHIR treatment dramatically increased nuclear β-catenin levels (
Wnt/β-Catenin Antagonism but not TGFβ Agonism Increased N-Cadherin Expression and Interactions with β-Catenin at AJs as Well as Enhancing Actin Cytoskeleton-Mediated Condensation
In addition to its role as a transcription factor, β-catenin is also a component of the AJ, a key structure of cell-cell adhesion which is a critical aspect during cartilage condensation (Alimperti and Andreadis, 2015). To investigate how Wnt/β-catenin modulation during MSC chondrogenesis affect AJs, we performed immunofluorescent staining for N-cadherin, a major AJ component in mesenchymal cell types like MSCs which is also essential during chondrogenesis (Quintana et al., 2009). We found N-cadherin expression to be strongly and most significantly upregulated in MSC micromass culture with XAV treatment after 24 hours, compared to all other conditions (
To validate interactions between N-cadherin and β-catenin in MSC chondrogenic differentiation, we first examined the N-cadherin pathway using the Pathway Interaction Database (PID_NCADHERIN_PATHWAY) and found that along with N-cadherin (CDH2), β-catenin (CTNNB1) is a core participant within the signaling pathway, along with numerous components of the AJ: α-catenin (CTNNA1), p120/δ-catenin (CTNND1), and plakoglobin (JUP). Moreover, the three major GTPases responsible for actin cytoskeleton organization—RhoA, CDC42, and Racl—are all found as downstream effectors in the signaling pathway (
Significant Downregulation of Wnt/β-Catenin and TGFβ-Related Pathways in Transcriptomes of Human Primary Chondrocytes Compared to Osteoblasts
To assess the physiological relevance of our findings, we performed bioinformatics analyses using human primary BM-MSC, chondrocyte, and osteoblast transcriptome data from public database (GSE108186 for human primary BM-MSCs; GSE68038 for human primary chondrocytes; and GSE121892 for human primary osteoblasts). Initial analyses using principal component analysis (PCA) demonstrated that chondrocytes, osteoblasts, and MSCs are three highly distinct populations (
Using Upstream Regulator Analysis in Ingenuity Pathway Analysis (IPA) which can provide a more causal relationship between transcription factors/master regulators and downstream pathways (Kramer et al., 2014), we found that TGFβ1, Smad2/3, WNT3A, TCF4, BMP2, and TGFBR2 were all predicted to be downregulated transcription factors/master regulators in human primary chondrocytes compared to MSCs. Furthermore, TGFβ1, CTNNB1, and WNT3A were predicted to be downregulated regulators in human primary chondrocytes compared to osteoblasts. Specifically, α-catenin—a core components of the AJ—was predicted as an upregulated regulator in chondrocytes compared to both MSCs and osteoblasts (
Discussion
MSC therapy likely offers the best possibility of a curative treatment for cartilage and joint diseases, which currently lacks such significant disease-modifying treatments. However, the relatively more difficult 3D pellet differentiation protocol and the need for using a protein-based growth factor, TGFβ, continue to be obstacles for robust clinical applications. TGFβ is a cytokine with complex functions, and has pleomorphic roles in specification of multiple mesenchymal lineages. In addition, this cytokine is part of a superfamily of ligands which includes the BMP subfamily, factors essential in ossification and bone biology. Therefore, it may be difficult to avoid osteogenic specification with use of proteins from this family during chondrogenic differentiation. While some early in vitro stem cell differentiation studies did not consistently find Wnt inhibition to promote chondrogenesis, these reports utilized biologically derived Wnt inhibitory ligands which are known to have off-target effects as well as potency issues (Im and Quan, 2010; Yano et al., 2005). In addition, some studies utilized non-MSCs which may not be the appropriate system to investigate chondrogenic lineage specification through Wnt/β-catenin (Yano et al., 2005). Using highly potent small molecule agonists and antagonists of the Wnt/β-catenin pathway, we demonstrated that Wnt/β-catenin antagonism efficiently induce in vitro chondrogenesis in human MSCs from multiple sources, and in vivo using murine BM-MSCs. Moreover, in comparison with TGFβ3, the most potent member of the family for induction of chondrogenesis, Wnt/β-catenin antagonism more rapidly induced chondrogenesis without inducing other non-chondrogenic, off-target, lineages. Our findings are further supported by transcriptome analysis of primary human MSCs, chondrocytes, and osteoblasts, which demonstrated that the Wnt/β-catenin and TGFβ family signaling pathways were downregulated in chondrocytes relative to the other 2 cell types (
We found that the efficient chondrogenic commitment mediated by Wnt/β-catenin antagonism involved strong upregulation of N-cadherin expression and N-cadherin/β-catenin interaction at the AJ which enhanced pellet condensation (
Our study revealed the many off-target lineages specification by TGFβ during MSC chondrogenesis. One of the most important molecules/pathways in developmental biology, TGFβ is known to be involved in specification of multiple mesodermal lineages, as well as mediate pathological fibrotic processes. In the development of the skeletal system, TGFβ not only induces chondrogenic differentiation and modulates the process of hypertrophy (Mueller et al., 2010) but also osteogenic differentiation as well (Wu et al., 2016). In addition, TGFβ promotes differentiation of MSCs into smooth muscle cells (Kurpinski et al., 2010); this was clearly reflected in our transcriptomic analysis which showed that signaling by TGFβ family was highly enriched in SMCs compared to MSCs (
The master transcription factor for chondrogenesis is SOX9 (Bi et al., 1999), however, we did not find further upregulation of this transcription factor with Wnt inhibition during MSC chondrogenic induction using 3D pellet culture (
In summary, using multiple sources of human MSCs including ESC-MSCs and iPSC-MSCs we found that replacement of TGFβ agonism with Wnt/β-catenin antagonism resulted in robust and specific in vitro and in vivo MSC chondrogenesis by eliminating off-target lineage specification into osteogenesis/hypertrophic cartilage and smooth muscle. Wnt/β-catenin antagonism also more efficiently induced MSC chondrogenesis by increasing N-cadherin levels as well as N-cadherin-β-catenin interactions at the AJ to enhance condensation (
While the present invention is disclosed by reference to the preferred embodiments and examples detailed above, it is to be understood that these examples are intended in an illustrative rather than in a limiting sense. It is contemplated that modifications and combinations will readily occur to those skilled in the art, which modifications and combinations will be within the spirit of the invention and the scope of the following claims and its equivalent systems and methods.
| Number | Date | Country | |
|---|---|---|---|
| 63162778 | Mar 2021 | US |
