CN114555101A - Multipotent articular progenitor cells, compositions thereof and methods of making - Google Patents

Abstract

A method of treatment using a multipotent Joint Progenitor Cell (JPC) is provided. Also provided are JPC populations, their use in the preparation of medicaments and methods of preparation thereof.

Description

Multipotent articular progenitor cells, compositions thereof, and methods of making

Cross Reference to Related Applications

This application claims priority to provisional patent application No. 62/892,067, entitled multi-point Joint reagent Cells, Compositions and Methods Thereif, filed on 27.8.2019, the contents of which are incorporated herein by reference in their entirety.

Technical Field

The present disclosure provides a method of treatment using a multipotent Joint Progenitor Cell (JPC). Also provided are JPC, compositions, and methods of using and making JPC.

Background

The knee joint is composed of three major structures, articular cartilage (articular cartilage), meniscus (menisci), and Anterior Cruciate Ligament (ACL). Articular cartilage is composed of deep-band chondrocytes and superficial cells, which express lubricin (lubricin) to reduce friction and make movement smoother. Meniscus is a fibrocartilage tissue that acts to disperse our body weight and reduce friction. The ACL provides stability against angulation and rotation at the knee joint. Trauma, sports injuries and aging of these tissues are the most common causes of Osteoarthritis (OA), one of the leading causes of disability and chronic pain, affecting millions of people worldwide. The self-repair capacity of these tissues is very limited, probably due to the depletion of adult progenitor cells. Cell-based therapies have been developed, such as joint injection of exogenous stem cells (e.g., bone marrow MSCs), but the results may not be ideal because the environmental cues for proper differentiation and repair of cells are not sufficient. Therefore, it is important to identify a more optimized progenitor cell type that initiates an efficient repair process.

Disclosure of Invention

Provided herein is a method of treating a joint disease comprising administering a multipotent joint progenitor cell (joint progenit) to a joint of a subject in need thereofor cells, JPC), wherein the JPC obtained from the interzone (interbone) is Lgr5⁺And/or Col22a1⁺。

Provided herein is the use of multipotent Joint Progenitor Cells (JPCs) in the manufacture of a medical product for treating joint disease in a subject in need thereof, wherein the JPCs obtained from the intermediate zone are Lgr5+ and/or Col22a1 +.

Provided herein are pluripotent Joint Progenitor Cells (JPCs) for use in treating joint disease, comprising administering pluripotent Joint Progenitor Cells (JPCs) at a joint of a subject in need thereof, wherein the JPCs obtained from the intermediate region are Lgr5+ and/or Col22a1 +.

In one embodiment, the joint disease is a trauma or associated degenerative disease of ligaments and articular cartilage. In one embodiment, the joint disease is associated with osteoarthritis or with damage associated with joint tissue.

In one embodiment, JPC differentiates into tenocytes, cruciate ligaments, synovium, meniscus and/or articular chondrocytes.

Provided herein is a method of treating a ligament defect (ligament defect) or tendon defect (tendon defect) comprising administering a multipotent Joint Progenitor Cell (JPC) at a joint of a subject in need thereof, wherein said JPC obtained from the interregion expresses Mkx and Scx.

Provided herein is the use of multipotent articular progenitor cells (JPCs) for the manufacture of a medical product for treating a ligament or tendon defect in a subject in need thereof, wherein said JPCs obtained from the intervehicular region express Mkx and Scx.

Provided herein are pluripotent Joint Progenitor Cells (JPCs) for use in treating a ligament or tendon defect, comprising administering the pluripotent Joint Progenitor Cells (JPCs) at a joint of a subject in need thereof, wherein the JPCs obtained from the interregion express Mkx and Scx.

Provided herein is a method of treating an articular chondrocyte or meniscal defect comprising administering pluripotent Joint Progenitor Cells (JPCs) at a joint of a subject in need thereof, wherein the JPCs obtained from the intercompartments express Pax9, Cdh13, Lrrc17, and Col26a 1.

Provided herein is the use of multipotent articular progenitor cells (JPCs) in the manufacture of a medical product for treating articular chondrocyte or meniscal defects in a subject in need thereof, wherein the JPCs obtained from the intermediate zone express Pax9, Cdh13, Lrrc17 and Col26a 1.

Provided herein are pluripotent Joint Progenitor Cells (JPCs) for use in treating a defect of articular chondrocytes or meniscus, comprising administering the pluripotent Joint Progenitor Cells (JPCs) at a joint of a subject in need thereof, wherein the JPCs obtained from the interregion express Pax9, Cdh13, Lrrc17, and Col26a 1.

Provided herein is a method of generating a cell line of Joint Progenitor Cells (JPCs) comprising the steps of

(i) Providing induced pluripotent stem cells (ipscs) or expanded potential stem cells (epscs);

(ii) culturing ipscs or epscs in the presence of a glycogen synthase kinase-3 β inhibitor;

(iii) (iii) activating TGF- β signalling from the ipscs or epscs of step (ii);

(iv) (iv) culturing the ipscs or epscs from step (iii) in the presence of Fgf2 and Mmp2, wherein the culturing from (ii) to (iv) steps is for about 5 days to form mesendoderm;

(v) (iii) removing the glycogen synthase kinase-3 β inhibitor of step (ii) from the iPSC or ePSC culture;

(vi) inhibiting TGF- β signaling, and culturing ipscs or epscs for about 4 days to form mesoderm;

(vii) differentiating the mesoderm into JPC in iPSC or ePSC cultures;

(viii) activating Wnt signaling for about 5 days; and

(ix) the hedgehog (hh) concentration in iPSC or ePSC cultures is modulated.

In one embodiment, the glycogen synthase kinase-3 β inhibitor is CHIR 99021.

In one embodiment, TGF- β signaling is activated by activin a (activin a).

In one embodiment, TGF- β signaling is inhibited by Fgf2, Bmp2, and Sb 431542.

In one embodiment, the mesoderm differentiates into JPC in the presence of Fgf2 and Gdf 5.

In one embodiment, Wnt signaling is modulated by the addition of R-spondin.

In one embodiment, the iPSC or ePSC comprises a LGR5-GFP reporter construct capable of expressing GFP in the iPSC or ePSC.

In one embodiment, the method further comprises the step of monitoring GFP expression in the ipscs or epscs.

Provided herein are populations of JPCs produced by the methods of the disclosure.

Provided herein is a method of treating joint disease comprising administering the JPC.

Drawings

The foregoing and other objects, features and advantages of particular embodiments of the present disclosure will be apparent from the following description and examples of the drawings.

FIG. 1: expression of Lgr5 in developing fingers and knee joints

(a) Overall images of hind limbs from Lgr5-GFP embryos (E13.5-E18.5). (b) Sagittal section of boxed area in (a) showing expression of Lgr5 (GFP). (c) GFP (green) immunostaining and Gdf5 (red) in situ hybridization of adjacent sagittal sections from E14.5 hind limb, finger III, showed developmental Lgr5 expression later than Gdf 5. In the P1/P2 and M/P1 interparticular regions, Lgr 5-labeled cells are centered in the region of cells expressing Gdf 5. (d) Higher magnification of the P2/P3 joint, showing Gdf5 expression at E14.5 but no Lgr5 expression, and its expression over time in development (circles), as shown in the same joint at E16.5(E) and E17.5 (f). (g) Global images of knees from E16.5 embryos. (h) A graphical representation of the location and structure of the slice selected for analysis is displayed. From E13.5 to E18.5, Lgr5 expression during the formation of articular cartilage/meniscus (i) and cruciate ligament (j). M, metacarpal (metacarpal); p1, proximal phalanx (proximal pharange); p2, middle phalanx (middle phalange); p3, distal phalanx (digital pharange); f, femur; t, tibia; ac, articular cartilage. Scale bar 100 μm.

FIG. 2: fate of Lgr5+ compartment cells in developing synovial joints

Carrying Lgr5-GFP at E13.5; R26R knock-in pregnant mice were injected with tamoxifen (tamoxifen) and offspring were collected for analysis of finger and knee joints at E15.5(a) and (b), E17.5(c) and (d), and 3 weeks (E) and (f) after birth. The cells in the middle zone (IZ) were labeled as indicated in the whole embryo and sagittal section at E15.5 stained for LacZ + cells (black arrows). Progeny cells that lineage track at different stages at the articular surface (Ac) of the finger and knee joints (blue arrows). In the knee, LacZ + cells were also detected in the meniscus (Me; red arrow), cruciate ligament (Cl; yellow arrow), intra-patellar fat pad (Ifp; green arrow) and synovium (Sm; orange arrow). Scale bar 100 μm.

FIG. 3: dynamic spatiotemporal expression of Lgr5 and collagen XXII in knee joint development

(a) By referring to the bulk transcriptome, there were higher expressed matrix genes in Lgr5+ compared to Lgr 5-. "+ ∞" indicates that the gene is not expressed in the Lgr 5-cell population. The expression data for developing joints are available in Eurexpress with the asterisked genes. (b & c) Co-expression of Lgr5(GFP) and Col22a1mRNA in finger (b) and knee joint (c). (d) Lgr5-GFP (Green) and ColXXII (Red) double immunofluorescent staining in sagittal sections of developing knee joints of Lgr5-GFP embryos at E14.5 and E18.5 (see also FIGS. 12a and b). Articular cartilage formation proceeds from Lgr5+ cells (white open arrows), to cells expressing both Lgr5 and ColXXII (white filled arrows), to cells expressing only ColXXII (filled arrows). (e) Colocalization of Lgr5 and ColXXII in developing articular cartilage and meniscus. The ColXXII stroma pools in a very narrow region on the superficial surface of the fully developed articular cartilage and meniscus. (f) LacZ + daughter cells (arrows) of Lgr5 (injected with Tamoxifen (TM) at E13.5 and harvested at E17.5) were detected at ColXXII + superficial layers (brackets) of articular cartilage and meniscus. (g) All cells in this layer (brackets) expressed Col22a1 in comparable sections. ColXXII co-localisation with Lgr5(GFP) (h) and Scx (GFP) driver (i). Histology of the same sections stained with alcian blue (alcian blue) and nuclear fast red (nuclear fast red) (lower panel of h & i). F, femur; t, tibia; cl, cruciate ligament. Scale bar 200 μm.

FIG. 4: characterization and molecular characterization of Lgr5+ cells in the intergonal region of E14.5 by Single cell transcriptome (molecular signature)

Analysis of gene expression in 5,460 cells using tSNE mapping of 2d (a) or 3d (b) identified 6 major clusters. (c & D) 3D view of Gdf5+ and Lgr5+ cell distribution. The relative distribution of Lgr5 expression in the different clusters is shown in (d) as a pie chart. (e & f) contour plots (contour plot) show the density of cells expressing Lgr5 shown in (b) and Gdf5 shown in (c), with corresponding color density plots. (g)2D-tSNE shows the distribution of Lgr5+ in cells expressing Gdf5 +. Higher magnification of the boxed area in cluster 1 is shown in the right panel, highlighting the opposing Lgr5+/Gdf5- (cyan) and Lgr5+/Gdf5+ (purple) cells. (h) The Venn plot shows the distribution and percentage of the Lgr5-/Gdf5+ (green), Lgr5+ Gdf5- (cyan) and Lgr5+/Gdf5+ (purple) subpopulations of cells. (i) The 2D-tSNE plot highlights the distribution of Lgr5+/Ki67+ in cluster 5 (relative to the predominance of Lgr5+/Ki 67-cells in cluster 1), and the percentage of Lgr5+/Ki67+ is shown in the pie chart. (j) Double immunofluorescence detection of Lgr5-GFP and Ki67 expressing cells in E14.5 Knee joints. The periphery of the Lgr5 cell population is marked with dashed lines, and the arrows indicate double positive cells, which are shown in box area at higher magnification. The relative percentages of Lgr5+/Ki67+ determined from the three embryos are also shown in the pie chart. F, femur; t, tibia. (k) The Venn plot shows DEG for cluster 1 versus non-cluster 1 (n-110, blue circles) and for Lgr5+ cells versus Lgr 5-cells (n-75, yellow circles). The two gene lists were used to identify 48 common DEG. Transcription factors in each sector are shown. A complete gene list is provided in fig. 20.

FIG. 5: pedigree trajectory of knee joint

(a) The heatmap shows clusters of the top 5% dispersed genes (whole genome) of the spectrum of 200 cells randomly selected as a representative from each cluster. Gene clusters associated with joint development, ligaments, cell cycle and cartilage were identified. (b) Summary of the features of each cluster, where clusters 1, 2, 5 and 6 are defined as compartment cells. (c) Pseudo-timing analysis (pseudotime analysis) of cells from clusters 1, 2, 5 and 6 identified eight cell states and showed distribution of cells from a single cluster and a merged profile along the pseudo-timing line. (d) Distribution of all Lgr5+ cells along the pseudo-time line, and relative percentages of distribution in each of the cell states shown, along with proliferating Lgr5+/Ki67+ cells. (e) The pseudo-temporal trajectory predicts a gradient of "early" (dark blue) to late (light blue) cells, mainly differentiating into two trajectories a and B, with the arrows indicating the direction of prediction. The expression profiles of ligament (f) and chondrocyte (g) genes were mapped onto 2D tSNE. The size of each circle is a relative reflection of the gene expression level, and Lgr5+ cells are indicated in green. (h) The major lineage differentiation of cells between Lgr5 is shown graphically. (i & j) distribution of cells expressing ligament (i) and cartilage (j) genes along pseudo-time lines. The box highlights a specific comparison of cells in state II expressing the mature ligament marker Tnmd instead of Col22a 1.

FIG. 6: ability of region tissue between Lgr5+ to repair cartilage defects (suspension)

(a) Experimental design for the isolation and transplantation of Lgr5+ compartment tissues (labeled with both GFP and tdTomato) into articular cartilage defects for repair. The right panel shows tdTomato + tissue within the defect after transplantation. (b) At 15 days after implantation, the implanted interregional tissue (tdTomato +) was integrated with the host cartilage and differentiated into cells in the articular cartilage and flat cells in the superficial layer (arrows). Histological (c) and ECM marker analysis (d-g) were performed after 15 days on the defect area with and without Lgr5+ implantation. Data for other animals are shown in figure 14.

FIG. 7: model for contribution and differentiation (divergence) of progenitor cells in joint formation

Schematic representation of the knee joint development process is shown. (a) Formation of cartilaginous mesenchymal anlage (cartilage anlagen) containing chondrocytes expressing Sox 9. (b) Sox9+ chondrocytes dedifferentiate into Gdf5+ spacer cells at the putative joint site. (c) By recruiting mesenchymal cells and partially proliferating Lgr5+ cells from the periphery, the compartment is expanded and stratified into specific regions. (d & e) Lgr5+ compartment cells are multipotent progenitor cells involved in the formation of all internal structures of the knee joint, including the cruciate ligament, articular cartilage and meniscus. Lgr5+/Col22a1+ cells are committed progenitors that can build articular cartilage and meniscus, while Lgr5+/Scx +/Col22a 1-cells are limited to the lineage of cells that build ligaments.

FIG. 8: human Joint Progenitor Cells (JPC) are distinguished from induced pluripotent stem cells (ipscs) or expanded pluripotent stem cells (epscs).

FIG. 9: quantitative PCR revealed the expression of Gdf5 and Lgr5 in the inter-phalangeal (IZ) region

mRNA was extracted from the interphalangeal and surrounding non-interventricular regions of the E14.5 embryo to generate cDNA for QPCR. Expression of Gapdh was normalized to 1.0, values above 1.0 indicate that these genes are up-regulated, and vice versa. Of all the selected stem/progenitor markers, the known interarticular region marker Gdf5 and the novel marker identified herein, Lgr5, are upregulated in the interregional region.

FIG. 10: normal knee and finger joints developed by Lgr5-GFP mutant

Representative tissue sections stained with arl blue and nuclear fast red showed no observable differences between the developing knee (a) and finger (b) joints of Lgr5+/+, Lgr5GFP/+ and Lgr5GFP/GFP E18.5 embryos (n ═ 3 per genotype). (c) Articular cartilage appears to have formed normally, with ColXXII being expressed and localized in the superficial layer as expected. F femur, T tibia, Cl cruciate ligament, M metacarpal bone, P1 proximal phalanx, P2 medial phalanx, P3 distal phalanx, and scale bar 200 μ M.

FIG. 11: bulk transcriptomics analysis of cell populations in joint development.

(a) Three cell populations isolated. One population was from Sox9-eGFP mice of E13.5, indicating cells expressing Sox9 in the bones, and the other two populations were from the developing Lgr5-GFP mice, the Lgr5+ interphalangeal region (red arrow) and the surrounding non-GFP region (yellow arrow), respectively

(b) In that respect (c) Schematic illustration of the protocol for selecting the appropriate cell population from all isolated cells for subsequent RNA-seq to generate three data sets. (d) And (3) a workflow of Bulk RNAseq data processing. (e) Venn plots show the determined number of genes and the overlap between the data sets. Genes with higher expression in the Lgr5+ population are shown on the right. (f) The heat map depicts the relative expression levels of genes involved in higher expression levels in a particular molecular pathway. The + Fold Change (FC) indicates an increase in expression in Lgr5+ cells, while-FC indicates a decrease in expression in Lgr5+ cells. "+ ∞" indicates a gene expressed only in Lgr5+ cells. Genes for which expression data were available from the Eurexpress database were marked with asterisks.

FIG. 12: expression of Lgr5 and ColXXII in the finger and knee joints.

Expression of Lgr5 (green) and ColXXII (red) co-localized in E18.5(a) and neonatal mouse (P0) (b) finger joints. (c) P10 in M/P1, Ta/M and knee. M, metatarsal bones. P1, proximal phalanx. P2, middle phalanx. P3, distal phalanx. Ta/M, tarsometatarsal joint. (d) Cells embedded in the ColXXII layer were flattened compared to round chondrocytes (yellow arrows) in the deeper regions of P6 mice. Focal adhesion (β 1-integrin) was found on both sides of the cells in direct contact with the ColXXII matrix (white arrows). The scale bar is 100 μm in (a-c) and 10 μm in (d).

FIG. 13 is a schematic view of: additional information on Single cell analysis

(a) Volcano plots (volcano plot) show that Gdf5 (red circle) is the only Differentially Expressed Gene (DEG) between the Lgr5+/Gdf 5-and Lgr5+/Gdf5+ subpopulations, and that these cells are essentially identical. (b) The volcano plots show DEG between Lgr5+ cells from clusters 1 and 5, also indicating that they are the same cells, except that the cells in cluster 5 are proliferating. (c) GO term analysis of DEG (circles) in panel (b). (d-g) Principal Component Analysis (PCA) of 5,460E 14.5 knee joint cells. (d) The profile shows the density of the cells, and two main peaks are found. (e) Peak 1 corresponds to the interregional cell expressing Gdf5 and Lgr5 and peak 2 corresponds to the non-interregional cell expressing the mature chondrocyte markers Comp and Epyc. (f) Clusters 1, 2, 5 and 6 of t-SNE were located at peak 1, while clusters 3 and 4 were located at peak 2, consistent with the finding of 2D t-SNE. (g) The profile of the cell cycle genes shows the presence of proliferative subpopulations of cells in the interregional cluster (cluster 5) and the non-interregional cluster (cluster 4). (h) Violin plots (violin plots) show expression profiles of representative cartilage, ligament and cell cycle genes in each cluster, providing basis for their cellular and molecular characteristics.

FIG. 14: lgr for the repair of cartilage defects⁵⁺Intercalary tissue transplantation

Two additional animals receiving an intercalary tissue transplant were analyzed. Similar positive results were obtained in all three-handed animals, including the differentiation of the compartment into collagen II-expressing chondrocytes, the reconstitution of superficial layers (CILP 1-expressing) and the inhibition of collagen I expression and fibrotic tissue. Please refer to fig. 6.

FIG. 15: genes expressed by only one cell population (top 100 in expression level) (referred to as bulk).

FIG. 16: a transcription factor (bulk transcriptome of a finger joint) expressed in a specific cell population.

FIG. 17: transcription factors are expressed in all three populations and expression in the Lgr5+ cell population is 1.5-fold higher than in the other two populations (bulk transcriptome of the finger joints).

FIG. 18 is a schematic view of: genes involved in molecular signaling pathways, cell-matrix contacts, ECM degradation and synthesis (E14.5 refers to the bulk transcriptome of the joint).

FIG. 19: detailed information and parameters for single cell transcriptome sequencing and analysis.

FIG. 20: DEG of "cluster 1vs. non-cluster 1" and "Lgr 5+ vs. Lgr 5-" (single cell transcriptome of E14.5 knee joint).

FIG. 21: primers are used herein.

Detailed Description

The interzone is a transient structure during the process of embryonic stage joint formation. Mesenchymal cell aggregates dedifferentiate to form cells of the Gdf5+ compartment, marking the location of future joint formation. Cells in the center of the compartment expressed Lgr 5(a marker we newly identified). At this stage, approximately 12% of the Lgr5+ cells are proliferative, with they being located predominantly in the outer region of the Lgr5 cluster. Together with cells recruited from the surroundings, the compartment expands and begins to separate into different compartments. Our lineage tracing experiments indicate that cells in the Lgr5+ compartment contribute to the formation of articular cartilage, meniscus and ACL. After birth, the number of Lgr5+ cells in these compartments is significantly reduced, which may be responsible for the poor healing capacity of the joint tissue. These Lgr5+ compartment cells promote cartilage defect repair and regenerate hyaline cartilage like structures (hyaline cartilage like structures) expressing markers for articular chondrocytes. A differentiation protocol for generating unlimited multipotent joint progenitor cells in the laboratory and studies on the properties of these Joint Progenitor Cells (JPCs) are disclosed.

The knee joint-forming region and the surrounding tissue containing differentiated chondrocytes were dissected out of the E14.5 embryo. 5.6K cells were sequenced and showed only one type of JPC as they shared similar gene expression profiles and clustered together in cluster analysis (tSNE and PCA). By comparing JPC with non-JPC (differentiated articular cells and chondrocytes), 137 tag genes (signature genes) of JPC have been identified, 18 of which are TF. JPCs can differentiate into two lineage cells because they express ligament-specific TF, Mkx, Scx and Meox2, and chondrogenic TF, Osr2 and Trps 1. All articular cells were sorted on a virtual timeline using the Monocle2 algorithm, along which they show the continuous variation of the transcriptome. There is only one type of JPC, which are pluripotent because they express TF in both ligament and cartilage lineages. More than 80% of JPCs appear to be in the youngest/original state before lineage specification to ligaments or articular chondrocytes. Thus, they are primitive cells and remain pluripotent until lineage specification. The fate of a JPC is likely to be controlled by external signals in its microenvironment. Disclosed herein is the expression of TF as a traceable JPC lineage-specialized cell surface and matrix molecule. Mkx and Scx expression can mark cells committed to differentiate into ligaments, while Pax9, Cdh13, Lrrc17 and Col26a1 mark cells committed to differentiate into articular cartilage and meniscus.

Synovial joints develop from a pool of progenitor cells in the area where the joint will form (i.e., the intervarietal region). Expression of Gdf5 and Wnt9a have been used to label the earliest cellular processes in the formation of internodes and progenitor cells. However, we have an inadequate understanding of the lineage specification and development processes of different tissues of the joint. Here, through lineage tracing studies, we identified a population of Lgr5+ compartment cells that contributed to the formation of cruciate ligaments, synovium and articular chondrocytes of the joint. Col22a1 (marker for early articular chondrocytes) is disclosed herein as an important lineage marker for differentiation into articular chondrocytes, co-expressed with Lgr5+ cells prior to formation of the joint cavity. Lgr5+ cells contribute to the repair of joint defects by reconstituting the superficial layer expressing Col22a 1.

Provided herein are populations of cells expressing Lgr5 in the intergenic regions of developing joints that contribute to the formation of articular cartilage, cruciate ligaments, and menisci. Col22a1 was expressed by Lgr5+ compartment cells prior to joint cavity formation, supporting lineage progression from Lgr5+ compartment cells to Lgr5+/Col22a1+ double positive cells, which are committed progenitors of Col22a1+ primary chondrocytes at the joint surface.

Examples

1. Materials and methods

1.1 mouse strains

Maintaining Lgr5-eGFP-IRES-CreER^T2(Lgr5-GFP) mice (Barker et al, 2007), ROSA26-LacZ (R26R) reporter mice (Soriano,1999) and Scx-GFP (Pryce et al, 2007) mice in the C57bl/6 genetic background. The PCR primers used to genotype these mice are listed in FIG. 21. All animal work was approved by the Committee for Use of Live Animals in education and Research (Committee on the Use of Live Animals in Teaching and Research of the University of Hong Kong) at Hong Kong University.

1.2 real-time quantitative PCR

mRNA was extracted from the E14.5 interphalangeal region and surrounding non-interjacent regions using TRIzol reagent (Thermofoisher). The cDNA was generated using PrimeScript RT kit (Clonetech). qPCR was performed using the LightCycler480 SYBR Green I Master kit (Roche). All primers are listed in FIG. 21.

1.3 in situ hybridization and immunohistochemistry

Using [ alpha ] for Gdf5³⁵S]UTP-labeled riboprobe (riboprobe),in situ hybridization was performed as previously described (Gao et al, 2009). Fluorescence in situ hybridization was performed as previously described (Shwartz and Zelzer,2014) using DIG labeled probes against Col22a 1. For immunohistochemistry, the sections were combined with goat anti-GFP (ab6673), guinea pig anti-ColXXII (produced by Roguel Koch), sheep anti-CILP 1 (R)&D systems) were incubated with primary rabbit anti-Ki 67(ab1558) and detected using relevant secondary antibodies (Alexa Fluor488 anti-rabbit (ThermoFisher), Alexa Fluor488 anti-goat (abcam), Alexa Fluor488 anti-sheep (abcam) and Cy3 anti-guinea pig igg (jackson immunoresearch)).

1.4 pedigree tracing and X-gal staining

Tamoxifen (Sigma) was administered by intraperitoneal injection (0.2mg/g body weight) to pregnant mice carrying the Lgr5-GFP/R26R knock-in gene of pregnancy E13.5. Whole embryo X-gal staining of the extremities was performed to detect lacZ activity. Samples aged 3 weeks were decalcified in 0.5M EDTA (pH 7.5) overnight. 7 μm paraffin sections were counterstained with Eosin (Eosin).

1.5bulk transcriptome analysis

Sox9+ cells were isolated from phalanges of E13.5 Sox9-GFP embryos (Nakamura et al, 2011). Lgr5+ and surrounding non-Lgr 5(Lgr5-) cells were derived from E14.5 Lgr5^GFP/+Isolated in the interphalangeal area of the mouse forelimb (FIGS. 11a, b and c). Cells were separated with a mixture of TrypLE Express (Gibco) and 0.1% DNase I for 20 min, filtered through a 40 μm cell filter, and sorted by FACS using an Aria I flow cytometer (BD Biosciences). Using mirVana^TMTotal RNA was extracted from The miRNA Isolation Kit (Ambion), a cDNA library was constructed with 10ng of RNA using The SMARTer Ultra Low Input Kit (v3, Illumina), and sequenced using The Illumina HiSeq 1500 platform (Centre for Genomic Sciences, The University of Hong Kong). The cDNA fragment sequences were aligned with the mouse genome (mm 10). Fragment numbers Per Million map reads Of exons Per Kilobase (FPKM) values were generated for comparison (FIG. 11 d). Mixing FPKM>The gene of 5 is considered to be an expression gene. Using DAVID (Huangda et al, 2009) and Euroxpress (Diez-Roux et al, 2011) databasesLane and expression analysis. The data sets were uploaded to a Gene Expression integration database (Gene Expression Omnibus, GEO) for public access (GSE 110281).

1.6 Single cell transcriptome analysis

Expression from E14.5 Lgr5 by GFP^GFP/+The embryo dissects the interknee area and surrounding cartilage tissue. The isolated cells were pooled and Single Cell RNA sequencing was performed using a 10Xgenomics chromosome Single Cell Controller (10Xgenomics Inc.). According to the manufacturer's instructions (Chromium)^TMSingle Cell 3' Reagent kit v2 and Chromium^TMSingle Cell a Chip Kit) and then sequenced on the Illunina HiSeq 1500 platform. The raw data were processed using Cell-anger pipeline (version 2.1.0; 10X Genomics Inc.) for alignment to mm10, UMI (Unique Molecular Identifier) quantification, and dimension reduction (tSNE) analysis. Combining data with CreER^T2The sequences were aligned to evaluate Lgr5-eGFP-CreER^T2Expression of an allele. Will express Lgr5 and/or Lgr5-eGFP-CreER^T2Allelic cells are considered to be Lgr5+ cells. Clustering and pseudo-temporal trajectory analysis were performed using Cell-range and Monocle2, respectively. The detailed information of the parameter setting is shown in fig. 19. This data set is deposited on a GEO (GSE 130919).

1.7 cartilage puncture and repair Using Lgr5+ interzone tissue

Hybridizing an Lgr5-GFP mouse to a ROSA26-tdTomato mouse to produce a double hybrid Lgr 5-GFP; tdTomato embryo. GFP + tissues were dissected from the knee joint during E13.5 embryonic development and transplanted into the defect area punctured with a 27G needle at the trochlear groove (trochlear groove) of an 8 week old C56bl/6 mouse (n-3, fig. 6 a). As a control, a punctured animal without tissue transplantation was used.

1.8 differentiation of human articular progenitor cells (JPC)

Human Joint Progenitor Cells (JPCs) will be differentiated from Induced pluripotent Stem cells (ipscs, iXCells Biotechnologies) or Expanded Potential Stem cells (epscs, gift from professor of Pentao Liu, XueFei Gao et al, 2019) by the following protocol, see fig. 8.

First stage (days 1-5) from Stem cells to mesendoderm

Human stem cells were prepared for mesendoderm cells by providing CHIR99021, a glycogen synthase kinase-3 β inhibitor, and activin A, which activates TGF- β signaling, followed by Fgf2 and Bmp 2.

Second stage (days 5-9) from mesendoderm to mesoderm

Cells will be differentiated into mesodermal cells by removing CHIR99021 and activin a from the medium and replacing with Fgf2, Bmp2 and Sb431542(TGF- β signalling inhibitors).

Third stage (days 9-14) from mesoderm to JPC

Cells will be differentiated into JPC by addition of Fgf2 and Gdf 5. Based on observations of embryonic joint development, Wnt signaling was activated by addition of R-spondin on days 9-11, followed by HH regulation at the final stage (by titration of HH concentration in the medium).

Evaluation of differentiation protocols

Genetically engineering stem cells to include LGR5-GFP reporter genes

Differentiated JPCs will express GFP and we have a panel of JPC markers to evaluate the efficiency of the protocol.

Example 1: lgr5 as a novel marker for different cells in developing synovial joints

Since the spacer cells were progenitor cells, we screened these cells with a panel of stem cell markers and detected Lgr5 expression by q-RT-PCR (fig. 9). Using GFP expression in Lgr5-eGFP-IRES-CreERT2(Lgr5-GFP) mice, we confirmed that Lgr5 is a marker for the mesenchymal cells. Lgr5-GFP is a null allele (null allele) in which GFP expression replaces Lgr5(Barker et al, 2007). Heterozygote mice for this allele are normal and viable, while homozygote mice die perinatally (Barker et al, 2007). However, we did not observe abnormalities in limb development or synovial joint formation in homozygotes (fig. 10). All analyses of Lgr5/GFP expression in synovial joints were performed in mice heterozygous for this allele. The development of the knuckles is from proximal to distal, thereby providing information about progression. By whole-body embryo analysis of Lgr5-GFP mice, we detected GFP in the knuckles from E13.5 to E18.5 (FIG. 1 a). At E13.5, the proximal M/P1 joint was clearly GFP positive, whereas the P1/P2 joint showed only weak signal and the P2/P3 joint showed no signal (FIG. 1a), a result confirmed by histological analysis (FIG. 1 b). At the M/P1 joint in finger III, the signal was detected in E13.5 as scattered in the compartment cells in a "salt and pepper" distribution pattern, starting from E14.5, with a more intense and uniform distribution in the centre of the compartment. As the joint cavity is formed, Lgr5+ cells are detected in the marginal areas of the future articular cartilage and the strength and number of the expressed cells are significantly reduced.

Example 2: lgr5 expression begins after Gdf5 expression in knuckle formation

Gdf5 is a marker for the compartment cells (Merino et al, 1999; Storm and Kingsley, 1999). We compared the expression of Lgr5 with Gdf5 in adjacent sections in finger III (FIGS. 1c-1 f). The last joint formed in the P2/3 compartment, i.e.in E14.5, had been expressed with Gdf5 (FIG. 1c), but had not expressed Lgr5 (FIG. 1c), indicating that the latter was expressed later. In the more proximal P1/P2 and M/P1 joints, both Gdf5 and Lgr5 are expressed. Interestingly, expression of Lgr5 was localized to the central, peripheral position of the interregional cells expressing GDF5 in each joint (fig. 1 d). At E16.5, just prior to the formation of the joint cavity, Gdf5 was expressed continuously in the interzone region and presented a distinct horseshoe-shaped distribution (fig. 1E) with Lgr5+ cells located in the center of the horseshoe (fig. 1f, circle) flanked by distinct Gdf5+ cells. As the joint cavity is formed, the number of Lgr5+ cells decreases, while some Gdf5+ cells remain on the joint surface (fig. 1 f). Thus, Lgr5 marks a subpopulation of cells in the compartment that express Gdf5, with a unique temporal and spatial pattern in joint development.

Example 3: expression of Lgr5 in developing knee joints

The knee joint is more complicated by its extra structure with menisci and cruciate ligaments. Specific Lgr5 expression was seen in whole embryo imaging of E16.5 (FIG. 1 g). We examined tissue sections from the peripheral (fig. 1i) and central (fig. 1j) regions of the developing joint from E13.5 to E18.5, as shown in fig. 1 h. Lgr5 was expressed in the interzone as early as E13.5, earlier than the formation of meniscus, articular cartilage and cruciate ligaments. From E16.5, with early joint cavity formation and meniscus and cruciate ligament formation, to E18.5 joint development maturity, expression of Lgr5 becomes localized to the future articular surface of the knee joint and attenuated (FIG. 1 i; lateral section). However, at this stage, many Lgr5+ cells became evident in the developing lateral and medial menisci (fig. 1i, lateral section). These Lgr5+ cells are located at the central free rim and thinner regions of the meniscus. Postnatally, Lgr5 expression decreased and was barely or undetectable in articular cartilage or meniscal cells on day 10 (P10) (fig. 12 c). The formation of the cruciate ligament also begins in the interzone. Strong Lgr5 expression was detected in the cruciate ligament (fig. 1j, central section), extending from ligament origin to cartilage element full length (fig. 1 j).

Example 4: lgr5+ compartment cells are progenitor cells of the internal structure of the knee joint

We used Lgr5-eGFP-IRES-CreERT2 mice to label and track Lgr5+ cells in developing joints. A single injection of tamoxifen into Lgr5-GFP at E13.5; rosa26-LacZ (R26R) pregnant mice, showing galactosidase-labeled (LacZ +) cells in the interphalangeal regions of the finger (fig. 2a) and knee (fig. 2b) joints at E15.5. At E17.5, progeny of Lgr5+ cells persist through the formation of joint cavities (fig. 2c), and at P21, the knuckle shows Lgr5+ progeny throughout the entire thickness of the articular cartilage (fig. 2E). In the developing knee, progeny of Lgr5+ cells could be detected at E17.5 near the articular cartilage surface (fig. 2d, blue arrow), meniscus (fig. 2d, red arrow), intra-patellar fat pad (fig. 2d, green arrow) and developing cruciate ligament (fig. 2d, yellow arrow). They can also be detected when tracing to P21 (fig. 2 f). Interestingly, there were much more progeny of Lgr5+ cells detected in the ligament than expected from the restricted compartment cells marked with E15.5, indicating that there was cell proliferation in this lineage. Therefore, Lgr5+ cells in the ligament originate from Lgr5+ compartment cells, which persist to P21 (fig. 2f), and can be localized in the synovium (fig. 2f, orange arrows). In summary, our findings support that Lgr5+ compartment cells are progenitor cells contributing to all joint structures.

Example 5: marker Col22a1 of articular chondrocyte lineage

To better understand Lgr5+ cells, we performed transcriptome analysis using RNA-seq. We used fluorescence-activated cell sorting (FACS) to isolate three cell types: sox9-GFP + cells from Sox9IRES-eGFP/+ (Sox9-GFP) embryos at E13.5 aggregating mesenchymal metacarpal cartilage mesenchymal leaf primordia, Lgr5+ (GFP +) cells from the developing interarticular area of E14.5, and Lgr5- (GFP-) cells from the surrounding tissue of the interregional area of the Lgr5GFP/+ embryos (FIGS. 11a, b and c). By setting the positive judgment value (cut-off) to FPKM.gtoreq.5, about 8,000 genes were identified in each pool: of these 7,356 genes were common to all three datasets, some were common to both datasets, and each dataset contained uniquely expressed genes (fig. 11 e). The most commonly expressed Lgr5+ bulk dataset-specific genes include transcription factors (Glis1, Barx2 and Pknox2) and ECM proteins (Cilp and Col22a 1). Several transcription factors associated with joint formation were reported to be specifically or more strongly expressed in Lgr5+ cells from finger joints, such as Gata3(Singh et al, 2018), Barx1/2(Makarenkova and Meech,2012), Irx1/2(Zulch et al, 2001) and Sox5/6(Dy et al, 2010) (fig. 11e, 15, 16 and 17). Our Lgr5+ dataset also showed abundant known pathways regulating interregional differentiation, such as WNT, TGF and MAPK (Decker et al, 2014; Gunnell et al, 2010) (FIGS. 11f and 18). Our data are consistent with the literature descriptions of the cells in the interphase, confirming the quality of our data set.

Next, we looked for new information and potential markers of progression to the articular chondrocyte lineage. ECM environmental analysis of Lgr5+ cells can provide clues to lineage progression that accompany changes in the cellular microenvironment (cell niche). Cilp and Col22a1 are the most differentially expressed ECM genes in Lgr5+ cells (fig. 3a), with Col22a1 being cell-specifically expressed in Lgr5+ (fig. 11 e). The ECM-associated gene expressed in Lgr5+ cells indicates that it is a chondroprogenitor phenotype that has not expressed Comp or Prg4 (fig. 3 a). Thus, Cilp and Col22a1 are potential markers of lineage progression of ECM cells. We focused on Col22a1(ColXXII) because it is located at the tissue junction and is detected on the surface of the mature synovial joint (Koch et al, 2004). To compare Lgr5 and Col22a1 expression during joint development, we used in situ hybridization on E14.5 to P0 fingers and Col22a1 in the knee joint (fig. 3b and 3c), and immunostaining on ColXXII (fig. 3d and 3E) and GFP (Lgr5) proteins therein. In the E14.5P 1/P2 knuckle, Col22a1/ColXXII expression in the Lgr5+ compartment region was limited (FIGS. 3b and 3 d); however, higher levels of ColXXII were observed in the more mature M/P1 joint (fig. 3 d). This indicates that Col22a1 expression begins later than Lgr 5. Prior to joint cavity formation, many cells co-expressed Lgr5 and ColXXII (fig. 3 d). With joint cavity formation (E16.5), there were fewer Lgr5+ cells and more Lgr 5-cells expressing ColXXII (FIG. 3d, E16.5). Taking the P1/P2 joint of E16.5 as an example, we clearly identified cells expressing Lgr5 but not Col22a1 (FIG. 3d, open arrows), double positive cells (FIG. 3d, closed arrows) and many cells expressing only ColXXII (FIG. 3d, closed arrows). Since ColXXII is an ECM protein, we defined cells that are GFP + (intracellular) and have staining for ColXXII around the cell as double positive cells. This was supported by direct co-localization of Col22a1mRNA in Lgr5 expressing cells (fig. 3 b). By E18.5, there were only a few Lgr5+ cells, which were likely double positive for ColXXII because they were embedded in the ColXXII-rich ECM layer (fig. 12 a). At this stage, cells expressing ColXXII marked the surface of the future articular cartilage. By birth (P0), the thin layer of the colexiii-containing ECM became clear and the proximal/distal expression difference was no longer evident (fig. 12 b). Similarly, at P10 (fig. 12c), Lgr5+ cells were no longer detected along the entire surface of the knuckle.

In the knee, a similar expression relationship was observed (fig. 3c and 3 e). Lgr5 is expressed at E13.5 (fig. 3E) and E14.5 (fig. 3c and 3E) because of the aggregation of the compartment cells to form articular cartilage and meniscus. Lgr5+ cells at E13.5 first labeled the structure of the future meniscus, and then cells expressing ColXXII from E15.5 to E18.5 were aggregated along the surface layer (fig. 3E). Differentiation of meniscal superficial cells is also likely to involve a transition from Lgr5 to cells expressing ColXXII. Indeed, Lgr5/ColXXII double positive cells were detected at the border by immunostaining at E15.5 (fig. 3E) and in situ hybridization at E17.5 (fig. 3 c). By E18.5 (FIGS. 3c and 3E), a distinct ColXXII-containing stromal layer with some Lgr5+ cells appeared deep in the meniscus. Similar to the knuckles, Lgr5+ cells were no longer detected at P10, but the ColXXII layer persisted (FIG. 12 c). At E17.5, we detected Lgr5 progeny cells labeled at E13.5 (LacZ +) in the superficial layer containing ColXXII (fig. 3f), with Col22a1 transcript also detected (fig. 3g), indicating that some cells in this layer are from Lgr5+ spacer cells. Furthermore, cells embedded in this layer interacted with the ECM, which was shown by aggregation of β 1-integrin to form focal adhesions (fig. 12 d).

Since Lgr5 is also expressed in developing ligaments, we analyzed its expression/localization relationship between E15.5 and ColXXII when cruciate ligament formation began (fig. 3h and 3 i). Interestingly, although Lgr5+ cells were detectable throughout the central region of the interzone, ColXXII expression was localized in the flanking region outside the developing ligament (fig. 3 h). Furthermore, analysis of ColXXII expression in Scx-GFP mice showed that significant expression of Scx (GFP) was restricted to developing ligaments and not in the flanking ColXXII positive regions of the intergenic region (fig. 3 i). This supports lineage differentiation of Lgr5+ progenitor cells into ligament cells and articular chondrocytes (with mutually different expression patterns), and the unique ECM microenvironment of these two lineages exists. The microenvironment containing ColXXII will support the formation of articular cartilage and meniscus.

Example 6: differential labelling of Lgr5+ compartment cells against chondrocyte or ligament lineages

To investigate the relationship of different cell populations in the developing knee joint, we performed a single cell RNA-seq analysis of cells from the interknee region of E14.5 Lgr5GFP/+ embryos. After the blood cells were depleted, 5,460 spacer and surrounding cells were sequenced (FIG. 19). T-distributed stochastic neighbor embedding (tSNE) analysis divides cells with similar expression profiles into six clusters. When the t-SNE profile (2D-tSNE) was observed in two dimensions, clusters 4 and 5 appeared to be distant from the major clusters (1, 2, 3, 6) (FIG. 4 a). The 3D view (3D-tSNE) shows a horseshoe shape with cluster 1 and cluster 5 at the tip of the horseshoe shape (fig. 4 b). The marker Gdf5 for all the cells of the compartment was expressed in cells scattered in six clusters (fig. 4c), but appeared to be concentrated in clusters 1, 2, 5 and 6 (fig. 4 e). A total of 207 cells expressing Lgr5 were identified, with cells expressing endogenous Lgr5 (n-94), Lgr5-eGFP-CreERT2 allele (n-87), or both (n-26). Interestingly, Lgr5 expression was concentrated in cluster 1 (64% of all Lgr5+ cells) and cluster 5 (13%) at the top of the horseshoe (fig. 4D) and is well shown in the 2D-tSNE heatmap (fig. 4 f). Other Lgr5+ cells were scattered throughout the cluster map (fig. 4 d).

Next, we evaluated the relationship between Lgr5+ and Gdf5+ cells. According to our in vivo analysis at E14.5, Lgr5+ cells were expected to be Gdf5+ (fig. 1c and 1 d). We observed Gdf5+/Lgr 5-and Gdf5+/Lgr5+ cells, but also Gdf5-/Lgr5+ cells (FIGS. 4g and 4 h). Analysis of Differentially Expressed Genes (DEG) in Lgr5+/Gdf 5-and Lgr5+/Gdf5+ cells revealed that the only difference was the absence or presence of Gdf5 expression (FIG. 13 a). Thus, the two cell populations are identical, and the difference may result from a "drop" event in scRNAseq (khachenko et al, 2014) by Gdf5 when expression of Gdf5 is low in some Lgr5+ cells. Similar DEG analysis of Lgr5+ cell clusters 1 and 5 showed that the key difference was the additional expression in cluster 5 of a gene that maps to GO terms corresponding to cell cycle events (fig. 13b and c). We selected Ki67 as one of the most differentially expressed cell cycle genes and mapped its expression in a 2D-tSNE profile. A total of 14% of Lgr5+ cells expressed Ki67 in all clusters; most of them are in cluster 5, but not in cluster 1 (fig. 4 i). Similarly, we found that 12.2% ± 2.1% (n ═ 3) of Lgr5+ cells in the E14.5 interknee region expressed Ki 67; these cells were located in the periphery of the Lgr5+ compartment region (fig. 4 j).

Since most of the Lgr5+ cells were in cluster 1, we performed DEG analysis on cluster 1 relative to the other five clusters. We identified 110 DEG (fig. 4k and 20) including transcription factors associated with joint development, such as Osr2, Trps1, Barx1 and Sox 4. When we improved the differential screening criteria for Lgr5+ cells in cluster 1 compared to Lgr 5-cells in the entire population, we identified 75 DEG (fig. 20), 48 of which overlapped the previous gene set, including transcription factor Mkx and Scx. 27 genes were specifically expressed in Lgr5+ cells, including the transcription factor Meox2 (FIG. 4 k). Taken together, these analyses described the characteristics of Lgr5+ cells in the intergenic region for the chondrocyte and ligament lineages.

Example 7: trajectory of articular pedigree specialization

To annotate these clusters, the first 5% (whole genome of all 5,460 cells) of the dispersed genes were clustered into one heatmap. Gene modules associated with the intergenic region, ligament, cell cycle and cartilage were identified (fig. 5 a). As expected from the 2D-tSNE heatmap of Gdf5 expression (fig. 4e), the cells in clusters 1, 2, 5, and 6 expressed genes associated with the interzone cells, including Gdf5, Osr2, Sfrp2, Sulf1, and Sox 4. Cells in clusters 5 and 6 expressed higher levels of ligament-associated genes, such as Lox, Col1a1, Aspn, and Scx, relative to other clusters. The main difference between cluster 5 and cluster 6 is the high abundance of cell cycle associated genes in cluster 5, such as Ki67, Ccna2, Birc5 and Top2a, which are also expressed in cluster 4. Cells in clusters 2, 3 and 4 express cartilage related genes such as Epyc, Matn1, Acan, Lect1 and Sox 9. When we analyzed the same data set using principal component analysis, we found that clusters of interregional and non-interregional cells were similar, and the cell cycle status of the cells in these clusters was similar (FIG. 13 d-g). The expression profile in each cluster was consistent with a violin map of representative genes of cartilage and ligaments and cell cycle genes (fig. 13 h). Thus, clusters 1, 2, 5 and 6 are considered "inter-zone clusters", while 3 and 4 are defined as "non-inter-zone clusters" (fig. 5 b). Cluster 1 is rich in Lgr5+/Gdf5+ internode cells and maps between clusters with ligament (cluster 6) and chondrocyte (cluster 2) tags, with some cell "mixing" at the boundary, indicating potential differentiation of the internode cells of cluster 1 into chondrocyte or ligament lineages (fig. 5 b).

To study cell lineage differentiation, we performed a quasi-timeline analysis of the compartment clusters (1, 2, 5 and 6), mapping to eight potential cell states (fig. 5 c). The cells in cluster 1 map primarily in state I, with some cells interspersed in other states. Cells in ligament-tagged clusters 5 and 6 were distributed in states I, II and III, while cells in chondrocyte-tagged cluster 2 were predominantly distributed in cell states V, VI, VII and VIII (fig. 5 c). State IV mixed cells from all four clusters. Next, we evaluated the distribution of Lgr5+/Ki76+ cells, finding that they are distributed within Lgr5+/Ki 67-cells, but most of the Lgr5+ cells map to state I (fig. 5d), which represents the most primitive/young state predicted in the pseudo-timeline (fig. 5 e). Two main traces branching from state I: trace A consists of cells in states II/III (FIG. 5e, green arrows) and trace B includes cells in states V/VI/VII/VIII (FIG. 5e, red arrows).

To characterize the trajectory, we evaluated the expression of the identified differentially expressed transcription factors (fig. 4 k). Scx, Meox2 and Mkx were expressed in ligament cells, and their expression was enriched in cells of clusters 1 and 6 (fig. 5 f; green oval), but less in cells of cluster 2 (fig. 5f, pink oval). Osr2 and Barx1 were expressed in cartilage development, and their expression was enriched in clusters 1 and 2, but less in cluster 6 (FIG. 5 g). Sox9 is a key chondrogenic transcription factor whose expression does not differ in cluster 1 or in Lgr5+ cells: overall, its expression was upregulated in cluster 2, but not in cluster 6 (fig. 5 g). This supports the "bipotency" differentiation status of the cells of cluster 1, which can differentiate towards the ligament or articular cartilage lineage (fig. 5 h). Next, we mapped the transcription factors and ECM genes specific for ligament (fig. 5i) and cartilage (fig. 5j) onto pseudo-temporal lines, finding that they are enriched in the cellular state of the relevant tissue tracks supported (fig. 5 e). Based on the algorithm, some cells express both ligament and cartilage transcription factors prior to lineage differentiation. For example, cells from cluster 6 were distributed at the end of state I, expressing both Meox2 (blue circle in fig. 5I, left panel) and Osr2 (blue circle in fig. 5j, left panel). As support for the in vivo data (fig. 3h), the cells of state II of track a do not express Col22a1, but do express the mature ligament marker Tnmd (fig. 5i and 5j, blue boxes). Col22a1 was expressed in the cells of track B, which supports our hypothesis that Col22a1 indicates that the chondrocyte lineage is distinct from the ligament lineage. Thus, our single-cell transcriptome data together with our lineage tracing experiments showed that the Lgr5+ compartment cells are likely to be pluripotent and represent a stage of joint formation at which the lineage divides into ligament and articular chondrocytes (fig. 5 h).

Example 8: lgr5+ interzone tissue repair of cartilage defects

Cells in the Lgr5+ compartment have "primitive" and multipotency properties and are therefore suitable for cartilage repair. To test this ability, we dissected the GFP from E13.5 Lgr 5; lgr5-GFP + interzone tissues of ROSA-tdTomato embryos were transplanted to full-thickness needle defects (n-3) at the trochlear groove of the knee of 8-week-old mice (FIGS. 6a and 14). At 15 days post-acupuncture, tdTomato + cells were detected at the defect, with round chondrocytes in differentiation in the middle and deep regions of articular cartilage (fig. 6b and 6c, arrows), and flattened cells in differentiation in the superficial region (fig. 6b and 6c, arrows). The healed defect comprises cells derived from the transplanted tissue, which produces a proteoglycan-rich matrix (fig. 6c) and collagen II deposition (fig. 6 d). The new tissue binds well to the host cartilage (fig. 6c, box) where the new superficial layer containing collagen XXII and CILP1 is embedded in the superficial region of the host articular cartilage (fig. 6d and 6 e). The implanted tissue inhibited the expression of collagen I (fig. 6g) at the defect, which could lead to unwanted fibrosis during cartilage repair. In summary, our findings indicate that cells in the Lgr5+ compartment have the ability to repair articular cartilage.

2. Discussion of the related Art

Various tissues of the synovial joint are derived from mesenchymal cells of the developing interzone, most or all of which express Gdf5(Koyama et al, 2008). For Gdf5-CreER^T2Follow-up studies in mice have shown that after the primary de-differentiation of chondroblasts (chondrogenic cells) into cells of the Gdf5+ compartment, further expansion of the Gdf5+ compartment is primarily through recruitment of regional mesenchymal cells rather than proliferation of cells of the Gdf5+ compartment (Shwartz et al, 2016). Furthermore, Shwartz et al (2016) suggested that cells in the compartment respond to signals from the surrounding environment and that they produce different joint tissuesIs controlled by the position effect. Here we identified a new subset of Gdf 5-expressing interregional cells, differentiated by co-expressing Lgr5, Lgr5 being a known marker of highly proliferating stem cells (Leung et al, 2018), although the cells within the interregional are rarely dividing (shuwartz et al, 2016). Notably, this subpopulation of Lgr 5-expressing cells in the Gdf5+ population can also produce a variety of joint tissues including synovium, cruciate ligament, meniscus, and articular cartilage.

Lgr5+ compartment cells contribute to the formation of different joint structures

The Lgr5+ cell pool appears shortly after the initial appearance of Gdf5 expressing cells, which mark future joint sites. Therefore, Lgr5+ cells are likely to be derived from Gdf5+ cells that have undergone de-differentiation, consistent with the results of cell fate mapping using Gdf5-Cre mice (Koyama et al, 2008), and progressive differentiation from Gdf5+ to Lgr5+ cells is a continuous event. In the knuckle, Gdf5+ cells, proposed in a continuous inflow model (Shwartz et al, 2016), were considered peripheral cells of the central Lgr5+ cell pool, as Lgr5+ cells appeared and remained in the center of the interzone. Our single cell transcriptome showed that the expansion of the Lgr5+ pool is partly through differentiation and proliferation of cells in the peripheral intergenic region, manifested by the appearance of Lgr5+/Ki67+ cells in developing joints, and that there was a difference in the cell clusters shown in the tSNE map only in the Lgr5+ cell population expressing active cell cycle related genes. In more complex interknee regions, other mechanisms may exist.

In our mapping study, activation of LacZ at E13.5 labeled this early Lgr5+ cell pool, and we observed that they contributed to all structures of the joint. The labeling of only a few cells expressing Lgr5+ by a single tamoxifen injection allowed us to assess the level of contribution to different structures of the joint. In the developing knee joint, there was no observable difference in the contribution of Lgr5+ cells to articular cartilage, meniscus, and synovium. Interestingly, more daughter cells were found in the cruciate ligament, indicating that they had proliferated. Furthermore, Lgr5 is expressed continuously in cells of the developing ligament, consistent with expansion of Lgr5+ cells marked by the E13.5 compartment, with a large number of progeny cells in the ligament of E17.5. This is supported by the identification of ligament cell clusters with active cell cycle genes in the tSNE map from the single cell transcriptome data of the E14.5 interregion.

Lgr5+ cells are pre-progenitor cells at the onset of lineage differentiation

Consistent with in vivo analysis, transcriptomics data from both bulk and single cell analyses supported Lgr5+ cells as progenitor cells, ready for differentiation to the articular chondrocyte or ligament lineages. Using clusters representing the compartment cells to generate a pseudo-timeline, we identified eight cell states, putting together cluster 1 with most of the Lgr5+ cells as the most "initial" state I of the "prepared" progenitor cells (fig. 5 d). Many Lgr5+ cells in state I express both ligament and chondrocyte gene signatures, and then specify more distinct signatures of the ligament or chondrocyte lineage. Changes in the balance of lineage specific transcription factors can control this differentiation.

Interestingly, about 10% of Lgr5+ cells were found to be in state V, which is predicted to be derived from state I, and the reason for this is not yet clear. These cells may be due to cells recruited in different ways in joint development (Shwartz et al, 2016), without excluding this. There are multiple cellular states in each ligament or chondrocyte trajectory, indicating that there are subsets of cells in each lineage, which may be related to their location along the cruciate ligament, or to the differences between chondrocyte locations in the joint or meniscal regions. For example, the multipotent Scx +/Sox9+ progenitor cell pool at the cartilage-tendon/ligament junction produces tenocytes/ligament cells and chondrocytes (Sugimoto et al, 2013). Based on what we have now found is shown in the model created, we propose that positional information for these cells may exist within the developing knee joint (fig. 7).

Role of Lgr5 and Wnt signaling in joint development

Although Lgr5-GFP homozygous mice died perinatally (Barker et al, 2007), they showed no histological abnormalities in the joints (fig. 10). This is probably due to functional redundancy between the three LGR proteins (LGR4, 5 and 6) (Ruffner et al, 2012). There was expression in our transcriptome dataset for Lgr4, but not Lgr 6. Expression of Lgr5 likely reflects the positional effect of appropriate levels of Wnt ligands on their activation, and the enhancement of Wnt signaling by Lgr5 in turn maintains higher signaling levels through interaction with R-spondin (de Lau et al, 2011), which is necessary to progress to the next stage, in preparation for the formation of other structures and joint cavity formation.

Col22a1 markers of the articular cartilage lineage from cells expressing the interregion of Lgr5

As articular cavity formation Lgr5 expression is reduced in early articular surface cells, this is consistent with the need to reduce Wnt signaling that inhibits chondrogenesis for articular chondrocyte differentiation. Little is known about the molecular control of this lineage. Cartilage-forming BMP signaling plays an important role (roundree et al, 2004), and supporting the notion that continued expression of Gdf5 and decreased Lgr5 predisposed the balance to cartilage formation, we identified Col22a1 as an intermediate marker for these lineages, and that cells co-expressed Col22a1 and Lgr 5. Col22a1/ColXXII expression starts proximally and then lies near the edge of the future articular surface in the middle region of development. This is best illustrated in the less complex finger joints and is consistent with the proposed model, i.e. the developing intermediate zone is divided into three layers, the two outer layers containing cells that eventually become articular chondrocytes, the cells in the intermediate layer contributing to other structures or undergoing apoptosis (mitroviic, 1977). Shortly before joint cavity formation, the layers of cells expressing ColXXII in the intergenic zone are widespread. The overlap between the region expressing ColXXII and Lgr5+ cells at the earliest stages of joint cavity formation indicates that most of the cells expressing Col22a1 are derived from Lgr5+ cells. Interestingly, the Lgr5+/Col22a1+ co-expressing cells were located primarily in the medial region of the articular surface throughout the formation of the joint cavity, and the Col22a1+/Lgr 5-cells occupied the lateral wing regions of the entire articular surface. If cells expressing Col22a1 are derived from Lgr5+ cells, expansion of the articular surface is mediated by cellular differentiation at the midpoint, which promotes lateral growth on the articular surface. In fact, this may have reflected the various cell states determined in the chondrocyte trajectories predicted from the pseudo-timeline of the single-cell transcriptome (fig. 5d and e).

We propose a model in which cells express Gdf5, progressing to Gdf5/Lgr5 double positive progenitor cells, then Lgr5/Col22a1 double positive committed articular cartilage progenitor cells, and finally early articular cartilage cells expressing Col22a1 (fig. 7). The continuous influx model suggests that recruitment causes expansion of the cell pool between Gdf5+ (Shwartz et al, 2016). We propose that the spatial effects of multiple committed progenitor pools determine the location of the joint structure. Single cell transcriptomes with more cells from additional developmental time points will provide key markers for validation and further implications on molecular control and cell status subdivision.

Cartilage repair capacity of Lgr5+ cells: reconstitution of collagen-containing XXII superficial layer

The superficial region contains cells (Kozhemyakina et al, 2015) and progenitor cells (Dowthwaite et al, 2004) that produce lubricin/Prg 4 for joint lubrication. Recent cell-tracking studies have identified self-renewing progenitors in the superficial regions of mouse articular cartilage that may undergo symmetric and asymmetric expansion in early joints (Decker et al, 2017; Li et al, 2017). Our analysis results show that ColXXII defines the outermost surface of the joint. Cells embedded in the ColXXII-containing layer have a pronounced flat morphology and show direct interaction with the ECM by clustering with β 1 integrin receptors to form local adhesive plaques. These cells may provide a special microenvironment for cell maintenance and serve as a source of progenitor cells. Finally, we found that Lgr5+ cells collected from the knee joint of the E13.5 embryo can repair full thickness articular cartilage defects. Importantly, the collagen XXII-containing superficial layer is reconstituted at the healing defect and rapidly integrates with host cartilage, suggesting that these Lgr5+ cells are candidates for cell therapy for cartilage and ligament trauma or related degenerative diseases.

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The foregoing description of the specific embodiments reveals the general nature of the disclosure sufficiently that others can, by applying knowledge within the skill of the relevant art (including the contents of the references cited herein and the contents of the documents incorporated by reference), readily modify and/or adapt for various applications such specific embodiments, without undue experimentation, and without departing from the general concept of the present disclosure. Therefore, such adaptations and modifications are intended to be within the meaning and range of equivalents of the disclosed embodiments, based on the teaching and guidance presented herein. It is to be understood that the phraseology or terminology herein is for the purpose of description and not of limitation, such that the terminology or phraseology of the present specification is to be interpreted by the skilled artisan in light of the teachings and guidance presented herein, in combination with the knowledge of one of ordinary skill in the relevant art.

While various embodiments of the present disclosure have been described above, it should be understood that they have been presented by way of example only, and not limitation. It will be apparent to persons skilled in the relevant art that various changes in form and detail can be made therein without departing from the spirit and scope of the disclosure. Thus, the present disclosure should not be limited by any of the above-described exemplary embodiments, but should be defined only in accordance with the following claims and their equivalents.

Claims (16)

1. A method of treating joint disease comprising administering multipotent Joint Progenitor Cells (JPCs) at a joint of a subject in need thereof, wherein the JPCs obtained from the intermediate zone are Lgr5⁺And/or Col22a1⁺。

2. The method of claim 1, wherein the joint disease is ligament and articular cartilage trauma or associated degenerative disease.

3. The method of claim 1, wherein the joint disease is associated with osteoarthritis or damage associated with joint tissue.

4. The method of claim 1, wherein said JPC differentiates into tenocytes, cruciate ligaments, synovium, meniscus and/or articular chondrocytes.

5. A method of treating a ligament or tendon defect comprising administering a pluripotent Joint Progenitor Cell (JPC) at a joint of a subject in need thereof, wherein the JPC obtained from the interregion expresses Mkx and Scx.

6. A method of treating an articular chondrocyte or meniscus defect comprising administering pluripotent Joint Progenitor Cells (JPCs) at a joint of a subject in need thereof, wherein the JPCs obtained from the intercompartments express Pax9, Cdh13, Lrrc17, and Col26a 1.

7. A method for producing a cell line of Joint Progenitor Cells (JPC), comprising the steps of

(i) Providing induced pluripotent stem cells (ipscs) or expanded pluripotent stem cells (epscs);

(ii) culturing ipscs or epscs in the presence of a glycogen synthase kinase-3 β inhibitor;

(iii) (iii) activating TGF- β signalling from the ipscs or epscs of step (ii);

(iv) (iv) culturing the ipscs or epscs from step (iii) in the presence of Fgf2 and Mmp2, wherein the culturing from (ii) to (iv) steps is for about 5 days to form mesendoderm;

(v) (iii) removing the glycogen synthase kinase-3 β inhibitor of step (ii) from the iPSC or ePSC culture;

(vi) inhibiting TGF- β signaling, wherein the ipscs or epscs are cultured for about 4 days to form mesoderm;

(vii) differentiating the mesoderm into JPC in the iPSC or ePSC culture;

(viii) activating Wnt signaling for about 5 days; and

(ix) modulating the Hedgehog (HH) concentration in the iPSC or ePSC culture.

8. The method of claim 7, wherein the glycogen synthase kinase-3 β inhibitor is CHIR 99021.

9. The method of claim 7, wherein the TGF- β signaling is activated by activin a.

10. The method of claim 7, wherein the TGF- β signaling is inhibited by Fgf2, Bmp2, and Sb 431542.

11. The method of claim 7, wherein the mesoderm differentiates into JPCs in the presence of Fgf2 and Gdf 5.

12. The method of claim 7, wherein said Wnt signaling is modulated by the addition of R-spondin.

13. The method of claim 7, wherein the iPSC or ePSC comprises an LGR5-GFP reporter construct capable of expressing GFP in the iPSC or ePSC.

14. The method of claim 13, further comprising the step of monitoring GFP expression in the ipscs or epscs.

15. A population of JPCs produced by the method of any one of claims 7 to 14.

16. A method of treating joint disease comprising administering a population of JPCs of claim 15.

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