Markers for skeletal stem cells and uses thereof

Abstract

The present application relates to stem cell biology and regenerative medicine. Disclosed herein are methods for the isolation of skeletal stem cells, related methods, related compositions, related products, and related uses.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Application No. 63 / 311,692, filed February 18, 2022. The contents of this application are incorporated herein by reference in their entirety.

[0002] Government Interests This invention was made with Government support under DE015654 and DE026936 awarded by the National Institutes of Health. The Government has certain rights in this invention.

[0003] The present invention relates to stem cell biology and regenerative medicine. [Background technology]

[0004] Skeletal stem cells (SSCs) are a type of self-renewing, multipotent, and skeletal lineage-committed progenitor cell that can give rise to cartilage, bone, and bone marrow stroma, including bone marrow adipocytes and stromal cells. With the trilineage potential to differentiate into chondrocytes, osteoblasts, bone marrow stromal cells, or adipocytes, SSCs play a key role in bone tissue development, homeostasis, and regeneration. For example, SSCs derived from suture mesenchyme, termed suture stem cells (SuSCs), exhibit long-term self-renewal, clonal expansion, and multipotency. These SuSCs reside at the suture midline and function as a skeletal stem cell population responsible for calvarial development, homeostasis, injury repair, and regeneration. However, the in vivo identity of SuSCs and SSCs remains largely elusive. There is a need for cell markers, such as cell surface markers, and related cell identification and isolation methods. Summary of the Invention

[0005] The present application addresses the above needs in several aspects.

[0006] In one aspect, the present application provides a method for identifying or isolating or enriching skeletal stem cells. The method includes obtaining a starting cell population and identifying from the starting cell population a first marker expressed by, on or in the cells, the first marker being selected from the group consisting of those listed in Tables 1 and 2. In some embodiments, the first marker is selected from the group consisting of those listed in Table 1. In some embodiments, the first marker is selected from the group consisting of Bmpr1a, Bmpr2, Fdz1, Fgfr2, Lrig3, Itgbl1, Apoe, Gpc3, Lpl, Sulf2, Cdon, Tgfbr2, Tgfbr3, Lrrc15, Mif, Axl, Itgav, Fgfr1, Jag1, Acvr1, Acvr2a, Acvr2b, Bmpr2, Erg, Six2, Pthlh, Twist1, Alpl, Msx1, Efnb1, Zic1, Spry1, Abcc9, Erf, Bmp2, Bmp3, Bmp4, Bmp5, Bmp6, Bmp7, Bmp8a, Bambi, Bmper, and Col3a1. In some embodiments, the first marker is selected from one, two, three, or four of Group 1, Group 2, Group 3, and / or Group 4 disclosed herein.

[0007] In one embodiment, the method further comprises isolating a cell expressing the first marker, hi one embodiment, the method further comprises collecting a plurality of cells expressing the first marker to obtain an enriched skeletal stem cell population.

[0008] In some embodiments, the cell(s) can be identified, isolated, or enriched using a first agent (such as a first protein, a first polypeptide, a first nucleic acid, or a first composition) that specifically binds to a first marker.

[0009] In some embodiments, the method may further comprise identifying, from the starting cell population, a second marker expressed by, on, or in the cells expressing the first marker. The second marker is different from the first marker. The second marker may be a marker selected from the group above. The cells may then be identified, isolated, or enriched using a second agent (e.g., a second protein, a second polypeptide, a second nucleic acid, or a second composition) that specifically binds to the second marker. In some examples, the method may further comprise collecting a plurality of cells expressing the first marker and the second marker to obtain an enriched skeletal stem cell population. In other examples, the method may further comprise identifying, from the starting cell population, one or more additional markers expressed on or in the cells that co-express both the first marker and the second marker. Each of the one or more additional markers may be identified individually using the same method as the first marker and the second marker. Thus, the method may result in cells that co-express the first, second, and one or more additional markers. In one embodiment, the method may further include collecting the first marker, the second marker, and a plurality of cells expressing the one or more markers to obtain an enriched skeletal stem cell population.

[0010] In each of the above methods, the starting cell population can be derived from the tissue of a subject, such as a vertebrate, a mammal (including human and non-human mammal). The starting cell population can include one or more selected from the group consisting of bone marrow, umbilical cord blood cells, embryonic stem cells or their progeny, mesenchymal stem cells or their progeny, and induced pluripotent stem cells (iPSCs) or their progeny. In a preferred embodiment, the mesenchymal stem cells are suture mesenchymal stem cells.

[0011] The present application further provides a composition comprising (i) a carrier and (ii) one or more cells identified, isolated or enriched according to the above method, or cells derived therefrom. The composition may be a pharmaceutical composition, in which the carrier is a pharma- ceutically acceptable carrier. The composition may be an in vitro cell culture composition, in which the carrier may comprise a culture medium or a maintenance medium. The present application also provides a bone regeneration product or formulation comprising (i) the above composition and (ii) a scaffold.

[0012] Also provided is a method for generating or regenerating cartilage or bone in a subject, the method comprising administering to a subject in need thereof an effective amount of the composition described above or the bone regeneration product or formulation described above to a site where bone or cartilage regeneration is desired.

[0013] Further provided is a method of generating skeletal, stromal or cartilage tissue, comprising (i) providing one or more cells obtained according to the method described above, and (ii) inducing differentiation of the one or more cells or cells derived therefrom, wherein the one or more cells may express a second distinct marker selected from the group described above.

[0014] The details of one or more embodiments of the application are set forth in the specification below. Other features, objects, and advantages of the application will become apparent from the specification and the claims. [Brief description of the drawings]

[0015] The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.

[0016] [Figure 1A-1C]Single-cell RNA sequencing analysis of mouse calvarial sutures. Figures 1A and 1B show heatmaps (Figure 1A) and tSNE plots (Figure 1B) of the combined P10 coronal and lambdoid suture datasets showing the top five marker genes for each cell mass and the grouping of suture cells into 10 clusters (0-9), respectively. Figure 1C shows a feature plot of whole suture cells grouped into three major lineages. Runx2+ skeletal cell lineages (clusters 0, 1, 2, 4, and 9), Mcam+ vascular cell lineages (clusters 7 and 8), and Rac2+ hematopoietic cell lineages (clusters 3, 5, and 6) are depicted as dashed blue, brown, and red boxes, respectively. The color scale represents centered natural log transformation between cells. [Figures 2A-2D] Characterization of Runx2+ skeletal cell lineage in suture mesenchyme. Figure 2A shows that tSNE plots of cells from P10 coronal and lambdoid sutures show Runx2+ skeletal lineage (depicted as blue dashed box). Runx2+ lineage can be subdivided into five clusters (MC, PMC, OB, PCH, CH). Figures 2B, 2C, and 2D show the expression distribution of representative osteoblast (B), chondrocyte (C), and proliferation (D) gene markers projected onto the tSNE plots. The MC cluster shows low expression of osteoblast, chondrocyte, and proliferation genes. Proliferation marker genes classify the PMC and PCH clusters containing proliferating mesenchymal cells and chondrocytes. The color scale in (B), (C), and (D) represents the cell-centered natural log transformation. [Figure 3A-3E]Characterization of Rac2+ hematopoietic cell lineages. Color scales in (B), (C), and (D) represent cell-centered natural logarithm transformations. Figure 3A shows that tSNE plots of cells from P10 coronal and lambdoid sutures containing Rac2+ hematopoietic cell lineages, depicted as red dashed box, can be subdivided into three clusters (HC1, HC2, and HC3). Figures 3B and 3C show the expression distribution of representative monocyte / macrophage and dendritic cell genes (B) and osteoclast genes (C) projected onto the tSNE plot classifying the HC1 cluster. Figure 3D shows that the HC2 cluster represents T and B lymphocytes based on the projected distribution of lymphocyte genes. Figure 3E shows that the HC3 cluster contains NK cells and neutrophils expressing their specific genes. [Figure 4A-4B] Characterization of the Mcam+ vascular cell lineage is shown. The color scale represents the cell-centered natural logarithm transformation. Figure 4A shows that the tSNE plot of cells from P10 coronal and lambdoid sutures containing the Mcam+ vascular cell lineage, depicted as a brown dashed box, can be subdivided into two clusters (VC1 and VC2). Figure 4B shows that the expression distribution of representative endothelial cell markers projected onto the tSNE plot classifies the VC1 cluster, while the VC2 cluster contains cells expressing pericytes and vascular smooth muscle cell (SMC) markers. [Figure 5A-5H] Figure 5 shows tSNE analysis of skeletal stem and progenitor cell markers. Color scale represents cell-centered natural log transformation. Figure 5A shows tSNE and violin plot of Axin2, a marker of skeletal stem cells of the calvarial suture, showing expression in MC clusters. Figure 5B-5H show that other skeletal stem and progenitor cell genes Gli1 (B), Lepr (C), Prrx1 (D), Grem1 (E), Erg (F), Pthlh (G), and Six2 (H) also mark MC clusters. [Figure 6A-6C]Figure 6A shows the isolation of Axin2 expressing cells from calvarial sutures. Figure 6A shows cells isolated from control or Axin2GFP mice are subjected to FACS-based sorting based on the parameters used to separate cells with differential expression of GFP. Figure 6B shows post-sorting analysis demonstrating successful isolation of cells expressing high levels of GFP (population P5). Figure 6C shows a graph depicting the Axin2+ cell population, which constitutes approximately 3-7 percent of total suture cells, in three independent experiments. [Figure 7A-7C] Quality control for scRNA-seq analysis of Axin2-expressing suture mesenchymal cells. Figure 7A shows that nFeature_RNA and nCount-RNA indicate the number of genes and the total number of molecules detected in the cells, respectively, and the comparison of two independent datasets. Figure 7B shows a UMAP plot illustrating the differential distribution of Axin2-expressing cells between two independent experiments and their division into seven subpopulations. Figure 7C shows a heatmap visualizing the most diverse genes and the top five DEGs for PCs within cell clusters based on PCA scores. [Figure 8A-8C] Figure 8A shows the cell type specific identification in Axin2 expressing cells. Figure 8A shows the UMAP plot showing the distribution of 11,090 Axin2 expressing cells isolated from mouse calvarial sutures and their visualization in seven subclusters. Figure 8B shows dot plots showing the cell type specific markers enriched in each cluster. Figure 8C shows binomial and UMAP plots showing the enrichment of BMPR1A expressing cells in the SC cluster. NEUT: neutrophils, DC: dendritic cells, PHC: proliferating hematopoietic cells, NK: natural killer cells, MONO: monocytes, SC: skeletogenic cells, LYM: lymphocytes. The color scale in (B) and (C) represents the cell-centered natural log transformation. [Figure 9]Figure 1. Enriched expression of genes associated with human craniosynostosis in the SC cluster. A total of 83 genes with mutations linked to the sutural processes in humans were examined, of which 27 showed enriched expression in the SC cluster. NEUT: neutrophils, DC: dendritic cells, PHC: proliferating hematopoietic cells, NK: natural killer cells, MONO: monocytes, SC: skeletogenic cells, LYM: lymphocytes. [Figure 10A-10C] Figure 10 shows a reclustering analysis of skeletogenic cells in the Axin2+ population. Figure 10A shows a UMAP plot showing the distribution of 4,696 Axin2 expressing cells isolated from mouse calvarial sutures and their visualization in five subclusters. Figure 10B shows dot plots showing cell type specific markers enriched in each cluster. The color scale represents the cell-centered natural log transformation. Figure 10C shows plots showing intra-cluster signaling activity of the BMP pathway and associated BMP signaling molecules expressed within the SC cluster. MONO: monocytes, DC: dendritic cells, MP: macrophages, SC: skeletogenic cells, ENDO: endothelial cells. [Figure 11] Figure 1 shows enriched expression of BMP signaling molecules in SC clusters. Violin plots show distribution of genes related to BMP signaling within SC clusters. MONO: monocytes, DC: dendritic cells, MP: macrophages, SC: skeletogenic cells, ENDO: endothelial cells. [Figure 12] Enriched distribution of genes associated with human craniosynostosis to the SC cluster. A total of 83 genes with mutations linked to the sutural processes in humans were examined, of which 27 showed enriched expression in the SC cluster. MONO: monocytes, DC: dendritic cells, MP: macrophages, SC: skeletogenic cells, ENDO: endothelial cells. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0017] This application relates to stem cell biology and regenerative medicine. Certain aspects of the invention are based, at least in part, on the identification and use of one or more cell markers, e.g., surface markers, of SSCs and / or SuSCs. The ability of SSCs and / or SuSCs to engraft at injury sites and replace damaged scaffolds can be used in stem cell-based therapy.

[0018] Cells and markers Suture mesenchyme-derived skeletal stem cells (SSCs), also called suture stem cells (SuSCs), exhibit long-term self-renewal, clonal expansion, and multipotency. These SuSCs reside at the sutural midline and serve as a skeletal stem cell population responsible for calvarial development, homeostasis, injury repair, and regeneration. The ability of SuSCs to engraft at injury sites and replace damaged skeleton supports their potential use for stem cell-based therapy.

[0019] Shown below in Table 1 are examples of certain preferred SSC markers. Exemplary amino acid sequences of these makers are listed below. Additional cell surface markers are listed below in Table 2. Those in bold shown in Table 2 are also listed in Table 1. Table 1. Preferred SSC markers TIFF2025506727000002.tif132170

[0020] Group 1: cell surface marker genes expressed within the SC cluster 1. Bmpr1a Protein sequence of human BMPR1A NCBI reference sequence: NP_004320.2 (SEQ ID NO: 1) TIFF2025506727000003.tif321702.Bmpr2 (SEQ ID NO:2) TIFF2025506727000004.tif721703.Fdz1 (SEQ ID NO:3) TIFF2025506727000005.tif461704.Fgfr2 FGFR2 [Homo sapiens] Accession CAA96492 (SEQ ID NO: 4) TIFF2025506727000006.tif56170FGFR2 protein [Homo sapiens] ACCESSION AAH39243 (SEQ ID NO:5) TIFF2025506727000007.tif48170 Fibroblast growth factor receptor 2 isoform 1 precursor [Homo sapiens] ACCESSION NP_000132 (SEQ ID NO:6) TIFF2025506727000008.tif56170 Fibroblast growth factor receptor 2 isoform 3 precursor [Homo sapiens] ACCESSION NP_001138385 (SEQ ID NO: 7) TIFF2025506727000009.tif53170 Fibroblast growth factor receptor 2 isoform 4 precursor [Homo sapiens] ACCESSION NP_001138386 (SEQ ID NO: 8) TIFF2025506727000010.tif48170 Fibroblast growth factor receptor 2 isoform 5 precursor [Homo sapiens] ACCESSION NP_001138387 (SEQ ID NO: 9) TIFF2025506727000011.tif48170 Fibroblast growth factor receptor 2 isoform 6 precursor [Homo sapiens] ACCESSION NP_001138388 (SEQ ID NO: 10) TIFF2025506727000012.tif48170 Fibroblast growth factor receptor 2 isoform 7 precursor [Homo sapiens] ACCESSION NP_001138389 (SEQ ID NO: 11) TIFF2025506727000013.tif48170 Fibroblast growth factor receptor 2 isoform 8 precursor [Homo sapiens] ACCESSION NP_001138390 (SEQ ID NO: 12) TIFF2025506727000014.tif48170 Fibroblast growth factor receptor 2 isoform 9 precursor [Homo sapiens] ACCESSION NP_001138391 (SEQ ID NO: 13) TIFF2025506727000015.tif48170 Fibroblast growth factor receptor 2 isoform 11 precursor [Homo sapiens] ACCESSION NP_075418 (SEQ ID NO: 14) TIFF2025506727000016.tif53170 Fibroblast growth factor receptor 2 isoform 15 [Homo sapiens] ACCESSION NP_001307583 XP_011537812 (SEQ ID NO: 15) TIFF2025506727000017.tif41170 Fibroblast growth factor receptor 2 isoform 16 precursor [Homo sapiens] ACCESSION NP_001307587 XP_006717772 (SEQ ID NO: 16) TIFF2025506727000018.tif561705.Lrig3 (SEQ ID NO: 17) TIFF2025506727000019.tif781706.Itgbl1 (SEQ ID NO: 18) TIFF2025506727000020.tif361707.Apoe (SEQ ID NO: 19) TIFF2025506727000021.tif241708.Gpc3 (SEQ ID NO: 20) TIFF2025506727000022.tif411709.Lpl (SEQ ID NO: 21) TIFF2025506727000023.tif3417010.Sulf2 (SEQ ID NO: 22) TIFF2025506727000024.tif2117011.Cdon (SEQ ID NO: 23) TIFF2025506727000025.tif8817012.Tgfbr2 (SEQ ID NO: 24) TIFF2025506727000026.tif2417013.Tgfbr3 (SEQ ID NO: 25) TIFF2025506727000027.tif6017014.Lrrc15 (SEQ ID NO: 26) TIFF2025506727000028.tif4117015.Mif (SEQ ID NO: 27) TIFF2025506727000029.tif917016.Axl (SEQ ID NO: 28) TIFF2025506727000030.tif6017017.Itgav (SEQ ID NO: 29) TIFF2025506727000031.tif68170 Group 2 (Craniosynostosis genes) from Figures 9 and 12: 18. Fgfr1 (SEQ ID NO: 30) FGFR1 protein [Homo sapiens] ACCESSION AAH15035 TIFF2025506727000032.tif56170 Fibroblast growth factor receptor 1 isoform 1 precursor [Homo sapiens] ACCESSION NP_075598 NP_000595 (SEQ ID NO: 31) TIFF2025506727000033.tif56170 Fibroblast growth factor receptor 1 isoform 3 precursor [Homo sapiens] ACCESSION NP_001167537 (SEQ ID NO: 32) TIFF2025506727000034.tif53170 Fibroblast growth factor receptor 1 isoform 4 precursor [Homo sapiens] ACCESSION NP_075594 (SEQ ID NO: 33) TIFF2025506727000035.tif53170 Fibroblast growth factor receptor 1 isoform 5, part [Homo sapiens] ACCESSION ALQ33527 (SEQ ID NO: 34) TIFF2025506727000036.tif53170 Fibroblast growth factor receptor 1 isoform 6, part [Homo sapiens] ACCESSION ALQ33528 (SEQ ID NO: 35) TIFF2025506727000037.tif53170 Fibroblast growth factor receptor 1 isoform 10 precursor [Homo sapiens] ACCESSION NP_001167534 (SEQ ID NO: 36) TIFF2025506727000038.tif56170 Fibroblast growth factor receptor 1 isoform 11 precursor [Homo sapiens] ACCESSION NP_001167535 XP_005273506 (SEQ ID NO: 37) TIFF2025506727000039.tif56170 Fibroblast growth factor receptor 1 isoform 14 precursor [Homo sapiens] ACCESSION NP_001167538 (SEQ ID NO: 38) TIFF2025506727000040.tif60170 Fibroblast growth factor receptor 1 isoform 15 precursor [Homo sapiens] ACCESSION NP_001341296 XP_006716369 (SEQ ID NO: 39) TIFF2025506727000041.tif56170 Fibroblast growth factor receptor 1 isoform 16 precursor [Homo sapiens] ACCESSION NP_001341297 XP_016868717 (SEQ ID NO: 40) TIFF2025506727000042.tif53170 Fibroblast growth factor receptor 1 isoform 17 precursor [Homo sapiens] ACCESSION NP_001341298 XP_016868712 (SEQ ID NO: 41) TIFF2025506727000043.tif56170 Fibroblast growth factor receptor 1 isoform 18 precursor [Homo sapiens] ACCESSION NP_001341299 XP_006716376 (SEQ ID NO: 42) TIFF2025506727000044.tif5317019.Jag1 Jagged1[Homo sapiens] ACCESSION AAC51731 NP_000205 EAX10341 AAI26208 AAI26206 AAH98393 (SEQ ID NO: 43) TIFF2025506727000045.tif85170 Group 3 (BMP signaling) from Figures 10 and 11 20. Acvr1 (SEQ ID NO: 44) TIFF2025506727000046.tif3617021.Acvr2a (SEQ ID NO: 45) TIFF2025506727000047.tif2817022.Acvr2b (SEQ ID NO: 46) TIFF2025506727000048.tif2417023.Bmpr2 (see above) Group 4: Stem cell markers, not cell surface markers 24. Erg Transcriptional regulator ERG isoform 1 [Homo sapiens] ACCESSION NP_891548 (SEQ ID NO: 47) TIFF2025506727000049.tif33170 Transcriptional regulator ERG isoform 2 [Homo sapiens] ACCESSION NP_004440 (SEQ ID NO: 48) TIFF2025506727000050.tif32170 Transcriptional regulator ERG isoform 3 [Homo sapiens] ACCESSION NP_001129626 (SEQ ID NO: 49) TIFF2025506727000051.tif36170 Transcriptional regulator ERG isoform 4 [Homo sapiens] ACCESSION NP_001129627 (SEQ ID NO: 50) TIFF2025506727000052.tif28170 Transcriptional regulator ERG isoform 5 [Homo sapiens] ACCESSION NP_001230358 (SEQ ID NO:51) TIFF2025506727000053.tif28170 Transcriptional regulator ERG isoform 6 [Homo sapiens] ACCESSION NP_001230361 (SEQ ID NO:52) TIFF2025506727000054.tif24170 Transcriptional regulator ERG isoform 7 [Homo sapiens] ACCESSION NP_001278320 (SEQ ID NO:53) TIFF2025506727000055.tif24170 Transcriptional regulator ERG isoform 8 [Homo sapiens] ACCESSION NP_001317954 XP_016883777 (SEQ ID NO: 54) TIFF2025506727000056.tif3217025.Six2 SIX2 [Homo sapiens] ACCESSION AAK16583 (SEQ ID NO:55) TIFF2025506727000057.tif21170Homeobox protein SIX2 isoform X1 [Homo sapiens] ACCESSION XP_005264157 (SEQ ID NO:56) TIFF2025506727000058.tif2117026.Pthlh Parathyroid hormone-related protein isoform 1 preproprotein [Homo sapiens], ACCESSION NP_945317.1 (SEQ ID NO: 57) TIFF2025506727000059.tif12170 Parathyroid hormone-related protein isoform 2 preproprotein [Homo sapiens], ACCESSION NP_945315 (SEQ ID NO: 58) TIFF2025506727000060.tif1217027.Twist1 twist-related protein 1 [Homo sapiens] ACCESSION NP_000465 (SEQ ID NO:59) TIFF2025506727000061.tif16170twist homolog 1 (acromanthodysis 3, Säthle-Hochschen syndrome) (Drosophila) [Homo sapiens] ACCESSION EAW93711 (SEQ ID NO: 60) TIFF2025506727000062.tif1617028.Alpl (SEQ ID NO: 61) TIFF2025506727000063.tif3617029.Msx1 (SEQ ID NO: 62) TIFF2025506727000064.tif2117030.Efnb1 Ephrin-B1 precursor [Homo sapiens] ACCESSION NP_004420 (SEQ ID NO: 63) TIFF2025506727000065.tif2417031.Zic1 Zinc finger protein ZIC1 [Homo sapiens] ACCESSION NP_003403 (SEQ ID NO: 64) TIFF2025506727000066.tif3217032.Spry1 ACCESSION NP_001362339 XP_005262743 (SEQ ID NO: 65) TIFF2025506727000067.tif2417033.Abcc9 (SEQ ID NO: 66) TIFF2025506727000068.tif1217034.Erf (SEQ ID NO: 67) TIFF2025506727000069.tif4017035.Bmp2 Bone morphogenetic protein 2 [Homo sapiens] ACCESSION ACV32596 (SEQ ID NO: 68) TIFF2025506727000070.tif28170 Bone morphogenetic protein 2, isoform CRA_a [Homo sapiens] ACCESSION EAX10386 (SEQ ID NO: 69) TIFF2025506727000071.tif2417036.Bmp3 (SEQ ID NO: 70) TIFF2025506727000072.tif3217037.Bmp4 (SEQ ID NO: 71) TIFF2025506727000073.tif3317038.Bmp5 (SEQ ID NO: 72) TIFF2025506727000074.tif3417039.Bmp6 (SEQ ID NO: 73) TIFF2025506727000075.tif3617040.Bmp7 (SEQ ID NO: 74) TIFF2025506727000076.tif3317041.Bmp8a (SEQ ID NO: 75) TIFF2025506727000077.tif1617042.Bambi (SEQ ID NO: 76) TIFF2025506727000078.tif2117043.Bmper (SEQ ID NO: 77) TIFF2025506727000079.tif4817044.COL3A1 COL3A1 protein [Homo sapiens] ACCESSION AAH28178 (SEQ ID NO: 78) TIFF2025506727000080.tif80170Collagen alpha-1(III) chain preproprotein [Homo sapiens] ACCESSION NP_000081 AAL13167 (SEQ ID NO: 79) TIFF2025506727000081.tif100170

[0021] In some embodiments, examples of useful positive markers for SSCs include BMPR1A, Axin2, BMP2, BMP3, BMP4, BMP6, BMP7, BMP8b, BMP15, Gremlin1, CD200, AlphaV, Cathepsin K, Gli1, Ltf, Camp, Ear1, Lcn2, Ngp, Chil3, Anxa1, Prg2, Elane, and Alox15.

[0022] All of the markers described herein and their respective homologs may be used as markers to identify, enrich, or isolate skeletal and suture stem cells in an animal subject, such as a human or mouse.

[0023] Identification, isolation, and enrichment of SSCs In one aspect, the present application relates to the identification, isolation, and enrichment of SSCs, and in particular SuSCs. For example, these cells can be separated from a complex mixture of cells by techniques that identify or enrich cells that express one marker alone or in combination with other markers and characteristics described herein. Mammalian SSCs, such as mouse SSCs and human SSCs, can be characterized with functional homologs of one or more markers disclosed herein. Methods and compositions are provided for the separation and characterization of SSCs and osteoprogenitor cells. Cells can be separated from other cells by the expression of these specific cell markers, such as surface markers.

[0024] Cells of interest, i.e., cells expressing a marker of choice, can be isolated or enriched, i.e., separated from the rest of a cell population, by several methods well known in the art. For example, flow cytometry, e.g., fluorescence-activated cell sorting (FACS), can be used to separate a cell population based on the intrinsic fluorescence of the marker, or the binding of the marker to a specific fluorescent reagent, e.g., a fluorophore-conjugated antibody, as well as other parameters such as cell size and light scattering. In other words, cell selection can be achieved by flow cytometry. Although the absolute level of staining may vary depending on the specific fluorescent dye and reagent preparation, the data can be normalized to a control. To normalize the distribution to a control, each cell is recorded as a data point with a particular intensity of staining. These data points can be displayed according to a logarithmic scale, with the unit of measurement being any staining intensity. In one example, the most brightly stained cells in a sample can be 4 logs more intense than unstained cells. When displayed in this manner, it is clear that the cells that fall into the highest logarithm of staining intensity are bright, and the cells with the lowest intensity are negative. "Low" positive staining cells have a staining level above the brightness of the isotype-matched control, but not as intense as the most brightly staining cells normally found in the population. Alternative controls may utilize substrates with a defined density of markers on their surface, such as fabricated beads or cell lines that provide an intense positive control. Other methods of marker-based separation, i.e., cell selection, may be accomplished, for example, by magnetic activated cell sorting (MACS), immunopanning, and laser capture microdissection.

[0025] A population that is enriched by selecting for expression of one or more markers will usually have at least about 50%, e.g., 80% of the cells of the selected phenotype, more usually at least 90% of the cells, and may be 95% or more of the cells of the selected phenotype.

[0026] In some instances, for isolation of cells from tissue, a suitable solution may be used for dispersion or suspension. Such a solution may be a balanced salt solution, such as normal saline, PBS, Hank's balanced salt solution, etc., supplemented with fetal bovine serum or other naturally occurring factors, generally at low concentrations, generally 5-25 mM, of an acceptable buffer. Convenient buffers include HEPES, phosphate buffer, lactate buffer, etc. The tissue may be enzymatically and / or mechanically dissociated. In some embodiments, bone tissue is treated with a mild protease, such as a dissociation enzyme, for a period of time sufficient to dissociate the cells, and then gently mechanically dissociated.

[0027] Initial separation may select cells by various methods known in the art, including elution, Ficoll-Hypaque, or flow cytometry using forward and obtuse angle scatter parameters. Separation of the SSC population would then use affinity separation to provide a substantially pure population. Techniques for affinity separation may include magnetic separation using antibody-coated magnetic beads, affinity chromatography, cytotoxic agents conjugated to monoclonal antibodies or used with monoclonal antibodies, e.g., complement and cytotoxins, and "panning" with antibodies bound to solid matrices, e.g., plates, or other convenient techniques. Techniques that provide accurate separation include fluorescence-activated cell sorters, which may have various degrees of sophistication, such as multiple color channels, low and obtuse angle light scatter detection channels, impedance channels, etc. Cells may be selected against dead cells by using a dye that associates with dead cells (propidium iodide, 7-AAD). Any technique that is not overly detrimental to the viability of the selected cells may be used.

[0028] The affinity reagent can be a specific receptor or ligand for the above cell surface molecule. Of particular interest is the use of antibodies as affinity reagents. The preparation details of antibodies and their suitability for use as specific binding members are well known to those skilled in the art. Depending on the particular population of cells selected, an antibody having specificity for the markers described herein can be contacted with the starting population of cells. Optionally, a reagent specific for one or more of the other markers disclosed herein can also be included.

[0029] As is known in the art, antibodies can be selected to have specificity for the relevant species, i.e., antibodies specific for human markers are used to select human cells, antibodies specific for mouse markers are used to select mouse cells, etc.

[0030] These antibodies can be conjugated with labels for use in separation. Labels include magnetic beads that allow direct separation, biotin that can be removed with avidin or streptavidin bound to a support, fluorescent dyes that can be used in fluorescence activated cell sorters and the like to facilitate the separation of specific cell types. Examples of fluorescent dyes that can be used include phycobiliproteins, such as phycoerythrin and allophycocyanin, fluorescein, and Texas Red. Often, each antibody is labeled with a different fluorescent dye to allow independent sorting for each marker.

[0031] The antibody may be added to the suspension of cells and incubated for a period of time sufficient to bind to available cell surface antigens. Incubation may usually be at least about 5 minutes, and usually less than about 2 hours. It is desirable to have a sufficient concentration of antibody in the reaction mixture so that the efficiency of separation is not limited by a lack of antibody. The appropriate concentration may be determined by titration. The medium in which the cells are separated may be any medium that maintains the viability of the cells. An exemplary medium may be phosphate buffered saline containing about 0.1-20% BSA or FBS. A variety of media are commercially available and may be used according to the nature of the cells, including those mentioned above, such as Dulbecco's Modified Eagle's Medium (DMEM), Hank's Basic Salt Solution (HBSS), Dulbecco's Phosphate Buffered Saline (dPBS), RPMI, Iscove's Medium, PBS with 5 mM EDTA, etc., often supplemented with fetal bovine serum, BSA, HSA, etc.

[0032] The labeled cells are then separated for the above phenotypes. The separated cells can be collected in any suitable medium that maintains the viability of the cells, usually with a cushion of serum at the bottom of the collection tube. A variety of media are commercially available and can be used according to the nature of the cells, including DMEM, HBSS, dPBS, RPMI, Iscove's medium, etc., often supplemented with fetal bovine serum.

[0033] A composition highly enriched for SSCs can be achieved in this manner. The cell population can contain about 50% or more of SSCs, usually about 90% or more of SSCs, and can be about 95% or more of SSCs. The enriched cell population can be used immediately or frozen at liquid nitrogen temperature and stored for a long period of time, and can be thawed and reused. The cells can usually be stored in 10% DMSO, 50% FCS, 40% medium (e.g., RPMI1640 medium). When thawed, the cells can be expanded by the use of growth factors for proliferation and differentiation.

[0034] In some embodiments, cells are identified, isolated, or enriched from tissue. The tissue can be from any animal of interest. Examples include vertebrates, such as mammals and non-mammals, such as fish, amphibians, and birds, that have similar skeletal structures and skeletal stem cells (Mork and Crump, Curr Top Dev Biol, 2015). Examples of mammals include humans, primates, domestic and livestock animals, and zoo, laboratory, or pet animals, such as dogs, cats, cows, horses, sheep, pigs, goats, rabbits, rats, and mice. The cells can be established cell lines or primary cells, and "primary cells," "primary cell lines," and "primary cultures" are used interchangeably herein to refer to cells and cell cultures derived from a subject and grown in vitro for a limited number of passages.

[0035] SSCs can be isolated from fresh or frozen cells, which may be from neonates, juveniles or adults, as well as from tissues including skin, muscle, bone marrow, peripheral blood, umbilical cord blood, spleen, liver, pancreas, lungs, intestines, stomach, fat, and other tissues. Tissues may be obtained by biopsy or apheresis from live donors, or from dead or dying donors within about 48 hours of death, or from freshly frozen tissue, tissue frozen within about 12 hours of death and maintained indefinitely at below about -20°C, usually about liquid nitrogen temperature (-190°C). For isolation of cells from tissues, a suitable solution may be used for dispersion or suspension. Such solutions will generally be balanced salt solutions, such as normal saline, PBS, Hank's balanced salt solution, etc., supplemented with fetal bovine serum or other naturally occurring factors, along with low concentrations, generally 5-25 mM, of an acceptable buffering agent. Convenient buffers include HEPES, phosphate buffers, lactate buffers, etc.

[0036] Cell culture, proliferation, and differentiation The SSCs described above can be cultured and maintained in vitro under various culture conditions. Suitable culture or nutrient media can be liquid or semi-solid (e.g., containing agar, methylcellulose, etc.). The cell population can be suspended in a suitable nutrient medium, such as DMEM, Iscove's modified DMEM, or RPMI-1640, typically supplemented with fetal bovine serum (about 1-25%), L-glutamine, thiols, particularly 2-mercaptoethanol, and antibiotics. In one embodiment of the present invention, the SSCs can be maintained in culture, such as in the absence of feeder layer cells or in the absence of serum. Examples of culture media or nutrient media include DMEM, DMEM / F12, DMEM High Glucose, MEM, IMDM, Gibco StemPro™-34 SFM, Gibco Essential 8, Gibco Essential 6, Gibco MesenPRO, Gibco StemPro MSC, Gibco CTS KnockOut SR XenoFree, Corning NutriStem hPSC XF, and HyClone AdvanceSTEM.

[0037] The culture may contain antibiotics to prevent bacterial and fungal growth. Examples of antibiotics include penicillin, streptomycin, amphotericin B, gentamicin, puromycin, hygromycin B, ciprofloxacin, chloramphenicol, kanamycin, neomycin, blasticidin S, G418 sulfate, ampicillin, and carbenicillin.

[0038] The culture or medium may contain growth factors to which the cells respond. Growth factors, as defined herein, are molecules capable of promoting the survival, growth, and / or differentiation of cells, either in culture or in intact tissues, through specific effects on transmembrane receptors. Growth factors include polypeptide and non-polypeptide factors.

[0039] The growth factor can be any suitable growth factor, including, for example, bone morphogenetic proteins (BMPs), Indian hedgehog (IHH), transforming growth factor beta (TGFβ), bone morphogenetic proteins (BMPs), such as BMP-2, 4, 6, and 9, fibroblast growth factors (FGFs) such as FGF-1 and 2, epidermal growth factor (EGF), Wnt ligand and β-catenin, insulin growth factors (IGFs) such as IGF-1 and IGF-2, collagen-1, Runx2, osteopontin, osterix, vascular endothelial growth factor (VEGF), platelet-derived growth factor (PDGF), osteoprotegerin (OPG), NEL-like protein 1 (NELL-1), or any combination thereof.For detailed disclosure of such growth factors, see, for example, Devescovi, V. et al., “Growth factors in bone repair,” Chir Organi Mov 92, 161, 2008; James AW, “Review of Signaling Pathways Governing MSC Osteogenic and Adipogenic Differentiation,” Scientifica (Cairo) 2013; 2013: 684736; Carofino BC et al., “Gene therapeutic applications for fracture healing,” J Bone Joint Surg Am, 90 (Suppl 1) (2008), pp. 99-110; Javed A. et al., “Genetic and transcriptional control of bone formation,” Oral Maxillofac Surg Clin North Am. 2010; 22: 283-93; Chen G. et al., “TGF-β and BMP signaling in osteoblast differentiation and bone See, Ornitz, D. M. et al., "FGF signaling pathways in endochondral and intramembranous bone development and human genetic disease. Genes Dev. 16, 1446-1465 (2002); Krishnan V. et al., "Regulation of bone mass by Wnt signaling" J. Clin. Invest. 116 1202-1209 (2006); and Boyce B. F. et al., "Regulation of bone mass by Wnt signaling" J. Clin. Invest. 116 1202-1209 (2006). The entire contents of each of these publications are incorporated herein by reference.

[0040] SSCs can be directed to differentiate along specific pathways under conditions known in the art. For example, SSCs can be directed to chondrogenesis by differentiation factors, which may be referred to herein as chondrogenic factors. In some examples, SSCs can be differentiated into desired skeletal lineage cells. Certain embodiments include distorting the differentiation of skeletal stem cells into chondrocytes by contacting with an effective dose of one or both of a VEGF inhibitor and a TGFβ inhibitor, and their use in tissue repair. Other examples of skeletal cells that may be generated by the methods described herein include pre-osteochondral and stromal precursors (pre-BCSP), BCSP, definitive chondrocyte precursors (CCP), osteoprogenitors, B-cell lymphocytic stromal precursors (BLSP), 6C3 stroma, hepatic leukemia factor expressing stromal cells (HEC), and their progeny.

[0041] Cells contacted in vitro with a factor, such as a factor that promotes reprogramming and / or promotes chondrocyte growth and / or differentiation, may be incubated in the presence of the agent for about 30 minutes to about 24 hours, which may be repeated about every day to about every 4 days, e.g., every 2 days, every 3 days, etc. The agent may be provided to the cells one or more times, allowing the cells to be incubated with the agent for some time after each contacting event, e.g., 16 to 24 hours, after which period the medium is replaced with fresh medium and the cells are further cultured.

[0042] After contacting the cells with the factors, the contacted cells can be cultured to promote survival and differentiation of the population of skeletal stem cells, chondrocytes, or progenitor cells defined herein. Methods and reagents for culturing cells are known in the art, any of which can be used herein to grow and isolate the cells. For example, the cells (either before or after contacting with the factors) can be seeded on matrigel or other substrates, as known in the art. The cells can be cultured in a medium supplemented with factors. Alternatively, the contacted cells can be frozen at liquid nitrogen temperature and stored for long periods of time, ready for use upon thawing. When frozen, the cells will typically be stored in 10% DMSO, 50% FCS, 40% RPMI1640 medium. Upon thawing, the cells can be expanded by the use of growth factors and / or stromal cells associated with skeletal survival and differentiation.

[0043] The induced skeletal or chondrogenic cells produced by the above methods can be used in cell replacement or transplantation therapy to treat disease. Specifically, the cells can be transferred to subjects suffering from a wide range of diseases or disorders that have a skeletal or cartilaginous component.

[0044] In some cases, the cells of interest or a subpopulation of cells of interest can be purified or isolated or enriched from the remainder of the cell culture before being transferred to the subject. In some cases, one or more antibodies specific to a marker of cells of the skeletal / chondrogenic lineage or a marker of a subpopulation of cells of the skeletal lineage are incubated with the cell population, and those bound cells are isolated. In other cases, the cells or subpopulation of cells express a marker that is under the control of a specific promoter and is then used to purify or isolate the cells or subpopulations thereof, such as a reporter gene, e.g., EGFP, dsRED, lacz, etc.

[0045] sphere In one aspect, the present application relates to cell spheres comprising one or more cells expressing one or more of the makers described herein. The spheres may be 20-200 μm in diameter or may have about 10-500 (e.g., 50-400, 100-300, etc.) cells per sphere. The spheres may comprise Axin2+ cells. To generate such spheres, a population of suture or skeletal stem cells may be seeded in the maintenance medium described above on a low or ultra-low attachment surface and the cells or their progeny may be cultured for a period of time to form one or more spheres. The period may be 5-20 days, such as 5-15 days and 7-10 days.

[0046] Low attachment or ultra-low attachment surfaces refer to surfaces for cell culture that have been treated to reduce or minimize cell attachment. Such surfaces may have a neutral hydrophilic hydrogel coating that significantly reduces the binding of adhesive proteins. This minimizes cell attachment and spreading. The covalent hydrogel layer effectively inhibits cell attachment and minimizes protein absorption, enzyme activation, and cell activation. Cell culture with such ultra-low attachment surfaces is available from manufacturers such as CORNING.

[0047] The terms "sphere" and "sphere-like cell aggregate" are used interchangeably to refer to cell aggregates having a three-dimensional shape that is close to a sphere. Examples of three-dimensional shapes that are close to a sphere include a spherical shape, which has a three-dimensional structure and shows, for example, a circular or elliptical shape when projected onto a two-dimensional surface, and a shape formed by fusing multiple spherical shapes (for example, shows a shape formed by overlapping two to four circular or elliptical shapes when projected onto a two-dimensional surface). The term "cell aggregate" refers to a ball-shaped cluster of cells or a block of cells, including pluripotent stem cells such as SSCs. The cell aggregate may have a spherical shape. The cell aggregate may be a sphere. The sphere or aggregate is preferably formed by suspension culture. The sphere or cell aggregate is a cluster of cells including undifferentiated pluripotent stem cells. The sphere or cell aggregate has the ability to produce various types of cells when the sphere / cell aggregate is cultured.

[0048] About 5% of mouse suture mesenchymal cells can be Bmpr1A+ cells, and about 0.5% of suture mesenchymal cells can form spheres. The majority of spheres contain Bmpr1a+ cells. Thus, approximately 10% of Bmpr1a+ cells form spheres. For human stem cells, about 5% of suture mesenchymal cells are BMPR1A+ cells, and about 0.1% of suture mesenchymal cells form spheres under current culture conditions. The majority of spheres contain BMPR1A+ cells. Thus, approximately 2% of BMPR1A+ suture mesenchymal cells form spheres.

[0049] As disclosed herein, in primary sphere culture, 68% of the spheres contain Axin2+ cells. In subsequent secondary and tertiary cultures, 100% of the spheres contain Axin2+ cells. In theory, all spheres should be formed by Axin2+ cells, since there are stem cells with "unlimited self-renewal" capacity that require spheres to form in sequential culture for the true definition of stem cells. The explanation for primary culture is that some spheres (32%) are formed by progenitor cells with limited proliferation capacity, and therefore can only form spheres in primary culture, but not in secondary culture. Axin2+ stem cells are quiescent and give rise to spheres by asymmetric cell division. Spheres that do not contain Axin2+ cells imply the absence of Axin2+ stem cells with quiescent / low cycling function.

[0050] The cells, spheres, and methods described herein are useful for the development of in vitro or in vivo models for bone function, gene therapy, and artificial organ construction. For example, developing bone can serve as a source of growth factors and pharmaceuticals, for the production of viruses or vaccines, for in vitro toxicity and metabolism testing of drugs and industrial compounds, for gene therapy experiments, for the construction of artificial, implantable bone, and for bone mutagenesis and carcinogenesis.

[0051] bone regeneration products The cells described herein may be provided in combination with other types of cells, drugs, materials, and structures for various uses, such as tissue engineering and treatment of any bone with defects. Thus, the present application provides a bone regeneration product or formulation that includes at least one skeletal stem cell and, optionally, at least one of such other types of cells, drugs, materials, and structures. For that purpose, the cells may be three-dimensional (3D) synthetic, semi-synthetic, or living biological tissue.

[0052] In one example, the bone regeneration product / formulation comprises at least one SSC and at least one scaffold suitable for carrying cells. The scaffold may comprise any 3D printed scaffold suitable for carrying cells. The bone regeneration product / formulation is suitable for dense bone regeneration, spongy bone regeneration, or a combination thereof. The bone regeneration product / formulation may further comprise growth factors as described above.

[0053] The scaffold may include a variety of suitable materials, such as hydroxyapatite (HA), tricalcium phosphate (TCP), and polymers, which may be prepared by using photocurable polymers and / or monomers.

[0054] The scaffold may comprise a porous 3D network of interconnected void spaces. The scaffold may be any scaffold suitable for incorporating the cells and / or growth factors disclosed herein to help form direct and / or indirect contacts between these cells and / or growth factors and tissue (e.g., bone) for regeneration of said tissue (e.g., bone). The scaffold may incorporate the cells and / or growth factors in any form, for example, by carrying, supporting, adsorbing, absorbing, encapsulating, holding, and / or attaching the cells and / or growth factors.

[0055] The scaffold may have any shape or geometry. The scaffold may have any pore size. The scaffold may have any porosity (i.e., void volume). The scaffold may have any morphology. The scaffold may have any mechanical strength.

[0056] Bone defects can be formed in different parts of the animal or human body. These defects can have any shape and size. A scaffold suitable for treating such defects can have a shape, volume, and size that can, for example, match or resemble the shape and size of the defect. Such scaffolds can also have a pore volume, pore size, and / or pore shape similar to the bone that the bone regeneration product containing such scaffolds is designed to treat. Such scaffolds can also have pores with a pore size small enough that these scaffolds can contain the cells described herein within their porous structure and allow the bone regeneration product to be implanted and successfully perform the treatment. The bone regeneration product / formulation can have sufficient mechanical strength to handle the load-bearing conditions of implantation into the body. It can also have sufficient mechanical strength to handle the load-bearing conditions and / or body weight of the bone during movement (e.g., walking).

[0057] The scaffold may include any material. For example, the scaffold may include a non-resorbable material, a resorbable material, or a mixture thereof. A resorbable material may be resorbed by the patient's body and eventually replaced with healthy tissue. A "resorbable" material may include, for example, a biocompatible, bioabsorbable, biodegradable polymer, any similar material, or a mixture thereof.

[0058] A biocompatible material is one that can be accepted by and function in a patient's body without eliciting a significant foreign body response (such as, for example, an immune, inflammatory, thrombotic, or similar response) and / or is not clinically contraindicated for administration to a tissue or organ. Biodegradable materials can include materials that are absorbable or degradable when administered in vivo and / or under in vitro conditions. Biodegradation can occur either directly or indirectly through the action of biological agents.

[0059] The scaffold may comprise a solid, a liquid, or a mixture thereof. For example, the scaffold may be a paste. For example, the scaffold may comprise a paste comprising a mixture of hydroxyapatite and tricalcium phosphate (HA / TCP). The scaffold may be prepared, for example, by mixing hydroxyapatite (HA) and tricalcium phosphate (TCP) with a formulation comprising a liquid to prepare a paste. For example, the mesenchymal stem cell preparation may comprise a liquid, and mixing such a mesenchymal stem cell preparation with hydroxyapatite (HA) and tricalcium phosphate (TCP) may form a paste. This type of scaffold is referred to herein as a HA / TCP scaffold.

[0060] A mixture comprising HA and TCP (HA / TCP) may be formed of equal amounts of HA and TCP by weight, e.g., 50% HA and 50% TCP, unless otherwise specified. However, a mixture comprising HA and TCP may have any composition varying, e.g., from 0% HA to 100% TCP by weight. For example, HA concentrations of greater than 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% by weight are within the scope of the present application.

[0061] The scaffolds described herein may comprise any biodegradable polymer. For example, the scaffolds may comprise synthetic polymers, naturally occurring polymers, or mixtures thereof. Examples of suitable biodegradable polymers may be polylactide (PLA), polyglycolide (PGA), poly(lactide-co-glycolide) (PLGA), poly-E-caprolactone, polydioxanone trimethylene carbonate, polyhybroxyalkonates (e.g., poly(hydroxybutyrate)), poly(ethyl glutamate), poly(DTH iminocarbony (bisphenol A iminocarbonate), poly(orthoesters), polycyanoacrylates, fibrin, casein, serum albumin, collagen, gelatin, lecithin, chitosan, alginates, polyamino acids (such as polylysine), and mixtures thereof.

[0062] The scaffold may further comprise one or more of the above growth factors. The growth factors may be continuously released into the surrounding (bone) tissue after the bone regeneration product is implanted into the bone defect site. The release rate of the growth factors may be controlled via the chemical composition and / or pore structure of the scaffold.

[0063] The scaffold may be manufactured by any technique. For example, the scaffold may be manufactured manually and / or using a machine. For example, the scaffold may be manufactured by additive manufacturing and / or manufacturing. For example, the scaffold may be manufactured using a combination of more than one such manufacturing techniques.

[0064] In one embodiment, the cells are used in a "bioprinting" process to generate spatially controlled cell patterns using 3D printing technology. Any bioprinting or biofabrication process known in the art can be used, for example, as described in U.S. Patent Application Publication Nos. 2014 / 0099709, 2014 / 0093932, 2014 / 0274802, 2014 / 0012407, 2013 / 0345794, 2013 / 0190210, and 2013 / 0164339, and U.S. Patent No. 8,691,974.

[0065] For example, in one embodiment, a printer cartridge is filled with a suspension of SSC or SSC spheres and a gel. Alternating patterns of gel and cells or spheres are printed using a standard printing nozzle. In an alternative embodiment, a NOVOGEN (San Diego, Calif.) MMX.TM. or Organovo Holdings, Inc. bioprinter can be used for 3D bioprinting. These and equivalent "bioprinters" can be optimized to "print" or fabricate bone, cartilage, and other tissues, all of which are suitable for surgical therapy and implantation.

[0066] Any 3D printing technique can be used to manufacture the scaffold. 3D printing or additive manufacturing (AM) can be a process for creating a physical object from a 3D digital model, typically by laying down many successive thin layers of material. Such thin layers of material can be formed under computer control. Examples of 3D printing techniques can be stereolithography (SLA), digital light processing (DLP), fused deposition modeling (FDM), selective laser sintering (SLS), selective laser melting (SLM), electron beam melting (EBM), laminated object manufacturing (LOM), binder jetting (BJ), material jetting (MJ), or wax casting (WC), or a combination thereof.

[0067] Scaffolds, including 3D printed scaffolds, can be fabricated, for example, by using formulations including polycaprolactone dimethacrylate (PCLDA), calcium phosphate, HA, TCP, polyethylene glycol diacrylate (PEGDA), gelatin methacryloyl (GeIMA), or mixtures thereof.

[0068] The bone regeneration product / formulation can be used for bone regeneration. Such bone regeneration methods can include implanting the bone regeneration product / formulation in or across a bone defect. The bone defect can be any bone defect, such as a bone defect in a long bone. The size of the bone defect can be any size. For example, the size of the bone defect can be a critical size. The bone defect can be formed due to congenital bone malformation. The congenital bone defect can be a defect associated with cleft lip and / or cleft palate. The bone defect can be formed due to surgery, accident, and / or disease. The bone defect can be formed as a result of surgery performed to treat craniosynostosis.

[0069] Pharmaceutical Compositions The present application further provides a composition comprising (i) a carrier and (ii) one or more cells obtained according to the methods described herein, or cells derived therefrom (such as progeny cells). The composition may be a pharmaceutical composition, where the carrier is a pharma- ceutically acceptable carrier.

[0070] Compositions for pharmaceutical use, such as scaffolds or implants with cells and / or factors, may contain non-toxic carriers of pharma- ceutically acceptable diluents, which are defined as vehicles commonly used to formulate pharmaceutical compositions for animal or human administration, depending on the desired formulation. Diluents may be selected so as not to affect the biological activity of the combination. Examples of such diluents are distilled water, buffered water, physiological saline, PBS, Ringer's solution, dextrose solution, and Hank's solution. In addition, pharmaceutical compositions or formulations may contain other carriers, adjuvants, or non-toxic, non-therapeutic, non-immunogenic stabilizers, excipients, and the like. The compositions may also contain additional substances that approximate physiological conditions, such as pH adjusting and buffering agents, toxicity adjusting agents, wetting agents, and detergents.

[0071] The composition may also include any of a variety of stabilizing agents, such as, for example, antioxidants. When the pharmaceutical composition includes a polypeptide (e.g., a growth factor), the polypeptide may be complexed with a variety of well-known compounds that enhance the in vivo stability of the polypeptide or otherwise enhance its pharmacological properties (e.g., increase the half-life of the polypeptide, reduce its toxicity, enhance solubility or uptake). Examples of such modifying or complexing agents include sulfates, gluconates, citrates, and phosphates. The polypeptides of the composition may also be complexed with molecules that enhance their in vivo attributes. Such molecules include, for example, carbohydrates, polyamines, amino acids, other peptides, ions (e.g., sodium, potassium, calcium, magnesium, manganese), and lipids.

[0072] Further guidance regarding formulations suitable for various types of administration can be found in Remington's Pharmaceutical Sciences, Mace Publishing Company, Philadelphia, Pa., 17th ed. (1985). For a brief review of methods for drug delivery, see Langer, Science 249:1527-1533 (1990).

[0073] The pharmaceutical compositions described herein, such as SSCs, alone or in combination with various agents, can be administered for prophylactic and / or therapeutic treatment. Toxicity and therapeutic efficacy of the active ingredients can be determined according to standard pharmaceutical procedures in cell cultures and / or experimental animals, including, for example, determining LD50 (the dose lethal to 50% of the population) and ED50 (the dose therapeutically effective in 50% of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index, which can be expressed as the ratio LD50 / ED50. Compounds that exhibit large therapeutic indices are preferred.

[0074] The data obtained from cell culture and / or animal studies can be used in formulating a range of dosages for humans. The dosage can vary within this range depending on the dosage form employed and the route of administration utilized.

[0075] Components used to formulate pharmaceutical compositions are preferably of high purity and substantially free of potentially harmful contaminants (e.g., at least National Food (NF) grade, generally at least analytical grade, and more typically at least pharmaceutical grade). Furthermore, compositions intended for in vivo use are usually sterile. To the extent that a given compound must be synthesized prior to use, the resulting product is typically substantially free of any potentially toxic agents, particularly any endotoxins, that may be present during the synthesis or purification process. Compositions for parental administration are also sterile, substantially isotonic, and made under GMP conditions.

[0076] The effective amount of a therapeutic composition given to a particular patient will depend on a variety of factors, some of which will vary from patient to patient. A competent clinician will be able to determine the effective amount of a therapeutic agent to administer to a patient to halt or reverse the progression of a disease state, if necessary. Using animal data and other information available about the agent, the clinician can determine the maximum safe dose for an individual, depending on the route of administration. For example, a dose administered intravenously may be higher than a dose administered topically, given the larger fluid into which the therapeutic composition is being administered. Similarly, compositions that are rapidly cleared from the body may be administered at higher doses or in repeated doses to maintain therapeutic concentrations. Using routine techniques, a competent clinician will be able to optimize the dosage of a particular therapeutic agent during the course of routine clinical trials.

[0077] Mammalian species that may be treated with the present methods include canines and felines, equines, bovines, ovines, etc., as well as primates, particularly humans. Animal models, particularly small mammals, such as murines, lagomorphs, etc., may be used for experimental investigations.

[0078] use The cells, compositions, formulations, and products disclosed herein can be used for a variety of purposes, including treatment of associated disorders and tissue engineering.

[0079] In some embodiments, the cells, compositions, formulations, and products disclosed herein may be used in the treatment of a subject, such as a human patient, in need of bone or cartilage replacement therapy. Examples of such subjects may be subjects suffering from conditions associated with cartilage or bone loss due to osteoporosis, osteoarthritis, genetic defects, disease, etc. Patients with diseases and disorders characterized by such conditions would greatly benefit from the treatment protocols of the present invention as claimed in the pending patents.

[0080] An effective amount of a pharmaceutical composition is an amount that will result in an increase in the number of chondrocytes, skeletal cells, cartilage, or bone mass at the implant site and / or an amount that will result in a measurable reduction in the rate of disease progression in vivo. For example, an effective amount of a pharmaceutical composition will increase bone or cartilage mass by at least about 5%, at least about 10%, at least about 20%, preferably about 20% to about 50%, and even more preferably by more than 50% (e.g., about 50% to about 100%) compared to a suitable control, which is typically a subject not treated with the composition.

[0081] The above methods may be used in combination with therapies, such as those known in the art, to provide relief of symptoms associated with the aforementioned diseases, disorders, and conditions. The pharmaceutical compositions described herein and their combination with other agents may have the advantage that lower dosages of the individual drugs are required and the effects of the different drugs are complementary.

[0082] In some embodiments, an effective dose of SSCs, preferably SuSCs, as described herein is provided in an implant or scaffold for regeneration of skeletal or cartilage tissue. The effective cell dose may depend on the purity of the population. In some embodiments, the effective dose is at least about 10 2 , about 10 3 , about 10 4 , about 105 , about 10 6 , about 10 7 , about 10 8 , about 10 9 A cell dose of cells or more is delivered, and the stem cells may be present in the cell population at a concentration of about 1%, about 5%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90% or more.

[0083] The present application provides methods and compositions for differentiating SSCs, including SuSCs, into cells such as chondrocytes. The cells produced by the methods are useful to provide a source of fully differentiated functional cells for research, transplantation, and development of tissue engineered products for the treatment of human disease and traumatic injury repair.

[0084] tissue engineering Tissue engineering is the use of a combination of cells, engineering and material methods, and suitable biochemical and physicochemical factors to improve or replace biological functions. For example, cells can be implanted or seeded into artificial structures that can support three-dimensional tissue formation. These structures, referred to herein as matrices or scaffolds, allow cell attachment and migration, deliver and retain cells and biochemical factors, and allow diffusion of important cell nutrients and expressed products. High porosity and appropriate pore size are important to facilitate cell seeding and diffusion throughout the structure of both cells and nutrients. Biodegradability is often a factor, as the scaffold can be absorbed by the surrounding tissue without the need for surgical removal. The rate at which degradation occurs needs to be as contemporaneous as possible with the rate of tissue formation, meaning that the scaffold can provide structural integrity in the body while the cells fabricate their own natural matrix structure around themselves, eventually degrading leaving behind neotissue, the newly formed tissue that takes over the mechanical load. Injectability is also important for clinical use.

[0085] Many different materials (natural and synthetic, biodegradable and permanent) have been investigated and may be used for tissue engineering matrices or scaffolds. Examples include Puramatrix, polylactic acid (PLA), polyglycolic acid (PGA), and polycaprolactone (PCL), as well as combinations thereof. Scaffolds may also be constructed from natural materials, e.g., proteins such as collagen, fibrin; polysaccharide materials such as acytosan; glycosaminoglycans (GAGs) such as alginate, hyaluronic acid. Functional groups on the scaffold may be useful for the delivery of small molecules (drugs) to specific tissues. Another form of scaffold under investigation is decellularized tissue extracts, whereby the remaining cellular remnants / extracellular matrix act as the scaffold.

[0086] Treatment method The above cells, compositions, formulations, products, and methods can be used to treat various bone defects, such as large craniofacial defects, craniosynostosis, and fractures. Examples include: (A) fractures caused by various conditions, such as osteoporosis and osteopenia; (B) large bone defects caused by various conditions, including cancer surgery, congenital malformations (e.g., Cleidocranial Dysplasia, Cleft Palate, Facial Cleft, Treacher-Collins, fibrous dysplasia), trauma, and progressive degenerative diseases; and (C) stem cell-based therapy can be used to replace any treatment, including bone grafts.

[0087] Large craniofacial bone defects caused by various conditions including trauma, infection, tumors, congenital diseases, and progressive degenerative diseases are major health problems. Autologous bone grafts are the recommended treatment for extensive skeletal repair, but their success remains very challenging due to several limitations. Therefore, alternative approaches are being explored. Stem cell-based therapy is particularly attractive and promising in light of the characterization of skeletal stem cells in the craniofacial and somatic skeletons. Craniofacial bones are primarily formed via intramembranous ossification, a process that is distinct from the endochondral ossification required for the somatic skeleton. Due to the different properties of stem cells in the craniofacial and somatic skeletons, there is a need to study various types of skeletal stem cells. Suture stem cells (SuSCs) are a stem cell population that are naturally programmed to form intramembranous bone during craniofacial skeletal formation. The lack of cell markers, and in particular cell surface markers for stem cell isolation, as well as the inability to maintain stemness characteristics ex vivo are two important hurdles that limit further progress in the field of skeletal regeneration. The cells, compositions, and methods disclosed herein address this unmet need.

[0088] Craniosynostosis, affecting approximately 1 in 2,500 individuals, is one of the most common congenital deformities and is caused by premature suture closure. Sutures, which serve as growth centers for the morphogenesis of the cranial vault, are the equivalent of the growth plates of long bones. Excessive intramembranous ossification caused by genetic mutations promotes suture fusion. One example is the genetic loss of function of AXIN2, which causes craniosynostosis in mice and humans. In 2010, we found that craniosynostosis can also be caused by a switch in the fate of mesenchymal cells, resulting in suture closure via endochondral ossification. By regulating the interaction between bone morphogenetic protein (BMP) and fibroblast growth factor (FGF) pathways, Axin2-mediated Wnt signaling determines the osteogenic decision towards osteogenic or chondrogenic lineages. Multipotency further supports the presence of skeletal stem cells within the suture mesenchyme. Expression of Axin2 in the putative niche sites was tightly associated with suture patency, thus identifying Axin2-expressing SuSCs as essential for calvarial development, homeostasis, and injury-induced repair. Axin2-positive (Axin2+) SuSCs fit the contemporary and rigorous stem cell definition: they exhibit long-term self-renewal, clonal expansion, differentiation, and multipotency, as well as the ability to repair skeletal defects by direct engraftment and replacement of damaged tissue.

[0089] When used for tissue engineering or therapeutic methods, the combination of SSCs and / or factors for lineage differentiation may be provided for in vivo use in a cell solution, which is a hydrated solution, suspension, or other fluid containing cells or factors capable of differentiating into bone or cartilage.

[0090] Bone or cartilage graft devices and compositions can be provided that are optimized for use in both load-bearing and non-load-bearing cartilage or bone graft applications in terms of one or more of composition, bioactivity, porosity, pore size, protein binding potential, degradability, or strength. Preferably, the graft material is formulated to promote one or more of the processes involved in bone or cartilage healing that can occur with the application of a single graft material: chondrogenesis, osteogenesis, osteoinduction, and osteoconduction. Chondrogenesis is the formation of new cartilage structures. Osteogenesis is the formation of new bone by cells contained within the graft. Osteoinduction is a chemical process in which molecules contained within the graft (e.g., bone morphogenetic proteins and TGF-β) convert the patient's or other bone precursor cells into cells capable of forming bone. Osteoconduction is a physical effect in which the matrix of the graft forms a scaffold on which the recipient's bone-forming cells can form new bone.

[0091] The inclusion of factors and / or cells described herein may be used to facilitate replacement and filling of cartilage or bone material in and around pre-existing structures. In some embodiments, the cells first produce chondrocytes, followed by deposition of extracellular matrix and bone formation. Bone grafts may provide an osteoconductive scaffold comprising calcium phosphate ceramics, which provides a framework for implanted progenitor cells and local bone cells to differentiate into osteogenic cells and deposit new bone. The use of calcium phosphate ceramics may slow the degradation of the ceramic, resulting in a localized source of calcium and phosphate for bone formation. Thus, new bone may be formed without loss of calcium and phosphate from the host bone surrounding the defect site. Calcium phosphate ceramics are chemically compatible with those of the mineral components of bone tissue. Examples of such calcium phosphate ceramics include calcium phosphate compounds and salts, and combinations thereof.

[0092] In some embodiments, the cells and / or factors may be prepared as an injectable paste. A cell suspension may be added to one or more cells to form an injectable hydrated paste. The paste may be injected into the implant site. In some embodiments, the paste may be prepared prior to implantation and / or the paste may be stored in a syringe at sub-ambient temperature until needed. In some embodiments, application of the composite by injection may be similar to bone cement, which may be used to bond and hold bone fragments in place or improve the adhesion of, for example, artificial hip joints, such as for replacing damaged cartilage in joints. Implantation in a closed surgical environment may also be performed.

[0093] In other embodiments, the cells and / or factors can be prepared as a moldable putty. A cell suspension can be added to one or more powdered minerals to form a putty-like hydrated graft composite. The hydrated graft putty can be prepared and shaped to approximate any implant shape. The putty can then be pressed into place to fill a void in cartilage, bone, dental socket, or another site. In some embodiments, the graft putty can be used to repair defects in non-union bones or other situations where the fracture, hole, or void to be filled is large and requires some mechanical integrity from the implant material to both fill the gap and retain its shape.

[0094] The methods described herein may be used to treat cartilage or bone lesions or injuries in humans or other animal subjects, and include applying to a site a composition comprising the cells and / or factors described herein, which may be provided in combination with cements, factors, gels, and the like. As referred to herein, such lesions include any condition involving skeletal tissue, including cartilage, tissue, that is unsuitable for physiological or cosmetic purposes. Such defects include defects that are congenital, result from disease or trauma, and result from surgical or other medical procedures. Such defects include, for example, bone defects resulting from injury, defects caused during the course of surgery, osteoarthritis, osteoporosis, infections, malignancies, developmental deformities, as well as simple fractures, compound fractures, transverse fractures, pathological fractures, avulsion fractures, greenstick fractures, and comminuted fractures. In some embodiments, the bone defect is a void in the bone that needs to be filled with a bone precursor composition.

[0095] The cells described herein can also be genetically modified to enhance their ability to participate in tissue regeneration or to deliver therapeutic genes to the site of administration. To that end, vectors can be designed using the known coding sequence of the desired gene operably linked to a promoter that is either pan-specific or specifically active in differentiated cell types. Of particular interest are cells genetically modified to express bone morphogenetic proteins such as BMP-2 or BMP-4. See WO99 / 39724. The production of these or other growth factors at the site of administration can enhance the beneficial effects of the administered cells or increase the proliferation or activity of host cells adjacent to the treatment site.

[0096] Research Use and Drug Screening The cells described herein can also be used as research or drug discovery tools, e.g., to assess the phenotype of a genetic disease, e.g., to better understand the pathogenesis of the disease, to identify target proteins for therapeutic treatment, to identify candidate agents with disease-modifying activity, e.g., to identify agents that would be effective in treating a subject. For example, a candidate agent can be added to a cell culture containing SSCs derived from a subject, and the effect of the candidate agent assessed by monitoring output parameters such as survival, ability to form bone or cartilage, by methods described herein and in the art.

[0097] A parameter is a quantifiable component of a cell, one that can be accurately measured, especially in a desirably high throughput system. A parameter can be any cellular component or cell product, including cell surface determinants, receptors, proteins or their conformational or post-translational modifications, lipids, carbohydrates, organic or inorganic molecules, nucleic acids, such as mRNA, DNA, etc., or moieties derived from such cellular components, or combinations thereof. Most parameters will provide a quantitative readout, but in some cases semi-quantitative or qualitative results will be acceptable. The readout may include a single determined value, or may include the mean, median, or variance, etc. Characteristically, a range of parameter readouts will be obtained for each parameter from multiple identical assays. Variability is expected, and the range of values ​​for each of the set of test parameters will be obtained using standard statistical methods, with a common statistical method used to provide a single value.

[0098] Candidate agents of interest for screening include known and unknown compounds from many chemical classes, primarily organic molecules, which may include organometallic molecules, inorganic molecules, genetic sequences, etc. An important aspect of the present invention is the evaluation of candidate drugs, including toxicity testing, etc.

[0099] Examples of candidate agents include organic molecules that contain functional groups necessary for structural interactions, particularly hydrogen bonding, typically at least amine, carbonyl, hydroxyl, or carboxyl groups, and often at least two of the functional chemical groups. Candidate agents may include cyclic carbon or heterocyclic structures and / or aromatic or polyaromatic structures substituted with one or more of the above functional groups. Candidate agents may be biomolecules including peptides, polynucleotides, saccharides, fatty acids, steroids, purines, pyrimidines, derivatives, structural analogs, or combinations thereof. Further examples include pharmacologically active drugs, genetically active molecules, and the like. Compounds of interest include chemotherapeutic agents, hormones, or hormone antagonists, and the like. Exemplary pharmaceutical agents suitable for the present invention are those described in "The Pharmacological Basis of Therapeutics," Goodman and Gilman, McGraw-Hill, New York, NY, (1996), Ninth edition.

[0100] In one example, the cells and methods described herein are useful for screening candidate agents for activity modulating cell conversion to cells of the skeletal or chondrogenic lineage, e.g., chondrocytes, osteoblasts, or their precursor cells. In a screening assay for a bioactive agent, cells can be contacted with a candidate agent of interest in the presence of a cellular reprogramming or differentiation system or an incomplete cellular reprogramming or differentiation system, and the effect of the candidate agent is assessed by monitoring an output parameter, such as expression levels of genes specific to the desired cell type, as known in the art, or the ability of the cells to be induced to function like a desired cell type, as known in the art.

[0101] kit Provided herein are kits for identifying, isolating or enriching suture or skeletal stem cells.

[0102] The kit may include one or more agents (e.g., antibodies or nucleic acid probes) specific for one or more makers described herein. In one example, the kit includes a system for contacting a biological sample with one or more marker-specific antibodies or antigen-binding fragments thereof, and instructions for use thereof. In one embodiment, the kit further includes a culture system (e.g., cell culture medium or cell culture devices, or both) for culturing, maintaining, or expanding a population of cells, and instructions for use thereof. The kit may include one or more agents for measuring marker protein or mRNA transcription levels.

[0103] The agent may be, for example, a reagent for performing an analytical method for detecting a protein or measuring a protein level. Examples of such analytical methods include, but are not limited to, FACS, immunohistochemical staining assay, Western blotting, ELISA, radioimmunoassay, radial immunodiffusion, Ouchterlony immunodiffusion assay, rocket immunoelectrophoresis assay, immunoprecipitation assay, complement fixation assay, protein chip assay, and the like. The kit may include reagents for detecting antibodies forming antigen-antibody complexes, such as labeled secondary antibodies, chromophores, enzymes (e.g., conjugated with antibodies) and their substrates. In addition, it may include an antibody specific to a control protein for quantification. The amount of antigen-antibody complexes formed may be quantitatively determined by measuring the signal intensity of the detection label. Such detection labels may be selected from the group consisting of, but are not limited to, enzymes, fluorescent substances, ligands, luminescent substances, microparticles, redox molecules, and radioisotopes.

[0104] To detect mRNA transcripts, the kit may include reagents (e.g., probes or PCR primers) for extracting mRNA and measuring the expression level of the mRNA of the markers described herein. The expression of mRNA may be measured by any one selected from the group consisting of, but not limited to, in situ hybridization, polymerase chain reaction (PCR), reverse transcription polymerase chain reaction (RT-PCR), real-time PCR, RNase protection assay (RPA), microarray, and northern blotting.

[0105] A kit is also provided that includes the SSC, sphere, composition, product, or formulation described herein. Optionally, the kit includes one or more of the scaffolds described herein. The SSC, sphere, composition, product, or formulation is provided in a kit, pack, or dispenser that can contain one or more unit dosage forms containing the active ingredient. The kit can include, for example, a metal or plastic foil such as a blister pack. The kit, pack, or dispenser can be accompanied by instructions for administration.

[0106] definition The terms "polypeptide" or "protein" are used interchangeably herein and refer to a polymer of amino acid residues and their derivatives. The terms apply to amino acid polymers in which one or more amino acid residues are analogs or mimetics of the corresponding naturally occurring amino acids, as well as to naturally occurring amino acid polymers. The terms can also encompass amino acid polymers that have been modified, for example, by the addition of carbohydrate residues to form glycoproteins, or that have been phosphorylated. Polypeptides and proteins can be produced by naturally occurring non-recombinant cells, or can be produced by genetically engineered or recombinant cells, and include molecules having the amino acid sequence of a native protein, or molecules having deletions, additions, and / or substitutions of one or more amino acids of the native sequence. The terms "polypeptide" and "protein" specifically encompass antigen binding proteins, antibodies, or sequences that have deletions, additions, and / or substitutions of one or more amino acids of the antigen binding protein. The term "polypeptide fragment" refers to a polypeptide that has an amino-terminal deletion, a carboxyl-terminal deletion, and / or an internal deletion compared to the full-length protein. Such fragments may contain modified amino acids compared to the full-length protein. In certain embodiments, the fragments are about 5-500 amino acids in length. For example, fragments can be at least 5, 6, 8, 10, 14, 20, 50, 70, 100, 110, 150, 200, 250, 300, 350, 400, or 450 amino acids in length. Useful polypeptide fragments include immunologically functional fragments of antibodies that contain the binding domain.

[0107] The term "isolated polypeptide" refers to a polypeptide that is separated from at least about 50 percent of the polypeptides, peptides, lipids, carbohydrates, polynucleotides, or other materials with which the polypeptide is naturally found when isolated from a source cell. Preferably, an isolated polypeptide is substantially free of any other contaminating polypeptides or other contaminants found in its natural environment that would interfere with its therapeutic, diagnostic, prophylactic, or research uses.

[0108] The term "antibody" as referred to herein includes whole antibodies and any antigen-binding fragment or single chain thereof. Whole antibodies are glycoproteins comprising at least two heavy (H) chains and two light (L) chains interconnected by disulfide bonds. Each heavy chain comprises a heavy chain variable region (herein referred to as V H The heavy chain constant region is abbreviated as C H 1. C H 2, and C H Each light chain is composed of three domains: a light chain variable region (herein referred to as V L The light chain constant region consists of one domain, C L It consists of. V H and V L The regions can be further subdivided into regions of hypervariability, called complementarity determining regions (CDRs), interspersed with more conserved regions, called framework regions (FRs). H and V L consists of three CDRs and four FRs arranged in the following order from amino terminus to carboxy terminus: FRl, CDRl, FR2, CDR2, FR3, CDR3, FR4. The heavy chain variable region CDRs and FRs are HFRl, HCDRl, HFR2, HCDR2, HFR3, HCDR3, HFR4. The light chain variable region CDRs and FRs are LFRl, LCDRl, LFR2, LCDR2, LFR3, LCDR3, LFR4. The variable regions of the heavy and light chains contain binding domains that interact with antigens. The constant region of the antibody can mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (CIq) of the classical complement system.

[0109] Thus, the term "antibody(s)" includes full length antibodies, antigen-binding fragments of full length antibodies, and molecules comprising an antibody CDR, VH region, or VL region. Exemplary antibodies include monoclonal antibodies, recombinantly produced antibodies, monospecific antibodies, multispecific antibodies (including bispecific antibodies), human antibodies, humanized antibodies, chimeric antibodies, immunoglobulins, synthetic antibodies, tetrameric antibodies comprising two heavy and two light chain molecules, antibody light chain monomers, antibody heavy chain monomers, antibody light chain dimers, antibody heavy chain dimers, antibody light chain-antibody heavy chain pairs, intrabodies, heteroconjugate antibodies, single domain antibodies, monovalent antibodies, single chain antibodies or single chain Fvs (scFvs), scFv-Fcs, camelid antibodies (e.g., llama antibodies), camelized antibodies, affibodies, Fab fragments, F(ab')2 fragments, disulfide-linked Fvs (sdFvs), anti-idiotypic (anti-Id) antibodies (including, for example, anti-anti-Id antibodies), and antigen-binding fragments of any of the above. In certain embodiments, the antibodies disclosed herein refer to polyclonal antibody populations. The antibody may be of any type (e.g., IgG, IgE, IgM, IgD, IgA, or IgY), any class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, or IgA2), or any subclass (e.g., IgG 2a or IgG 2b ) immunoglobulin molecule. In certain embodiments, the antibodies disclosed herein are IgG antibodies, or classes (e.g., human IgG1 or IgG4) or subclasses thereof. In certain embodiments, the antibodies are humanized monoclonal antibodies.

[0110] The term "antigen-binding fragment or portion" of an antibody (or simply "antibody fragment or portion"), as used herein, refers to one or more fragments of an antibody that retain the ability to specifically bind to an antigen. It has been shown that the antigen-binding function of an antibody can be performed by fragments of a full-length antibody. Examples of binding fragments encompassed by the term "antigen-binding fragment or portion" of an antibody include (i) Fab fragments, V L , V H , C L and C H(ii) a F(ab')2 fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; and (iii) a Fab' fragment, which is essentially a Fab with part of the hinge region (FUNDAMENTAL IMMUNOLOGY (Paul ed., 3 rd ed. 1993), (iv) V H and C H (v) an Fd fragment consisting of an I domain, and (v) a V of a single arm of an antibody L and VH domains, (vi) dAb fragments consisting of the VH domain (Ward et al., (1989) Nature 341:544-546), (vii) isolated CDRs, and (viii) nanobodies, a heavy chain variable region containing a single variable domain and two constant domains. Furthermore, although the two domains of the Fv fragment, VL and VH, are encoded by separate genes, they can be joined by a synthetic linker that allows them to be produced using recombinant methods as a single protein chain that allows the VL and VH domains to pair to form a monovalent molecule (known as single-chain Fv or scFv); see, e.g., Bird et al. (1988) Science 242:423-426; and Huston et al. (1988) Proc. Natl. Acad. Sci. USA 85:5879-5883). Such single chain antibodies are also intended to be encompassed within the term "antigen-binding fragment or portion" of an antibody. These antibody fragments are obtained using conventional techniques known to those of skill in the art, and the fragments are screened for utility in the same manner as are intact antibodies.

[0111] An "isolated antibody," as used herein, is intended to refer to an antibody that is substantially free of other antibodies having different antigenic specificities (e.g., an isolated antibody that specifically binds to a particular antigen is substantially free of antibodies that specifically bind to antigens other than the particular antigen). An isolated antibody can be substantially free of other cellular material and / or chemicals.

[0112] As used herein, the term "administering" refers to the delivery of the composition of the present invention by any suitable route. Cells can be administered in many ways, including but not limited to parenteral (such term refers to intravenous and intraarterial, as well as other suitable parenteral routes), intrathecal, intracerebroventricular, intraparenchymal, intracisternal, intracranial, intrastriatal, intranigra, intranasal, intraperitoneal, intramuscular, subcutaneous, intradermal, transdermal, or transmucosal administration, among others, which allows the cells to migrate to the final target site as necessary. Multiple units of cells can be administered to a patient simultaneously or sequentially (e.g., over the course of minutes, hours, or days).

[0113] The terms "grafting" and "transplanting" as well as "graft" and "transplantation" are used to describe the process by which cells are delivered to the site where they are intended to have a desired effect, such as repairing damage to a patient's bone or cartilage. Cells may also be delivered to distant regions of the body by any of the modes of administration described above, depending on cell migration to the appropriate area to achieve engraftment.

[0114] The term "therapeutic composition" or "pharmaceutical composition" refers to a combination of an active agent with a carrier, inert or active, that makes the composition particularly suitable for diagnostic or therapeutic uses, in vivo or ex vivo.

[0115] As used herein, "therapeutic cells" refers to a cell population that ameliorates a patient's condition, disease, and / or injury. Therapeutic cells can be autologous (i.e., derived from the patient), allogeneic (i.e., derived from an individual of the same species but different from the patient), or xenogeneic (i.e., derived from a species but different from the patient). Therapeutic cells can be homogeneous (i.e., composed of a single cell type) or heterogeneous (i.e., composed of multiple cell types). The term "therapeutic cells" includes both therapeutic active cells and progenitor cells that can differentiate into therapeutic active cells.

[0116] The phrase "pharmacologically acceptable" is used herein to refer to those compounds, materials, compositions, and / or dosage forms that are suitable for use in contact with human and animal tissues, within the scope of sound medical judgment, without excessive toxicity, irritation, allergic response, or other problems or complications commensurate with a reasonable benefit / risk ratio. A "pharmacologically acceptable carrier" does not cause undesirable physiological effects after administration to or on a subject. A carrier in a pharmaceutical composition should also be "acceptable" in the sense that it may be compatible with and capable of stabilizing the active ingredient. One or more solubilizing agents may be utilized as pharmaceutical carriers to deliver the active compound. Examples of pharma-ceutical acceptable carriers include, but are not limited to, biocompatible vehicles, adjuvants, additives, and diluents to achieve a composition that can be used as a dosage form. Other examples of carriers include colloidal silicon oxide, magnesium stearate, cellulose, and sodium lauryl sulfate.

[0117] As used herein, the term "subject(s)" may refer to any vertebrate, including, but not limited to, mammals (e.g., cows, pigs, camels, llamas, horses, goats, rabbits, sheep, hamsters, guinea pigs, cats, dogs, rats, and mice, non-human primates (e.g., monkeys such as cynomolgus monkeys and chimpanzees), and humans). The term "subject" includes humans and non-human animals. A preferred subject for treatment is a human. As used herein, the terms "subject" and "patient" are used interchangeably, regardless of whether the subject has undergone or is currently undergoing any form of treatment. In one embodiment, the subject is a human. In another embodiment, the subject is a non-human animal for experiments or an animal suitable as a disease model.

[0118] The term "patient" is used herein to describe an animal, preferably a human, to whom therapy, including prophylactic treatment, with cells according to the invention is provided. The term "donor" is used to describe an individual (animal, including human) who provides cells or tissues for use in a patient.

[0119] The term "primary culture" refers to a mixed cell population of cells derived from an organ or tissue within an organ. The term "primary" has its usual meaning in the field of tissue culture.

[0120] "Tissue" refers to a group or layer of similarly specialized cells that together perform some particular specialized function.

[0121] The terms "treatment", "treating", "treating" and the like are generally used herein to refer to obtaining a desired pharmacological and / or physiological effect. The effect may be preventative in that it completely or partially prevents the disease or its symptoms, and / or it may be therapeutic in that it partially or completely stabilizes or cures the disease and / or the adverse effects caused by the disease. As used herein, "treatment" encompasses any treatment of a disease in a mammal, particularly a human, and includes (a) preventing the disease or condition from occurring in a subject who may be predisposed to the disease or condition, but has not yet been diagnosed as having it, (b) inhibiting a disease symptom, i.e., arresting its development, or (c) eliminating a disease symptom, i.e., causing regression of the disease or condition. The terms "prevent", "preventing", "prevention", "prophylactic treatment", and the like refer to reducing the probability of developing a disorder or condition in a subject who does not have the disorder or condition, but who is at risk of or susceptible to developing the disorder or condition. "Ameliorating" generally refers to reducing the number or severity of the signs or symptoms of a disease or disorder.

[0122] "Prophylactic treatment" includes treatment administered to a subject who does not exhibit signs or symptoms of a condition, such that the treatment is administered with the intent to alleviate, prevent, or reduce the risk of developing the condition. "Therapeutic treatment" includes treatment administered to a subject who exhibits signs or symptoms of a condition, with the intent of reducing the severity or progression of the condition. Therapeutic treatment may also partially or completely eliminate the condition.

[0123] "Effective amount" generally refers to an amount that provides a desired local or systemic effect. For example, an effective amount is an amount that is sufficient to achieve a beneficial or desired clinical result. An effective amount can be provided all at once in a single administration, or in a divided amount that provides an effective amount in several administrations. The exact determination of the amount that will be considered an effective amount can be based on individual factors for each subject, including the subject's size, age, injury, and / or disease or injury being treated, and the amount of time since the injury occurred or the disease began. A person skilled in the art will be able to determine the effective amount for a given subject based on these considerations, which are routine in the art. As used herein, "effective dose" means the same as "effective amount."

[0124] As used herein, the term "stem cell" refers to a cell that has the ability to both replace itself and differentiate into more specialized cells. Their self-renewal ability generally persists for the life of an organism. Pluripotent stem cells can give rise to all of the various cell types of the body. Multipotent stem cells can give rise to a limited subset of cell types. For example, hematopoietic stem cells can give rise to the various types of cells found in the blood, but cannot give rise to other types of cells. Multipotent stem cells can also be referred to as somatic stem cells, tissue stem cells, lineage-specific stem cells, and adult stem cells. The non-stem cell progeny of multipotent stem cells are progenitor cells (also referred to as restricted progenitor cells). Progenitor cells give rise to fully differentiated cells, but give rise to a more restricted set of cell types than stem cells. Progenitor cells also have a relatively limited ability to self-renew, and as they divide and differentiate, they eventually wear out and are replaced by new progenitor cells derived from their upstream multipotent stem cells.

[0125] The term "skeletal stem cells" refers to multipotent and self-renewing cells that can generate bone marrow stromal cells, skeletal cells, and chondrogenic cells. Self-renewal means that when they undergo mitosis, they produce at least one daughter cell that is a skeletal stem cell. Multipotent means that they can give rise to precursor cells (skeletal precursors) that give rise to all cell types of the skeletal system. They are not multipotent, i.e., they cannot give rise to cells of other organs in vivo.

[0126] Skeletal stem cells can be reprogrammed from non-skeletal cells, including but not limited to mesenchymal stem cells and adipose tissue containing such cells. Reprogrammed cells can be referred to as induced skeletal stem cells, or iSSCs. "iSSCs" arise from non-skeletal cells by experimental manipulation. Induced skeletal cells have the characteristics of naturally occurring functional SSCs, i.e., they can give rise to the same lineage. Human SSC cell populations can be negative for the expression of certain markers, such as CD45, CD235, Tie2, and CD31, and positive for the expression of other markers, such as podoplanin (PDPN). Mouse skeletal lineages can be characterized as CD45-, Ter119-, Tie2-, αv integrin+. Mouse SSCs can be further characterized as Thy1-6C3-CD105-CD200+.

[0127] Suture stem cells (SuSCs) refer to a population of skeletal stem cells derived from suture mesenchyme that exhibit long-term self-renewal, clonal expansion, and multipotency. These SuSCs reside at the sutural midline and function as a skeletal stem cell population responsible for calvarial development, homeostasis, injury repair, and regeneration. Suture stem cells are a stem cell population that are naturally programmed to form intramembranous bone during craniofacial skeletogenesis.

[0128] "Chondrocytes (cartilage cells)" refers to cells capable of expressing characteristic biochemical markers of chondrocytes, including but not limited to, type II collagen, chondroitin sulfate, keratan sulfate, and characteristic morphological markers of chondrocytes, including but not limited to, the rounded morphology observed in culture, and capable of secreting type II collagen, including but not limited to, producing tissue or matrix with the hemodynamic properties of cartilage in vitro.

[0129] As used herein, the phrase "maintaining stem cells" refers not only to culturing stem cells in a manner that maintains their viability, but also to retaining their functionality as stem cells, i.e., being self-renewing and capable of giving rise to the full range of precursor lineages appropriate for a particular type of stem cell (these two together function as "regenerative activity"). One way to show that stem cells are successfully maintained is through engraftment experiments, where all appropriate cell types (with genetic markers that distinguish them from the host) are observed to arise from the graft and continue to exist for an extended period of time, e.g., four months.

[0130] As used herein, the phrase "expanding stem cells" refers to culturing stem cells in a manner that increases the number of stem cells in culture, as well as maintaining stem cells.One way to show that stem cells are successfully expanded is an engraftment experiment that compares the percentage of donor-derived cells obtained from transplantation of cultured stem cells and freshly isolated stem cells.The comparison is based on transplanting the same number of freshly isolated stem cells as were originally placed in culture.An increase in the percentage of donor-derived cells in recipients of cultured stem cells compared to recipients of freshly isolated stem cells is consistent with successful expansion of stem cells in culture.

[0131] A "marker" or "biomarker" is a molecule useful as an indicator of a biological state in a subject. The markers or biomarkers disclosed herein can be polypeptides that exhibit a change in expression or state, which can correlate with cell development, differentiation, or fate. In addition, the markers disclosed herein include messenger RNA (mRNA) that encodes a marker polypeptide, since measuring changes in expression of the mRNA can correlate with changes in expression of the polypeptide encoded by the mRNA. Thus, determining the amount of a biomarker in a biological sample includes determining the amount of the polypeptide biomarker and / or the amount of the mRNA encoding the polypeptide biomarker, either by measuring the mRNA directly or indirectly (e.g., by measuring complementary DNA (cDNA) synthesized from the mRNA).

[0132] In the context of skeletal stem cells, "marker" or "biomarker" means that in a culture or tissue containing cells that are programmed to become skeletal stem cells, the marker is expressed only by cells of the culture or tissue that will, are developing, and / or have developed into skeletal stem cells. It will be understood by those skilled in the art that the expression level specified reflects the detectable amount of marker protein or mRNA on or in the cell. Cells that stain negatively (the binding level of the marker-specific reagent is not detectably different from an isotype-matched control) may still express small amounts of the marker. Also, while it is common in the art to refer to cells as "positive" or "negative" for a particular marker, the actual expression level is a quantitative property. The number of molecules on the cell surface may vary by several logs, but still be characterized as "positive".

[0133] Standard gene / protein nomenclature guidelines generally stipulate that for humans and all non-human primates, livestock species, and all but mouse, rat, fish, worm, or fly, the default gene name abbreviation / symbol is capitalized and italicized (e.g., BMPR1A) and the protein name abbreviation is capitalized but not italicized (e.g., BMPR1A). Additionally, standard gene / protein nomenclature guidelines generally stipulate that for mouse, rat, and chicken, the default gene name abbreviation / symbol is capitalized and italicized (e.g., Bmpr1a) and the protein name abbreviation is capitalized but not italicized (e.g., Bmpr1a).

[0134] In contrast, the gene / protein nomenclature used herein when referring to specific biomarkers includes the biomarker abbreviations in all capital letters (e.g., BMPR1A, PTHLH, ERG, SIX2, ALX4, BMP2, EFNB1, FGFR1, FGFR2, SMAD6, SPRY1, TWIST1, ZIC1, CREB3L1, ENG, CTGF, TEAD2, WWTR1, AMOTL1, SAV1, AMOTL2, TEAD1, HES1, PDGFRB, SIX1, SOX4, JAG1, MYLK, TBX2, and COL 3A1), or those including lowercase letters (e.g., Bmpr1a, Pthlh, Erg, Six2, Alx4, Bmp2, Efnb1, Fgfr1, Fgfr2, Smad6, Spry1, Twist1, Zic1, Creb311, Eng, Ctgf, Tead2, Wwtr1, Amotl1, Sav1, Amotl2, Tead1, Hes1, Pdgfrb, Six1, Sox4, Jag1, Mylk, Tbx2, and Col3a1) are intended to include genes (including mRNA and cDNA) and proteins across animal species. That is, when referring to manufacturers in this application, capitalized and non-capitalized name abbreviations / symbols are used interchangeably unless the context dictates otherwise (e.g., in the Examples below, associated figures, and associated description). Thus, the exemplary human or mouse biomarkers described herein are not intended to limit the subject matter to only human or mouse polypeptide or mRNA biomarkers, but rather, the subject matter encompasses biomarkers across animal species that are associated with SSCs.

[0135] As disclosed herein, several ranges of values ​​are provided. It is understood that each intervening value, to the tenth of the unit of the lower limit, between the upper and lower limits of the range is also specifically disclosed, unless the context clearly dictates otherwise. Each smaller range between any stated or intervening value in a stated range and any other stated or intervening value in that stated range is encompassed within the invention. The upper and lower limits of these smaller ranges may be independently included or excluded within the range, and each range in which either, neither, or both limits are included within the smaller range is also encompassed within the invention, subject to any specifically excluded limits in the stated range. If a stated range includes one or both of the limits, then ranges excluding either or both of those included limits are also included in the invention.

[0136] The term "about" or "approximately" means within an acceptable range for a particular value as determined by one of ordinary skill in the art, which depends in part on how the value is measured or determined, e.g., the limitations of the measurement system. For example, "about" can mean a range of up to 20%, preferably up to 10%, more preferably up to 5%, and even more preferably up to 1% of a given value. Unless otherwise specified, the term "about" means within an acceptable range of error for a particular value. EXAMPLES

[0137] Using single-cell RNA sequencing analysis, we first investigated the heterogeneity of the suture mesenchyme by defining the presence of multiple cell lineages. Visualization of the high-dimensional t-distributed stochastic neighbor embedding (tSNE) algorithm grouped all suture cells into 10 distinct clusters (Figure 1A and Figure 1B).

[0138] We then used expression of the Runx2, Mcam, and Rac2 genes to subgroup them into three major lineages: skeletal, vascular, and hematopoietic cell lineages, respectively ( Fig. 1C ).

[0139] We next further investigated the cell types present in the Runx2+ skeletal cell lineage, revealing the identity of five clusters (Figure 2A). Markers expressed in various osteoblasts and chondrocytes were used to identify their corresponding clusters (Figures 2B and 2C). Clusters negative for osteoblast and chondrocyte markers represent mesenchymal cell (MC) clusters.

[0140] Cell proliferation markers were able to identify proliferating mesenchymal cells and proliferating chondrocyte clusters (Figure 2D). Using a similar approach, we identified three clusters, HC1-3, in the hematopoietic cell lineage (Figure 3A). The HC1 cluster was composed of closely related monocytes, macrophages, dendritic cells, and osteoclasts (Figures 3B and 3C). The HC2 cluster contained T and B lymphocytes, while NK cells and neutrophils were included in the HC3 cluster (Figures 3D and 3E). The vascular cell lineage contains three major cell types: endothelial cells, pericytes, and vascular smooth muscle cells. Using genes expressed in these cells, we characterized the two VC1-2 clusters of the vascular cell lineage (Figure 4A). Endothelial cell markers were highly expressed in the VC1 cluster, whereas pericytes and vascular smooth muscle cells were concentrated in the VC2 cluster (Figure 4B).

[0141] After successfully identifying all 10 clusters, several known skeletal stem and progenitor cell markers were used to test whether their positive cells were present in the expected MC clusters (Figure 5). Expression of all of these marker genes, including Axin2, could be found in the MC clusters. However, their expression was not restricted to MCs and could also be identified in other clusters.

[0142] Axin2-expressing cells have previously been shown to be bona fide skeletal stem cells capable of generating bone at ectopic sites and repairing large bone defects by direct engraftment and replacement of damaged tissue. Therefore, we performed assays to further characterize the Axin2+ cell population using single-cell RNA sequencing (scRNA-seq) to purify the stem cell population, thereby identifying potential stem cell markers and elucidating the mechanisms underlying stem cell regulation.

[0143] First, Axin2+ cells were isolated from calvarial sutures using FACS-based cell sorting (Figure 6A). Consistent with previous findings, Axin2+ cells constituted 3-7% of the suture cell population (Figure 6B). Next, single-cell RNA-seq analysis was performed to further examine Axin2-expressing suture cells. Visualization with the high-dimensional uniform manifold approximation and projection (UMAP) algorithm grouped the Axin2+ cell population into seven clusters (Figure 7). These clusters consisted of neutrophils, dendritic cells, proliferating hematopoietic cells, natural killer cells, monocytes, skeletogenic cells, and lymphocytes based on the expression of genetic markers (Figure 8A-B).

[0144] As we have previously shown that the BMP1 receptor Bmpr1a is a stem cell marker, we performed an assay to determine which clusters contained Bmpr1a-expressing cells. Surprisingly, we found that Bmpr1a+ cells were highly concentrated within the SC (skeletogenic cell) cluster, which contained only 244 cells, suggesting an enrichment of highly potent skeletal stem cells within this cluster (Figure 8C).

[0145] Thus, these results allow us to identify genes expressed in the SC cluster as additional markers and potential regulators of skeletal stem cells in sutures. First, we performed an assay to examine genes whose mutations are associated with human patients with craniosynostosis, a devastating childhood disease caused by suturing abnormalities. Of the 83 craniosynostosis genes examined, 27 of them, such as Fgfr1, Fgfr2, Twist1, Smo, Alx4, Bmp2, Bmper, Efnb1, Smad6, Spry1, Msx1, and Sox6, are highly enriched in the SC cluster (Figure 9). The remaining 56 genes can be grouped into high enrichment in the SC but also present in other clusters (6 genes), low expression in the SC and other clusters (12 genes), and uniform expression in all clusters (38 genes).

[0146] To further examine the SC cluster, reclustering was performed to exclude cells from the hematopoietic lineage and refine the remaining cells into five subclusters (Figures 10A and 10B). Analysis of BMP signaling reveals its close connection to the SC subcluster (Figure 10C), consolidating previous findings of Bmpr1a being essential to maintain stemness of stem cells. Thus, skeletal stem cells are included in this cluster, enriched for Bmpr1a expression. In addition, expression of Bmp2, Acvr1, and Bmpr2 is highly restricted to the SC subcluster. A more detailed examination of genes related to BMP signaling shows expression within the SC subcluster, e.g., Bmpr1a, Acvr1, Bambi, Bmp2, Bmp3, Bmp4, Bmp6, and Bmp7 (Figure 11). Similar to those described in Figure 9, 27 of the 83 craniosynostosis genes also showed enriched distribution in the SC subcluster in the recluster plot (Figure 12). 95 genes were identified that are specifically restricted to the SC subcluster (Table 2). Their gene products are membrane-associated cell surface proteins and therefore can be used for skeletal stem cell purification. Table 2. Exemplary cell surface markers for SSCs TIFF2025506727000082.tif242170TIFF2025506727000083.tif243170TIFF20255067270 00084.tif243170TIFF2025506727000085.tif251170TIFF2025506727000086.tif215170

[0147] The above examples and description of the preferred embodiments should be considered as illustrative, rather than limiting, the present invention as defined by the claims. As will be readily understood, numerous variations and combinations of the features defined above may be utilized without departing from the invention as defined in the claims. Such variations are not considered to depart from the scope of the present invention, and all such variations are intended to be included within the scope of the following claims. All references cited herein are incorporated by reference in their entirety.

Claims

1. 1. A method for identifying or isolating or enriching skeletal stem cells, comprising obtaining a starting cell population; From the starting cell population, a first marker expressed by cells, the first marker being Bmpr1a, Bmpr2, Fdz1, Fgfr2, Lrig3, Itgbl1, Apoe, Gpc3, Lpl, Sulf2, Cdon, Tgfbr2, Tgfbr3, Lrrc15, Mif, Axl, Itgav, Fgfr1, Jag1, Acvr1, Acvr2a, A and identifying a first marker selected from the group consisting of cvr2b, Erg, Six2, Pthlh, Twist1, Alpl, Msx1, Efnb1, Zic1, Spry1, Abcc9, Erf, Bmp2, Bmp3, Bmp4, Bmp5, Bmp6, Bmp7, Bmp8a, Bambi, Bmper, and Col3a1.

2. The method of claim 1 , further comprising isolating the cells that express the first marker.

3. 2. The method of any one of the preceding claims, further comprising collecting a plurality of cells expressing said first marker to obtain an enriched skeletal stem cell population.

4. 2. The method of any one of the preceding claims, wherein the cells are identified, isolated or enriched using a first protein, first polypeptide, first nucleic acid, or first composition that specifically binds to the first marker.

5. 2. The method of any one of the preceding claims, further comprising identifying from the starting cell population a second marker expressed by the cells expressing the first marker, wherein the second marker is selected from the group.

6. 6. The method of claim 5, wherein the cells are identified, isolated, or enriched using a second protein, a second polypeptide, a second nucleic acid, or a second composition that specifically binds to the second marker.

7. 7. The method of claim 6, further comprising collecting a plurality of cells expressing said first and second markers to obtain an enriched skeletal stem cell population.

8. 7. The method of claim 6, further comprising identifying from the starting cell population one or more additional markers expressed by cells that co-express both the first marker and the second marker, each of the one or more additional markers being individually identified using the same method as the first marker and the second marker.

9. 9. The method of claim 8, resulting in a cell that co-expresses the first marker, the second marker, and one or more additional markers.

10. 10. The method of claim 9, further comprising collecting a plurality of cells expressing the first marker, the second marker, and one or more markers to obtain an enriched skeletal stem cell population.

11. 10. The method of any one of the preceding claims, wherein the starting cell population is derived from a tissue of a subject.

12. The method of claim 11 , wherein the subject is a mammal.

13. The method of claim 12, wherein the mammal is a human.

14. 2. The method of any one of the preceding claims, wherein the starting cell population comprises one or more selected from the group consisting of bone marrow, umbilical cord blood cells, embryonic stem cells or their progeny, mesenchymal stem cells or their progeny, and induced pluripotent stem cells (iPSCs) or their progeny.

15. The method of claim 14 , wherein the mesenchymal stem cells are suture mesenchymal stem cells.

16. A composition comprising (i) a carrier and (ii) one or more cells identified, isolated or enriched according to the method of claims 1 to 15, or cells derived therefrom.

17. The composition of claim 16, wherein the composition is a pharmaceutical composition and the carrier is a pharma- ceutically acceptable carrier.

18. 20. The composition of claim 17, wherein the composition is an in vitro cell culture composition and the carrier comprises culture medium.

19. A bone regeneration product or formulation comprising: (i) the composition according to any one of claims 16 to 18; and (ii) a scaffold.

20. A method for generating or regenerating cartilage or bone in a subject, the method comprising administering to a subject in need thereof an effective amount of a composition according to any one of claims 16 to 18, or a bone regeneration product or formulation according to claim 15, to a site where bone or cartilage regeneration is desired.

21. 1. A method for producing skeletal, stromal, or cartilage tissue, comprising: (i) providing one or more cells obtained according to any one of claims 1 to 15; (ii) inducing differentiation of said one or more cells or cells derived therefrom.

22. 22. The method of claim 21 , wherein the one or more cells express a second, different marker selected from the group.

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