Isolation of 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 Patent Application No. 63 / 154,293, filed February 26, 2021, the contents of which 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. Possessing the "tri-lineage" potential to differentiate into chondrocytes, osteoblasts, bone marrow stromal cells, or adipocytes, SSCs play a crucial role in the development, homeostasis, and regeneration of bone tissue. For example, suture mesenchyme-derived SSCs, 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 identities of SuSCs and SSCs remain largely elusive. Currently, the lack of cell surface markers for the isolation of such stem cells and the inability to maintain stemness properties ex vivo are two significant hurdles limiting further progress in the field of skeletal regeneration. Thus, there is a need for cell surface markers, associated cell identification and isolation methods, and in vitro or ex vivo maintenance methods. Summary of the Invention

[0005] The present application addresses the above needs in several aspects. In one aspect, the present application provides a method for identifying, isolating, or enriching skeletal stem cells or suture stem cells. The method includes obtaining a starting cell population, isolating from the starting cell population cells that express a BMPR1A polypeptide (BMPR1A + In one embodiment, the method comprises identifying a cell expressing an identified BMPR1A polypeptide, or a cell expressing mRNA encoding the BMPR1A polypeptide. + In one embodiment, the method further comprises isolating the cells. + The method further includes harvesting the cells to obtain an enriched suture stem cell population or an enriched skeletal stem cell population.

[0006] In the above method, the cells may express one or more markers selected from the group consisting of Axin2, BMP2, BMP3, BMP4, BMP6, BMP7, BMP8b, and BMP15. The cells may also be positive for one or more positive markers selected from the group listed in Table 1 below. The cells may also be negative for one or more negative markers selected from the group listed in Table 2 below.

[0007] In some embodiments, BMPR1A + The cells can be isolated using a protein, polypeptide, or composition that specifically binds to BMPR1A. In one embodiment, the protein comprises an anti-BMPR1A antibody or an antigen-binding fragment thereof. In another embodiment, the protein, peptide, or composition comprises a ligand of BMPR1A or a BMPR1A-binding fragment thereof. Examples of ligands include BMP2, BMP4, BMP6, BMP7, and GDF6. BMPR1A + Cells may be identified, isolated, or enriched by fluorescence-activated cell sorting.

[0008] In the methods described herein, the starting cell population can be derived from a tissue of a subject, such as a vertebrate, mammal (including human and non-human mammal). The starting cell population can comprise 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.

[0009] In a second aspect, the present application provides a method for maintaining suture or skeletal stem cells in vitro, the method comprising: (i) providing a population of suture or skeletal stem cells, (ii) plating the cells in maintenance medium on a low or ultra-low attachment surface, and (iii) culturing the cells or their progeny for a period of time to form one or more spheres.

[0010] The population may be derived from tissue of a subject, such as a vertebrate, mammal (including humans and non-human mammals). The period may be 5 to 20 days, such as 5 to 15 days and 7 to 10 days. The maintenance medium may include (i) a culture or nutrient medium and (ii) one or more of the following: about 5 to 125 μg / ml insulin, about 20 to 500 μg / ml transferrin, about 4 to 100 nM progesterone, about 5 to 150 nM sodium selenate, about 10 to 300 nM putrescine, about 4 to 100 ng / ml EGF, about 4 to 100 ng / ml bFGF, and about 4 to 100 ng / ml B27 supplement. The method for maintaining stemness may further include (iv) obtaining cells from the spheres formed from step (iii) above, and then (v) repeating steps (ii) and (iii) above.

[0011] In one embodiment, the maintenance medium comprises one or more of the following: about 10-50 μg / ml insulin, about 50-200 μg / ml transferrin, about 10-50 nM progesterone, about 10-100 nM sodium selenate, about 10-100 nM putrescine, about 10-50 ng / ml EGF, about 10-50 ng / ml bFGF, and about 10-50 ng / ml B27 supplement. In a further embodiment, the maintenance medium comprises one or more of the following: about 25 μg / ml insulin, about 100 μg / ml transferrin, about 20 nM progesterone, about 30 nM sodium selenate, about 60 nM putrescine, about 20 ng / ml EGF, about 20 ng / ml bFGF, and about 20 ng / ml B27 supplement. In yet another embodiment, the maintenance medium comprises about 1-30% FBS, such as about 5-25% and about 20%. + The cells may be seeded in a single cell suspension. To this end, the cells may be seeded at a density of about 500 cells / ml to about 10,000 cells / ml.

[0012] The present application also provides a method for detecting one or more BMPR1A + Cell spheres containing cells are provided. 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 contain Axin2+ cells.

[0013] The present application provides a method for detecting a BMPR1A gene comprising: (i) a carrier; and (ii) one or more BMPR1A genes. + The present application also provides a composition comprising a cell, or one or more spheres described above, or cells (such as progeny cells) derived from one or more spheres. The composition may be a pharmaceutical composition, in which the carrier is a pharmaceutically 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 composition described above, and (ii) a scaffold.

[0014] 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.

[0015] Further provided is a method for producing skeletal, stromal, or cartilage tissue, the method comprising: (i) detecting one or more BMPR1A genes as described above; + (ii) providing a cell, or one or more spheres thereof, or a cell (e.g., a progeny cell) derived from one or more spheres thereof; and + and inducing differentiation of the cells, or cells from one or more spheres.

[0016] 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 claims. [Brief explanation of the drawings]

[0017] [Figures 1A-1G]We demonstrate that stem cell-mediated calvarial development and homeostasis requires Bmpr1a. The Bmpr1aAx2, Bmpr1a- / -, and Acvr1Ax2 mouse models investigated the role of BMP type I receptors in calvarial morphogenesis. (A) The diagram shows the procedure for inducing Bmpr1a or Acvr1 deletion during calvarial development in Axin2+ SuSCs using Axin2Cre-Dox (Axin2-rtTA, TRE-Cre) mice. Control mice were Axin2-rtTA;Bmpr1aFx / Fx or TRE-Cre;Bmpr1aFx / Fx mice with Dox for Bmpr1aAx2, Bmpr1b+ / - mice for Bmpr1b- / -, and Axin2-rtTA;Acvr1Fx / Fx or TRE-Cre;Acvr1Fx / Fx mice with Dox for Acvr1Ax2. Mice were examined at 2 months of age (n = 3 mice per group). (B) Photographs of the mouse head and face. (C) μCT images of the skulls of the indicated mice. Arrowheads indicate abnormal suture closure. (D) Hematoxylin and eosin staining of calvarial tissue at the sagittal suture. Scale bar, 400 μm. (E) Schematic showing the loss of Bmpr1a in mouse SuSCs after calvarial development. Control mice were Axin2-rtTA;Bmpr1aFx / Fx or TRE-Cre;Bmpr1aFx / Fx mice with Dox. Mice were examined after 3 months (n = 3 mice per group). (F) μCT images of the skulls of the indicated mice. Arrowheads indicate abnormal suture closure. (G) Hematoxylin and eosin staining of calvarial tissue at the sagittal and lambdoid sutures. Asterisks indicate abnormal suture closure. Scale bar, 200 μm. COR, coronal suture; LAM, lambdoid suture; SAG, sagittal suture. [Figure 2A-2B]Craniosynostosis caused by SuSC-specific disruption of Bmpr1a is associated with an abnormal suture closure process. Bmpr1aAx2 mice (Axin2-rtTA;TRE-Cre) were induced with Dox from E16.5 to P3. Control mice were Axin2-rtTA;Bmpr1aFx / Fx or TRE-Cre;Bmpr1aFx / Fx mice with Dox. Skulls from mice with the indicated conditions were analyzed at P0, P7, or P14 (n = 3 mice per group). (A) Alizarin red staining of intact skull. (B) Goldner's trichome staining of calvarial sections along the sagittal suture. Arrowheads indicate mineralization between calvarial bone plates. Arrows indicate the bone formation front. Scale bar, 400 μm. [Figures 3A-3D] We show that loss of Bmpr1a in SuSCs leads to abnormal intramembranous ossification within the suture mesenchyme. Bmpr1aAx2 mice (Axin2-rtTA;TRE-Cre) were induced with Dox from E16.5 to P3. Control mice were Axin2-rtTA;Bmpr1aFx / Fx or TRE-Cre;Bmpr1aFx / Fx mice with Dox. Calvarial sections were analyzed from mice at either P0 or P3, as indicated (n = 3 mice per group). (A) Ki67 immunostaining at the osteogenic front (OF, indicated by the dotted line). (B) Ki67 immunostaining in the suture mesenchyme. Scale bars for A and B are 60 μm. (C) Osterix (Osx) immunostaining. Scale bars for the top four images are 400 μm; scale bars for the bottom two images are 100 μm. (D) In ​​situ hybridization for type 1 collagen (Col1). Scale bar, 200 μm. In C and D, arrows indicate the osteogenic front. [Figures 4A-4D]Bmpr1a regulates SuSC and stem cell-dependent bone formation. (A-C) To investigate SuSC frequency, limiting dilution analysis was performed on control and Bmpr1aAx2 cells isolated from P5 suture mesenchyme of mice administered Dox from E16.5 to P3, followed by kidney capsule transplantation (n = 3 mice per group). Control mice were Axin2-rtTA;Bmpr1aFx / Fx or TRE-Cre;Bmpr1aFx / Fx mice with Dox. Ectopic bone formation was assessed by whole-mount von Kossa staining (A) and histology in sections (B). Scale bars, 4 mm (A), 200 μm (B). Quantification of bone formation rate following transplantation of 10, 10, 10, and 10 cells with quantitative estimation of stem cell frequency using ELDA software (C). (D) Immunostaining of Axin2 in sections of the sagittal suture at P7 quantified the mean percentage of Axin2+ cells. Sections were counterstained with DAPI. The dashed line defines the calvarial bone (scale bar, 100 μm). Quantified data are from three mice per group and are presented as mean + SEM (*, p<0.01 by Student's t-test). [Figures 5A-5D]This demonstrates that SuSC stemness is preserved in sphere culture. (A, B) Genetic cell labeling in Axin2Cre-Dox;R26R Tomato mice was used to track the fate of Axin2+ SuSCs in sphere culture. Dox was administered to Axin2Cre-Dox;R26R Tomato mice from P7 to P10. Panel A shows fluorescent images of Axin2+ SuSCs at day 1 and day 14 of culture. Scale bar, 100 μm. Panel B shows assessment of the proportion of spheres derived from Axin2+ cells (arrows) and Axin2- cells (arrowheads). The proportion of spheres derived from Axin2+ and Axin2- cells was quantified (p<0.01, n=3, mean + SEM, Student's t-test). Scale bar, 400 μm. (C) Whole-mount imaging of Tomato fluorescence of (Axin2+-derived) SuSC spheres 4 weeks after transplantation into the kidney capsule. Scale bar, 1 mm. (D) Whole-mount von Kossa staining of ectopic bone generated by SuSC spheres 8 weeks after transplantation. (E) Comparison of ectopic bone grown from SuSC spheres 8 weeks after transplantation into the kidney capsule (left) and the calvarial bone plate (right) from a 2-month-old mouse skull. Sections were stained with hematoxylin and eosin. Scale bar, 200 μm. In C–E, images are representative of three independent experiments. [Figures 6A-6N]We demonstrate that Bmpr1a is essential for SuSC self-renewal. (A-C) Ex vivo pulse-chase labeling analysis of cells isolated from Axin2GFP mouse sutures labeled in vivo at P7-P10 by Dox administration and then cultured in the absence of Dox. Spheres from 10, 20, and 30 cultures were examined for the presence of cells with GFP fluorescence (arrows). (D) Graph of the percentage of spheres with (Axin2+) or without (Axin2-) label-retaining GFP+ cells at the indicated passages. (E) Diagram showing the distribution of GFP intensity in cells generated by asymmetric or symmetric division of SuSCs. (F-G) Ex vivo pulse-chase labeling of Axin2-positive cells (as described for A-C) was followed by immunostaining for Axin2 (F) or by incubating cells with EdU to label proliferating cells in suture spheres (G). Arrows indicate single GFP+ label-retaining cells that are positive for Axin2 immunofluorescence (F) or negative for EdU labeling (G). Scale bar, 100 μm, for F; scale bar, 50 μm, for G. (H) Ex vivo pulse-chase labeling of Axin2-positive cells (as described for A–C) was followed by immunostaining for Bmpr1a in suture spheres. Arrows indicate single GFP+ label-retaining cells that are positive for Bmpr1a. Scale bar, 50 μm. (I, J) In vitro self-renewal was investigated by serial culture of spheres. Sphere number (I) and size (J) were assessed in spheres derived from suture cells from control or Bmpr1aAx2 mice. For sphere number, data are presented as mean + SEM (n = 3; *, p < 0.05, Student's t-test). Sphere size is shown as the mean (middle line), 75th percentile (top line), and 25th percentile (bottom line) values ​​for individual spheres. Statistical significance was determined by a two-tailed Student's t-test [n = 10 control, 236 spheres, and Bmpr1aAx2, 188 spheres; 20 control, 124 spheres, and Bmpr1aAx2, 66 spheres (N = 3 independent experiments)]. (K-N) Ten spheres cultured from control or Bmpr1aAx2 cells isolated from the suture mesenchyme at P5 in mice treated with Dox from E16.5 to P3 were transplanted into the kidney capsule (n = 3 mice per group).Control mice were Axin2-rtTA;Bmpr1aFx / Fx or TRE-Cre;Bmpr1aFx / Fx mice with Dox. Tissues were evaluated by whole-mount von Kossa staining (K-L) and histological analysis (M-N). Scale bars for K and L are 2 mm; for M and N, scale bars are 800 μm. [Figures 7A-7K] Self-renewal and osteogenic potential of human SuSCs are shown. (A-C) Sections of 14-month-old human coronal sutures were examined by hematoxylin and eosin (H&E) staining (A) or by immunostaining for AXIN2 (B) or BMPR1A (C). Dashed lines demarcate the calvaria at the osteogenic front (OF). Scale bars, 500 μm in the main image and 100 μm in the enlarged images from B and C. Images represent more than five individuals. (D-F) Characterization of spheres formed by cells isolated from human suture tissue. A representative primary sphere is shown in (D). Scale bar, 100 μm. Graphs of the average number (E, n = 5, mean + SD) and size (F, n = 5, >15 spheres at each passage, mean + SD) of spheres formed by the indicated cultures of human suture cells starting with 10 cells at each passage. (G-I) Immunostaining for AXIN2 (arrow) and EdU labeling of human spheres. Scale bar, 100 μm. (J, K) Thirty primary human spheres were transplanted into the kidney capsule and evaluated by von Kossa staining of whole mounts (J) and sections (K). In J, scale bar, 300 μm; in K, scale bar, 100 μm. Data in G-K are representative of at least five independent experiments. [Figures 8A-8L]The osteogenic potential of mouse Bmpr1a-positive and human BMPR1A-positive suture cells is shown. (A) Cell sorter isolation of Bmpr1a / BMPR1A-high and Bmpr1a / BMPR1A-low cell populations derived from P10 mouse or human suture mesenchyme. (B-F) Sorted mouse cells (5 x 103) were transplanted into the kidney capsule and assessed by von Kossa (VK) staining to identify bone tissue or immunostaining for Osterix (Osx) to identify osteoprogenitor cells. (G-L) Sorted human cells (5 x 103) were transplanted into the kidney capsule and assessed by von Kossa (VK) staining to identify bone tissue or immunostaining for Osterix (Osx) to identify osteoprogenitor cells. In L, tissues were counterstained with DAPI, and the dotted area represents bone. Images are representative of at least five independent experiments. Scale bars, 50 μm (B–C, H–M), 100 μm (D–F). [Figures 9A-9C] Mouse genetic models for identifying Axin2-expressing cells and their derivatives are shown. (A) Schematic of the genetic cell labeling strategy for identifying Axin2-expressing cells by GFP analysis. (B) Schematic of the genetic cell labeling strategy for identifying descending cells from Axin2-expressing cells. (C) Fluorescence images show GFP analysis of Axin2-rtTA;TRE-H2BGFP mice at P9, revealing Axin2-expressing cells in the midline of the suture mesenchyme (n = 3). The fate of these cells can be tracked using a cell tracking model based on β-galactosidase (β-gal) positivity (Axin2Cre-Dox;R26RlacZ) or red fluorescence from Tomato (Axin2Cre-Dox;R26RTomato). Dashed lines demarcate the calvarium and the osteogenic front. Scale bar, 100 μm. [Figures 10A-10E]Enhanced BMP signaling in suture stem cells. A) Gene expression profiles of Axin2-positive (+) and -negative (-) cells isolated from suture mesenchyme were analyzed by activity Z-score using IPA software to predict signaling pathway activity. (B) Heatmap showing the expression of genes related to the BMP pathway-7 ligand, the receptor subunit Bmpr1a, and two negative regulators, Smad7 and Smurf1, in Axin2+ and Axin2- suture cell populations. (C-E) Sagittal suture sections expressing lacZ in Axin2+ cells were analyzed at postnatal day (P) 28. Sections were processed for β-gal activity and Bmpr1a immunoreactivity and counterstained with DAPI. In C, Axin2-expressing cells were identified in Axin2Cre-Dox;R26RlacZ by staining for β-gal. In D, cells were stained with an antibody recognizing Bmpr1a, and nuclei were labeled with DAPI. In E, positivity for both β-gal and Bmpr1a is shown. The dashed line demarcates the calvarium at the osteogenic front (OF). Images are representative of three independent experiments. Scale bar, 100 μm. [Figures 11A-11D] Efficacy of Cre-mediated ablation of Bmpr1a and Bmpr1a antibody specificity. Dox was administered from E16.5 to P3 to activate Cre expression in Axin2+ SuSCs. Sagittal suture sections were obtained at P7. (A) Absence of LacZ expression in β-gal-labeled calvaria from TRE-Cre;R26RlacZ mice. (B) Presence of LacZ expression in β-gal-labeled calvaria from Axin2Cre-Dox;R26RlacZ mice. In A and B, sections were counterstained with nuclear fast red. (C, D) Bmpr1a staining of control and Bmpr1aAx2 calvaria. Sections were counterstained with DAPI. Dashed lines delineate the calvaria and osteogenic front. Scale bar, 100 μm. [Figures 12A-12F]Effect of loss of Bmpr1a function on the skull. Dox was administered from E16.5 to P3 to activate Cre expression in Axin2+ SuSCs. Control mice were Axin2-rtTA;Bmpr1aFx / Fx or TRE-Cre;Bmpr1aFx / Fx mice with Dox for Bmpr1aAx2, Bmpr1b+ / - mice for Bmpr1b- / -, and Axin2-rtTA;Acvr1Fx / Fx or TRE-Cre;Acvr1Fx / Fx mice with Dox for Acvr1Ax2. n = 4 mice per group. (A, B) Graphs of skull length and width at P28 for control mice or the indicated mutants. (C, D) Graphs of skull length and width for control mice or Bmpr1aAx2 mutants at the indicated postnatal days. In A–C, statistical significance was determined by Student's t test (n = 4, *, p < 0.05). (E, F) μCT images of the skulls of 2-month-old control and Bmpr1aAx2 mutant mice. Scale bar, 2 mm. [Figures 13A-13H] Multiple suture synostosis in the skull of a mouse with SuSC-specific deletion of Bmpr1a is shown. Dox was administered from E16.5 to P3 to activate Cre expression in Axin2+ SuSCs. Control mice were Axin2-rtTA;Bmpr1aFx / Fx or TRE-Cre;Bmpr1aFx / Fx mice with Dox. n = 3 mice per group. Skulls from 2-month-old mice of the indicated genotypes were evaluated. (A, B) Whole-mount skeletal staining with alizarin red. Scale bar, 2 mm. (C-H) Sections were stained with hematoxylin and eosin. Scale bar, 200 μm. Arrowheads and asterisks indicate examples of calvarial bone fusion. COR, coronal; LAM, lambdoid; SQU, squamosal. [Figure 14]Identification of multiple suture synostosis in the skull of mice with SuSC-specific deletion of Bmpr1a by μCT analysis is shown. Dox was administered from E16.5 to P3 to activate Cre expression in Axin2+ SuSCs. Control mice were Axin2-rtTA;Bmpr1aFx / Fx or TRE-Cre;Bmpr1aFx / Fx mice with Dox. n = 3 mice per group. Images are shown as 3D reconstructions (blue background) and 2D images (black background) of the indicated sections of the skull from 2-month-old control and Bmpr1aAx2 mice. Asterisks indicate examples of abnormal suture closure. AF, anterior frontal; FN, frontonasal; IN, intranasal; LAM, lambdoid; NM, nasomaxillary; SAG, sagittal; SQU, squamosal. [Figure 15] Chondrocyte analysis in Bmpr1aAx2 mice is shown. For Bmpr1aAx2 mice, Dox was administered from E16.5 to P3 to activate Cre expression in Axin2+ SuSCs. Control mice were Axin2-rtTA;Bmpr1aFx / Fx or TRE-Cre;Bmpr1aFx / Fx mice with Dox. n = 3 mice per group. Sagittal suture sections were obtained from the indicated mice and stained for type II collagen (Col2) and counterstained with DAPI. Sections were obtained at P3 for control and Bmpr1aAx2 mice and at P7 for Axin2- / -;Fgfr1+ / - mice. Dashed lines delineate the calvaria and bone formation front. Scale bar, 100 μm. [Figures 16A-16C]The effect of Bmpr1a ablation on signaling pathways in the developing suture is shown. Dox was administered from E16.5 to P3 to activate Cre expression in Axin2+ SuSCs. Control mice were Axin2-rtTA;Bmpr1aFx / Fx or TRE-Cre;Bmpr1aFx / Fx mice with Dox. n = 3 mice per group. Calvarial sections were obtained at P3. (A) Immunostaining of phosphorylated Smad1, Smad5, and Smad8 using an antibody that recognizes all three phosphorylated Smads (pSmad1 / 5 / 8). The bottom image is an enlargement of the boxed area in the top image. Scale bar for the top image: 100 μm; scale bar for the bottom image: 50 μm. (B) Immunostaining of phosphorylated Tak1. Scale bar: 100 μm. (C) Immunostaining of the activated forms of the indicated mitogen-activated protein kinase (MAPK) family members: pJNK, pP38, and pErk. Scale bar, 200 μm. All images were counterstained with DAPI. Arrows or dashed lines indicate the osteogenic front. [Figures 17A-17S] Ex vivo culture and differentiation of SuSCs. (A) Schematic diagram showing isolation of suture cells from mouse sagittal sutures, followed by sphere culture at primary (10), secondary (20), tertiary (30), and up to 40-50 passages. (B) Images of suture cells prior to sphere culture. Scale bar, 50 μm. (C-E) Representative images of sphere cultures at the indicated passages. Scale bar, 50 μm. (F-K) Time course analysis of sphere formation from days 1 to 12 of culture. Scale bar, 50 μm. (L) Quantification of sphere size at the indicated passages (n = 3, >150 spheres at each passage, mean + SD). (M-O) Differentiation of 30 sphere cells into ALP+ osteoblasts (M), mineralized nodules positive for von Kossa staining (N), and Alcian blue-positive chondrocytes (O). Scale bar, 100 μm. (P-S) Von Kossa-stained whole mounts and sections of kidney capsules implanted with 30 spheres or unimplanted control kidney capsules. Arrows indicate ectopic bone formation. In P and Q, the scale bar is 250 μm; in R and S, the scale bar is 100 μm. [Figures 18A-18G]Lentiviral gene delivery to investigate the loss of Bmpr1a in sphere-mediated bone formation. (A) Cells isolated from the suture mesenchyme of Bmpr1aFx / Fx mice were infected with lentivirus expressing red fluorescent protein (RFP) at MOIs of 0, 1, and 5. Infection efficiency was monitored by fluorescence imaging. Scale bar, 1 mm. (B, C) Analysis of sphere size (B) and number (C) at the indicated MOIs (n = 3, mean + SD). (D) The efficiency of Cre-mediated recombination was investigated by PCR [primers: 5'-ggtttggatcttaaccttagg-3' and 5'-tggctacaatttgtctcatgc-3'], generating a 180-bp product. The unmodified Bmpr1aFx allele was detected by PCR [primers: 5'-gcagctgctgctgcagcctcc-3' and 5'-tggctacaatttgtctcatgc-3'], generating a 230-bp product. (E-G) Whole-mount von Kossa staining of ectopic bone formation in the kidney capsule implanted with Bmpr1aFx / Fx10 spheres infected with lentivirus expressing GFP (E, lenti-GFP) or Cre (F, lenti-Cre). Quantitative assessment of the size of the von Kossa-stained area (asterisk, p < 0.05, n = 3, mean + SEM, Student's t-test) (G). Images are representative of three independent experiments. Scale bar, 2 mm (D-E). [Figures 19A-19E] Identification of cells positive for AXIN2 and BMPR1A in the human lambdoid suture. Sections of a 10-month-old human lambdoid suture were examined. (A) Hematoxylin and eosin (H&E) staining. Scale bar, 400 μm. (B-E) Immunostaining for AXIN2 or BMPR1A. Panels C and E show enlargements of the boxed areas in panels B and D. Dashed lines demarcate the calvarial bone. Scale bars in B and D, 400 μm; in C and E, 100 μm. [Figures 20A-20C]Purification of mouse and human Bmpr1a / BMPR1A-expressing cells. (A) The specificity of the antibody used for cell sorting was determined by FACS of wild-type (WT) and Bmpr1aAx2 suture cells from P10 mice. (B, C) Cell sorting of Bmpr1a / BMPR1A-high and Bmpr1a / BMPR1A-low cell populations from mouse (B) and human (C) suture mesenchyme. Human suture mesenchyme was isolated from patients aged 4–12 months. FACS was performed with (top row) and without (bottom row) the addition of antibody. Data are representative of three independent experiments. In mice / humans, approximately 5–6% of suture cells have high levels of Bmpr1a / BMPR1A. DETAILED DESCRIPTION OF THE INVENTION

[0018] This application relates to stem cell biology and regenerative medicine. Certain aspects of the present invention are based, at least in part, on the identification and use of one or more cell surface markers of SSCs and / or SuSCs. The ability of SSCs and / or SuSCs to engraft at the site of injury and replace the damaged skeleton can be used in stem cell-based therapies.

[0019] Cells and markers Suture mesenchyme-derived skeletal stem cells (SSCs), also known as suture stem cells (SuSCs), exhibit long-term self-renewal, clonal expansion, and multipotency. These SuSCs reside at the midline suture and function 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 skeletons supports their potential use for stem cell-based therapies.

[0020] As disclosed herein, BMPR1A has been identified as essential for SuSC self-renewal and SuSC-mediated bone formation. Additionally, SuSC-specific disruption of Bmpr1a in mice led to premature differentiation, resulting in craniosynostosis initiated at the midline suture, the stem cell niche. Exemplary amino acid and nucleic acid sequences of human and mouse BMPR1A are provided below. [Table 1] TIFF2024508806000002.tif249170TIFF2024508806000003.tif142170TIFF2024508806000004.tif249170TIFF2024508806000005.tif48170

[0021] More notably, BMPR1A was found to be a cell surface marker for human SuSCs. Using an ex vivo system, we demonstrated that SuSCs maintained stem cell properties over a long period of time without losing their osteogenic potential. This study advances the knowledge base for congenital deformities and regenerative medicine mediated by skeletal stem cells. In addition to BMPR1A, additional positive and negative markers are disclosed herein.

[0022] Examples of 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. Table 1 below lists additional examples of positive markers. Examples of positive markers for SSCs include CD31, CD45, Ter-119, Tie 2, Thy, and CD105. Table 2 below lists additional examples of negative markers. The markers and their respective homologs can be used to identify, enrich, or isolate skeletal stem cells and suture stem cells from animal subjects, such as humans or mice. [Table 2] [Table 3]

[0023] Identification, isolation, and enrichment of SSCs In one aspect, the present application relates to the identification, isolation, and enrichment of SSCs, particularly SuSCs. For example, these cells can be separated from a complex mixture of cells by techniques to identify or enrich for cells expressing BMPR1A alone or in combination with other markers and characteristics described herein. Mammalian SSCs, such as mouse SSCs and human SSCs, can be characterized by functional homologs of BMPR1A and, optionally, other markers, including CD200, 6C3, PDPN, CD105, CD90, CD45, Tie2, αv integrin, and others disclosed herein. Methods and compositions for the isolation and characterization of SSCs and osteoprogenitor cells are provided. Expression of these specific cell surface markers allows cells to be separated from other cells.

[0024] Cells of interest, i.e., cells expressing a selected marker, 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 scatter. 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, data can be normalized to a control. To normalize the distribution to a control, each cell is recorded as a data point with a specific intensity of staining. These data points can be displayed according to a logarithmic scale, with the unit of measurement being arbitrary staining intensity. In one example, the most brightly stained cells in a sample may be 4 logs more intense than unstained cells. When displayed in this manner, it is clear that cells corresponding to the highest logarithm of staining intensity are bright, and cells with the lowest intensity are negative. "Low" positive staining cells have a staining level that exceeds the brightness of the isotype-matched control, but is not as intense as the most brightly staining cells normally found in the population. Alternative controls can utilize substrates with a defined density of markers on their surface, such as fabricated beads or cell lines that provide an intensity positive control. Other marker-based separation methods, i.e., cell selection, can be achieved, for example, by magnetic-activated cell sorting (MACS), immunopanning, and laser capture microdissection.

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

[0026] In some instances, for isolation of cells from tissue, an appropriate solution can be used for dispersion or suspension. Such a solution can be a balanced salt solution, such as normal saline, PBS, Hank's balanced salt solution, or the like, supplemented with fetal bovine serum or other naturally occurring factors, typically at a low concentration, typically 5-25 mM, of an acceptable buffer. Convenient buffers include HEPES, phosphate buffer, lactate buffer, and the like. Tissue can 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 sufficient to dissociate the cells, followed by gentle mechanical dissociation.

[0027] Initial separation may involve cell selection 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 may 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" using 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-angle and obtuse-angle light scatter detection channels, and impedance channels. Cells may be selected against dead cells by using a dye (propidium iodide, 7-AAD) that associates with dead cells. Any technique that is not overly detrimental to the viability of the selected cells may be used.

[0028] The affinity reagent may be a specific receptor or ligand for the above-mentioned cell surface molecule. Of particular interest is the use of an antibody as an affinity reagent. Details of antibody preparation 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 with specificity for BMPRIA 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 for human cells, antibodies specific for mouse markers are used to select for mouse cells, etc.

[0030] These antibodies can be conjugated with labels for use in separation. Labels include magnetic beads, which allow for direct separation; biotin, which can be removed with support-bound avidin or streptavidin; and fluorescent dyes, which 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 is added to the cell suspension and allowed to incubate for a period of time sufficient to bind to available cell surface antigens. Incubation typically lasts at least about 5 minutes, usually less than about 2 hours. It is desirable to have a sufficient concentration of antibody in the reaction mixture so that separation efficiency is not limited by a lack of antibody. The appropriate concentration can be determined by titration. The medium in which the cells are separated can be any medium that maintains cell viability. An exemplary medium is phosphate-buffered saline containing approximately 0.1-20% BSA or FBS. Various media are commercially available and can 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, and PBS containing 5 mM EDTA, often supplemented with fetal bovine serum, BSA, or HSA.

[0032] The labeled cells are then separated for the above-mentioned phenotypes. The separated cells can be collected in any suitable medium that maintains cell viability, usually with a serum cushion at the bottom of the collection tube. Various 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 SSCs, usually about 90% or more SSCs, and can even be about 95% or more SSCs. The enriched cell population can be used immediately or frozen at liquid nitrogen temperature and stored for a long period of time, thawed, and reused. Cells are typically stored in 10% DMSO, 50% FCS, and 40% medium (e.g., RPMI 1640 medium). Once 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, which have similar skeletal structures and skeletal stem cells, such as fish, amphibians, and birds (Mork and Crump, Curr Top Dev Biol, 2015). Examples of mammals include humans, primates, domestic and farm animals, as well as zoo, laboratory, or pet animals, such as dogs, cats, cows, horses, sheep, pigs, goats, rabbits, rats, and mice. Cells can be established cell lines or primary cells; the terms "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 derived from neonates, juveniles, or adults, as well as from tissues including skin, muscle, bone marrow, peripheral blood, umbilical cord blood, spleen, liver, pancreas, lung, intestine, stomach, adipose, and other tissues. Tissues may be obtained by biopsy or aphoresis from living donors, or from deceased or dying donors within about 48 hours of death, or from freshly frozen tissue, or tissue frozen within about 12 hours of death and maintained indefinitely at temperatures below about -20°C, usually about liquid nitrogen temperature (-190°C). For isolation of cells from tissue, an appropriate 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 buffer. Convenient buffers include HEPES, phosphate buffer, lactate buffer, 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.). Cell populations can be suspended in an appropriate nutrient medium, such as DMEM, Iscove's modified DMEM, or RPMI-1640, typically supplemented with fetal bovine serum (approximately 1-25%), L-glutamine, thiols (e.g., 2-mercaptoethanol), and antibiotics. In one embodiment of the present invention, SSCs can be maintained in culture in the absence of feeder layer cells or 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, including 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 cell survival, growth, and / or differentiation, either in culture or in intact tissue, 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 β (TGFβ), bone morphogenetic proteins (BMPs), e.g., 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 therapy 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, for example, "FGF signaling pathways in endochondral and intramembranous bone development and human genetic disease," Int. J. Biol. Sci. 2012;8:272-288; 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., Arch. Biochem. Biophys. 473(2):139-146 (2008). 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 toward 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. Particular embodiments include distorting the differentiation of skeletal stem cells into chondrocytes by contact 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 can be produced by the methods described herein include pre-osteochondral and stromal precursors (pre-BCSPs), BCSPs, committed chondrocyte precursors (CCPs), osteoprogenitors, B-cell lymphocytic stromal precursors (BLSPs), 6C3 stromal cells, hepatic leukemia factor-expressing stromal cells (HECs), 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, can be incubated in the presence of the agent for about 30 minutes to about 24 hours, which can be repeated about daily to about every four days, e.g., every two days, every three days, etc. The agent can be provided to the cells one or more times, allowing the cells to incubate with the agent for some time after each contacting event, e.g., 16 to 24 hours, after which time 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 the 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 expand and isolate the cells. For example, the cells (either before or after contact 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 the 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, and 40% RPMI 1640 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 cell transplantation therapy to treat diseases. 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, isolated, or enriched from the remainder of the cell culture before being transferred to a subject. In some cases, one or more antibodies specific for 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 binding cells are isolated. In other cases, the cells or subpopulation of cells express a marker that is under the control of a particular promoter, which is then used to purify or isolate the cells or subpopulation thereof, such as a reporter gene, e.g., EGFP, dsRED, lacz, etc.

[0045] sphere In one aspect, the present application provides one or more BMPR1A +The present invention relates to cell spheres containing cells. The spheres may be 20-200 μm in diameter or may have approximately 10-500 cells per sphere (e.g., 50-400, 100-300, etc.). The spheres may contain Axin2+ cells. To generate such spheres, a population of suture stem cells or skeletal stem cells can be seeded in the maintenance medium described above on a low-attachment or ultra-low-attachment surface, and the cells or their progeny can be cultured for a period of time to form one or more spheres. The period can 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 can have a neutral hydrophilic hydrogel coating that significantly reduces the binding of adhesive proteins, thereby minimizing cell attachment and spreading. The covalently bonded hydrogel layer effectively inhibits cell attachment, minimizing protein absorption, enzyme activation, and cell activation. Cell culture surfaces with such ultra-low-attachment surfaces are 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 similar to a sphere. Examples of three-dimensional shapes similar to a sphere include spherical shapes, which have a three-dimensional structure and, when projected onto a two-dimensional surface, appear, for example, circular or elliptical, and shapes formed by fusing multiple spherical shapes (e.g., when projected onto a two-dimensional surface, appear to be formed by overlapping two to four circular or elliptical shapes). The term "cell aggregate" refers to a ball-shaped cluster of cells or a block of cells containing 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 containing 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] As disclosed in the Examples below, approximately 5% of mouse suture mesenchymal cells can be Bmpr1A+ cells (Figure 8A), and approximately 0.5% of suture mesenchymal cells can form spheres (Figure 6I, control). The majority of spheres contain Bmpr1a+ cells (Figure 6H). Thus, approximately 10% of Bmpr1a+ cells form spheres. In the case of human stem cells, approximately 5% of suture mesenchymal cells are BMPR1A+ cells (Figure 8A), and approximately 0.1% of suture mesenchymal cells form spheres under current culture conditions (Figure 7E). The majority of spheres contain BMPR1A+ cells. Thus, approximately 2% of BMPR1A+ suture mesenchymal cells form spheres.

[0049] As disclosed in the Examples below, in primary sphere cultures, 68% of spheres contain Axin2+ cells. In subsequent secondary and tertiary cultures, 100% of spheres contain Axin2+ cells. Theoretically, all spheres should be formed by Axin2+ cells, since there are stem cells with "unlimited self-renewal" capacity that require sphere formation in sequential cultures to truly define stem cells. The explanation for primary culture is that some spheres (32%) are formed by progenitor cells with limited proliferative 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 indicate the absence of Axin2+ stem cells with quiescent / low-cycling function. See Figure 6.

[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, as well as 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 can 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. Accordingly, the present application provides bone regeneration products or formulations that include at least one skeletal stem cell and, optionally, at least one of such other types of cells, drugs, materials, and structures. For this purpose, the cells can be three-dimensional (3D) synthetic, semi-synthetic, or living biological tissue.

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

[0053] The scaffold may comprise 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 contact 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, retaining, 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 an animal or human body. These defects can have any shape and size. Scaffolds suitable for treating such defects can have, for example, a shape, volume, and size that can match or resemble the shape and size of the defect. Such scaffolds can also have pore volumes, pore sizes, and / or pore shapes similar to the bones that bone regeneration products containing such scaffolds are designed to treat. Such scaffolds can also have pores with sufficiently small pore sizes so 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 treatment. Bone regeneration products / formulations can have sufficient mechanical strength to handle the load-bearing conditions of implantation into the body. They can also have sufficient mechanical strength to handle the load-bearing stress and / or body weight of the bone during movement (e.g., walking).

[0057] The scaffold can include any material. For example, the scaffold can include a non-resorbable material, a resorbable material, or a mixture thereof. A resorbable material can be resorbed by the patient's body and eventually replaced with healthy tissue. A "resorbable" material can 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 (e.g., an immune response, an inflammatory response, a thrombotic response, or the like) 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 factors.

[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 containing a mixture of hydroxyapatite and tricalcium phosphate (HA / TCP). This scaffold may be prepared, for example, by mixing hydroxyapatite (HA) and tricalcium phosphate (TCP) with a formulation containing a liquid to prepare a paste. For example, a 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 an HA / TCP scaffold.

[0060] Unless otherwise specified, a mixture containing HA and TCP (HA / TCP) may be formed from equal amounts of HA and TCP by weight, e.g., 50% HA and 50% TCP by weight. However, a mixture containing HA and TCP may have any composition ranging, for example, 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 can comprise any biodegradable polymer. For example, the scaffolds can comprise synthetic polymers, naturally occurring polymers, or mixtures thereof. Examples of suitable biodegradable polymers can 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), polycyanoacrylate, fibrin, casein, serum albumin, collagen, gelatin, lecithin, chitosan, alginate, polyamino acids (such as polylysine), and mixtures thereof.

[0062] The scaffold may further comprise one or more of the above-mentioned 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 by hand 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 technique.

[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™ or Organovo Holdings, Inc. bioprinter can be used for 3D bioprinting. This and similar "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 technology can be used to manufacture the scaffold. 3D printing technology or additive manufacturing (AM) can be a process for creating a physical object from a 3D digital model, typically by depositing many successive thin layers of material. Such thin layers of material can be formed under computer control. Examples of 3D printing technologies 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 by using formulations including, for example, polycaprolactone dimethacrylate (PCLDA), calcium phosphate, HA, TCP, polyethylene glycol diacrylate (PEGDA), gelatin methacryloyl (GeIMA), or mixtures thereof.

[0068] The bone regeneration product / preparation can be used for bone regeneration. The bone regeneration method can include implanting the bone regeneration product / preparation into 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 critical. The bone defect can be formed due to a 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 Composition The present application provides a method for detecting a BMPR1A gene comprising: (i) a carrier; and (ii) one or more BMPR1A genes. + Further provided is a composition comprising a cell, or one or more spheres described above, or a cell (such as a progeny cell) derived from one or more spheres. The composition can be a pharmaceutical composition, wherein the carrier is a pharmaceutically acceptable carrier.

[0070] Compositions for pharmaceutical use, such as scaffolds or implants bearing cells and / or factors, may contain, depending on the desired formulation, a non-toxic carrier, a pharmaceutically acceptable diluent, which is defined as a vehicle commonly used to formulate pharmaceutical compositions for animal or human administration. The diluent may be selected so as not to affect the biological activity of the combination. Examples of such diluents include 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, etc. The compositions may also contain additional substances that approximate physiological conditions, such as pH adjusters and buffers, toxicity adjusters, 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., increasing the polypeptide's half-life, reducing its toxicity, enhancing solubility or uptake). Examples of such modifying or complexing agents include sulfate, gluconate, citrate, and phosphate. 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 of drug delivery, see Langer, Science 249:1527-1533 (1990).

[0073] The pharmaceutical compositions described herein, for example, SSCs, can be administered alone or in combination with various agents for prophylactic and / or therapeutic treatment. The 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 the LD50 (the dose lethal to 50% of the population) and the 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 exhibiting a large therapeutic index 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). Moreover, 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 parenteral administration are also sterile, substantially isotonic, and made under GMP conditions.

[0076] The effective amount of a therapeutic composition given to a particular patient depends 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, as needed. Using animal data and other available information about the drug, 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 volume of fluid into which the therapeutic composition is being administered. Similarly, compositions that are rapidly cleared from the body may be administered at higher or repeated doses to maintain therapeutic concentrations. Using routine skills, a competent clinician will be able to optimize the dosage of a particular therapeutic agent during 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 the treatment of related disorders and tissue engineering.

[0079] In some embodiments, the cells, compositions, formulations, and products disclosed herein can 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 can be those 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 greater than 50% (e.g., about 50% to about 100%) compared to a suitable control, which typically is 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 of requiring lower dosages of the individual drugs and of complementary effects of the different drugs.

[0082] In some embodiments, an effective dose of SSCs, preferably SuSCs, described herein is provided in an implant or scaffold for the 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 1 or more cells 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] This application provides methods and compositions for differentiating SSCs, including SuSCs, into cells such as chondrocytes. The cells produced by this method are useful for providing 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 transplanted or seeded into artificial structures capable of supporting three-dimensional tissue formation. These structures, referred to herein as matrices or scaffolds, allow cell attachment and migration, deliver and retain cellular and biochemical factors, and allow the diffusion of important cellular nutrients and expressed products. High porosity and appropriate pore size are important to facilitate cell seeding and the diffusion of both cells and nutrients throughout the structure. Biodegradability is often a factor, as the scaffold can be absorbed by surrounding tissue without the need for surgical removal. The rate at which degradation occurs should coincide as closely as possible with the rate of tissue formation, meaning that the scaffold can provide structural integrity in the body while cells fabricate their own natural matrix structure around themselves, eventually degrading to leave behind neotissue, the newly formed tissue that takes over mechanical loads. Injectability is also important for clinical use.

[0085] Many different materials (natural and synthetic, biodegradable and permanent) have been investigated and can 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 can also be constructed from natural materials, such as proteins such as collagen and fibrin; polysaccharide materials such as acytosan; and glycosaminoglycans (GAGs) such as alginate and hyaluronic acid. Functional groups on the scaffold can 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 function as the scaffold.

[0086] Treatment method The above-described 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 therapies can be used to replace any treatment involving bone grafts.

[0087] Large craniofacial bone defects caused by a variety of conditions, including trauma, infection, tumors, congenital disorders, and progressive degenerative diseases, are a major health problem (1). Autologous bone grafts are the recommended treatment for extensive skeletal repair, but their success remains extremely challenging due to several limitations (1, 2). Therefore, alternative approaches are being explored (3, 4). Stem cell-based therapy is particularly attractive and promising in light of the characterization of skeletal stem cells in the craniofacial and corporal skeletons (5-11). Craniofacial bones are primarily formed via intramembranous ossification, a process distinct from the endochondral ossification required for the corporal skeleton (12). Due to the distinct properties of stem cells in the craniofacial and corporal skeletons (5, 13), there is a need to study various types of skeletal stem cells. Suture stem cells (SuSCs) are a stem cell population that is naturally programmed to form intramembranous bone during craniofacial skeletogenesis (5). The lack of cell surface markers for stem cell isolation and the inability to maintain stemness properties ex vivo are two significant hurdles limiting further progress in the field of skeletal regeneration. The cells, compositions, and methods disclosed herein address this unmet need.

[0088] Craniosynostosis, which affects approximately 1 in 2,500 individuals, is one of the most common congenital deformities and is caused by premature suture closure (14). Sutures, which function as growth centers for calvarial morphogenesis, are equivalent to the growth plates of long bones. Excessive intramembranous ossification caused by genetic mutations promotes suture fusion (15). One example is the genetic loss of function of AXIN2, which causes craniosynostosis in mice and humans (16, 17). In 2010, we found that craniosynostosis can also be caused by mesenchymal cell fate switching, resulting in suture closure via endochondral ossification (18). By regulating the interaction between bone morphogenetic protein (BMP) and fibroblast growth factor (FGF) pathways, Axin2-mediated Wnt signaling determines bone formation toward either the osteogenic or chondrogenic lineage. Multipotency further supports the existence of skeletal stem cells within the suture mesenchyme (18). Because Axin2 expression in the putative niche was closely associated with suture patency, we identified Axin2-expressing SuSCs as essential for calvarial development, homeostasis, and injury-induced repair (5). Axin2-positive (Axin2+) SuSCs conform to the modern, strict definition of stem cells. They not only exhibit long-term self-renewal, clonal expansion, differentiation, and multipotency, but also the ability to repair skeletal defects by direct engraftment and replacement of damaged tissue.

[0089] When used for tissue engineering or therapeutic methods, the SSCs and / or combination of factors for lineage differentiation can 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 optimized for use in both load-bearing and non-load-bearing cartilage or bone graft applications can be provided 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 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 the physical effect in which the graft matrix forms a scaffold upon which the recipient's bone-forming cells can form new bone.

[0091] The inclusion of factors and / or cells described herein can be used to facilitate the replacement and filling of cartilage or bone material in and around pre-existing structures. In some embodiments, the cells initially produce chondrocytes, followed by extracellular matrix deposition and bone formation. Bone grafts can provide an osteoconductive scaffold containing 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 can slow the degradation of the ceramic, resulting in a localized source of calcium and phosphate for bone formation. Thus, new bone can form without the loss of calcium and phosphate from the host bone surrounding the defect site. Calcium phosphate ceramics are chemically compatible with the mineral components of bone tissue. Examples of such calcium phosphate ceramics include calcium phosphate compounds and salts, as well as 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 subambient temperature until needed. In some embodiments, application of the composite by injection may be similar to bone cement, used to bond and hold bone fragments in place or to improve the adhesion of, for example, artificial hip joints, such as for replacing damaged cartilage in a joint. Implantation in a closed surgical setting 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, a dental socket, or another site. In some embodiments, the graft putty can be used to repair non-union bone or other defects where the fracture, hole, or void to be filled is large and requires a degree of mechanical integrity from the implant material to both fill the gap and maintain its shape.

[0094] The methods described herein can be used to treat cartilage or bone lesions or injuries in humans or other animal subjects, and involve applying to the site a composition comprising the cells and / or factors described herein, which may be provided in combination with cements, factors, gels, etc. As referred to herein, such lesions include any condition involving skeletal tissue, including cartilage, that is unsuitable for physiological or cosmetic purposes. Such defects include those that are congenital, result from disease or trauma, and those that 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, infection, malignancy, developmental deformities, as well as simple fractures, compound fractures, transverse fractures, pathological fractures, avulsion fractures, greenstick fractures, and communuted fractures. In some embodiments, the bone defect is a void in 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 a desired gene operably linked to a promoter that is either pan-specific or specifically active in a differentiated cell type. Of particular interest are cells genetically modified to express bone morphogenetic proteins such as BMP-2 or BMP-4. See WO 99 / 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 evaluate 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 may 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, and is preferably one that can be accurately measured, especially in high-throughput systems. A parameter can be any cellular component or cellular 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, or moieties derived from such cellular components, or combinations thereof. Most parameters will provide a quantitative readout, although 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, variance, or the like. Typically, 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 a set of test parameters will be obtained using standard statistical methods, with common statistical methods used to provide a single value.

[0098] Candidate drugs of interest for screening include known and unknown compounds from many chemical classes, primarily encompassing 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 containing functional groups necessary for structural interactions, particularly hydrogen bonding, typically at least an amine, carbonyl, hydroxyl, or carboxyl group, and often at least two of the functional chemical groups. Candidate agents may include cyclical 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. Examples of 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 that modulates cell conversion into cells of the skeletal or chondrogenic lineage, e.g., chondrocytes, osteoblasts, or their precursor cells. In screening assays for bioactive agents, cells can be contacted with a candidate agent of interest in the presence of a cell reprogramming or differentiation system or an incomplete cell reprogramming or differentiation system, and the effect of the candidate agent is assessed by monitoring an output parameter, such as the expression level of a gene 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 an agent specific to BMPR1A protein or mRNA transcript. In one example, the kit includes a system for contacting a biological sample containing one or more anti-BMPR1A antibodies or antigen-binding fragments thereof, or a ligand of BMPR1A or a BMPR1A-binding fragment thereof, and instructions for use thereof. In one embodiment, the kit further includes a culture system (e.g., cell culture medium or a cell culture device, 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 BMPR1A protein levels.

[0103] The agent may be, for example, a reagent for performing an analytical method for detecting a protein or measuring protein levels. Examples of such analytical methods include, but are not limited to, FACS, immunohistochemistry, Western blotting, ELISA, radioimmunoassay, radial immunodiffusion, Ouchterlony immunodiffusion assay, rocket immunoelectrophoresis assay, immunoprecipitation assay, complement fixation assay, protein chip assay, etc. The kit may include reagents for detecting antibodies that form antigen-antibody complexes, such as labeled secondary antibodies, chromophores, enzymes (e.g., conjugated to the antibodies), and their substrates. In addition, it may include an antibody specific to a control protein for quantification. The amount of antigen-antibody complex formed can be quantitatively determined by measuring the signal intensity of the detection label. Such detection labels may be selected from the group consisting of, but 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 BMPR1A mRNA. mRNA expression 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] Kits are also provided that include the SSCs, spheres, compositions, products, or formulations described herein. Optionally, the kits include one or more scaffolds described herein. The SSCs, spheres, compositions, products, or formulations are provided in kits, packs, or dispensers that may contain one or more unit dosage forms containing the active ingredient. The kits may, for example, include metal or plastic foil such as a blister pack. The kits, packs, or dispensers may be accompanied by instructions for administration.

[0106] definition The terms "polypeptide" and "protein" are used interchangeably herein and refer to a polymer of amino acid residues and their derivatives. The terms also apply to amino acid polymers in which one or more amino acid residues are analogs or mimetics of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers. The terms also include amino acid polymers that have been modified, for example, by the addition of carbohydrate residues to form glycoproteins, or phosphorylated. Polypeptides and proteins can be produced by naturally occurring non-recombinant cells or by genetically engineered or recombinant cells, and include molecules having the amino acid sequence of a native protein or molecules with deletions, additions, and / or substitutions of one or more amino acids from 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 also contain modified amino acids compared to the full-length protein. In certain embodiments, fragments are between about 5 and 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% of the polypeptides, peptides, lipids, carbohydrates, polynucleotides, or other materials with which the polypeptide is naturally found when isolated from source cells. 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 composed of a C H 1. C H 2, and C H Each light chain consists 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 V H and V L The region 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 from the amino terminus to the carboxy terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The heavy chain variable region CDRs and FRs are HFR1, HCDR1, HFR2, HCDR2, HFR3, HCDR3, HFR4. The light chain variable region CDRs and FRs are LFR1, LCDR1, LFR2, LCDR2, LFR3, LCDR3, LFR4. The heavy and light chain variable regions contain binding domains that interact with antigens. The antibody constant region 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" includes full-length antibodies, antigen-binding fragments of full-length antibodies, and molecules comprising antibody CDRs, VH regions, or VL regions. Examples of 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 chain 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, antibodies disclosed herein refer to polyclonal antibody populations. Antibodies 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 within 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) a single arm V 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, heavy chain variable regions containing a single variable domain and two constant domains. Furthermore, although the two domains of an Fv fragment, VL and VH, are encoded by separate genes, they can be joined using recombinant methods by a synthetic linker that allows the VL and VH domains to be produced as a single protein chain that pairs to form a monovalent molecule (known as a 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 antigen 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 compositions 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 needed. 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" and "graft" and "transplantation" are used to describe the process by which cells are delivered to a site where they are intended to have a beneficial effect, such as repairing damage to a patient's bone or cartilage. Cells can 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 an inert or active carrier that makes the composition particularly suitable for diagnostic or therapeutic use in vivo or ex vivo.

[0115] As used herein, "therapeutic cells" refers to a population of cells that ameliorate 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 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 "pharmaceutically acceptable" is used herein to refer to those compounds, materials, compositions, and / or dosage forms that are, within the scope of sound medical judgment, suitable for use in contact with human and animal tissues without excessive toxicity, irritation, allergic response, or other problems or complications commensurate with a reasonable benefit / risk ratio. A "pharmaceutically acceptable carrier" does not cause undesired physiological effects after administration to or onto a subject. A carrier in a pharmaceutical composition must also be "acceptable" in the sense of being compatible with and capable of stabilizing the active ingredient. One or more solubilizing agents may be utilized as pharmaceutical carriers for delivering the active compound. Examples of pharmaceutically acceptable carriers include, but are not limited to, biocompatible vehicles, adjuvants, additives, and diluents to achieve a usable composition in 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" can 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 received or is currently receiving any form of treatment. In one embodiment, the subject is a human. In another embodiment, the subject is an experimental non-human animal 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 present invention is provided. The term "donor" is used to describe an individual (animal, including a human) who provides cells or tissue 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 can be preventative, in that a disease or its symptoms are completely or partially prevented, and / or therapeutic, in that a disease and / or its resulting adverse effects are partially or completely stabilized or cured. As used herein, "treatment" encompasses any treatment of a disease in a mammal, particularly a human, and includes (a) preventing a disease or condition from occurring in a subject who may be predisposed to, but has not yet been diagnosed as having, the disease or condition; (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. "Amelioration" generally refers to a reduction in the number or severity of 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 to reduce 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 produces a desired local or systemic effect. For example, an effective amount is an amount sufficient to achieve a beneficial or desired clinical result. An effective amount can be provided all at once in a single administration, or can be provided in divided amounts that provide an effective amount in several administrations. The exact determination of what 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. One of ordinary skill in the art would be able to determine an 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 lifespan of an organism. Pluripotent stem cells can give rise to all of the body's various cell types. 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 cell types. Multipotent stem cells can also be referred to as somatic stem cells, tissue stem cells, lineage-specific stem cells, and adult stem cells. Non-stem cell descendants of multipotent stem cells are progenitor cells (also referred to as restricted progenitor cells). Progenitor cells can 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 self-renewal ability; 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. Multipotency means that they can give rise to precursor cells (skeletal progenitors) 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 may be referred to as induced skeletal stem cells, or iSSCs. iSSCs are derived from non-skeletal cells through experimental manipulation. Induced skeletal cells possess the characteristics of naturally occurring functional SSCs, i.e., they can give rise to the same lineage. Human SSC cell populations may 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 may be characterized as CD45-, Ter119-, Tie2-, and αv integrin+. Mouse SSCs may be further characterized as Thy1-6C3-CD105-CD200+.

[0127] Suture stem cells (SuSCs) are a population of skeletal stem cells derived from suture mesenchyme that exhibit long-term self-renewal, clonal proliferation, and multipotency. These SuSCs reside at the midline suture 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 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 smooth muscle, 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 preserving their functionality as stem cells, i.e., being self-renewing and able to give rise to the full range of progenitor lineages appropriate for a particular type of stem cell (these two together constitute "regenerative activity"). One way to demonstrate successful stem cell maintenance is through engraftment experiments in which all appropriate cell types (bearing genetic markers that distinguish them from the host) are observed to arise from the graft and persist over an extended period of time, e.g., four months.

[0130] As used herein, the term "expanding stem cells" refers not only to maintaining stem cells but also to culturing stem cells so as to increase the number of stem cells in culture. One way to demonstrate successful expansion of stem cells is through engraftment experiments, which compare 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 initially 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 may correlate with cell development, differentiation, or fate. In addition, the markers disclosed herein include messenger RNA (mRNA) that encodes a marker polypeptide, as measuring changes in mRNA expression 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 mRNA encoding the polypeptide biomarker, either by measuring 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 programmed to become skeletal stem cells, the marker is expressed only by cells in the culture or tissue that will, are developing, and / or have developed into skeletal stem cells. Those skilled in the art will understand that the specified expression level reflects the detectable amount of marker protein or mRNA on or in the cells. Cells that stain negative (the level of binding 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 characteristic. The number of molecules on the cell surface can vary by several logs and still be characterized as "positive."

[0133] Standard gene / protein nomenclature guidelines generally provide 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). Furthermore, standard gene / protein nomenclature guidelines generally provide 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, when referring to specific biomarkers, the gene / protein nomenclature used herein uses biomarker abbreviations with all capital letters (e.g., BMPR1A) or lowercase letters (e.g., Bmpr1a) and is intended to encompass 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 following examples, related figures, and related description). Thus, the exemplary human or mouse biomarkers described herein do not limit the present subject matter to only human or mouse polypeptide or mRNA biomarkers. Rather, the present subject matter encompasses biomarkers across animal species related to SSCs.

[0135] As disclosed herein, several ranges of values ​​are provided. Unless the context clearly dictates otherwise, it is understood that each intervening value, to the tenth of the unit of the lower limit, between the upper and lower limits of that range is also specifically disclosed. 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 independently be included or excluded in the range, and each range in which either, neither, or both limits are included in the smaller ranges is also encompassed within the invention, subject to any specifically excluded limits in the stated range. When a stated range includes one or both of the limits, 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 more preferably still up to 1% of a given value. Unless otherwise specified, the term "about" means within an acceptable range of error for a particular value. [Example]

[0137] Example 1 Study Design, Materials, and Methods This example describes the study design, materials, and methods used in Examples 2-11 below.

[0138] research design This study was designed to identify regulators of SuSCs. Using a genomic approach, we found that BMP signaling is potentially involved in SuSC regulation. We generated loss-of-function mouse models to individually investigate three BMP type I receptors and revealed that Bmpr1a is essential for SuSC-mediated calvarial morphogenesis. We used an inducible knockout system to specifically disrupt Bmpr1a in Axin2+ SuSCs in developing mice, assessed the effects of disruption on suture fusion, and investigated the mechanism of abnormal suture closure. To test whether stem cell depletion caused premature differentiation that mediated abnormal suture closure, we investigated the stemness, self-renewal, quiescence, stem cell frequency, proliferation, clonal expansion, and osteogenic potential of SuSCs using ex vivo culture and in vivo transplantation assays. To provide a translational approach to humans, we analyzed human SuSC populations to determine whether BMPR1A served as a cell surface marker for the isolation of both mouse and human SuSCs with skeletal stem cell characteristics. To ensure scientific reproducibility, all studies were performed multiple times with appropriate controls, including wild-type mice or mice carrying the appropriate transgene. Mice of both sexes were tested for potential sensitivity of skeletogenesis to sex hormones. For mouse studies, at least three or five mice were used per group. For culture and transplantation studies, at least three to five independent experiments were performed. Human samples were obtained from nonsyndromic patients with synostosis, and at least five independent samples were used in this study. Analysis of samples by μCT was performed by technicians blinded to their condition. No randomization, statistical methods for predetermining sample size, or sample inclusion / exclusion criteria were used.

[0139] Animals and Models Axin2-rtTA, TRE-H2BGFP, TRE-Cre, R26RTomato, Bmpr1a Fx , Acvr1 Fx , Bmpr1b - / -The SCID mouse strain and genotyping methods were previously described (18, 25, 46-54). GFP To generate the Axin2 strain (18, 55), mice carrying the Axin2-rtTA and TRE-H2BGFP transgenes were obtained and treated with Dox (2 mg / ml plus 50 mg / ml sucrose) for 3 days as described (46, 47, 52). Cre-Dox The strain was generated by obtaining mice carrying the Axin2-rtTA and TRE-Cre transgenes. Cre-Dox Mice were transfected with Bmpr1a Fx , Acvr1 Fx and R26R Tomato mice, respectively. Ax2 , Acvr1 Ax2 , and Axin2 Cre-Dox R26R Tomato mice were obtained. Cre expression in Axin2-expressing cells was then induced by Dox treatment (18, 56). Both male and female mice were used in this study. The care and use of experimental animals described in this study complied with the guidelines and policies of the University Committee on Animal Resources at the University of Rochester.

[0140] Cell isolation and purification Primary suture mesenchymal cells containing SuSCs were isolated from mouse calvaria as described (5). Briefly, an approximately 1.5 mm wide tissue containing the sagittal suture and adjacent crestal bone was dissected, and the suture was separated from the crestal bone. The suture was then incubated with 0.2% collagenase in PBS (pH 7.0–7.6, product code 21-031-CV, Corning) at 37°C for 1.5 h. Dissociated cells were filtered using a CELL STRAINER 40 μm Nylon (No. 352340, FALCON) and then resuspended in DMEM medium for transplantation analysis, DMEM containing 5% fetal bovine serum (FBS) for cell sorting, or DMEM containing 25 μg / ml insulin, 100 μg / ml transferrin, 20 nM progesterone, 30 nM sodium selenate, 60 nM putrescine, 20 ng / ml EGF, 20 ng / ml bFGF, 20 ng / ml B27 supplement, and 1% penicillin-streptomycin for sphere culture.

[0141] For in vitro culture as spheres, cells were grown on ultra-low attachment surface plates (number 2023-03-23, Corning) for 7–10 days while the cells formed spheres (1 0 Spheres were dissociated with 0.25% trypsin-EDTA and cultured for the next passage (2 0 The cells were dissociated and seeded as a single cell suspension onto ultra-low attachment plates for culture. This dissociation and seeding was continued for up to five passages (5 0 For differentiation, spheres were transferred to 24-well plates with treated surfaces (662160, GREINER BIO-ONE, Monroe, NC) to enhance attachment and cultured for 3 weeks in differentiation α-MEM medium containing ascorbic acid (50 μg / ml) and 4 mM β-glycerophosphate.

[0142] For the isolation of human suture cells, we obtained calvarial waste containing unfused sutures from patients (aged 3-14 months) with nonsyndromic craniosynostosis. Bone fragments were removed to obtain suture mesenchyme, and the tissue was incubated with 0.2% collagenase in PBS (pH 7.0-7.6, product number 21-031-CV, Corning) at 37°C for 1.5 hours. Dissociated cells were then filtered through a 40 μm strainer and subsequently resuspended in DMEM medium containing 20% ​​FBS for sphere culture or in PBS containing 3% FBS for cell purification.

[0143] To purify Bmpr1a and Bmpr1a cell populations, freshly isolated suture cells were stained with a primary mouse monoclonal Bmpr1a antibody (MA5-17036, Thermo Fisher Scientific, Waltham, MA) and subsequently sorted according to the intensity of the secondary antibody-conjugated Texas Red using a FACSAria-II (BD Biosciences, San Jose, CA). The specificity of the Bmpr1a antibody for isolating cells with abundant Bmpr1a was determined by fluorescence-activated cell sorting (FACS) (Figure 20).

[0144] renal capsule transplantation Transplantation of freshly isolated suture cells or cultured sphere cells into the kidney capsule was performed as described (5). Freshly isolated cells were obtained from P5 mice. For limiting dilution analysis, 10 2 ~10 5 The stem cell frequency was calculated using Extreme Limiting Dilution Analysis (ELDA) software with validation of the likelihood ratio test for single-hit models (57). By transplanting 30 spheres per kidney capsule, 1 0 and 3 0 Passaged spheres were tested.

[0145] Staining and analysis Preparation, fixation, and embedding of skulls for paraffin and frozen sections were performed as described (16, 18, 56, 58). Samples were subjected to hematoxylin / eosin staining, Goldner trichrome staining, GFP analysis, β-gal staining, van Kossa staining, or immunological staining using avidin:biotinylated enzyme complex for histology (16, 18, 46, 47, 58-62). For antigen retrieval, samples were incubated in antigen unmasking solution (H3300, VECTOR) for 10 min during pressure cooking or in 20 mM Tris-HCl (pH 9) for 16 h at 70°C. For in vitro deletion of Bmpr1a, mouse Bmpr1a was used. Fx / Fx Cells isolated from suture lines were infected with lenti-GFP or lenti-Cre viruses (MOI = 1). Whole-mount von Kossa staining, immunohistochemistry, in situ hybridization, and double-labeling analysis were performed as described (5, 58, 63). For von Kossa staining and double-labeling immunohistochemistry, samples were fixed with 2% paraformaldehyde and 0.02% NP-40 at room temperature for 1 hour, followed by incubation with 1% silver nitrate for 30 minutes under UV light and 5% sodium thiosulfate for 5 minutes. Stained samples were then processed for paraffin sectioning and subsequent immunohistochemistry.

[0146] To detect proliferating cells, after 4 days of culture, EdU was added to the spheres for 16 hours, followed by attachment using CYTOSPIN (THERMO FISHER SCIENTIFIC). After fixation with 95% ethanol for 5 minutes on ice and 2% paraformaldehyde for 20 minutes at room temperature, the spheres were treated with 0.5% TRITON-X100 for 10 minutes and then incubated in EdU reaction buffer for 30 minutes according to the manufacturer's protocol (THERMO FISHER SCIENTIFIC). Rabbit polyclonal antibodies OSTERIX (ab22552, ABCAM, Cambridge, MA, 1:200), Bmpr1a (ABP-PAB-10536, ALLELE, San Diego, CA, 1:100), phospho-Tak1 (arb191688, BIORBYT, St Louis, MO, 1:200), phospho-ERK1 / 2 (4370, CELL SIGNALING TECHNOLOGY, 1:50), rabbit monoclonal antibodies Ki67 (RM-9106, THERMO FISHER SCIENTIFIC, 1:200), Axin2 (2151, CELL SIGNALING TECHNOLOGY, 1:500), phospho-p38 MAPK (4511, CELL SIGNALING TECHNOLOGY, 1:200), and phospho-JNK (4668, CELL SIGNALING TECHNOLOGY, 1:500). A 1:100 antibody (Bmpr1a, Thermo Fisher Scientific, MA5-17036) was used for immunostaining. A Bmpr1a antibody (Bmpr1a, Thermo Fisher Scientific, MA5-17036) was used for FASC sorting and immunostaining studies. Images were captured using a ZEISS AXIO OBSERVER microscope (Carl Zeiss, Thornwood, NY) or a LEICA DM2500 microscope equipped with a DFC7000T digital imaging system (LEICA BIOSYSTEMS Inc., Buffalo Grove, IL).

[0147] statistical analysis Statistical analysis was performed using R software version 3.2.1 or Microsoft Excel 2010. Significance was determined by a two-tailed Student's t-test. A p-value of less than 0.05 was considered statistically significant. Before performing the t-test, the normality of data distribution was first verified by the Shapiro-Wilk normality test. Signaling pathway activity in SuSCs was estimated by activity Z-score using IPA software (Ingenuity® Systems). Statistical data were expressed as mean + SEM or SD. Stem cell frequency was examined using Extreme Limiting Dilution Analysis (ELDA) software with validation of the likelihood ratio test for single-hit models (57).

[0148] Example 2 Identification of the BMP pathway in Axin2-expressing SuSCs To identify and isolate Axin2-positive (Axin2+) cells in the suture mesenchyme and trace the progeny of these cells, we genetically engineered mice that inducibly express green fluorescent protein (GFP), reflecting activity from the Axin2 promoter in a spatiotemporal-specific manner (Axin2). GFP ) (Fig. 9A). Using a system similar to inducible expression of Cre driving either lacZ for β-galactosidase (β-gal) labeling or the fluorescent protein Tomato, we established a mouse model that allows tracking of Axin2+ cells (Fig. 9B). Axin2+ cells at postnatal day 28 (P28) were detected. GFP From mice, we isolated an Axin2+ cell population with a high-intensity GFP signal (Axin2 + / GFP hi ) (Figure 9C), and a non-expressing cell population that was GFP-negative (Axin2 - / GFP - ) were isolated from suture mesenchyme.

[0149] Microarray analysis comparing SuSCs (Axin2+) and non-SuSCs (Axin2-) revealed approximately 9,000 genes with significant differences (p-value < 0.05, n = 3). We performed pathway analysis using the INGENUITY PATHWAY (IPA) software to obtain two scores: an enrichment score, which represents the statistically significant accumulation of genes in each pathway, and a Z-score, which represents the activation status of signals by matching the observed and predicted patterns of up- and down-regulation (19). In SuSCs, most of the identified signaling pathways were inactive, except for the BMP pathway, which showed significant activation (Figure 10A, p-value < 10). -13 , z-score >2.3). Detailed analysis of the expression of genes encoding BMP signaling components showed that seven BMP ligands and the type I receptor Bmpr1a were upregulated, and two negative regulators (Smad7 and Smurf1) were downregulated in SuSCs (Fig. 10B). The results suggested that BMP ligands signal through Bmpr1a to activate pathways in SuSCs. Therefore, we hypothesized that SuSCs express Axin2, which is marked by lacZ. Cre We probed SuSCs for Bmpr1a in the R26RlacZ model (Figure 9B). Double labeling identified Bmpr1a in Axin2+ SuSCs at P28 (Figures S10C-S), consistent with a role for BMP Bmpr1a signaling in SuSC regulation.

[0150] Example 3 Identification of the requirement for Bmpr1a for SuSC-mediated calvarial development and homeostasis To address the functional importance of BMP signaling in SuSCs, we investigated type I receptors in calvarial morphogenesis (20, 21). Most BMP family members signal through one of three type I receptors: Bmpr1a, Bmpr1b, and Acvr1 (20, 21). We focused on receptors because many BMP ligands exist, making genetic studies difficult. While mice with global inactivation of Bmpr1b are viable, null mutations of Bmpr1a or Acvr1 are associated with embryonic lethality due to defective mesoderm formation (22-25). Therefore, we hypothesized that Bmpr1a is involved in the signaling pathway. Ax2 (Axin2 Cre-Dox ;Bmpr1a Fx / Fx ) and Acvr1 Ax2 (Axin2 Cre-Dox ;Acvr1 Fx / Fx We developed a model to allow doxycycline (Dox)-induced deletion of Bmpr1a or Acvr1 in Axin2+ SuSCs. To study calvarial formation, Dox was administered from embryonic day (E) 16.5 to P3 to initiate Cre-dependent gene deletion (Figure 1A). The efficiency of Cre-mediated recombination in Axin2+ SuSCs and their progeny cells was demonstrated using the R26RlacZ reporter strain (Figure 11A,B). Immunostaining demonstrated not only the effectiveness of Bmpr1a ablation in the mutants but also the specificity of the Bmpr1a antibody (Figure 11C,D).

[0151] Bmpr1a Ax2 Mice showed craniofacial abnormalities at 2 months, but not Bmpr1b- / - or Acvr1 Ax2 This was not the case in mice (Fig. 1B and Fig. 12A-B). Ax2 Mutants were easily identified by their abnormal skull shape. Micro-computed tomography (μCT) analysis and histology revealed abnormalities in the calvarial bones and suture closure specifically caused by the loss of Bmpr1a (Fig. 1C, D and Fig. S12). Ax2The skull was significantly shorter, although there was no significant difference in width, throughout the first 14 days of postnatal development (Figure 12A-D, p < 0.05). Ax2 The skulls of mutant mice were dome-shaped compared to the flatter skulls of control mice (Figures 12E, F). Analysis of the skulls stained with Alizarin Red (Figures 13A, B), histological analysis (Figures 13C-H), and μCT analysis (Figure 14) revealed that Bmpr1a Ax2 Multiple synostosis was revealed in the nasal, prefrontal, sagittal, lambdoid, and squamosal sutures in mice. The results indicated a specific requirement for Bmpr1a in SuSCs during calvarial morphogenesis.

[0152] We previously demonstrated that Axin2+ cells function as skeletal stem cells in calvarial development and homeostasis (5). To test whether Bmpr1a regulates adult SuSCs, we induced its deletion in the adult skull. In humans, skull growth reaches 90% of adult size in the first year and 95% of adult size by age 6 (26). The size of the teenage skull is identical to that of an adult. In mice, 90% of skull development is completed by P28, and SuSCs are restricted to the midline suture (5, 27). Therefore, we hypothesized that P28 Bmpr1a Ax2 Mice were administered Dox for 7 days (Figure 1E). Three months after Dox treatment, mutants were examined by μCT and histology. Deletion of Bmpr1a in adult SuSCs resulted in abnormal suture morphogenesis and multiple suture synostosis (Figure 1F, G), suggesting an essential role for Bmpr1a in SuSC-mediated calvarial homeostasis. Thus, analysis of mice lacking Bmpr1a during early postnatal development, along with mice lacking Bmpr1a after skull maturation, together demonstrated that Bmpr1a is important for both skull development and homeostasis.

[0153] Example 4 Craniosynostosis is caused by Bmpr1a Ax2 Starts at the midline of the suture A time course study was performed to decipher the suture closure process. Dox-induced deletion of Bmpr1a was performed from E16.5 to P3. Skulls from mice were evaluated by Alizarin Red staining (Figure 2A) and Goldner Trichrome staining (Figure 2B) at P0, P7, and P14 (Figure 2). At P0, Bmpr1a deletion caused wider sutures. However, in the absence of Bmpr1a, abnormal ossification within the suture mesenchyme was evident at P7 and P14, ultimately leading to suture closure at 2 months (Figure 1C, D). This finding suggested that abnormal ossification initiated at the suture midline and moved toward the osteogenic front.

[0154] Calvarial bones form through osteoblast-mediated intramembranous ossification. To explain the abnormal ossification process caused by SuSC-specific deletion of Bmpr1a, we investigated osteoblast proliferation and differentiation. In the sutures of control mice at P3, immunostaining for Ki67, a marker for cells undergoing mitosis, revealed that most cells were quiescent in the suture mesenchyme but actively proliferated at the osteogenic front, the site of intramembranous ossification toward the suture midline (Figure 3A). Ax2 In mice, the number of Ki67+ cells was abnormally increased in the suture mesenchyme (Figure 3B). To investigate osteoprogenitor cells, we performed immunostaining for Osterix (Osx), and to detect osteoblasts, we performed in situ hybridization for type I collagen (Col1). At P0, Osx+ osteoprogenitors were significantly higher than those in control and Bmpr1a mice. Ax2 However, at P3, we detected an increase in the number of Osx+ osteogenic precursors in the suture mesenchyme in response to Bmpr1a deletion in SuSCs (Fig. 3C). As observed in the calvaria of control mice, Col1+ osteoblasts, rather than clusters of Col1+ osteoblasts at the osteogenic front, were detected only at both osteogenic fronts of the SuSCs (Fig. 3C). Ax2 It was found throughout the suture mesenchyme of the calvarium (Figure 3D). - / - ;Fgfr1 + / -Compared with mouse sutures, no type II collagen (Col2)-positive chondrocytes were detected in the mutants (Figure S15), suggesting that loss of Bmpr1a function did not promote stem cell fate changes and that abnormal suture closure was not caused by ectopic cartilage formation and endochondral ossification. This finding indicated that abnormal ossification was initiated in the suture mesenchyme, not at the osteogenic front.

[0155] Example 5 Signaling Effects of Bmpr1a on SuSCs in the Developing Suture To investigate downstream pathways affected by loss of Bmpr1a function in SuSCs, we analyzed "canonical" BMP signaling mediated by Smad proteins (28) and "non-canonical" kinase-mediated signaling (29). BMP signaling through Bmpr1a activates the transcriptional regulators Smad1, Smad5, or Smad8, or some combination thereof (collectively, Smad1 / 5 / 8). Immunostaining revealed that control and Bmpr1a-activated BMPs signaled through the Smad1 / Smad8 transcription factors, Smad1, Smad5, or Smad8 transcription factors, or some combination thereof (collectively, Smad1 / 5 / 8). Ax2 showed comparable amounts of phosphorylated Smad1 / 5 / 8 at the osteogenic front and periosteum (Fig. 16, top). However, the amount of phosphorylated Smad1 / 5 / 8 was significantly higher in Bmpr1a than in Bmpr1a. Ax2 It appeared to be less at the midline suture (Fig. 16A, bottom). Immunostaining for phosphorylated TAK1 showed activation of this kinase primarily at the osteogenic front, with similar amounts of phosphorylated TAK1 present in control and Bmpr1a. Ax2 Bmpr1a was present at the osteogenic front and suture region in mice (Fig. 16B). Examination of the activation of mitogen-activated protein kinases (MAPKs) downstream of Tak1 revealed strong activation (phosphorylation) of p38, but not JNK (Fig. 16C). Another MAP kinase, Erk, was also enriched in the mutant (Fig. 16C). These results indicated that loss of Bmpr1a reduced canonical but enhanced noncanonical signaling in SuSCs, leading to premature differentiation at the midline suture and craniosynostosis.

[0156] Example 6 Bmpr1a is required for the maintenance of SuSCs Bmpr1a Ax2 The enhanced cell proliferation and number of osteoprogenitors and osteoblasts in the suture mesenchyme (Figure 3) suggested a potential role for Bmpr1a in stem cell maintenance. We hypothesized that loss of Bmpr1a depletes the stem cell population, leading to premature osteoblast differentiation and intramembranous ossification. To test this hypothesis, we analyzed Bmpr1a Ax2 We investigated the SuSC characteristics of Bmpr1a. By in vivo clonal proliferation analysis, we demonstrated the ability of single Axin2+ SuSCs to generate calvarial bone when transplanted into the kidney capsule (5). Using limiting dilution analysis, we investigated stem cell clonal proliferation in transplanted kidneys and further established a quantitative method for measuring stem cell frequency (5). We used this assay to investigate whether Bmpr1a deficiency affects clonal proliferation and the number of SuSCs. Various amounts of cells isolated from the Bmpr1a Ax2 suture from control and P5 mice were transplanted into the kidney capsule, and the transplantation site was then evaluated by von Kossa staining to detect mineralized ectopic bone (Figure 4A) and bone structure was examined by histological analysis (Figure 4B). 3 ~10 5 Transplantation of 10 control cells had a 100% success rate in bone formation (Figure 4C). 2 At 10 cells, ectopic bone tissue formed less frequently than at higher cell numbers (Figure 4C). In contrast, ectopic bone tissue formed at 10 cells. 2 ~10 3 Bmpr1a Ax2 The cells were not detected in the transplanted kidneys, and 10 5 All kidneys showed osteogenesis only in cells from the ELDA gene (Figure 4C). Using ELDA software to estimate stem cell frequency (33), we found that loss of Bmpr1a significantly reduced SuSC frequency at the P5 suture (Figure 4C; control: 1 in 216 cells, and Bmpr1a Ax2 : 1 in 23,572 cells, p-value = 5.7 × 10 -6 ).

[0157] Furthermore, immunohistochemistry revealed that Bmpr1a Ax2revealed a significant loss of Axin2+ SuSCs at the suture line ( Fig. 4D ; control, 4.9 ± 0.3%, Bmpr1a Ax2 0.6±0.2%, p-value<0.01). These data suggested that Bmpr1a plays an essential role in maintaining SuSC stemness, as loss of Bmpr1a induces their premature differentiation and abnormal ossification at the midline suture, resulting in craniosynostosis.

[0158] Example 7 Preservation of SuSC stemness in culture Conventional culture methods for mesenchymal stromal cells (MSCs) do not preserve SuSC stemness, so protocols for maintaining SuSC stemness in vitro are needed. Sphere culture maintains the properties of neural and mammary stem cells and can recapitulate in vivo characteristics (34). We have established a culture protocol (Figure 17A) for cells isolated from suture mesenchyme that maintains stem cell properties. We have demonstrated that isolated suture cells can retain primary (1-cell) characteristics when grown in single-cell suspension culture at very low seeding densities. 0 ) and formed spheres (Fig. 17B, C). 0 When the spheres are dissociated into single cells, the cells form secondary (2 0 ) spheres were formed (Figure 17D). 4 When cells were seeded, suture cell spheres continued to form up to five passages without a significant decrease in number, suggesting the presence of SuSCs with self-renewal capacity (Figure 17E). Time course analysis suggested that each sphere was formed from a single cell (Figure 17F-K). The average sphere size remained comparable across different passages (Figure 17L).

[0159] To determine the cellular origin of sphere-forming cells, we investigated the expression of Axin2 Cre-Dox The R26R tomato model was used (Figure 9B). Axin2 was treated with Dox for 3 days from P7 to P10. Cre-DoxSuture cells from R26R Tomato mice were cultured in the absence of Dox. A small proportion of cells were positive for Tomato at the initiation of single-cell suspension culture (Fig. 5A, top row, 5 ± 0.3%, n = 3, mean ± SEM). After 2 weeks, most spheres consisted entirely of Tomato cells, suggesting that they were derived from a single Axin2 cell with clonal expansion potential (Fig. 5A, B). We did not detect chimeric spheres containing a mixture of Tomato and Tomato cells, indicating that suture spheres are not formed by cell aggregation.

[0160] We assessed the pluripotency of suture spheres by culturing the spheres under conditions that promote differentiation. These pluripotency tests were performed in 3 0 We demonstrated that the spheres differentiated into osteoblasts, forming mineralized nodules, or into chondrogenic cells (Figure 17M-O). To investigate the clonal growth and osteogenic potential of suture spheres in vivo, we performed kidney capsule transplantation assays. We found that Axin2 Cre-Dox Thirty spheres formed from cells isolated from the R26R tomato suture were transplanted into the renal capsule. The spheres successfully proliferated, colonized, and engrafted (Figure 5C). Transplanted 1, similar to transplanted freshly isolated suture cells (5), underwent intramembranous ossification. 0 spheres (Fig. 5D, E) or 3 0 The spheres (Figure 17P-S) generated bone tissue resembling calvarial bone (Figure 5E). This result demonstrated that the newly developed culture system preserves SuSC stemness and differentiation properties and allows for analysis in an ex vivo environment.

[0161] Example 8 Ex vivo characterization of SuSCs Previous in vivo examination of 1-month-old mouse SuSCs and data from P3 suture mesenchyme (Figure 3A) demonstrated their quiescence (5). These quiescent SuSCs must be included in the isolation procedure disclosed herein for sphere culture. To test whether a subpopulation of isolated SuSCs exhibits quiescence in culture, we performed pulse-chase labeling analysis by labeling cells in vivo and then tracking them in culture. Axin2 Dox-GFP Using the (Axin2-rtTA;TRE-H2BGFP) mouse model (Figure 9A), we performed pulse-chase analysis to investigate Axin2+ SuSCs. Axin2-expressing cells were labeled in vivo with GFP by administration of Dox at P7–P10 (5). Suture cells were isolated at P10 and cultured in the absence of Dox for sphere formation. 0 GFP analysis of the spheres revealed only single cells with strong fluorescence intensity (Figure 6A). 0 and 3 0 Similar results were obtained in cultures of 1 (Fig. 6B-C). 0 32% of the spheres in 2 were devoid of any GFP+ cells, whereas 2 0 and 3 0 All spheres found in passage 1 contained one strong GFP+ cell (Figure 6D). 0 The presence of some GFP-negative spheres in the culture of 1 0 This is likely due to the presence of skeletal precursors with limited proliferative capacity, which generate only spheres in the initial culture but not in subsequent passages. We hypothesized that the persistent presence of single cells that are strongly GFP-positive results from asymmetric division, whereas symmetric division dilutes the GFP signal (Figure 6E). We confirmed by immunofluorescence analysis that GFP+ cells were also positive for Axin2 (Figure 6F). Labeling of spheres of proliferating cells indicated that the GFP+ cells were not actively proliferating (Figure 6G). These results support the hypothesis that SuSCs undergo asymmetric division, with one daughter cell remaining undifferentiated and thus exhibiting label-retention capacity, and the remaining cells in the sphere arising from the other daughter cell (Figure 6E).

[0162] Example 9 Requirement of Bmpr1a for SuSC self-renewal and osteogenesis Using ex vivo pulse-chase labeling analysis, we investigated the distribution of Bmpr1a in spheres. Spheres immunostained for Bmpr1a showed that this receptor was present in GFP+ cells and some neighboring cells (Figure 6H). These results are consistent with in vivo double-labeling analysis, which showed only partial overlap of Bmpr1a and Axin2 reporter signals (Figure 10C-E). We then used continuous culture analysis to investigate the requirement for Bmpr1a for SuSC self-renewal. P5 control and Bmpr1a Ax2 Culture of cells isolated from sutures 0 The cultures showed comparable spheres (Figure 6I). 0 and 3 0 The number of spheres was significantly reduced in cultures derived from mutant mice, suggesting that the self-renewal capacity of SuSCs was impaired by the loss of Bmpr1a (Figure 6I, p-value < 0.05, n = 3, mean ± SEM, Student's t-test). The size of mutant spheres was also smaller compared to controls (Figure 6J). Thus, this data indicated that Bmpr1a plays an essential role in maintaining SuSC self-renewal and stemness properties in sphere cultures.

[0163] Our previous study showed that SuSC self-renewal is associated with clonal expansion and bone regeneration in vivo, especially when small numbers of cells are used in transplantation assays (5). To test whether clonal expansion and bone formation capacity are affected in Bmpr1a-deficient SuSCs, we transplanted 30 spheres into the kidney capsule. In this assay, we found that the formation of mutant spheres was impaired in subsequent passages, suggesting a 1-fold increase in the number of spheres. 0 spheres were used (Figure 6I). Ax2 1 0 Heterotopic bone formation mediated by the mutant suture spheres was severely impaired (Figure 6K-N). In one of three transplants with mutant spheres, we detected a small area stained with von Kossa.

[0164] Bmpr1a Ax2 To rule out potential non-cell-autonomous effects on SuSCs that occur before isolation from Bmpr1a-deficient mice using the model, we investigated the effect of Bmpr1a Fx / Fx Use suture line cells isolated from mice, infect the cells with lentivirus expressing GFP (control) or Cre in culture, grow the cells until they form spheres, and then transplant them into the kidney capsule for 1 min. 0 spheres were used. The efficiency of lentivirus-mediated expression with minimal toxicity was determined with lentivirus expressing RFP. At a multiplicity of infection (MOI) of 1, expression appeared optimal without significant changes in sphere size or number (Figure 18A-C). Cre-dependent deletion of Bmpr1a in suture spheres was highly efficient (Figure 18D) and significantly reduced the size of the generated bone tissue when transplanted into the kidney capsule (Figure 18E-G, p-value < 0.05, n = 3, mean ± SEM, Student's t-test).

[0165] Bmpr1a Ax2 These model-derived results suggested two important roles for Bmpr1a: it supports asymmetric division and clonal expansion of SuSCs (Fig. 6) and osteogenesis of SuSCs in a cell-autonomous manner (Fig. 6 and Fig. S18). Thus, the data indicated that Bmpr1a regulates not only SuSC self-renewal but also SuSC-mediated skeletal formation.

[0166] Example 10 Characterization of Human SuSCs To test for the presence of human SuSCs and the ability to isolate and maintain them in culture, we obtained discarded tissue containing unfused sutures from patients with craniosynostosis undergoing surgery. First, we detected AXIN2- and BMPR1A-positive cells in the human midline sutures by immunostaining of tissue sections (Figures 7A-C and 19). We found that isolated human suture cells, when cultured in single-cell suspension at very low seeding densities, were able to grow in 100% of the tissues. 0We determined that the spheres grew to 200 μm in size (Figure 7D). We observed no significant decrease in number or size after successive reseeding. 0 and 3 0 Obtain a sphere (1 0 ~3 0 About 10 4 These results indicate the presence of human SuSCs with self-renewal capacity (Figure 7E, F). Human suture spheres stained for AXIN2 and co-labeled with EdU revealed that in spheres with AXIN2+ cells, these cells were not actively proliferating (Figure 7G-I). Some human spheres did not contain AXIN2+ cells. These results indicated that human SuSCs are quiescent / low-cycling cells that maintain their stemness through asymmetric division.

[0167] Finally, transplantation of human cells into the mouse kidney capsule demonstrated von Kossa-positive ectopic bone formation in whole mounts and sections (Figure 7J-K) with an 80% success rate (n=5). The findings disclosed herein demonstrate the successful isolation and culture of human SuSCs, a major hurdle to overcome for translational research.

[0168] Example 11 Bone formation from mouse and human Bmpr1a-expressing cells The important role of Bmpr1a in stem cell regulation and the overlap in Bmpr1a and Axin2 positivity (Figure 10E) prompted us to test its use as a cell surface marker for SuSC isolation. Using a specific antibody and fluorescence-activated cell sorting (FACS), we isolated mouse-derived Bmpr1a 高 (BMPR1A of human origin 高 ) and Bmpr1a / BMPR1A derived from mouse / human sutures 低 Cell populations were purified (Figures 8A and 20). Mouse suture cells were derived from P10 C57 / BL6 mice, and human cells were derived from discarded calvarial tissue containing unfused sutures from a patient with craniosynostosis. 高 Successful bone formation was evident in animal recipients transplanted with Bmpr1a低 This was not the case in mouse suture cells (Figure 8B-C). Immunostaining for Osterix showed that mouse Bmpr1a 高 (Figure 8D-F) We identified osteoprogenitor cells surrounding the mineralized tissue generated by cell transplantation. 高 The same results were achieved with suture cells (Figure 8G-L). The results indicated that Bmpr1a / BMPR1A functions as a SuSC marker. Furthermore, these results demonstrate that Bmpr1a not only functionally regulates stemness of stem cells essential for suture patency and patellar synostosis in both mice and humans, but also that SuSCs express Bmpr1a. 高 It was confirmed that it was contained in the cell population.

[0169] Consideration This application provides evidence that Bmpr1a is essential for SuSC regulation. Loss of Bmpr1a in Axin2-expressing cells impairs SuSC self-renewal, clonal expansion, and osteogenic capacity. Therefore, Bmpr1a is required to maintain these functions related to stem cell stemness, implicating this receptor in suppressing differentiation or promoting asymmetric cell division. The inhibitory effect of BMP signaling on early bone formation is supported by previous reports showing that neonatal disruption or ablation of Bmpr1a in osteoprogenitor cells increases osteoblast numbers (35-37). Loss of Bmpr1a reduced Smad phosphorylation and enhanced p38 and ERK activation, suggesting that the balance between canonical and non-canonical BMP signaling cascades is altered in SuSCs. Alternatively, inactivation caused overactivation of downstream signaling of other BMP type I receptors. It has been proposed that Bmpr1a regulates this balance through modulation of Tak1 activity (38). Tak1 inhibits Bmpr1a Ax2 Since Bmpr1a was not activated in the mutant, the findings disclosed herein suggested that a non-canonical pathway distinct from Tak1-mediated MAPK signaling is involved in Bmpr1a-mediated SuSC stemness.

[0170] In kidney capsule transplants, only suture cells positive for Axin2, but not negative, can generate bone (5). This suggests that skeletal stem cells contained in the Axin2-positive cell population possess bone-forming potential in the kidney capsule. Despite the presence of osteogenic precursors or osteoblasts within the Axin2-negative cell population, these cells are unable to form ectopic bone (5). The requirement of Axin2-positive cells for ectopic bone formation may explain why direct engraftment and replacement of damaged tissue is difficult to achieve with most cell-based therapies. The number of Axin2-positive cells is too low in most therapies, and osteoblasts, despite being bone-forming cells in vivo, are ineffective in bone formation upon transplantation. For therapeutic success, the survival, engraftment, and proliferation of transplanted cells appear to be crucial factors. Only stem cells possess these properties, and we found that these properties were preserved by Bmpr1a using both in vivo ablation and culture deletion models. Further elucidation of the regulatory mechanisms underlying cell survival and engraftment promises important insights into Bmpr1a-mediated bone regeneration and provides new strategies for stem cell-based therapy.

[0171] SuSC-specific ablation of Bmpr1a resulted in premature differentiation and suture fusion. The findings disclosed herein reveal a novel etiology of craniosynostosis—stem cell depletion. This pathogenic mechanism is distinct from other known mechanisms of cell proliferation, differentiation, and apoptosis, all of which lead to excessive intramembranous ossification (14). It also differs from previous reports that suture fusion may be caused by a stem cell fate switch. SuSCs undergo chondrogenesis instead of osteoblast formation, resulting in craniosynostosis mediated by ectopic endochondral ossification (18). Stem cell depletion has previously been associated with ossification deficiency, potentially associated with patients with Cole-Carpenter syndrome, which exhibits wide, open midline sutures containing intrasuture bones (39). Intrasuture bones, also known as Worm's bones, frequently occur in disorders of reduced cranial ossification and are associated with craniosynostosis (40). The stem cell depletion mechanism we have identified herein may be used in patients with synostosis who do not have an enhanced ossification phenotype.

[0172] Although skeletal stem cells present at sutures have been identified, their role in craniosynostosis has not been investigated (5, 6). Axin2 and Gli1 have been used to identify skeletal stem cells in the calvaria (5, 6). However, deletion of Bmpr1a in Gli1+ cells does not induce craniosynostosis, despite enhancing osteoblast proliferation and differentiation (41). This discrepancy may be due to the presence of Axin2 in a more restricted cell population at the midline suture (5, 6). Furthermore, Bmpr1a colocalizes with Axin2 but not with Gli1 (41). The results disclosed herein demonstrate that Axin2+ SuSCs undergo asymmetric division to maintain quiescence. We believe that disruption of Bmpr1a-dependent regulation of SuSC quiescence is likely the trigger for craniosynostosis. We propose that SuSC stemness is maintained by Bmpr1a, and its deletion results in abnormal ossification initiated at the midline suture. Thus, craniosynostosis results from a deficiency of skeletal stem cells.

[0173] Preserving stemness in vitro is crucial for engineering bone tissue. Although bone marrow-derived sphere-forming cells have been reported, evidence regarding their in vivo origin and osteogenic potential is lacking (42-44). Whether their stemness is preserved in vitro remains unclear. We developed an ex vivo protocol for long-term culture of SuSCs. Cultured SuSCs generated bone tissue upon transplantation into ectopic sites. Furthermore, SuSC cultures provide an excellent system for examining skeletal stem cell properties, such as asymmetric division, cell fate determination, generation of skeletal progenitors, and skeletal differentiation. Therefore, this system represents a tool for advancing stem cell-based therapies for bone regeneration and repair.

[0174] For human applications, we identified human BMPR1A-positive SuSCs capable of generating ectopic bone tissue. Because Axin2 is an intracellular protein, identifying a surface marker for stem cell purification is essential. The BMP antagonist Gremlin1 marks skeletal stem cells that contribute to endochondral ossification; however, the functional significance of Gremlin1 remains unclear (8). The results disclosed herein demonstrate that Bmpr1a is not only a key regulator of SuSCs but also functions as a marker for their isolation. We have shown that BMPR1A, also known as CD292 (45), can be used to isolate human or mouse suture cells with skeletal stem cell properties for bone formation. This finding provides compelling evidence that BMPR1A positivity can be used to purify human SuSC populations.

[0175] The inclusion of SuSC niche cells is important for ectopic suture generation. The SuSC research disclosed herein facilitates the identification and isolation of niche cells. The cells, compositions, and methods disclosed herein can be used in preventive procedures for the prevention of suture resynthesis in surgical patients or for premature suture closure as an alternative to surgery in patients with craniosynostosis. The cells, compositions, and methods disclosed herein can also be used to elucidate the mechanisms underlying SuSC regulation and SuSC-mediated regeneration. They can also be used for the treatment or prevention of congenital deformities and skeletal repair.

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[0177] The foregoing examples and descriptions of preferred embodiments should be understood as illustrating, but not limiting, the present invention as defined by the claims. As will be readily understood, numerous variations and combinations of the features described above can be utilized without departing from the invention as set forth in the claims. Such variations will not be considered a departure 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. A method for obtaining an enriched skeletal stem cell population, comprising: Obtaining a starting cell population; From the starting cell population, cells expressing BMPR1A polypeptide (BMPR1A ＋ identifying a cell expressing the BMPR1A polypeptide, isolating the identified BMPR1A + cells; and harvesting a plurality of BMPR1A + cells to obtain an enriched skeletal stem cell population; A method comprising:

2. 2. The method of claim 1, wherein the cells express one or more markers selected from the group consisting of Axin2, BMP2, BMP3, BMP4, BMP6, BMP7, BMP8b, and BMP15.

3. The BMPR1A ＋ The method of claim 1 or 2, wherein the cells are isolated using a protein, polypeptide, or composition that specifically binds to BMPR1A.

4. The method of claim 3 , wherein the protein comprises an anti-BMPR1A antibody or an antigen-binding fragment thereof.

5. The method of claim 3, wherein the protein, peptide, or composition comprises a ligand of BMPR1A or a BMPR1A-binding fragment thereof, and optionally the ligand is selected from the group consisting of BMP2, BMP4, BMP6, BMP7, and GDF6.

6. The BMPR1A ＋ The method of any one of claims 1 to 5, wherein the cells are identified or isolated or enriched by fluorescence activated cell sorting.

7. The method of any one of claims 1 to 6, wherein the starting cell population is derived from a tissue of a subject, and optionally the subject is a mammal.

8. The method of claim 7, wherein the mammal is a human.

9. 9. The method of any one of claims 1 to 8, 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, and optionally, the mesenchymal stem cells are suture mesenchymal stem cells.

10. One or more BMPR1A ＋ A sphere of cells containing cells.

11. 11. The sphere of claim 10, wherein the one or more spheres are 20-200 μm in diameter, or have about 10-500 cells per sphere, or the spheres comprise Axin2+ cells.

12. (i) a carrier; and (ii) one or more BMPR1A ＋ 12. A composition comprising a cell, or one or more spheres according to claim 10 or 11, or progeny cells derived from said one or more spheres.

13. 13. The composition of claim 12, wherein the composition is a pharmaceutical composition and the carrier is a pharma- ceutically acceptable carrier, or the composition is an in vitro cell culture composition and the carrier comprises culture medium.

14. 14. A bone regeneration product or formulation comprising: (i) the composition of claim 12 or 13; and (ii) a scaffold.

15. 1. A method for producing skeletal, stromal, or cartilage tissue, comprising: (i) one or more BMPR1A ＋ Providing a cell or one or more spheres according to claim 10 or 11, or a progeny cell derived from said one or more spheres; (ii) the BMPR1A ＋ and inducing differentiation of a cell, or said cell derived from said one or more spheres.

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