A multi-stem cell basis for craniosynostosis and calvarial mineralization.

Craniosynostosis is a group of disorders of premature calvarial suture fusion. The identity of the calvarial stem cells (CSCs) that produce fusion-driving osteoblasts in craniosynostosis remains poorly understood. Here we show that both physiologic calvarial mineralization and pathologic calvarial fusion in craniosynostosis reflect the interaction of two separate stem cell lineages; a previously identified cathepsin K (CTSK) lineage CSC1 (CTSK+ CSC) and a separate discoidin domain-containing receptor 2 (DDR2) lineage stem cell (DDR2+ CSC) that we identified in this study. Deletion of Twist1, a gene associated with craniosynostosis in humans2,3, solely in CTSK+ CSCs is sufficient to drive craniosynostosis in mice, but the sites that are destined to fuse exhibit an unexpected depletion of CTSK+ CSCs and a corresponding expansion of DDR2+ CSCs, with DDR2+ CSC expansion being a direct maladaptive response to CTSK+ CSC depletion. DDR2+ CSCs display full stemness features, and our results establish the presence of two distinct stem cell lineages in the sutures, with both populations contributing to physiologic calvarial mineralization. DDR2+ CSCs mediate a distinct form of endochondral ossification without the typical haematopoietic marrow formation. Implantation of DDR2+ CSCs into suture sites is sufficient to induce fusion, and this phenotype was prevented by co-transplantation of CTSK+ CSCs. Finally, the human counterparts of DDR2+ CSCs and CTSK+ CSCs display conserved functional properties in xenograft assays. The interaction between these two stem cell populations provides a new biologic interface for the modulation of calvarial mineralization and suture patency.

This dropout of CTSK + CSCs was accompanied by a major shift in the form of bone formation in the sutures. Previously, we reported that CTSK + CSCs are specialized for the major physiologic form of calvarial bone formation, intramembranous bone formation 1 , where bone is deposited directly without a cartilage template. Accordingly, dropout of CTSK + CSCs was accompanied by the pathologic induction of endochondral bone formation, where a cartilage template precedes mineralization, a form of bone formation normally excluded from the sutures [4][5][6] (Fig. 1e). Cartilage clusters were observed in the sutures destined to fuse, but this cartilage was fully remodeled into the bone at later timepoints, distinguishing this endochondral ossi cation process from the formation of ectopic cartilage (Fig. 1b,Extended Data Fig. 1e). Taken together, this data pointed to the existence of a second calvarial lineage distinct from CTSK + lineage cells where these non-CTSK (CTSK -) lineage cells emerge to become the dominant cell type in the sutures during fusion and are associated with endochondral bone formation.
Dropout of CTSK + CSCs was su cient to drive the expansion of this alternative CTSKpopulation and suture fusion, as seen with inducible deletion of CTSK + CSCs using diphtheria toxin (DT) administration in iDTR;Ctsk-Cre (iDTR Ctsk ) mice. These mice not only displayed hypomineralization of the calvarium similar to that seen after conditionally deleting the Osterix (Osx) gene required for osteoblast generation in CTSK + cells (Osx Ctsk ) 1 , but also displayed partial suture fusion at similar sites to those fused in Twist1 Ctsk mice followed by compensatory expansion of CTSKlineage sutural cells Extended Data Fig. 2). The presence of a fusion phenotype only in iDTR Ctsk but not in Osx Ctsk indicates that it is the absence of CTSK + CSCs themselves and not the absence of more mature osterix-dependent osteoblast populations in the CTSK lineage that drive the fusion phenotype.
These results pointed to the existence of a second calvarial stem cell within the pool of CTSKlineage sutural cells. To identify this second CTSKcalvarial stem cell, candidate markers of this population were identi ed by transcriptional analysis and immunostaining . Among these candidates, the transmembrane collagen receptor DDR2 was prioritized based on reports that Ddr2 is expressed in the sutures and mutant mice display calvarial mineralization defects [7][8][9] . Our data showed that Ddr2 was signi cantly expressed on CTSKlineage sutural cells in Twist1 Ctsk mice (Extended Data Serial transplantation, analysis of differentiation hierarchy, lineage tracing, and label retention studies were used to identify that putative DDR2 + CSCs are indeed stem cells. Single clones of DDR2 + CSCs were capable of in vitro tri-lineage differentiation into osteoblasts, adipocytes, and, separately, chondrocytes (Extended Data Fig. 3g). In vivo transplantation showed that DDR2 + CSCs, but not other DDR2 + populations, were able to both self-renew and generate all of the other DDR2 + populations present in the native suture after the rst round of transplantation Extended Data Fig. 4). When re-isolated after the rst round of transplantation and subjected to the second round of transplantation, DDR2 + CSCs were capable of similar self-renewal and differentiation, demonstrating serial transplantation and selfrenewal capacity similar to other skeletal stem cell (SSC) populations identi ed in long bones 1,10 .
Transplantation also clari ed that DDR2 is present on the entire lineage of cells derived from DDR2 + CSCs. No evidence of interconversion between DDR2 + CSCs or CTSK + CSCs was observed (Fig. 2f, Extended Data Fig. 4a).
Another method used to distinguish stem from non-stem skeletal populations is in vivo pulse-chase lineage tracing, where targeting stem populations results in durable labeling that disseminates to other downstream populations but targeting non-stem populations results in transient labeling 11 . Ddr2-CreER;mTmG mice were used for pulse-chase labeling of DDR2-lineage calvarial cells. Ddr2-CreER labeled both suture resident cells and bone-adjacent osteoblasts and showed good concordance with DDR2 + ow cytometry (Fig. 2h,i,Extended Data Fig. 5). Consistent with DDR2 + cells including a stem population, a single early tamoxifen pulse provided durable labeling that was retained in the suture at least 3 months later.
Label retention in the H2B-GFP system has been used across many organ systems as a sensitive method to de ne slow-cycling stem cells [12][13][14][15] . Consistent with the result of transplantation-based stemness assays, H2B-GFP retention was observed in DDR2 + CSCs but not other DDR2 + populations at Extended Data Fig. 6a,b). In line with the model that the suture contains two separate stem cells, DDR2 + CSCs and CTSK + CSCs, additional, separate label-retaining cells were observed in the fraction of DDR2cells bearing markers consistent with CTSK + CSCs (Lin -/CD200 + /CD105 -/Thy-1.2 -/6C3cells). As it was not possible to use GFP-based reporters of the CTSK lineage together with the H2B-GFP system, we sought alternative markers of the CTSK + CSC lineage to con rm the additional presence of label-retaining CTSK + CSCs, identifying that Galectin-1 immunostaining shows high concordance with Ctsk-Cre based lineage reporters (Extended Data Fig. 6c). Using Galectin-1, the label retention in DDR2 -CSCs was con rmed to localize to CTSK + CSCs (Extended Data Fig. 6d). Thus, there are separate pools of label-retaining cells (LRCs) corresponding to both DDR2 + CSCs and CTSK + CSCs, consistent with the presence of a dual set of stem cells in the calvarium.
Next, we evaluated whether these DDR2 + CSCs are capable of differentiating into bone-forming osteoblasts and whether they mediate endochondral ossi cation accounting for the inappropriate sutural endochondral ossi cation seen in Twist1 Ctsk mice. Indeed, DDR2 + CSCs isolated by FACS and transplanted to the kidney capsule of hosts engrafted and formed robust bone organoids, forming signi cantly more bone on a per cell basis than CTSK + CSCs (Fig. 3a-c). This bone was derived from graft DDR2 + CSCs, as graft bone tissue was densely populated with osteocytes bearing the tdTomato marker of graft DDR2 + CSCs (Fig. 3d). Additionally, no interconversion between DDR2 + CSCs and CTSK + CSCs could be observed as there was no induction of Ctsk-Cre driven mGFP expression in the DDR2 + CSCs over time, consistent with the result of the serial transplantation study.
Whereas CTSK + CSCs mediated the expected intramembranous ossi cation after transplantation 1 , DDR2 + CSCs underwent endochondral ossi cation consistent with being mediators of the inappropriate sutural endochondral ossi cation seen in Twist1 Ctsk mice (Fig. 3e, Extended Data Fig. 7a). However, while endochondral ossi cation is intrinsically coupled with the recruitment of hematopoietic marrow elements 1,16 , DDR2 + CSCs mediate a distinct form of endochondral ossi cation without recruitment of hematopoietic cells (Fig. 3f, Extended Data Fig. 7b). While the etiology of this difference is likely multifactorial, DDR2 + CSC-derived organoids did not produce cells expressing the hematopoietic niche factor CXCL12 (Extended Data Fig. 7c) in contrast with organoids from long bone SSCs 17 . Taken together, this indicates that a third fundamental form of bone formation, endochondral ossi cation without hematopoiesis, exists alongside traditional intramembranous and endochondral ossi cation, and DDR2 + CSCs are specialized for this third form of bone formation.
Transcriptional pro ling of DDR2 + CSCs vs CTSK + CSCs established that these cells each display broadly distinct transcriptional pro les, reinforcing that DDR2 + CSCs vs CTSK + CSCs are distinct cell types (Fig.   3g). GLI1 was expressed in both CTSK + CSCs and DDR2 + CSCs (Extended Data Fig. 8a, b) suggesting that this schema resolves the pool of Gli1-lineage calvarial progenitors into multiple discrete stem cell types 18 . Consistent with the chondrogenic capacity of DDR2 + CSCs, these cells expressed higher levels of classically chondrocyte-associated transcripts such as Sox9, Col2a1, and Acan, though these transcripts were also present at lower levels in CTSK + CSCs. Both CTSK + CSCs and DDR2 + CSCs share the expression of several general stem cell, SSC-associated transcripts, including Myc, Runx2, Klf4, and Nes 1,[19][20][21] . They showed enrichment of many genes involved in embryonic suturogenesis including Msx1/2 22 , Acta2, Lgr5, and Lrig1 23 , and similarly co-expressed Axin2 24 , Prrx1 25 , and Gli1 26 , genes previously used to study calvarial stem cell populations (Fig. 3h,Extended Data Fig. 8c). Interestingly, many of these shared genes were also present in both periosteal and endosteal stem cells 1 , and growth plate resident SSCs 27 in long bones, indicating convergence on a set of SSC-de ning genes shared among multiple SSC types at different skeletal sites. Lastly, a number of genes associated with human craniosynostosis are expressed at higher levels in DDR2 + CSCs, including Efnb1, Zic1, Ihh, Msx2, Fgfr2, and Fgfr3 28-31 (Extended Data Fig. 8d).
In addition to a role in craniosynostosis, DDR2 + CSCs have an important contribution to calvarial mineralization distinct from that of CTSK + CSCs. Inducible postnatal deletion of DDR2 + CSCs in Ddr2-CreER;DTA mice resulted in enlargement of the anterior fontanelle due to hypomineralization of the frontal bones and parietal bones, and severe hypomineralization of the interparietal bones (Fig. 4a).
Similarly, deletion of Osx speci cally in DDR2 + CSCs (Osx / ;Ddr2-CreER) resulted in calvarial hypomineralization similar to that seen in Ddr2-CreER;DTA mice ( Fig. 4b). Interestingly, DDR2 itself appears to be functional on DDR2 + CSCs, as mice with the spontaneously occurring smallie mutation causing a loss of function in DDR2 (Ddr2 slie mice) display calvarial hypomineralization 8 especially evident in the parietal and frontal bones (Extended Data Fig. 9a). Ddr2 slie mice also displayed an absence or reduction in the cartilage structures in the lateral portions of the interparietal and occipital bones, implicating DDR2 + CSCs in the formation of the cartilaginous elements of the calvarium (Extended Data Fig. 9b, c).
Using an orthotopic transplantation model 32,33 where a native lambdoid suture is surgically ablated and then replaced with a cell pellet of de ned composition (Fig. 4c, Extended Data Fig. 10a), DDR2 + CSCs were su cient to promote fusion in a manner that is restrained by CTSK + CSCs (Fig. 4d). DDR2 + CSCs, but not CTSK + CSCs, were su cient to mediate fusion not only at the implantation site (the LAM suture), but also in an extended region of the SAG, OIP, and SQ sutures adjacent to the implantation site, despite both populations being capable of robust engraftment (Fig. 4e, Extended Data Fig. 10b-d). This fusion in adjacent regions re ected migration of graft DDR2 + CSCs to these sites, in addition to the retention of the graft cells at the primary implantation site (Extended Data Fig. 10e, f). Consistent with nding that depletion of CTSK + CSCs was su cient to induce suture fusion, co-implantation of CTSK + CSCs with DDR2 + CSCs prevented the fusion-promoting activity of DDR2 + CSCs, while not impacting their ability to engraft. This fusion-suppressing ability of CTSK + CSCs re ected continuous, direct regulation and not induced death or differentiation of DDR2 + CSCs as co-implantation of CTSK + CSCs and DDR2 + CSCs followed by post-transplantation ablation of the implanted CTSK + CSCs using a diphtheria toxin (DT) system was able to restore the fusion activity of DDR2 + CSCs ( Fig. 4f-h, Extended Data Fig. 11).
Lastly, human counterparts of DDR2 + CSCs are present in the sutures of patients with craniosynostosis, establishing the clinical relevance of this SSC type. Consistent with prior literature 34,35 and observations in Twist1 Ctsk mice, calvarial specimens from patients with sporadic craniosynostosis displayed inappropriate endochondral ossi cation with cartilage undergoing active remodeling into the bone and contained abundant DDR2 + cells (Fig. 5a, b). Human sutural tissue also included both DDR2 + and DDR2cells bearing a surface immunophenotype nearly identical to murine DDR2 + CSCs and CTSK + CSCs (Lin -/Thy-1 -/CD200 + /CD105cells that are DDR2 + or DDR2 -, hereafter hDDR2 + CSCs and hDDR2 -CSCs, Fig. 5c). Given the inability to utilize CTSK-based lineage reporters in humans, hDDR2 -CSCs were here utilized as a proxy for murine CTSK + CSCs. Interestingly, this hCSC de nition also largely overlapped with another set of markers used to de ne stem cells in human fetal long bones (PDPN + /CD146 -/CD164 + /CD73 + ) 36 (Extended Data Fig. 12a). Thus, hDDR2 + and hDDR2 -CSCs show a convergent immunophenotype that ful lls criteria from multiple schema for identifying SSCs. Both hDDR2and hDDR2 + CSCs were capable of forming bone in xenograft systems, with the hDDR2 + CSCs isolated from craniosynostosis patients showing a much greater per-cell bone tissue formation capacity ( Fig. 5d). Additionally, consistent with observations that murine DDR2 + CSCs are specialized for endochondral ossi cation without recruitment of hematopoietic elements, hDDR2 + CSCs mediated endochondral bone formation (Fig. 5e). Moreover, both DDR2 + CSCs and DDR2 -CSCs displayed evidence of stemness, as both were able to generate the populations present in the initial donor suture tissue in addition to self-renewing or maintaining the input cell type (Fig. 5f, Extended Data Fig. 12b, c).
In summary, we here nd that the calvarial sutures contain two distinct stem cell types. One is a CTSK + CSC that is specialized for intramembranous bone formation. A second identi ed here is a DDR2 + CSC that characteristically mediates endochondral ossi cation without marrow formation. Interactions between these two cells are critical for the pathogenesis of craniosynostosis and suture patency, as dropout of CTSK + CSCs triggers a maladaptive expansion and activation of DDR2 + CSCs, which in turn results in inappropriate endochondral ossi cation in the sutures and, ultimately, fusion. Both of these cell types are also present in human calvarial sutures (Fig. 5g). Given their role in craniosynostosis, agents that inhibit DDR2 + CSCs or otherwise block their compensatory expansion offer an attractive strategy for adjunct medical therapy of craniosynostosis. Moreover, this provides an example of how multiple skeletal stem cell lineages can co-exist and interact at a single site, with disruptions in these stem cell to stem cell interactions serving as a new mechanism for skeletal disease. MerCreMer-neoR cassette that was then ligated into the MluI/NotI sites of the pCR-BluntII-LHA vector using a MluI-HindIII-NcoI adapter. The pCR-BluntII-LHA vector with the MerCreMer-neoR insert was digested with NotI, dephosphorylated, and ligated into the Eag1 site upstream of the RHA in the pCR-BluntII-RHA vector. A diptheria toxin A (DTA) coding cassette (PspOMI/NotI fragment, provided by Dr. Ju Chen, UCSD) was ligated into the dephosphorylated NotI site distal to the RHA of the pCR-BluntII-LHA-MerCreMer-neoR-RHA vector to provide negative selection. The targeting vector was linearized with NotI and electroporated into 129R1 mouse ES cells. Individual G418-resistant clones were initially screened by PCR and then followed by Southern blot, using KpnI digestion with Probe C and BamHI digestion with Probe D, to identify positive clones (Extended Data Fig. 13b). One targeted clone was microinjected into C57Bl/6 blastocysts that were implanted into pseudopregnant female hosts to generate chimeric pups.

Declarations
Breeding of chimeric animals generated agouti pups that were screened for the targeted mutation. Targeted mice were bred with a FLatrigter mouse (provided by Dr. Ju Chen, UCSD) to remove the neomycin resistance (neoR) cassette prior to backcrossing onto C57Bl/6 for at least 10 generations. Cardiac broblasts from a tamoxifen-treated Ddr2-CreER mouse were used to validate protein expression of the MerCreMer (Extended Data Fig. 13c), then the mice crossed with mTmG mice. Recipient heterozygous MIP-GFP mice were generated by breeding homozygous MIP-GFP males with wildtype C57Bl6/J females. All mice were maintained on a C57Bl6/J background throughout the study. All animals were maintained in accordance with the NIH Guide for the Care and Use of Laboratory Animals and were handled according to protocols approved by the Weill Cornell Medical College subcommittee on animal care (IACUC).
Diphtheria toxin (DT) and tamoxifen (TAM) administration. iDTR;Ctsk-Cre;mTmG pups were given 50 ng of DT (Sigma, cat. D0564) intraperitoneally as indicated in Extended Data Fig. 2a. PBS was used as a vehicle. For lineage tracing analysis, Ddr2-CreER activity was induced at postnatal day 2 (P2). TAM (100 mg/kg; Sigma, cat. T5648) was prepared in corn oil and subcutaneously injected into animals for 2 consecutive days as described in Extended Data Fig. 5a. To investigate the physiologic contribution of DDR2 + CSCs, ROSA-DTA and Osx / mice were crossed with Ddr2-CreER;mTmG mice and TAM was delivered subcutaneously into P7 pups.
Human specimens. This study was carried out under the IRB protocol (no. 1402014802), with all patients providing informed consent through the Center for Neurogenetics at Weill Cornell Medical Center. Human calvarium materials used for scienti c analyses were obtained from 24 case specimens that were left over from craniosynostosis surgical repair procedures. Participants had an age range of less than 1 year old and were 80% male.
Isolation of suture cells. For murine samples, microdissected calvarial sutures were subjected to both mechanical and enzymatic digestion. The harvested tissues were minced using razor blades and digested for up to 20 minutes with digestion cocktail buffer containing Collagenase P (1 mg/ml; Roche, cat. 11213857001) and Dispase II (2 mg/ml; Roche, cat. 04942078001) in medium containing 2% serum at 37 °C with agitation. Medium containing 2% serum was added to the digest and the tubes were centrifuged to pellet cells. The supernatant was carefully removed, and the pellet was resuspended in DNase I (2 units/ml) solution and brie y incubated for 5 min at 37 °C. Media was added to the tube and the digested tissue was re-suspended thoroughly by pipetting and then ltered through a 70-micron nylon mesh. Tubes were centrifuged and the resulting cell pellet was subjected to FACS. For human calvarium specimens, tissues were subjected to mechanical and enzymatic digestion with Collagenase A (1 mg/ml; Roche, cat. 10103586001) and Dispase II (2 mg/ml) in a medium containing 2% serum for 30 min at 37°C under agitation, and the cells were then isolated following the same procedure as described above.
Fluorescence-activated cell sorting (FACS). Prepared single-cell suspensions were washed twice with icecold FACS buffer (2% serum + 1 mM EDTA in PBS) and incubated with Fc blocking buffer (1:100 dilution; BD bioscience, cat. 553142 for mouse and cat. 564765 for human) for 15 min at 4 °C. Primary antibody dilutions were prepared in Brilliant Stain Buffer (BD bioscience, cat. 563794). Cells were incubated in the dark for 40 min at 4 °C with primary antibody solution, washed 2 times with FACS buffer, and incubated with secondary antibody solution for 20 min, if needed. Cells were then washed several times and resuspended in FACS buffer with DAPI (BD bioscience, cat. 564907). FACS was performed using a Becton Dickinson Aria II equipped with 5 lasers (BD bioscience). Beads (Invitrogen, cat. 01-3333-42) were used to set initial compensation. Fluorescence minus one (FMO) controls were used for additional compensation and to assess background levels for each stain. Gates were drawn as determined by internal FMO controls to separate positive and negative populations for each cell surface marker. Typically, 2.5 million events were recorded for each FACS analysis, and the data were analyzed using FlowJo (v10.6.1). To better depict the FACS gating strategy, color boxes and arrows have been added to the plot to illustrate parent/daughter gates. Additionally, Extended Data Figures have been provided to show the full details of the gating strategies used (Extended Data Fig. 1a, 3f, 4b, 6a, 6b, 11a). 05502, 05500). After initial cell plating, cells were left undisturbed and allowed to grow at 37 °C under humidi ed conditions for a week. Half of the medium was replaced every 3 days. Cells were passaged once they were 60-70% con uent using cell dissociation reagent (Gibco, cat. A1110501). Cells sorted from human calvarial tissue were cultured under conditions similar to those described above using a commercial medium preparation (Stem Cell Technologies, cat. 05401) with stimulatory supplements (Stem Cell Technologies, cat. 05402). The differentiation potential of 10 colonies derived from single FACS-isolated cell populations was examined. In brief, single sorted cells were plated at a density of 100 cells in 6-well culture plates and allowed to form individual colonies. Initial dispersion of the plated cells as single cells was con rmed by light microscopy. Each of the selected colonies was extracted using a cloning cylinder. The extracted cells were regrown for 3 days in 12-well plates and then allowed to differentiate under both osteogenic and adipogenic conditions as described below. For osteogenic differentiation and Alizarin Red S staining, sorted cells were expanded and then allowed to differentiate using an osteogenic differentiation medium (Gibco, cat. A1007201) for 21 days. The medium was changed every other day. At the end of this period, cells were washed with cold PBS and xed with 4% PFA for 30 min on ice. Cells were washed with distilled water and stained with Alizarin Red S for 2 min.
Cells were then washed thoroughly with water and air-dried before microscopic visualization. Adipogenic differentiation studies were conducted with sorted cells using methods similar to those described above. In brief, cells were allowed to differentiate in adipogenic differentiation medium (Gibco, cat. A1007001).
Fresh medium was added every other day for a total of 14-20 days. The medium was removed, and cells were washed with PBS and xed with 4% PFA for 30 min at room temperature. Cells were rinsed again with PBS and stained for 30 min with Oil Red O working solution (3:2 dilution with water). After washing 5 times with PBS, cells were then observed under a light microscope. For chondrogenic differentiation and Alcian Blue staining, cells were grown as pellet culture. 1 x 10 4 cells in chondrogenic differentiation medium (Gibco, cat. A1007101) were centrifuged at 1500 rpm for 5 min in 15 ml conical tubes. Cultures were re-fed every 3 days and allowed to differentiate for a minimum of 14 days. At the endpoint of this study, the medium was removed, and pellets were harvested and washed carefully with PBS and xed with 4% PFA for 30 min, then embedded in OCT compound. After cryo-sectioning, slides were stained for 30 min with 1% Alcian Blue solution, washed three times with 0.1 N HCl, and then with PBS.
μCT scans and analysis. µCT analysis was conducted on a Scanco Medical µCT 35 system at the Citigroup Biomedical Imaging Core as previously described 43 . Brie y, a Scanco Medical μCT 35 system with an isotropic voxel size of 7 μm and 20 μm were used to image the distal femur/tibia and skull, respectively. Scans were conducted in PBS and used an X-ray tube potential of 55 kVp, an X-ray intensity of 0.145 mA, and an integration time of 600 ms. µCT analysis was performed by an investigator blinded to the genotypes of the animals.
Renal capsule transplant for bone organoids. Renal capsule transplantation was performed in 8-10 week old MIP-GFP mice as previously described 1 . To avoid potential immunogenicity of GFP itself, all transplantation studies were conducted in MIP-GFP hosts that have baseline immunologic tolerance to GFP variants. Brie y, mice were anesthetized and shaved on the left ank and abdomen before sterilization of the surgical site. The kidney was externalized through a 1 cm incision and a 2 mm pocket was made in the renal capsule. A 5 atrigelgel plug (Corning, cat.356231) containing the cells of interest was implanted underneath the capsule and the hole was sealed using a cauterizer before replacing the kidney back into the body cavity. Approximately more than 20 donor mice were sacri ced to harvest the desired number of CSCs for every organoid-based transplantation experiment. Animals were euthanized by CO2 after the indicated experimental duration. After sacri ce, kidneys were xed with 4% PFA for 6 hrs and bone formation was detected by µCT and uorescent stereomicroscopes. Samples were subjected to in ltration, embedding, and sectioning as described below.
Intramuscular transplant for differentiation hierarchy. Intramuscular transplantation was performed in 6 to 8-week-old MIP-GFP mice. A 1 mm longitudinal incision was made on the right hindlimb and the right anterolateral femur was exposed. The vastus lateralis muscle was dissected and a 2-3 mm muscle pouch was surgically created. A surgifoam absorbable gelatin sponge (Ethicon, cat. 1972) containing sorted cell populations of interest was placed into the muscle pouch. The overlying fascia was closed by using 4-0 polyglactin 910 absorbable sutures (Ethicon, cat. J386), and wound clips were used to close the skin incision. For serial transplantation studies, animals were euthanized by CO2 narcosis 7-10 days postsurgery, and the muscle was dissociated for FACS analysis.
Suture ablation model. 4-week-old MIP-GFP mice were anesthetized and had the skin overlying the skull shaved before sterilization of the surgical site. A scalpel was used to carefully remove the periosteum of the skull prior to removing the lambdoid sutures and surrounding bones on the right side of the skull using a micro hand drill with 0.2 mm drill bits (Stoelting, cat. 58610). Cell populations of interest (2 x 10 4 cells) were isolated by FACS from calvarial sutures of Ctks-Cre;mTmG or iDTR;Ctsk-Cre;mTmG mice and were mixed with 2 µl of a gel mixture (GelMA:Matrigel:Collagen I, 6:3:1) (Cellink, cat. IK3051020303, Corning, cat. 356231, Gibco, cat. A1048301, respectively) prior to implantation at the defect site. The gel mixture, with or without cells, was cross-linked by exposure to UV light for few seconds after transplantation and the skin incision was closed using an absorbable suture. Animals were euthanized by CO2 and the specimens were collected after 8 and 16 weeks.
Sample preparation for cryo-sectioning and imaging. Freshly extracted mouse samples were xed with 4% PFA for 4-6 hours at 4 °C. Samples were washed with PBS and decalci ed with daily changes of 0.5 M EDTA for 1-5 days depending on the age of the samples. Samples were incubated with in ltration medium (20% sucrose + 2% polyvinylpyrrolidone in PBS) with rocking until they sank to the bottom of the tube. Embedding was performed with a customized embedding medium (OCT + 15% sucrose in PBS) and samples were preserved at −80 °C. Sections, 10-20 μm in thickness, were cut using a Leica cryostat.
Immunohistochemistry. Immunohistochemical analysis was conducted on Zeiss instruments and systems at the Optical Microscopy Core using the previously described 44 . In brief, frozen samples were thawed at room temperature and rehydrated with PBS, permeabilized with 0.3% Triton X-100 in PBS for 10 min and blocked for 30 min with 5% donkey serum in PBS. Dilutions of primary antibodies (1:50-1:100) were freshly prepared in 0.3% Triton X-100 in PBS. Samples were incubated overnight with primary antibodies at 4 °C and, then washed three times with PBS. Secondary antibodies (1:1000 dilution) were added to samples for 1 hour, followed by washing three times with PBS. Samples were nally mounted with ProLong Gold antifade reagent with DAPI (Invitrogen, cat. P36931). Imaging was performed with a Zeiss LSM 880 laser scanning confocal microscope. All data were processed using Zeiss ZEN 2.3 SP1 software.
Bulk RNA-sequencing. Total mRNA was freshly extracted from FACS-isolated CSC populations from P7 Ctsk-Cre mice (n=5) using RNeasy Plus Micro Kit (Qiagen, cat. 74034). Total RNA integrity was checked using a 2100 Bioanalyzer (Agilent Technologies). cDNA synthesis and ampli cation were performed by SMART-Seq v4 ultra-low input RNA kit (Takara Bio, USA) starting with 1 ng of total RNA from each sample. 150 pg of quali ed full-length double strand cDNA was used and processed for Illumina library construction with the Nextera XT DNA Library Preparation Kits (Illumina). Then the normalized cDNA libraries were pooled and sequenced on an Illumina NovaSeq6000 sequencer with pair-end 50 cycles. Bulk RNA-sequencing was performed on a total of 10 FACS-isolated CSC populations (5 CTSK + and 5 DDR2 + ) from mice, and a counts matrix was obtained. DESeqDataSet was generated using this unnormalized counts matrix. Differential expression analysis was performed using the 'DESeq' function and results tables were generated using the 'results' function to compare the DDR2 + vs. CTSK + groups 45 .
Log fold change shrinkage was performed using the 'lfcShrink' function with the 'apeglm' method 46 for visualization and ranking of genes. Results were exported as .csv les (one with all the genes, and one with the subset of genes with adjusted P value < 0.05). 2-D PCA plots of the samples were generated by the rst two principal components using the 'plotPCA' function. Normalized read counts were used for generating heatmap plots. The data was visualized by heatmap with hierarchical clustering in R v3.6.2 with the pheatmap package v1.0.12. GO analysis was performed using DAVID Bioinformatics Resource tools v6.8 47,48 .