Gremlin 1 identifies a skeletal stem cell with bone, cartilage, and reticular stromal potential

Abstract

The stem cells that maintain and repair the postnatal skeleton remain undefined. One model suggests that perisinusoidal mesenchymal stem cells (MSCs) give rise to osteoblasts, chondrocytes, marrow stromal cells, and adipocytes, although the existence of these cells has not been proven through fate-mapping experiments. We demonstrate here that expression of the bone morphogenetic protein (BMP) antagonist gremlin 1 defines a population of osteochondroreticular (OCR) stem cells in the bone marrow. OCR stem cells self-renew and generate osteoblasts, chondrocytes, and reticular marrow stromal cells, but not adipocytes. OCR stem cells are concentrated within the metaphysis of long bones not in the perisinusoidal space and are needed for bone development, bone remodeling, and fracture repair. Grem1 expression also identifies intestinal reticular stem cells (iRSCs) that are cells of origin for the periepithelial intestinal mesenchymal sheath. Grem1 expression identifies distinct connective tissue stem cells in both the bone (OCR stem cells) and the intestine (iRSCs).

Figure 1. Grem1 Identifies Rare Adult Multipotent Mesenchymal Stromal Cells

(A) Protocol and (B) Grem1-creER^T;R26-LSL-TdTomato;Nes-GFP mouse femur, showing that metaphyseal Nes-GFP⁺ (green) and Grem1⁺ cells (red, white arrows) are distinct. (C) Adult Grem1-creER^T;R26-LSL-TdTomato bone marrow cells are rare and mesenchymal (CD45⁻CD31⁻Ter-119⁻). (D–G) Adult Grem1-creER^T;R26-LSL-TdTomato;Nes-GFP mice: Grem1 and mesenchymal Nes-GFP cells do not overlap, and clonogenicity is greater in the Grem1⁺ versus the mesenchymal Nes-GFP⁺ cells (CD45⁻CD31⁻Ter-119⁻). (E and F) 10 cm cell culture dish; (G) n = 5, data shown with mean ± SD, p = 0.013. (H–K) Grem1⁺ cells from Grem1-creER^T;R26-LSL-ZsGreen;Acta2-RFP mice could be clonally expanded in vitro and differentiated into bone (H) (alizarin red), cartilage (I) (toluidine blue), and myofibroblasts (J) (Grem1⁺ green-derived cells with coexpression of Acta2 [red]), but very limited adipogenesis (oil red) (K). Lower right insets show equivalent stain in a control marrow culture. In all graphs, the data are shown with the mean ± SD.

Figure 2. Grem1⁺ Cells Are Enriched for CD105 Bone Marrow Cells with Upregulated Osteochondral versus Adipogenic Gene Expression

(A–F) n = 3, Grem1⁺ cells from adult, collagenase-digested whole bone and bone marrow were compared to the Grem1-negative population. On average, 40% (95% CI 20%–60%) of all Grem1 cells were CD45⁻CD31⁻Ter-119⁻CD105⁺ compared to only 1.8% of Grem1-negative cells (F, p = 0.014). (C) Grem1⁺ cells, however, were not further enriched for other MSC markers CD140a and Sca-1. Grem1⁺ and Grem1-negative cells were compared across the increasingly specific immunophenotypes; data shown with mean ± SD. (G) Microarray was performed to compare the Grem1⁺ (red) to the nonrecombined stromal (CD45⁻CD31⁻Ter-119⁻) population (green); in qPCR, we also sorted and evaluated the CD45CD31Ter-119⁺ population (blue) that did not contain any recombined cells. (H) Grem1⁺ cells were not enriched for Nes expression. (I–L) qPCR confirmation of microarray revealed that Grem1⁺ cells had increased expressionof pericytic (Cpsg4;I) and osteochondral genes (Acan and Sp7; J and K) but no association with the adipogenic differentiation gene Pparg (L). In all graphs, the data are shown with the mean ± SD.

Figure 3. Endogenous Grem1 Cells Self-Renew and Lineage Trace Bone, Cartilage, and Stroma

(A) Protocol. (B–E) P1 induction in Grem1-creER^T;R26-LSL-ZsGreen;Acta2-RFP mice. (B) At 24 hr after tamoxifen, Grem1 recombined (green) only within the primary spongiosa of long bones distinct from the Acta2-RFP (red) cells in the marrow. But, over the following 96 hr (C), the Grem1 cells began organizing into chondrocytic columns (D) and differentiated into stromal cells that invade the bone marrow (E) intertwined with Acta2-positive (red) cells. (F–I) Grem1-creER^T;R26-LSL-TdTomato;2.3colGFP mice induced at P1, examined at 6 weeks, show that the Grem1⁺ cells generate reticular marrow stromal cells (G), chondrocytes in the epiphyseal plate (H), osteoblasts (2.3colGFP⁺, thus yellow) in the trabecular bone (I). (J) Grem1-creER^T;R26-Confetti P1 induction, examined at 6 weeks, revealed clonal populations of chondrocytes, and (K) serial sections confirm mixed clones, yellow clone shown, of chondrocytes and marrow stromal cells, low- and higher-power (inset) images. (L–P) Adult induction in Grem1-creER^T;R26-LSL-TdTomato analyzed 11 months after adult induction (L). Grem1⁺ cells had generated chondrocytes (M and N), reticular marrow stromal cells (O), and bone and periosteal cells (red) (P).

Figure 4. Nes-GFP Cells Make Little Contribution to Skeletal Tissues during Early Life

(A) Whole-mount in situ hybridization on mouse embryos at E9.5, E10.5, E11.5 and E12.5. These embryos were evaluated for Sox9, Runx2, Grem1, and Nes expression. Nes-creER^T; R26-LSL-TdTomato; Nes-GFP mice (n = 3) were generated to lineage trace from Nes-GFP-positive cells throughout the bone marrow. These mice were induced at P1 and examined 6–8 weeks after. (B) Protocol. (C) By flow cytometry, approximately 4% of Nes-GFP-positive cells recombined (that is, were both green and red). (D–H) This specific Nes-CreERT transgenic line recombined in all typical Nes-GFP populations, including perivascular cells immediately inferior to the growth plate (D), in periarteriolar cells (E), and in perisinusoidal Nes-GFP-positive cells (red arrows, F–H). The only osteochondral lineage tracing found were isolated osteocytes throughout the diaphyseal bone (H, white arrow).

Figure 5. Adult Grem1 Cells, Both Endogenous and Transplanted, Differentiate into Osteochondral Fracture Callus

(A) Protocol. (B and C) Grem1-creER^T;R26-LSL-TdTomato;2.3colGFP mice adult induction: Grem1⁺ (red) cells were not osteoblasts (green) but were adjacent to each other in situ (B) and during the first week of adherent bone marrow stromal culture (C). (D) is an X-ray of the femoral osteotomy and internal fixation of the bone. (E) and (F) show the serial histology and fluorescent microscopy sections from the resulting fracture callus after the osteotomy. (G) and (H) are magnified from areas shown in (F). Grem1⁺ cells (red) stream into the fracture site and differentiate into either osteoblasts (G, yellow cells, white arrows) or Sox9⁺ (white, nuclear stain) chondrocytes (yellow arrows) (H). (I) A Grem1⁺ clone, after adult induction, was expanded in vitro. An osteotomy with internal fixation was performed in wild-type mice, at which point 500 × 10⁶ clonal cells (red) were irrigated into the surgical field. Seven days later, the Grem1 clone had engrafted (J), the site of injury was identified by TdTomato fluorescence imaging, and recombined cells (fluorescent red) differentiated into osteoblastic cells (K) (alkaline phosphatase positive cells, red-brown, white arrows) in the callus (sequential fluorescence microscopy and ALP staining performed on the same slide). (L) Callus culture was performed, and the recombined Grem1 cells were easily recovered in vitro and serially transplanted into a secondary fracture (Figure S4E).

Figure 6. Grem1 Expression Identifies iRSCs

(A) Protocol. (B and C) Grem1-creER^T;R26-mT/mG adult induction identifies rare, single cells at the junction of the crypt and villus in the small intestine (green = Grem1-creER^T+, red = Grem1-creER^T-negative). Over 1 year, Grem1⁺ cells expand to renew the entire periepithelial mesenchymal sheath. By 6 months, they are immediately beneath the intestinal stem cells at the crypt base (B and C). Axes are provided to indicate the longitudinal and circumferential axes (x and y), and “CVA” to designate the crypt-villus, or radial, axis. (D) The mesenchymal expansion was plotted relative to the adjacent epithelial position; 20 well-orientated crypt-villus columns were quantified per mouse. n = 3– 5 mice at each time point; Kruskal-Wallis analysis (p < 0.0001) and post-hoc pairwise Mann-Whitney tests, corrected for multiple comparisons, revealed significant differences (****p ≤ 0.0001, *p ≤ 0.05). (E) The sheath was comprised of a reticulated population of stellate cells with long processes that encircled the entire intestinal gland. This cell encapsulated the very base of the intestinal crypt, similar to the position of the cell identified in (C). (F) The Grem1⁺ population self-renewed and was multipotent, generating both Acta2 positive (yellow cell, white arrow) and negative fibroblastic lineages. (G) Transmission electron microscopy: the Grem1 lineage (yellow arrows) is immediately beneath the epithelial cells. (H) Tissue engineering: representative images from n = 8 small-intestinal organoid unit transplants. Small intestines were harvested from 3-week-old, tamoxifen-induced donor mice. In the donor intestines, prior to harvest, there were single Grem1⁺ cells (red) near the isthmus of the intestinal gland. After digestion of the donor intestines into organoid units, rare Grem1⁺ mesenchymal cells (red) were found within individual organoid units (inset). (I) Four weeks after transplantation, TESIs develop, with the periepithelial mesenchymal sheath recapitulated from the donor Grem1⁺ cells (red). E-cadherin staining (green) was used to identify the epithelium.

Figure 7. A Model in which the OCR Stem Cell and the Perisinusoidal MSC Make a Complementary Contribution to Skeletal Development, Adult Homeostasis, and Repair