Pharmacological rescue of diabetic skeletal stem cell niches

Abstract

Diabetes mellitus (DM) is a metabolic disease frequently associated with impaired bone healing. Despite its increasing prevalence worldwide, the molecular etiology of DM-linked skeletal complications remains poorly defined. Using advanced stem cell characterization techniques, we analyzed intrinsic and extrinsic determinants of mouse skeletal stem cell (mSSC) function to identify specific mSSC niche-related abnormalities that could impair skeletal repair in diabetic (Db) mice. We discovered that high serum concentrations of tumor necrosis factor-α directly repressed the expression of Indian hedgehog (Ihh) in mSSCs and in their downstream skeletogenic progenitors in Db mice. When hedgehog signaling was inhibited during fracture repair, injury-induced mSSC expansion was suppressed, resulting in impaired healing. We reversed this deficiency by precise delivery of purified Ihh to the fracture site via a specially formulated, slow-release hydrogel. In the presence of exogenous Ihh, the injury-induced expansion and osteogenic potential of mSSCs were restored, culminating in the rescue of Db bone healing. Our results present a feasible strategy for precise treatment of molecular aberrations in stem and progenitor cell populations to correct skeletal manifestations of systemic disease.

Fig. 1. mSSC-dependent bone healing is impaired in Db mice

(A) Schematic of fracture creation and assessment by MST. (B) Maximal load to fracture (in newtons) of uninjured and healing femora from Leprdb (db/db, DbLR, or Db; red) versus wild-type (WT; blue) mice [n = 13 to 24; **P = 0.0018, one-way analysis of variance (ANOVA)]. (C) (i) Representative μCT images showing trabecular bone of healing femora from WT (left column) or DbLR (right column) mice. The Outlined area is magnified showing differences in trabecular spaces (red arrows). Scale bars, 500 μm (top) and 100 μm (bottom). (ii) Assessment of bone mineral density (BMD) of trabecular bone in healing femora from WT versus DbLR mice (n = 4). (iii) μCT images of calluses from WT and DbLR mice. (iv) Assessment of bone volume/total volume (BV/TV) of healing femora from WT or DbLR mice (n = 6 to 7). (D) Schematic of mSSC lineage hierarchy: mSSC; pre-bone cartilage, stromal progenitor (Pre-BCSP); BCSP; pro-chondrogenic cell (PCP); Thy⁺ osteogenic progenitor (Thy); B cell lymphocyte stromal progenitor (BLSP); 6C3⁺ stromal progenitor (6C3); hepatic leukemia factor-expressing cell (HEC). (E) Schematic of stem and progenitor cell isolation. mSSCs and BCSPs were isolated from whole uninjured femora and whole calluses at different time points using fluorescence-activated cell sorting (FACS). (F) FACS plots showing similar proportions of mSSCs and BCSPs in post-fracture day 7 calluses from WT (top row) versus DbLR (bottom row) mice. (G) Temporal differences in absolute cell numbers of mSSCs (top) and BCSPs (bottom) in WT versus DbLR mice (n = 5 per group). (H) Respective population percentages of 5-bromo-2′-deoxyuridine (BrdU)-labeled mSSCs (top) or BCSPs (bottom) from post-fracture day 3 calluses (n = 5 per group). (I) Respective population percentages of annexin V expression in mSSCs (left) and BCSPs (right) from post-fracture day 7 calluses of DbLR versus WT mice. FMO, full minus one stain; FITC, fluorescein isothiocyanate. Data and error bars represent means ± SEM. *P < 0.05, **P < 0.001, ****P < 0.0001, unpaired two-tailed t test.

Fig. 2. Exposure to a non-Db circulation does not fully restore Db fracture healing

(A) Schematic of parabiotic pairing, fracture creation, and assessment of healing using MST. Parabionts were rested for 4 weeks before further testing to ensure blood chimerism. Fractures were fixed with an intramedullary pin. (B) Representative FACS analysis confirming blood chimerism in parabiotic pairs. Blood chimerism was determined by WT green fluorescent protein-expressing (GFP⁺, green boxes) cells in nonfluorescent DbLR mice, (C) Blood glucose levels of each mouse in a chimeric pair at post-parabiosis week 8 show that parabiosis does not change the glycemic control of DbLR or WT mice in WT/DbLR chimeric pairs (n = 5 per group), (D) MST of healing femora from each parabiotic pair shows that a non-Db circulation increases the strength of DbLR femora but does not restore it to WT levels (n = 5 per group: **P = 0.0014, one-way ANOVA). Data and error bars represent means ± SEM. **P < 0.01, unpaired two-tailed t test.

Fig. 3. Altered mSSC skeletogenic activity is cell-extrinsic in Db mice

(A) Schematic of kidney capsule transplantation assays. FACS-sorted mSSCs (2 × 10⁴) from WT or DbLR calluses (top sequence) or from the appendicular skeleton of postnatal day 3 (P3) mice (bottom sequence) were transplanted into non-Db, immunodeficient (top) and WT or DbLR (bottom) mice. Heterotopic skeletal grafts were excised after 1 month for histological analysis. (B) Left: Representative micrographs of grafts produced in immunocompromised mice by mSSCs from WT (top row) or DbLR (bottom row) mice show no difference in graft size (scale bars, 1 mm; left column) or tissue composition (scale bars, 200 μm; right column). Right: Grafts produced in WT (top row) or DbLR (bottom row) mice by P3 mSSCs show no significant differences in graft size [scale bars, 1 mm (left column) and 200 μm (right column)]. Movat’s pentachrome stain: yellow, bone; blue, cartilage; brown, marrow; red, kidney tissue. (C) Quantification of kidney graft histomorphometry (n = 4 per group). Data and error bars represent means ± SEM. **P < 0.01, unpaired two-tailed t test.

Fig. 4. Db skeletal niches exhibit differential Hh signaling after fracture

(A) Heat map showing relative gene expression of skeletogenic factors in mSSCs and BCSPs from uninjured femora (left columns) or calluses (right columns) of WT versus DbLR mice. Genes related to (i) Hh signaling; (ii) skeletal development, growth, and repair; (iii) apoptotic processes; and (iv) proliferation are shown. Differential gene expression was seen for Hh signaling factors, apoptosis-related proteins, and proliferation-related proteins (black arrows). Blue, low expression; red, high expression. (B) Relative protein levels of Ihh in post-fracture day 7 calluses from DbLR versus WT mice. Western blot quantification determined by densitometry analysis (n = 8). A.U., arbitrary units. (C) Heat map showing relative gene expression of Hh signaling factors in mSSCs from post-fracture day 7 calluses of multiple Db and control mouse models. Models Include 10-week-old WT, 4-week-old pre-Db Leprdb (pre-DbLB), 4-week-old WT Leprdb (DbLR), streptozotocin-induced diabetes (DbSTZ), and diet-induced diabetes (DbDIO). Black arrows mark differentially expressed genes. (D) Alizarin red staining shows that XL139 reduces in vitro osteogenic potential of WT mSSCs from post-fracture day 7 calluses in a dose-dependent manner. (E) MST of healing femora at post-fracture week 4 from XL139-treated (gray) versus untreated (blue) WT mice (n = 12 to 13). (F) Absolute cell numbers of WT mSSCs and BCSPs from XL139-treated versus untreated post-fracture day 7 calluses (n = 5). (G) Pre- and post-fracture serum levels of TNFα in DbLR versus WT mice (n = 3). Data and error bars represent means ± SEM. *P < 0.05, **P < 0.001, ***P < 0.001, ****P < 0.0001, unpaired two-tailed t test.

Fig. 5. Increased levels of TNFα directly suppress Ihh expression in skeletal progenitors

(A) scRNA-seq shows cell-specific expression of TNFα receptor, TNF receptor superfamily 1α (Tnfrsf1α), in mSSCs and BCSPs from post-fracture day 7 calluses of DbLR mice. (B) scRNA-seq shows cell-specific expression of Ihh in mSSCs and BCSPs from post-fracture day 7 calluses of DbLR mice. (C) scRNA-seq shows cell-specific expression of Hh receptor, Ptch1, in mSSCs and BCSPs from post-fracture day 7 calluses of DbLR mice. (D) scRNA-seq shows cell-specific expression of Hh effector, Gli1, in mSSCs and BCSPs from post-fracture day 7 calluses of DbLR mice. (E) Schematic of procedure investigating the effects of glucose or TNFα on Ihh expression in vitro. FACS-sorted mSSCs and BCSPs were isolated from the appendicular skeleton of uninjured P3 WT mice. Protein quantification was measured using qRT-PCR. (F) qRT-PCR analysis shows that TNFα significantly diminishes Ihh expression in mSSC-derived cultures isolated from uninjured P3 mice. GAPDH, glyceraldehyde-3-phosphate dehydrogenase. (G) qRT-PCR analysis shows that glucose and TNFα reduce Ihh expression in BCSP-derived cultures isolated from uninjured P3 mice. (H) qRT-PCR analysis shows that TNFα diminishes Ihh expression in BCSP-derived cultures isolated from uninjured P3 mice in a dose-dependent manner. (I) Schematic of stem and progenitor cell cross-talk in the skeletal niche. BCSPs mediate mSSC activity through autocrine and/or paracrine signaling. Ihh expressed by BCSPs is recognized by Ptch1 on mSSCs, leading to signal transduction via Gli1. (J) Venn diagram of Ihh/Tnfrsf coexpression in DbLR mSSCs (left) and BCSPs (right) from post-fracture day 7 calluses shows that either autocrine or paracrine Ihh-mediated signaling could be repressed directly via TNFα signaling. Data and error bars represent means ± SEM. **P < 0.01, ****P < 0.0001, unpaired two-tailed t test.

Fig. 6. Ihh and Gli1 expression are repressed in Db human skeletal progenitors

(A) Representative image of osteoarthritic femoral head, with black arrow marking an area of cartilage degeneration from which cells were isolated for analysis. (B) FACS gating strategy to isolate human skeletal progenitor populations from collagenase-dissociated cells extracted from human femoral head and knee specimens. Both CD146⁺ and CD146⁻ skeletal progenitors are represented in nonhematopoietic CD45⁻CD235⁻ populations. (C) qRT-PCR analysis of relative Ihh expression in CD45⁻CD235⁻CD146^+/− human skeletal progenitors from non-Db versus Db patients (n = 5 to 6). (D) qRT-PCR analysis of relative Gli1 expression in CD45⁻CD235⁻CD146^+/− human skeletal progenitors from non-Db versus Db patients (n = 5 to 6). Data and error bars represent means ± SEM. *P < 0.05, unpaired two-tailed t test.

Fig. 7. Local delivery of Ihh restores the mSSC injury response

(A) Schematic of slow-release hydrogel placement on the fracture site in WT or DbLR mice. Hydrogel-treated mSSCs and BCSPs were isolated from post-fracture day 7 calluses using FACS for in vitro analyses, or post-fracture week 4 femora were harvested for MST. (B) MST of PBS-treated WT femora (blue) versus PBS-treated (black), Ihh-treated (red), or Shh-treated (green) femora from DbLR mice. Ihh or Shh treatment significantly increases DbLR femur strength compared to PBS-treated controls (n = 5). (C) Absolute cell numbers of mSSCs (top) and BCSPs (bottom) from PBS-treated WT femora versus PBS-, Ihh-, or Shh-treated calluses from DbLR mice. Calluses were harvested at post-fracture day 7 (n = 5). (D) Colony-forming assay of mSSCs isolated at post-fracture day 7 from each experimental group. The total number of colonies was measured (n = 3). (E) Respective population percentages of BrdU-labeled post-fracture day 3 callus mSSCs (left) or BCSPs (right) from each experimental group (n = 4). (F) Respective population percentages of annexin V expression in mSSCs from PBS-treated (left column) versus Ihh-treated (right column) calluses of WT (blue) or DbLR (red) mice. Calluses harvested at post fracture day 7. (G) Alizarin red staining shows osteogenic potential of mSSCs (top row) and BCSPs (bottom row) from PBS-treated WT (far left column) and PBS-treated (second column from left), Ihh-treated (second column from right), or Shh-treated (far right column) Db calluses. mSSCs and BCSPs were isolated from DbLR mice at post-fracture day 7. Bright-field microscopy, 10×. Scale bars, 200 μm. Data and error bars represent means ± SEM. *P < 0.05, **P < 0.01, unpaired two-tailed t test.

Fig. 8. Schematic showing the cellular and molecular mechanisms underlying impaired Db skeletal repair

(Left) In non-Db conditions, normal Hh-mediated cross-talk between mSSCs and BCSPs in skeletal niches coordinates an effective injury response. (Top right) in Db conditions, high serum levels of TNFα disrupt skeletal niche signaling, leading to impaired fracture repair. (Bottom right) Impaired fracture repair in Db conditions Can be rescued by supplying Ihh signaling.