The Msi2 expression level decreases during adipogenesis and increases during osteogenesis of BMSCs

BMSCs were able to differentiate into both osteoblasts and adipocytes. To explore the functions of Msi2 in BMSCs, we first surveyed the protein level of Msi2 during BMSC differentiation. When BMSCs were induced to differentiate into adipocytes that were stained with Oil Red O and BODIPY (Fig. 1a), both the mRNA and protein levels of Msi2 decreased, while the expression levels of adipocyte markers such as CCAAT/enhancer binding protein α (Cebpα), peroxisome proliferative activated receptor γ (Pparγ), fatty acid binding protein 4 (Fabp4) and perilipin increased, indicating efficient differentiation into adipocytes (Fig. 1b–d). In contrast, when BMSCs were induced to differentiate into osteoblasts that were stained with ALP and Alizarin red S (Fig. 1e), both the mRNA and protein levels of Msi2 increased during the differentiation process. Consistently, the expression levels of osteoblast markers, including Runt-related transcription factor 2 (Runx2), Sp7 transcription factor (Osterix), and Collagen type 1 alpha 1 (Col1α1), increased, suggesting efficient differentiation into osteoblasts (Fig. 1f–h). The dynamic changes in Msi2 expression levels during BMSC adipogenesis and osteogenesis indicate that Msi2 may play distinct roles in adipogenesis and osteoblastogenesis from BMSCs.

Fig. 1

Changes in Msi2 expression levels in the adipogenesis and osteogenesis of BMSCs. a BMSCs isolated from 4-week-old wild-type mice. Cultures were stained with Oil Red O and BODIPY as shown. Scale bar = 200 μm. b Western blot analysis of Msi2 levels during adipogenesis for different durations. c qPCR analysis of Msi2 expression in BMSCs during adipogenesis for the indicated durations. Data represent the mean ± SD, n = 4. d qPCR analysis of the expression of adipocyte markers, including perilipin, Fabp4, Pparγ and Cebpα, in BMSCs during adipogenesis for the indicated durations. Data represent the mean ± SD, n = 4. e BMSCs isolated from 6-week-old wild-type mice. Cultures were stained with ALP and Alizarin red S as shown. Scale bar = 3 mm. f Western blot analysis of Msi2 levels during osteogenesis for different durations. g qPCR analysis of Msi2 expression in BMSCs during osteogenesis for the indicated durations. Data represent the mean ± SD, n = 4. h qPCR analysis of the expression of osteoblast markers, including Runx2, Col1a1 and Osterix, in BMSCs during osteogenesis for the indicated durations. Data represent the mean ± SD, n = 4. i Immunostaining of Msi2 (green), CD105 (red) and DAPI (blue) in tibia from 6-week-old WT mice. Scale bar = 200 μm (left). Scale bar =30 μm (right). j Immunostaining of Msi2 (green) and DAPI (blue) in femurs from 6-week-old Prx1-Cre Tdtomato mice. Scale bar = 1 000 μm (left). Scale bar = 200 μm (right)

We next determined the MSI2 expression level in long bone in vivo and found that MSI2 was highly expressed in the growth plate and trabecular bone (Fig. S1A). Interestingly, MSI2 was also expressed in the internal and external periostea but was barely expressed in cortical bone (Fig. S1B). CD105 is a marker of MSCs. Further research found that MSI2 and CD105 can be colocalized (Fig. 1i). Further research was performed to determine whether MSI2 is expressed in Prx1-positive cells, which are mainly MSCs. We found that MSI2 expression colocalized with Prx1-positive cells (Fig. 1j). These results further suggested that Msi2 may have functions in MSC commitment and bone formation.

Msi2-deficient mice display increased bone marrow adipocytes and decreased bone mass

To investigate the function of Msi2 in BMSC differentiation, we generated Msi2 knockout mice using CRISPR-Cas9 technology to introduce a frameshift in the first intron of Msi2 (Fig. 2a). Immunofluorescence staining and western blotting confirmed the knockout of Msi2 in bone and BMSCs (Fig. 2b and Fig. 6j). We tested the knockout efficiency of Msi2 in the main organs of the knockout mice. The results showed that Msi2 was almost completely eliminated in the Msi2 knockout mice (Fig. S2A, B). In addition, we tested whether Msi1, a homolog of Msi2, has a compensatory effect in knockout mice, and the results showed that Msi1 expression in the BMSCs of knockout mice was not significantly different from that in the control mice. (Fig. S2C). The Msi2−/− mice survived normally after birth and had normal fertility. However, the Msi2−/− mice exhibited short stature and skeletal dysplasia regardless of sex (Fig. 2c and Fig. S2D). Compared with the control mice, the Msi2 knockout mice had reduced body weight, body length, and femur length (Fig. S2E-G). BODIPY staining results of the Msi2−/− mouse tibia revealed increased adipocyte accumulation in the tibia bone marrow of the Msi2−/− mice, and older Msi2 knockout mice had more fat vacuoles in the bone marrow cavity (Fig. 2d). Immunofluorescence staining of perilipin A, a mature adipocyte marker, also confirmed adipocyte accumulation in the Msi2−/− mice (Fig. 2e, f). Both the number and the size of adipocytes in the bone marrow cavity increased with age in the Msi2−/− mice (Fig. 2g, h).

Fig. 2

Msi2-deficient mice show increased bone marrow adipocytes. a Mouse construction strategy. b Immunostaining of MSI2 (green) and DAPI (blue) in tibiae from 6-week-old WT and Msi2−/− mice. Scale bar = 200 μm. c Representative view of the wild-type, Msi2+/−, and Msi2−/− 6-week-old mice. d BODIPY (green) staining of tibiae from the 6-week-old WT and Msi2−/− mice. Scale bar = 500 μm (top). Hematoxylin-eosin staining of femurs from the 30-week-old WT and Msi2−/− mice. Scale bar = 500 μm (middle). Scale bar = 500 μm (down). e Immunostaining of perilipin A/B (green) and OPN (red) of femurs from the 6-week-old WT and Msi2−/− mice. Scale bar = 50 μm. f Quantification of the relative areas of OPN and perilipin in (e). g Immunostaining of perilipin A/B (green) and OPN (red) of femurs from the 15-week-old WT and Msi2−/− mice. Scale bar = 50 μm. h Quantification of the relative areas of OPN and perilipin in (g)

We further investigated whether bone formation was affected. Microquantitative computed tomography (μ-CT) analysis was performed to compare the changes in bone-related elements in the long bones of the Msi2 knockout mice and the WT littermates. We found that the 6-week-old Msi2−/− mice showed significantly decreased bone mass (Fig. 3a). Trabecular bone per tissue volume (BV/TV) in the Msi2−/− mice was decreased compared to that in the age-matched WT littermates (Fig. 3c), accompanied by a reduction in trabecular number (Tb.N) (Fig. 3d), a reduction in trabecular bone thickness (Tb.Th) and an increase in trabecular bone spacing (Tb.Sp) (Fig. 3e, f). There was no significant difference in cortical bone thickness (Cor.Th) of the Msi2−/− mice compared with that of the WT mice, which is consistent with the observation that Msi2 is rarely expressed on cortical bone (Figs. 1j, 3b, g).

Fig. 3

Msi2-deficient mice show decreased bone mass. a Three-dimensional μ-CT images of trabecular bone of distal femurs isolated from the 6-week-old female WT and Msi2−/− mice (n = 6). b Three-dimensional μ-CT images of cortical bone of distal femurs isolated from the 6-week-old female WT and Msi2−/−mice (n = 6). c–g μ-CT analysis of distal femurs from the 6-week-old WT and Msi2−/− mice for trabecular bone volume per tissue volume (BV/TV) (c), trabecular number (Tb.N) (d), trabecular separation (Tb.Sp) (e), trabecular thickness (Tb.Th) (f) and cortical bone thickness (Cor.Th) (g). h Masson trichrome staining of the 6-week-old WT and Msi2−/− mice. Scale bar = 500 μm. i–n Histomorphometric analysis of distal femurs from the 5-week-old WT and Msi2−/− mice to determine the trabecular bone volume per tissue volume (BV/TV) (i), trabecular number (Tb.N) (j), trabecular thickness (Tb.Th) (k) trabecular separation (Tb.Sp) (l) and number of osteoblasts per bone perimeter (N.Ob/B.Pm) (m) and osteoblast surface per bone surface (Ob.S/BS) (n). Data represent the mean ± SD, n = 4. *P < 0.05, **P < 0.01, ns indicates no significance, unpaired Student’s t test. o Immunostaining of OPN (red) and DAPI (blue) in femurs from the 6-week-old WT and Msi2−/− mice. Scale bar = 500 μm. p Quantification of the relative area of OPN in (o). q Immunohistochemical staining of Col1α1 from the 6-week-old WT and Msi2−/− mice. Scale bar = 100 μm. r Quantification of the relative area of Col1α1 in (q)

To further explore the function of Msi2 in bone formation, we performed histomorphometric analysis to evaluate static and dynamic parameters of bone formation and resorption (Fig. 3h). Consistent with the μ-CT data, histomorphometric analysis also showed that the Msi2−/− mice had a significant decrease in both BV/TV and Tb.Th and also showed a significant increase in Tb.Sp but no changes in Tb.N (Fig. 3i–l). The numbers of osteoblasts per bone perimeter (N.Ob/B.Pm) and osteoblast surface per bone surface (Ob.S/BS) were decreased in the Msi2−/−mice compared to the WT control mice (Fig. 3m, n). Further immunofluorescence staining analysis of the distal femur of the Msi2−/− mice revealed decreased expression of the osteoblast markers osteopontin (OPN) and COL1α1 in the Msi2−/− mice (Fig. 3o–r).

Bone formation by osteoblasts and bone resorption by osteoclasts are essential for the maintenance of bone homeostasis. Our results showed that the osteoclast differentiation of the Msi2 knockout mice was weakened in vitro (Fig. S3A, B). Interestingly, no changes in the number of HSCs were detected in the bone marrow cells of the Msi2−/− mice (Fig. S3C). TRAP staining for osteoclast activity showed no significant difference between the WT and Msi2−/− mice in vivo (Fig. S3D, E). This finding indicates that the decrease in bone mass in the Msi2−/− mice is mainly due to decreased bone formation. Taken together, the above results suggested that Msi2 is required for proper bone formation.

Msi2 promotes osteoblastogenesis and inhibits BMSC adipogenesis

The accumulation of adipocytes and decreased bone formation in the bone of the Msi2−/− mice prompted us to further explore how Msi2 regulates BMSC differentiation. BMSCs were isolated from the WT or Msi2−/− mice and were differentiated in adipogenic medium for 7 days. Adipogenic differentiation was enhanced in the Msi2−/− BMSCs, as indicated by increased Oil Red O staining and BODIPY staining compared to that of the BMSCs from the WT mice (Fig. 4a, b). The expression levels of adipocyte markers such as Cebpα, Cebpβ, Fabp4, lipoprotein lipase (Lpl), perilipin and Pparγ increased in the Msi2 knockout BMSCs upon induction to adipogenesis compared to those of the WT BMSCs (Fig. 4c).

Fig. 4

Msi2 deficiency promotes adipogenesis and inhibits osteoblastogenesis in BMSCs. a Oil Red O and BODIPY staining of BMSCs cultured with adipocyte differentiation medium for 6 days. Data are representative of three independent experiments. Scale bar = 40 μm. b Statistical analysis of the percentage of Oil Red O-positive area via ImageJ. Data are presented as the mean ± SD, n = 4 in each group. Data represent the mean ± SD, ***P < 0.005, unpaired Student’s t test. c qPCR analysis of Cebpα, Cebpβ, Fabp4, Lpl, perilipin and Pparγ expression in BMSCs from the WT and Msi2−/− mice after adipocyte differentiation for 6 days. Data represent the mean ± SD, n = 4. *P < 0.05, **P < 0.01, ***P < 0.005, unpaired Student’s t test. d ALP staining and Alizarin red S staining after osteoblast differentiation for 7 days (upper) and 14 days (lower). Data are representative of three independent experiments. Scale bar = 3 mm. e ALP activity was measured by phosphatase substrate assays. Data represent the mean ± SD, n = 3. **P < 0.01, unpaired Student’s t test. f qPCR analysis of Runx2, Alp, Bsp, Col1α1, Osterix, and ATF4 expression after osteoblast differentiation for 7 days; BMSCs were from the WT and Msi2−/− mice. Data represent the mean ± SD, n = 4. *P < 0.05, **P < 0.01, ns: no significance; unpaired Student’s t test

We next examined the role of Msi2 in the osteoblast differentiation of BMSCs. BMSCs were isolated from the WT or Msi2−/− mice and were induced to differentiate in osteogenic medium for 1 week and 2 weeks. Alkaline phosphatase (ALP) activity assays and Alizarin red histochemical staining revealed reduced osteoblast differentiation in BMSCs from the Msi2−/− mice (Fig. 4d, e). The expression levels of osteoblast markers, such as Alp, bone sialoprotein (Bsp), Col1α1, Osterix and Atf4, also decreased in the Msi2−/− BMSCs (Fig. 4f).

Taken together, the above results revealed that Msi2 regulates the balance of BMSC fate commitment by repressing adipocyte differentiation and enhancing osteoblast differentiation.

MSI2 inhibits PPAR signaling in BMSCs

To explore the molecular mechanism by which Msi2 regulates osteoblast-adipocyte lineage commitment, we performed RNA sequencing analysis using BMSCs from the WT and Msi2−/− mice (7 days after osteoblast differentiation) and compared the gene expression profiles. Genes related to adipocyte differentiation showed upregulated expression, and genes related to osteoblast differentiation showed downregulated expression (Fig. 5a). Gene set enrichment analysis (GSEA) was then performed to identify significantly enriched Gene Ontology (GO) terms. Lipid localization or storage regulators and adipocyte differentiation markers showed upregulated expression in the Msi2−/− BMSCs (Fig. 5b). Ossification-, skeletal development- and bone development-related genes showed significantly downregulated expression (Fig. 5c). Kyoto Encyclopedia of Genes and Genomes pathway analysis indicated that the PPAR signaling pathway was significantly enhanced in the Msi2 knockout BMSCs (Fig. 5d). To further analyze the changes in the PPAR signaling pathway in the Msi2 knockout cells, we utilized GSEA to mine the RNA-seq data, and the results showed that Msi2 knockout increased the enrichment score for the PPAR signaling pathway module (Fig. 5e). Genes with upregulated expression that showed a significant difference in expression in the GSEA were visualized by a heatmap (Fig. 5f). The expression levels of the genes with upregulated and downregulated expression were further confirmed in the Msi2 knockout BMSCs by RT-PCR (Fig. 5g). As PPARγ is considered to be one of the major drivers of adipogenesis,10,11 these results suggested that Msi2 may regulate BMSC commitment by inhibiting the PPARγ signaling pathway.

Fig. 5

MSI2 inhibits PPAR signaling in BMSCs. a Heatmap of RNA sequencing data between the WT and Msi2−/− mouse BMSCs cultured in osteoblast differentiation medium for 7 days, n = 2 for each group. b Upregulated (red) GO analysis associated with significantly regulated genes (P < 0.05) in the Msi2 knockout versus WT control groups. c Downregulated (blue) GO analysis associated with significantly regulated genes (P < 0.05) in the Msi2 knockout versus WT control groups. d Upregulated (red) pathways associated with significantly regulated genes (P < 0.05) in the Msi2 knockout versus WT control groups. e GSEA of the enrichment of all genes in RNA sequencing. f Heatmap of genes with upregulated expression in the PPAR signaling pathway obtained by GSEA. g qPCR results of adipogenesis-related gene (Cebpα, Lpl, Perilipin, Pparγ) and osteogenesis-related gene (Alp, Bsp, Col1α1) expression in the WT and Msi2−/− mouse BMSCs

Msi2 inhibits Cebpα translation and PPARγ expression in BMSCs

Msi2 is an RNA-binding protein. Previous results demonstrated that three phenylalanine residues in Msi2 are essential for Msi2 RNA binding. To determine whether RNA binding is essential for the function of Msi2, we mutated three phenylalanine residues essential for Msi2 RNA binding to leucine (F64/66/69 L) to generate an RNA binding-deficient mutant of Msi2 (hereafter Msi2RBDmut) (Fig. 6a).16,27 We next compared the function of Msi2 with that of Msi2RBDmut. As shown in Fig. 6b, overexpression of Msi2 reduced the differentiation of BMSCs into adipocytes, but Msi2RBDmut overexpression did not reduce the differentiation of BMSCs into adipocytes (Fig. 6b). Moreover, overexpression of Msi2 enhanced the differentiation of BMSCs into osteoblasts, but Msi2RBDmut overexpression did not (Fig. 6c). These results suggest that the mRNA binding activity of Msi2 is required for BMSC commitment.

Fig. 6

Msi2 inhibits Cebpα translation and PPARγ activation in BMSCs. a Schematic illustration of Msi2 and the Msi2RBDmut mutation. b BMSCs isolated from 4-week-old wild-type mice and treated with Msi2 and Msi2RBD lentivirus. Cultures were stained with Oil Red O and BODIPY as shown. Scale bar = 200 μm. c BMSCs isolated from 4-week-old wild-type mice, and treated with Msi2 and Msi2RBD lentivirus. Cultures were stained with ALP, and ALP activity was quantified as shown. Scale bar = 3 mm. Data represent the mean ± SD, *P < 0.05, one-way ANOVA. d Western blot analysis of PPARγ and perilipin protein levels in the C3H10 cells overexpressing Flag-tagged Msi2 and Msi2RBDmut protein; GAPDH was used as a reference protein. e Schematic of the mouse Cebpα transcript. Bars, the putative MBEs (r(G/A)U1–3AGU). Two MBEs were identified within the 3′ UTR of Cebpα. CDS, coding sequence for mC/EBPα protein. f RIP with anti-Flag antibody from C3H10 cells expressing empty vector, Flag-tagged Msi2 or Flag–Msi2RBDmut. Coimmunoprecipitated RNAs were analyzed for the enrichment of Cebpα transcripts. n = 3 each. Data represent the mean ± SD, ***P < 0.001, ****P < 0.000 1, one-way ANOVA. g RIP with anti-Msi2 antibody or a control rabbit IgG from BMSCs. Coimmunoprecipitated RNAs were analyzed for the enrichment of Cebpα transcripts. n = 3 each. Data represent the mean ± SD, ***P < 0.001, ordinary one-way ANOVA. h qPCR results of Cebpα in the C3H10 cells overexpressing Flag-tagged Msi2 and Msi2RBDmut proteins. Data represent the mean ± SD, ns: no significance, one-way ANOVA. i qPCR results of Pparγ in the C3H10 cells overexpressing Flag-tagged Msi2 and Msi2RBDmut proteins. Data represent the mean ± SD, ****P < 0.000 1, one-way ANOVA. j Western blot analysis of C/EBPα, PPARγ, FABP4, LPL, perilipin and Msi2 protein levels in the WT and Msi2−/− mouse BMSCs. GAPDH was used as a reference protein. k The model of Msi2 regulating PPAR signaling

Transcriptional profiling analysis suggested that Msi2 may regulate BMSC commitment by inhibiting the PPARγ signaling pathway. Msi2 is considered to be a translational repressor by binding the 3′ UTR of the target mRNA.13 We next explored whether Msi2 regulates the PPARγ signaling pathway by repressing the translation of key components of PPARγ signaling. As shown in Fig. 6d, overexpression of Msi2 reduced the protein levels of PPARγ and perilipin when BMSCs were induced to differentiate into adipocytes (Fig. 6d). In contrast, Msi2RBDmut overexpression abolished the inhibitory effect of Msi2 (Fig. 6d). These results indicated that mRNA binding activity is required for Msi2 to inhibit the PPARγ signaling pathway.

We next examined how Msi2 relies on the mRNA binding ability to regulate PPAR signaling. The C/EBP family has been reported to be closely related to the regulation of PPAR signaling, and the mRNA level of Cebp factors was not changed significantly in our RNA sequencing data. We then examined the putative MBEs in the 3′ UTR of different Cebps and found that only Cebpα’s 3′ UTR has two MBEs; the Cebpβ and Cebpδ 3′ UTRs did not (Fig. 6e). We then performed an RNA immunoprecipitation (RIP) assay using C3H10 cells transfected with plasmids expressing Flag-tagged Msi2 or Flag-tagged Msi2RBDmut. Interestingly, Cebpα transcripts were significantly enriched by Flag immunoprecipitation when Flag-Msi2 was expressed. In contrast, Cebpα transcripts were not enriched when Flag-tagged Msi2RBDmut was expressed (Fig. 6f). These results suggested that Msi2 binds to the mRNA of Cebpα. Consistently, RIP with an anti-Msi2 antibody also specifically enriched Cebpα transcripts relative to that of an immunoglobulin-G (IgG) control (Fig. 6g), further confirming the interaction between Msi2 and Cebpα mRNA. Msi2 overexpression in C3H10 cells did not change the RNA level of Cebpα (Fig. 6h), However, the RNA level of Pparγ, which is regulated by Cebpα, was significantly downregulated when Msi2 was overexpressed in C3H10 cells (Fig. 6i). The protein level of Cebpα was increased significantly in the MSI2 knockout BMSCs, and PPARγ signaling markers were also significantly increased in the MSI2 knockout BMSCs (Fig. 6j). These data indicated that binding of Msi2 to Cebpα transcripts negatively regulates the translation of Cebpα. Regulation of PPARγ signaling by Msi2 is essential for the dynamic balance of the commitment between osteoblasts and adipocytes (Fig. 6k).

Msi2 expression is downregulated during aging

The depletion of Msi2 in mice led to decreased bone mass with increased marrow adipocytes, resembling aging-induced osteoporosis. We next examined whether Msi2 expression changed during aging. We isolated BMSCs from 2-month-old (young) or 24-month-old (old) mice and found that the Msi2 expression level was decreased in old BMSCs, as indicated by RT-qPCR assays (Fig. 7a). Immunohistochemical staining also showed that Msi2 protein expression levels were downregulated in the aged mice (Fig. 7b). μ-CT analysis confirmed that the bone mass of the old mice was significantly reduced (Fig. 7c, d), accompanied by increased Tb.Sp (Fig. 7e) and decreased Tb.N (Fig. 7f). Interestingly, compared with that in the young mice, cortical bone in the aging mice was thicker (Fig. S4A, B). Similar to the phenotype of the Msi2 knockout mice, abnormal accumulation of adipocytes in the bone marrow cavity of the aged mice was observed (Fig. 7g), suggesting the occurrence of aging-related osteoporosis. Immunofluorescence staining revealed decreased expression levels of the osteoblast marker OPN and increased expression levels of the adipocyte marker perilipin in the bone marrow cavity of the old mice (Fig. 7h, i). Similar to the scenario in Msi2−/− BMSCs, the RNA level of Cebpα remained unchanged in BMSCs isolated from the aged mice, and the RNA level of Pparγ increased in BMSCs isolated from the aged mice (Fig. 7j). Immunohistochemical staining also showed that PPARγ protein expression levels were upregulated in the aged mice (Fig. 7k, l). Consistent with previous reports,28 the mRNA level of the senescence marker p16 increased in the old BMSCs. In addition, the target genes of PPARγ increased significantly (Fig. S4C). The old BMSCs had a phenotype similar to that of the Msi2−/− BMSCs, which is consistent with the decreased expression level of Msi2 in the aged BMSCs.

Fig. 7

Msi2 expression is downregulated during aging. a qPCR results of Msi2 expression in BMSCs from 8-week-old and 24-month-old mice (n = 4). Data represent the mean ± SD, ***P < 0.001, unpaired Student’s t test. b Immunohistochemistry staining of Msi2 from 8-week-old and 24-month-old mice. Scale bar = 50 μm. c Three-dimensional μ-CT images of trabecular bone of distal femurs isolated from 8-week-old female and 24-month-old female mice (n = 6). d μ-CT analysis of trabecular bone volume per tissue volume (BV/TV) in the distal femur of 8-week-old female and 24-month-old female mice. e μ-CT analysis of the distal femur of 8-week-old female and 24-month-old female mice for trabecular separation (Tb.Sp). f μ-CT analysis of the trabecular number (Tb.N) of the distal femur of 8-week-old female and 24-month-old female mice. g Hematoxylin-eosin staining of femurs from wild-type mice at 8 weeks and 24 months. Scale bar = 500 μm. h Immunostaining of perilipin A/B (green) and OPN (red) of femurs from 8-week-old and 24-month-old mice. Scale bar = 50 μm. i Quantification of the relative areas of OPN and Perilipin in (h). j qPCR results of Cebpα and Pparγ expression in BMSCs from 8-week-old and 24-month-old mice (n = 4). Data represent the mean ± SD, ***P < 0.01, ns: no significance, unpaired Student t test. k Immunohistochemistry staining of PPARγ from 8-week-old and 24-month-old mice. Scale bar = 100 μm. l Quantification of the relative area of PPARγ in (k)

These results suggest that Msi2 could be one of the contributors to aging-induced osteoporosis. In old BMSCs, the reduction in the Msi2 expression level leads to a shift in the differentiation balance of BMSCs. Adipogenesis is enhanced, and osteoblastogenesis declines, which results in aging-induced osteoporosis.