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RNAseq for cultured ctrl and TgAPPsweOCN-Cre osteoblasts from skull bone

ABSTRACT: Purpose: To investigate how APPswe+ osteoblasts affect skull bone and skull bone marrow remodeling, we isolated tdTomato⁺ calvarial osteoblasts from the skulls of TgAPPswemOCN-Cre;Ai9 mice and control (mOCN-Cre;Ai9) mice at 6-MO using fluorescence-activated cell sorting (FACS) and subjected them to bulk RNA sequencing. Methods: (1) Calvariae from TgAPPswemOCN-Cre;Ai9 mice and control (mOCN-Cre;Ai9) mice were transferred to a dish containing PBS, and the soft tissues were carefully removed using tweezers. The cleaned bones were then cut into small fragments of approximately 1–2 mm² and sequentially digested with collagenase solution (37 °C, 30 min, twice) and trypsin solution (37 °C, 30 min) to remove residual soft tissue and adherent cells. After an additional collagenase digestion, the bone fragments were washed three times with culture medium and transferred to 60 mm culture dishes containing DMEM supplemented with 10% FBS, 1% penicillin/streptomycin, 10 mmol/L β-glycerophosphate, and 50 μmol/L L-ascorbic acid-2-phosphate. The culture medium was replaced three times per week. Osteoblasts began to migrate from the bone chips after 3–5 days. After two weeks, the monolayer cells were detached by incubation with trypsin solution and replated onto 100 mm tissue culture dishes in α-MEM supplemented with 10% FBS and 1% penicillin/streptomycin. (2) Total RNA was extracted from purified Td⁺ osteoblasts isolated by flow cytometry from mOCN-Cre; Ai9 and TgAPPswemOCN-Cre; Ai9 mice. RNA integrity was assessed for each sample, and only samples with a RNA Integrity Number (RIN) > 2 were included for further analysis. RNA-seq was performed by BGI America (Cambridge, MA) using the DNBseq platform. Gene expression levels were normalized as fragments per kilobase of transcript per million mapped reads (FPKM). Differentially expressed genes (DEGs) were identified using DESeq2 and PoissonDis algorithms, with P-values adjusted by the Benjamini–Hochberg (BH) method. Genes with adjusted P ≤ 0.05 and log₂ fold change ≥ 1 were considered significantly differentially expressed. Results: Transcriptomic profiling revealed 425 upregulated and 478 downregulated genes in APPswe-expressing osteoblasts compared to controls. Gene ontology (GO) pathway analysis showed that the upregulated genes were primarily enriched in pathways related to positive regulation of cell migration, cytokine production, Wnt/β-catenin signaling, angiogenesis, cell-cell adhesion, and negative regulation of cell proliferation, while downregulated genes were associated with cell-cell signaling, cell proliferation, and osteoclast differentiation. Conclusions: Early thinning of the skull in TgAPPswemOCN-Cre mice may result from reduced osteoblast proliferation.

ORGANISM(S): Mus musculus

PROVIDER: GSE310893 | GEO | 2026/07/15

REPOSITORIES: GEO

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Project description:Purpose: To investigate if and how APPswe in OCN-Cre+ OB-lineage cells gives rise to the brain and behavior phenotypes, we purified OCN-Cre+ BMSCs (marked by tdTomato+, believed to be OB progenitors) from both the 6-MO control (OCN-Cre; Ai9) and TgAPPsweOCN; Ai9 mice using fluorescence-activated cell sorting (FACS) and then subjected them to RNA-seq analysis Methods: (1)the whole bone marrow cells of 6-month-old OCn-Cre;Ai9 andTgAPPswe;OCn-Cre;Ai9 flushed out from long bones of mice with DMEM were filtered through a 70-mm filter mesh, washed, re-suspended, and then plated in 100-mm dishes with growth medium (DMEM plus 10% FBS), which were incubated at 37oC with 5% CO2. The non-adherent cells were removed 72 hours after changing the medium. The attached bone marrow cells were cultured with the growth medium for 7 days. These cells were then resuspended and plated at culture dishes and cultured for another 3-6 days with the same growth medium. These cells, so called BMSCs. (2)Then cell media were removed from culture dishes and cells were rinsed with PBS. Trypsin solution was added to incubate at 37°C for 2 min. The detached adherent cells were centrifuged, and the pellet cells were washed with 1 ml cold PBS, and finally resuspended in 0.5 ml PBS with 1% FBS for flow cytometry analysis. Flow cytometric analysis was performed by use of a flow cytometer in CWRU core facility. Acquisition and analysis were performed by using FACSDiva 8.0.1 software (BD).(3)Total RNAs were extracted from purified Td+ OB-progenitor cells from OCN-Cre; Ai9 and TgAPPsweOCN; Ai9 mice by flow cytometer. RNA Integrity Number (RIN) was accessed for every sample, and the samples were considered qualified with RIN> 2. These RNA samples were then subjected to RNA-seq analyses by BGI America (Cambridge, MA) using the DNBseq platform. Firstly, we removed the reads mapped to rRNA and obtained the raw data with 52.47 Mb reads. After filtering low-quality, adaptor-polluted and high content of unknown base reads in the sequencing reads, 51.9 Mb clean reads were obtained per sample on average. Then clean reads were mapped to reference genome using HISAT2. On average 92.91% reads were mapped and the uniformity of the mapping result for each sample suggests that the samples were comparable. Comparisons to RNAseq were normalized to fpkm Results: 917 up- and 1825 down-regulated genes were identified in APPswe+ OB progenitors. Among these genes, 154 up- and 269 down-regulated genes encode secreted proteins. Interestingly, go analysis showed that most up-regulated genes are involved in inflammatory response, cytokine production, and cytokine/chemokine-mediated signaling pathways; and most down-regulated genes are implicated in cell cycle, cell proliferation, and bone mineralization. Conclusions: Increased cytokines and chemokines in APPswe+ OB-progenitors, indicating that APPswe+ OB progenitors may undergo cellular senescence.

Project description:Runx1 is highly expressed in osteoblasts, however, its function in osteogenesis is unclear. We generated mesenchymal progenitor-specific (Runx1f/fTwist2-Cre) and osteoblast-specific (Runx1f/fCol1α1-Cre) conditional knockout (Runx1 CKO) mice. The mutant CKO mice with normal skeletal development displayed a severe osteoporosis phenotype at postnatal and adult stages. Runx1 CKO resulted in decreased osteogenesis and increased adipogenesis. RNA-sequencing analysis, Western blot, and qPCR validation of Runx1 CKO samples showed that Runx1 regulates BMP signaling pathway and Wnt/β-catenin signaling pathway. ChIP assay revealed direct binding of Runx1 to the promoter regions of Bmp7, Alk3, and Atf4, and promoter mapping demonstrated that Runx1 upregulates their promoter activity through the binding regions. Bmp7 overexpression rescued Alk3, Runx2, and Atf4 expression in Runx1-deficient BMSCs. Runx2 expression was decreased while Runx1 was not changed in Alk3 deficient osteoblasts. Atf4 overexpression in Runx1-deficient BMSCs did not rescue expression of Runx1, Bmp7, and Alk3. Smad1/5/8 activity was vitally reduced in Runx1 CKO cells, indicating Runx1 positively regulates the Bmp7/Alk3/Smad1/5/8/Runx2/ATF4 signaling pathway. Notably, Runx1 overexpression in Runx2-/- osteoblasts rescued expression of Atf4, OCN, and ALP to compensate Runx2 function. Runx1 CKO mice at various osteoblast differentiation stages reduced Wnt signaling and caused high expression of C/ebpα and Pparγ and largely increased adipogenesis. Co-culture of Runx1-deficient and wild-type cells demonstrated that Runx1 regulates osteoblast−adipocyte lineage commitment both cell-autonomously and non-autonomously. Notably, Runx1 overexpression rescued bone loss in OVX-induced osteoporosis. This study focused on the role of Runx1 in different cell populations with regards to BMP and Wnt signaling pathways and in the interacting network underlying bone homeostasis as well as adipogenesis, and has provided new insight and advancement of knowledge in skeletal development. Collectively, Runx1 maintains adult bone homeostasis from bone loss though up-regulating Bmp7/Alk3/Smad1/5/8/Runx2/ATF4 and WNT/β-Catenin signaling pathways, and targeting Runx1 potentially leads to novel therapeutics for osteoporosis. Using unbiased genome-wide RNA-seq data from Runx1f/fCol1α1-Cre, Runx1ffTwist2-cre, Runx1f/f;Col2α1-Cre and their control osteoblasts, we examined Runx1-mediated transcriptional targets that could account for osteoblast differentiation defects and increased adipocytes.

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RNAseq for cultured ctrl and TgAPPsweOCN-Cre osteoblasts from skull bone

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