scholarly journals Untargeted metabolomics in primary murine bone marrow stromal cells reveals distinct profile throughout osteoblast differentiation

Metabolomics ◽  
2021 ◽  
Vol 17 (10) ◽  
Author(s):  
Biswapriya B. Misra ◽  
Shobana Jayapalan ◽  
Alison K. Richards ◽  
Ron C. M. Helderman ◽  
Elizabeth Rendina-Ruedy
Keyword(s):  
Bone Marrow ◽  
Stromal Cells ◽  
Bone Marrow Stromal Cells ◽  
Osteoblast Differentiation ◽  
Marrow Stromal Cells ◽  
Untargeted Metabolomics ◽  
Coupled Processes ◽  
Intracellular Metabolite ◽  
Mineral Density ◽  
Murine Bone Marrow

Abstract Introduction Skeletal homeostasis is an exquisitely regulated process most directly influenced by bone resorbing osteoclasts, bone forming osteoblasts, and the mechano-sensing osteocytes. These cells work together to constantly remodel bone as a mechanism to prevent from skeletal fragility. As such, when an individual experiences a disconnect in these tightly coupled processes, fracture incidence increases, such as during ageing, gonadal hormone deficiency, weightlessness, and diabetes. While therapeutic options have significantly aided in the treatment of low bone mineral density (BMD) or osteoporosis, limited options remain for anabolic or bone forming agents. Therefore, it is of interest to continue to understand how osteoblasts regulate their metabolism to support the energy expensive process of bone formation. Objective The current project sought to rigorously characterize the distinct metabolic processes and intracellular metabolite profiles in stromal cells throughout osteoblast differentiation using untargeted metabolomics. Methods Primary, murine bone marrow stromal cells (BMSCs) were characterized throughout osteoblast differentiation using standard staining protocols, Seahorse XFe metabolic flux analyses, and untargeted metabolomics. Results We demonstrate here that the metabolic footprint of stromal cells undergoing osteoblast differentiation are distinct, and while oxidative phosphorylation drives adenosine triphosphate (ATP) generation early in the differentiation process, mature osteoblasts depend on glycolysis. Importantly, the intracellular metabolite profile supports these findings while also suggesting additional pathways critical for proper osteoblast function. Conclusion These data are the first of their kind to characterize these metabolites in conjunction with the bioenergetic profile in primary, murine stromal cells throughout osteoblast differentiation and provide provocative targets for future investigation.

scholarly journals LIF/STAT3/SOCS3 Signaling Pathway in Murine Bone Marrow Stromal Cells Suppresses Osteoblast Differentiation

2014 ◽  
Vol 115 (7) ◽  
pp. 1262-1268 ◽  
Author(s):  
Kenta Matsushita ◽  
Shousaku Itoh ◽  
Shun Ikeda ◽  
Yumiko Yamamoto ◽  
Yukako Yamauchi ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Signaling Pathway ◽  
Stromal Cells ◽  
Bone Marrow Stromal Cells ◽  
Osteoblast Differentiation ◽  
Marrow Stromal Cells ◽  
Murine Bone Marrow

scholarly journals Combination of hTERT and bmi-1, E6, or E7 Induces Prolongation of the Life Span of Bone Marrow Stromal Cells from an Elderly Donor without Affecting Their Neurogenic Potential

2005 ◽  
Vol 25 (12) ◽  
pp. 5183-5195 ◽  
Author(s):  
Taisuke Mori ◽  
Tohru Kiyono ◽  
Hideaki Imabayashi ◽  
Yukiji Takeda ◽  
Kohei Tsuchiya ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Life Span ◽  
Stromal Cells ◽  
Bone Marrow Stromal Cells ◽  
Marrow Stromal Cells ◽  
Murine Bone Marrow ◽  
Differentiated Cells ◽  
E6 And E7 ◽  
Old Donor

ABSTRACT Murine bone marrow stromal cells differentiate not only into mesodermal derivatives, such as osteocytes, chondrocytes, adipocytes, skeletal myocytes, and cardiomyocytes, but also into neuroectodermal cells in vitro. Human bone marrow stromal cells are easy to isolate but difficult to study because of their limited life span. To overcome this problem, we attempted to prolong the life span of bone marrow stromal cells and investigated whether bone marrow stromal cells modified with bmi-1, hTERT, E6, and E7 retained their differentiated capability, or multipotency. In this study, we demonstrated that the life span of bone marrow stromal cells derived from a 91-year-old donor could be extended and that the stromal cells with an extended life span differentiated into neuronal cells in vitro. We examined the neuronally differentiated cells morphologically, physiologically, and biologically and compared the gene profiles of undifferentiated and differentiated cells. The neuronally differentiated cells exhibited characteristics similar to those of midbrain neuronal progenitors. Thus, the results of this study support the possible use of autologous-cell graft systems to treat central nervous system diseases in geriatric patients.

The Transcription Repressor Gfi1 Directly Interacts With and Is Phosphorylated By Aurora A Kinase, Which Abrogates Myeloma-Induced Gfi1 Repression Of The Runx2 Promoter In Pre-Osteoblasts

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 1844-1844
Author(s):  
Jixin Ding ◽  
Fengming Wang ◽  
ShunQian Jin ◽  
Judy Anderson ◽  
Deborah L. Galson ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Dna Binding ◽  
Bone Disease ◽  
Stromal Cells ◽  
Protein Turnover ◽  
Bone Marrow Stromal Cells ◽  
Osteoblast Differentiation ◽  
Marrow Stromal Cells ◽  
Aurora A Kinase

Abstract Multiple myeloma (MM) is a plasma cell malignancy that is the most frequent cancer to involve the skeleton. MM bone disease is characterized by the formation of lytic bone lesions adjacent to MM cells that rarely heal even when patients are in long-term remission. This is due to the persistent suppression of bone marrow stromal cell (BMSC) differentiation into osteoblasts. We previously reported that MM cells induce long-lasting suppression of osteoblast differentiation by repression of the Runx2 gene through elevated expression of the transcriptional repressor Gfi1. However, how Gfi1 activity in BMSC is regulated by MM cells remains unclear. Using bioinformatics analysis, we found that there are three putative phosphorylation sites in the Gfi1 protein for Aurora A kinase (AurA) at S216, S326, and T418. We confirmed that Gfi1 was phosphorylated by AurA at multiple sites using an in vitro kinase assay. Co-immunoprecipitation assays revealed that AurA physically interacted with Gfi1 and phosphorylated Gfi1 protein. The interaction with AurA stabilized Gfi1 protein by blocking Gfi1 protein turnover, thereby extending the Gfi1 half-life from 2 hrs to 6 hrs. Further, co-transfection studies using wildtype and mutant AurA and Gfi1 showed that AurA inhibition of Gfi1 protein turnover was dependent on AurA kinase activity and phosphorylation of the S326 and T418 amino acid residues of Gfi1. Studies with co-transfected Myc-ubiquitin, FLAG-Gfi1, and HA-AurA revealed that AurA decreased Gfi1 ubiquitination, thereby leading to increased Gfi1 protein stability. Amino acids S326 and T418 are in Gfi1 zinc fingers (ZF) 3 and 6, respectively. It is known that Gfi1 ZF3, 4, and 5 are required for DNA binding, and that the K403R mutation in ZF6 interferes with DNA binding. Therefore we investigated if AurA phosphorylation of Gfi1 interferes with DNA binding. Chromatin immunoprecipitation and mRunx2 promoter oligo-pull down assays demonstrated that phosphorylated Gfi1 can still bind the Runx2 promoter. However, co-transfection studies with AurA and Gfi1 expression vectors with mRunx2-promoter luciferase reporters demonstrated that AurA phosphorylation of Gfi1 blocked repression of the Runx2 promoter. These data indicate that although AurA increased the amount of Gfi1 protein present on Runx2, AurA phosphorylation of Gfi1 appeared to lock Gfi1 in an “Off” (inactive) status and abrogated Gfi1 repression of Runx2 expression in osteoblast precursor cells. Since AurA phosphorylation of Gfi1 is not blocking DNA binding, the difference between Gfi1 “OFF” and “ON” status probably involves altered protein-protein interactions between Gfi1 and other factors that regulate Runx2 transcription. TNFa treatment, which we showed also represses Runx2 via Gfi1 activity, decreased the AurA protein level in MC-4 osteoblast precursor cells. Importantly, we found that AurA mRNA was decreased in both MC-4 cells treated with MM cells in vitro, and in bone marrow stromal cells isolated from MM patients. In conclusion, these data indicate that MM cells lower the levels of AurA in bone marrow stromal cells, thereby decreasing AurA phosphorylation of Gfi1. This helps to maintain Gfi1 in the “ON” status and allows Gfi1 repression of the Runx2 gene, thereby preventing osteoblast differentiation. These data suggest that AurA is an important regulator of Gfi1 function in MM bone disease. Disclosures: Roodman: Amgen: Membership on an entity’s Board of Directors or advisory committees; Eli Lilly: Research Funding.

scholarly journals Effects of Transplanting Bone Marrow Stromal Cells Transfected with CXCL13 on Fracture Healing of Diabetic Rats

2018 ◽  
Vol 49 (1) ◽  
pp. 123-133 ◽  
Author(s):  
Hui Jiang ◽  
Yicun Wang ◽  
Jia Meng ◽  
Shuo Chen ◽  
Jun Wang ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Fracture Healing ◽  
Stromal Cells ◽  
High Glucose ◽  
Bone Marrow Stromal Cells ◽  
Diabetic Rats ◽  
Marrow Stromal Cells ◽  
Area Measurement ◽  
Mineral Density

Background/Aims: Diabetic fracture have poor treatment and serious complications. Therefore, how to treat diabetic fracture is receiving increasing attention. This study aimed to investigate the effects of transplanting CXCL13-stimulated bone marrow stromal cells (BMSCs) on the fracture healing in diabetic rats. Methods: In vitro, RT-PCR was employed to examine the expression of CXCL13 in BMSCs in high glucose environment. MTT assay and apoptosis assay were utilized to determine the effects of CXCL13 overexpression on the proliferation and apoptosis of BMSCs respectively. ALP staining was applied to detect the ALP activity. In vivo, CXCL13-stimulated BMSCs were transplanted into the fracture sites of diabetic rats. At the 1st week, 2nd weeks, 4th week and 6th week after the operation, bone mineral density (BMD) and callus area measurement, ELISA detection, and HE staining were performed to evaluate the fracture healing. Results: Low BMD and less area of callus in diabetic rats showed that the recovery after fracture was worse in diabetic rats than in non-diabetic rats. Meanwhile, the expression of CXCL13 in serum was lower in diabetic rats than in non-diabetic rats. Overexpression of CXCL13 promoted the proliferation of BMSCs in vitro high glucose environment. After BMSCs transfected with CXCL13 being transplanted into the fracture sites of diabetic rats, it was found that the fracture healing was enhanced and ALP expression in serum became higher. HE staining results further verified the effects of transplantation of BMSCs transfected with CXCL13 on fracture healing in diabetic rats. Conclusion: These finding indicated that CXCL13 may play a critical role in the process of fracture healing, which could provide a deeper insight into molecular targets for the fracture healing in diabetic people.

scholarly journals The effects of Runx2 immobilization on poly (ɛ-caprolactone) on osteoblast differentiation of bone marrow stromal cells in vitro

Biomaterials ◽  
2010 ◽  
Vol 31 (12) ◽  
pp. 3231-3236 ◽  
Author(s):  
Ying Zhang ◽  
Xiaopei Deng ◽  
Erica L. Scheller ◽  
Tae-Geon Kwon ◽  
Joerg Lahann ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Stromal Cells ◽  
Bone Marrow Stromal Cells ◽  
Osteoblast Differentiation ◽  
Marrow Stromal Cells
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