12 research outputs found
Isolation and in vitro characterization of murine young-adult long bone skeletal progenitors
No full textSkeletal stem and progenitor cells (SSPCs) constitute a reservoir of bone-forming cells necessary for bone development, modeling and remodeling, as well as for fracture healing. Recent advances in tools to identify and isolate SSPCs have revealed that cells with multipotent properties are present not only in neonatal bone, but also in adult bone marrow and periosteum. The long bone metaphysis and endosteum have been proposed as an additional SSPC niche, although in vitro approaches to study their cellular and molecular characteristics are still limited. Here, we describe a comprehensive procedure to isolate and culture SSPCs derived from the metaphysis and endosteum of young-adult mice. Based on flow cytometry analysis of known SSPC markers, we found the presence of putative multipotent SSPCs, similar to neonatal bone tissue. In vitro, metaphyseal/endosteal SSPCs possess self-renewing capacity, and their multipotency is underscored by the ability to differentiate into the osteogenic and adipogenic lineage, while chondrogenic potential is limited. Expansion of metaphyseal/endosteal SSPCs under low oxygen conditions increases their proliferation capacity, while progenitor properties are maintained, likely reflecting their hypoxic niche in vivo. Collectively, we propose a validated isolation and culture protocol to study metaphyseal/endosteal SSPC biology in vitro
Just air? Spatial injustices and the politicisation of air pollution
No full textsponsorship: The author(s) disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: The work of the first author was supported by the Research Foundation Flanders (FWO). (Research Foundation Flanders (FWO))status: Publishe
Image_1_Isolation and in vitro characterization of murine young-adult long bone skeletal progenitors.tif
No full textSkeletal stem and progenitor cells (SSPCs) constitute a reservoir of bone-forming cells necessary for bone development, modeling and remodeling, as well as for fracture healing. Recent advances in tools to identify and isolate SSPCs have revealed that cells with multipotent properties are present not only in neonatal bone, but also in adult bone marrow and periosteum. The long bone metaphysis and endosteum have been proposed as an additional SSPC niche, although in vitro approaches to study their cellular and molecular characteristics are still limited. Here, we describe a comprehensive procedure to isolate and culture SSPCs derived from the metaphysis and endosteum of young-adult mice. Based on flow cytometry analysis of known SSPC markers, we found the presence of putative multipotent SSPCs, similar to neonatal bone tissue. In vitro, metaphyseal/endosteal SSPCs possess self-renewing capacity, and their multipotency is underscored by the ability to differentiate into the osteogenic and adipogenic lineage, while chondrogenic potential is limited. Expansion of metaphyseal/endosteal SSPCs under low oxygen conditions increases their proliferation capacity, while progenitor properties are maintained, likely reflecting their hypoxic niche in vivo. Collectively, we propose a validated isolation and culture protocol to study metaphyseal/endosteal SSPC biology in vitro.</p
Image_2_Isolation and in vitro characterization of murine young-adult long bone skeletal progenitors.tif
No full textSkeletal stem and progenitor cells (SSPCs) constitute a reservoir of bone-forming cells necessary for bone development, modeling and remodeling, as well as for fracture healing. Recent advances in tools to identify and isolate SSPCs have revealed that cells with multipotent properties are present not only in neonatal bone, but also in adult bone marrow and periosteum. The long bone metaphysis and endosteum have been proposed as an additional SSPC niche, although in vitro approaches to study their cellular and molecular characteristics are still limited. Here, we describe a comprehensive procedure to isolate and culture SSPCs derived from the metaphysis and endosteum of young-adult mice. Based on flow cytometry analysis of known SSPC markers, we found the presence of putative multipotent SSPCs, similar to neonatal bone tissue. In vitro, metaphyseal/endosteal SSPCs possess self-renewing capacity, and their multipotency is underscored by the ability to differentiate into the osteogenic and adipogenic lineage, while chondrogenic potential is limited. Expansion of metaphyseal/endosteal SSPCs under low oxygen conditions increases their proliferation capacity, while progenitor properties are maintained, likely reflecting their hypoxic niche in vivo. Collectively, we propose a validated isolation and culture protocol to study metaphyseal/endosteal SSPC biology in vitro.</p
Inhibition of the Oxygen Sensor PHD2 Enhances Tissue-Engineered Endochondral Bone Formation
No full textTissue engineering holds great promise for bone regenerative medicine, but clinical translation remains challenging. An important factor is the low cell survival after implantation, primarily caused by the lack of functional vasculature at the bone defect. Interestingly, bone development and repair initiate predominantly via an avascular cartilage template, indicating that chondrocytes are adapted to limited vascularization. Given these advantageous properties of chondrocytes, we questioned whether tissue-engineered cartilage intermediates implanted ectopically in mice are able to form bone, even when the volume size increases. Here, we show that endochondral ossification proceeds efficiently when implant size is limited (≤30 mm3 ), but chondrogenesis and matrix synthesis are impaired in the center of larger implants, leading to a fibrotic core. Increasing the level of angiogenic growth factors does not improve this outcome, because this strategy enhances peripheral bone formation, but disrupts the conversion of cartilage into bone in the center, resulting in a fibrotic core, even in small implants. On the other hand, activation of hypoxia signaling in cells before implantation stimulates chondrogenesis and matrix production, which culminates in enhanced bone formation throughout the entire implant. Together, our results show that induction of angiogenesis alone may lead to adverse effects during endochondral bone repair, whereas activation of hypoxia signaling represents a superior therapeutic strategy to improve endochondral bone regeneration in large tissue-engineered implants. © 2018 American Society for Bone and Mineral Research.sponsorship: The TCS SP8-MP system used for intravital imaging at the VIB-KU Leuven Centre for Cancer Biology was funded by a structural grant from the Hercules Foundation (AKUL/11/033). GC acknowledges funding support from the Fund for Scientific Research-Flanders (FWO: G.096414, G0A4216N). PJS is a fellow from the Agency for Innovation by Science and Technology in Flanders (IWT). SS is financed by a postdoctoral grant of the Research Foundation-Flanders (FWO/12H5917N). NvG is funded by BOF-KU Leuven GOA project 3M120209. This work is part of Prometheus, the KU Leuven R&D Division of Skeletal Tissue Engineering. We thank Karen Moermans, Ingrid Stockmans, and Shauni Loopmans for excellent technical assistance. The collagen type I-dsRed mouse and pJ320 plasmid were kindly provided by Sunichi Murakami. pWPXLd was a gift from Didier Trono (Addgene #12258). (Hercules Foundation|AKUL/11/033, Fund for Scientific Research-Flanders|FWO: G.096414, Fund for Scientific Research-Flanders|G0A4216N, Research Foundation-Flanders|FWO/12H5917N, BOF-KU Leuven GOA project|3M120209)status: Publishe
Adaptations in hepatic glucose metabolism after chronic social defeat stress in mice
No full textChronic stress has been shown to induce hyperglycemia in both peripheral blood and the brain, yet the detailed mechanisms of glucose metabolism under stress remain unclear. Utilizing 13C6-labeled glucose to trace metabolic pathways, our study investigated the impact of stress by chronic social defeat (CSD) on glucose metabolites in the liver and brain one week post-stress. We observed a reduction in 13C6-enrichment of glucose metabolites in the liver, contrasting with unchanged levels in the brain. Notably, hepatic glycogen levels were reduced while lactate concentrations were elevated, suggesting lactate as an alternative energy source during stress. Long-term effects were also examined, revealing normalized blood glucose levels and restored glycogen stores in the liver three weeks post-CSD, despite sustained increases in food intake. This normalization is hypothesized to result from diminished glucagon levels leading to reduced glycogen phosphorylase activity. Our findings highlight a temporal shift in glucose metabolism, with hyperglycemia and glycogen depletion in the liver early after CSD, followed by a later phase of metabolic stabilization. These results underscore the liver's critical role in adapting to CSD and provide insights into the metabolic adjustments that maintain glucose homeostasis under prolonged stress conditions
DataSheet_1_The Combination of the CDK4/6 Inhibitor, Palbociclib, With the Vitamin D3 Analog, Inecalcitol, Has Potent In Vitro and In Vivo Anticancer Effects in Hormone-Sensitive Breast Cancer, But Has a More Limited Effect in Triple-Negative Breast Cancer.docx
No full textActive vitamin D3, 1,25-dihydroxyvitamin D3 [1,25(OH)2D3], and its synthetically derived analogs possess potent anticancer properties. In breast cancer (BC) cells, 1,25(OH)2D3 blocks cell proliferation and induces apoptosis through different cell-type specific mechanisms. In this study, we evaluated if the combination of the potent vitamin D3 analog, inecalcitol, with a selective CDK4/6 inhibitor, palbociclib, enhanced the antiproliferative effects of both single compounds in hormone-sensitive (ER+) BC, for which palbociclib treatment is already approved, but also in triple-negative BC (TNBC). Inecalcitol and palbociclib combination treatment decreased cell proliferation in both ER+ (T47D-MCF7) and TNBC (BT20-HCC1143-Hs578T) cells, with a more pronounced antiproliferative effect in the former. In ER+ BC cells, the combination therapy downregulated cell cycle regulatory proteins (p)-Rb and (p)-CDK2 and blocked G1-S phase transition of the cell cycle. Combination treatment upregulated p-mTOR and p-4E-BP1 protein expression in MCF7 cells, whereas it suppressed expression of these proteins in BT20 cells. Cell survival was decreased after inecalcitol treatment either alone or combined in MCF7 cells. Interestingly, the combination therapy upregulated mitochondrial ROS and mitotracker staining in both cell lines. Furthermore, in vivo validation in a MCF7 cell line-derived xenograft mouse model decreased tumor growth and cell cycle progression after combination therapy, but not in a TNBC BT20 cell line-derived xenograft model. In conclusion, we show that addition of a potent vitamin D3 analog to selective CDK4/6 inhibitor treatment results in increased antiproliferative effects in ER+ BC both in vitro and in vivo.</p
DataSheet_2_The Combination of the CDK4/6 Inhibitor, Palbociclib, With the Vitamin D3 Analog, Inecalcitol, Has Potent In Vitro and In Vivo Anticancer Effects in Hormone-Sensitive Breast Cancer, But Has a More Limited Effect in Triple-Negative Breast Cancer.docx
No full textActive vitamin D3, 1,25-dihydroxyvitamin D3 [1,25(OH)2D3], and its synthetically derived analogs possess potent anticancer properties. In breast cancer (BC) cells, 1,25(OH)2D3 blocks cell proliferation and induces apoptosis through different cell-type specific mechanisms. In this study, we evaluated if the combination of the potent vitamin D3 analog, inecalcitol, with a selective CDK4/6 inhibitor, palbociclib, enhanced the antiproliferative effects of both single compounds in hormone-sensitive (ER+) BC, for which palbociclib treatment is already approved, but also in triple-negative BC (TNBC). Inecalcitol and palbociclib combination treatment decreased cell proliferation in both ER+ (T47D-MCF7) and TNBC (BT20-HCC1143-Hs578T) cells, with a more pronounced antiproliferative effect in the former. In ER+ BC cells, the combination therapy downregulated cell cycle regulatory proteins (p)-Rb and (p)-CDK2 and blocked G1-S phase transition of the cell cycle. Combination treatment upregulated p-mTOR and p-4E-BP1 protein expression in MCF7 cells, whereas it suppressed expression of these proteins in BT20 cells. Cell survival was decreased after inecalcitol treatment either alone or combined in MCF7 cells. Interestingly, the combination therapy upregulated mitochondrial ROS and mitotracker staining in both cell lines. Furthermore, in vivo validation in a MCF7 cell line-derived xenograft mouse model decreased tumor growth and cell cycle progression after combination therapy, but not in a TNBC BT20 cell line-derived xenograft model. In conclusion, we show that addition of a potent vitamin D3 analog to selective CDK4/6 inhibitor treatment results in increased antiproliferative effects in ER+ BC both in vitro and in vivo.</p
Skeletal progenitors preserve proliferation and self-renewal upon inhibition of mitochondrial respiration by rerouting the TCA cycle
No full textA functional electron transport chain (ETC) is crucial for supporting bioenergetics and biosynthesis. Accordingly, ETC inhibition decreases proliferation in cancer cells but does not seem to impair stem cell proliferation. However, it remains unclear how stem cells metabolically adapt. In this study, we show that pharmacological inhibition of complex III of the ETC in skeletal stem and progenitor cells induces glycolysis side pathways and reroutes the tricarboxylic acid (TCA) cycle to regenerate NAD(+) and preserve cell proliferation. These metabolic changes also culminate in increased succinate and 2-hydroxyglutarate levels that inhibit Ten-eleven translocation (TET) DNA demethylase activity, thereby preserving self-renewal and multilineage potential. Mechanistically, mitochondrial malate dehydrogenase and reverse succinate dehydrogenase activity proved to be essential for the metabolic rewiring in response to ETC inhibition. Together, these data show that the metabolic plasticity of skeletal stem and progenitor cells allows them to bypass ETC blockade and preserve their self-renewal
Adaptations in hepatic glucose metabolism after chronic social defeat stress in mice
No full textAbstract Chronic stress has been shown to induce hyperglycemia in both peripheral blood and the brain, yet the detailed mechanisms of glucose metabolism under stress remain unclear. Utilizing 13C6-labeled glucose to trace metabolic pathways, our study investigated the impact of stress by chronic social defeat (CSD) on glucose metabolites in the liver and brain one week post-stress. We observed a reduction in 13C6-enrichment of glucose metabolites in the liver, contrasting with unchanged levels in the brain. Notably, hepatic glycogen levels were reduced while lactate concentrations were elevated, suggesting lactate as an alternative energy source during stress. Long-term effects were also examined, revealing normalized blood glucose levels and restored glycogen stores in the liver three weeks post-CSD, despite sustained increases in food intake. This normalization is hypothesized to result from diminished glucagon levels leading to reduced glycogen phosphorylase activity. Our findings highlight a temporal shift in glucose metabolism, with hyperglycemia and glycogen depletion in the liver early after CSD, followed by a later phase of metabolic stabilization. These results underscore the liver’s critical role in adapting to CSD and provide insights into the metabolic adjustments that maintain glucose homeostasis under prolonged stress conditions
