Growth Plate Borderline Chondrocytes Behave as Transient Mesenchymal Precursor Cells

The growth plate provides a substantial source of mesenchymal cells in the endosteal marrow space during endochondral ossification. The current model postulates that a group of chondrocytes in the hypertrophic zone can escape from apoptosis and transform into cells that eventually become osteoblasts in an area beneath the growth plate. The growth plate is composed of cells with various morphologies; particularly at the periphery of the growth plate immediately adjacent to the perichondrium are “borderline” chondrocytes, which align perpendicularly to other chondrocytes. However, in vivo cell fates of these special chondrocytes have not been revealed. Here we show that borderline chondrocytes in growth plates behave as transient mesenchymal precursor cells for osteoblasts and marrow stromal cells. A single‐cell RNA‐seq analysis revealed subpopulations of Col2a1‐creER‐marked neonatal chondrocytes and their cell type–specific markers. A tamoxifen pulse to Pthrp‐creER mice in the neonatal stage (before the resting zone was formed) preferentially marked borderline chondrocytes. Following the chase, these cells marched into the nascent marrow space, expanded in the metaphyseal marrow, and became Col(2.3 kb)‐GFP+ osteoblasts and Cxcl12‐GFPhigh reticular stromal “CAR” cells. Interestingly, these borderline chondrocyte‐derived marrow cells were short‐lived, as they were significantly reduced during adulthood. These findings demonstrate based on in vivo lineage‐tracing experiments that borderline chondrocytes in the peripheral growth plate are a particularly important route for producing osteoblasts and marrow stromal cells in growing murine endochondral bones. A special microenvironment neighboring the osteogenic perichondrium might endow these chondrocytes with an enhanced potential to differentiate into marrow mesenchymal cells. © 2019 American Society for Bone and Mineral Research.Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/151266/1/jbmr3719_am.pdfhttps://deepblue.lib.umich.edu/bitstream/2027.42/151266/2/jbmr3719-sup-0001-Suppl_Info_JBMR_021819.pdfhttps://deepblue.lib.umich.edu/bitstream/2027.42/151266/3/jbmr3719.pd

Augmentation of natural killer activity by neuraminidase treatment of lymphocytes from tumor-bearing mice.

Spleen cells from tumor-bearing mice showed decreased natural killer (NK) activity and decreased binding to target cells with progression of the tumor. Treatment of spleen cells from tumor-bearing mice with vibrio cholerae neuraminidase (VCN) increased the cytotoxicity to a level twice or more as high as that of untreated cells, but the same treatment of spleen cells from normal mice had no or little effect. On the other hand, neither in spleen cells from tumor-bearing mice nor in those from normal mice, the VCN treatment had no effect on their binding to M-HeLa cells. The suppression of NK activity by preincubation with serum from tumor-bearing mice or prostaglandin E2 was completely abolished by VCN treatment. The above results indicate that VCN treatment of lymphocytes might augment NK activity by an antagonistic effect against an immune suppressive factor.</p

A Subset of Chondrogenic Cells Provides Early Mesenchymal Progenitors in Growing Bones

The hallmark of endochondral bone development is the presence of cartilaginous templates, in which osteoblasts and stromal cells are generated to form mineralized matrix and support bone marrow hematopoiesis. However, the ultimate source of these mesenchymal cells and the relationship between bone progenitors in fetal life and those in later life are unknown. Fate-mapping studies revealed that cells expressing cre-recombinases driven by the collagen II (Col2) promoter/enhancer and their descendants contributed to, in addition to chondrocytes, early perichondrial precursors prior to Runx2 expression and, subsequently, to a majority of osteoblasts, Cxcl12 (chemokine (C-X-C motif) ligand 12)-abundant stromal cells and bone marrow stromal/mesenchymal progenitor cells in postnatal life. Lineage-tracing experiments using a tamoxifen-inducible creER system further revealed that early postnatal cells marked by Col2-creER, as well as Sox9-creER and aggrecan (Acan)-creER, progressively contributed to multiple mesenchymal lineages and continued to provide descendants for over a year. These cells are distinct from adult mesenchymal progenitors and thus provide opportunities for regulating the explosive growth that occurs uniquely in growing mammals

Activation of NK activity and auto-reactive cytotoxicity after hepatectomy.

Spleen cells serially sampled from normal mice following partial hepatectomy were tested for antibody-dependent cellular cytotoxicity (ADCC) and natural killer (NK) activity. There was a marked augmentation of these activities of spleen cells from the hepatectomized animals, compared to cells from controls with a simple laparotomy. The augmentation of ADCC in the hepatectomized mice was largely attributable to the activity of T lymphocytes. When cultured with interleukin-2 (IL-2), the spleen cells from hepatectomized mice exhibited cytotoxicity to syngeneic lymphoblasts, which was found to be effected by T cells.</p

Dissociation of natural killer activity and antibody-dependent cellular cytotoxicity in spleen cells of tumor bearing mice.

Antibody-dependent cellular cytotoxicity (ADCC) increased with the development of tumors in C3H/He mice bearing spontaneous breast cancer or the syngeneic hepatoma MH-134 and in C57BL/6 mice bearing the syngeneic Lewis lung carcinoma 3LL. This cytotoxicity decreased after treatment with guinea pig, monoclonal IgM anti-Thy 1.2 serum and complement to the non-cancer level thus indicating that the increased ADCC in mice with cancer seems mainly attributable to cells with the Thy 1 antigen. On the other hand, NK activity decreased greatly when mice had tumors. Treatment with monoclonal IgM anti-Thy 1.2 serum and complement showed no significant influence on the natural killer (NK) activity of spleen cells of mice bearing MH-134 cancer, but in the 3LL-bearing mice the activity decreased significantly.</p

Diabetes-Related Ankyrin Repeat Protein (DARP/Ankrd23) Modifies Glucose Homeostasis by Modulating AMPK Activity in Skeletal Muscle.

Skeletal muscle is the major site for glucose disposal, the impairment of which closely associates with the glucose intolerance in diabetic patients. Diabetes-related ankyrin repeat protein (DARP/Ankrd23) is a member of muscle ankyrin repeat proteins, whose expression is enhanced in the skeletal muscle under diabetic conditions; however, its role in energy metabolism remains poorly understood. Here we report a novel role of DARP in the regulation of glucose homeostasis through modulating AMP-activated protein kinase (AMPK) activity. DARP is highly preferentially expressed in skeletal muscle, and its expression was substantially upregulated during myotube differentiation of C2C12 myoblasts. Interestingly, DARP-/- mice demonstrated better glucose tolerance despite similar body weight, while their insulin sensitivity did not differ from that in wildtype mice. We found that phosphorylation of AMPK, which mediates insulin-independent glucose uptake, in skeletal muscle was significantly enhanced in DARP-/- mice compared to that in wildtype mice. Gene silencing of DARP in C2C12 myotubes enhanced AMPK phosphorylation, whereas overexpression of DARP in C2C12 myoblasts reduced it. Moreover, DARP-silencing increased glucose uptake and oxidation in myotubes, which was abrogated by the treatment with AICAR, an AMPK activator. Of note, improved glucose tolerance in DARP-/- mice was abolished when mice were treated with AICAR. Mechanistically, gene silencing of DARP enhanced protein expression of LKB1 that is a major upstream kinase for AMPK in myotubes in vitro and the skeletal muscle in vivo. Together with the altered expression under diabetic conditions, our data strongly suggest that DARP plays an important role in the regulation of glucose homeostasis under physiological and pathological conditions, and thus DARP is a new therapeutic target for the treatment of diabetes mellitus

Trimming of a Migrated Biliary Nitinol Stent Using Argon Plasma

Metallic stent migration is a well-known complication which cannot always be managed by removal or repositioning, especially in case of uncovered stent. We report a patient who developed obstructive jaundice due to migration of an expandable metallic stent (EMS) inserted in the lower bile duct. Trimming of the EMS using argon plasma was performed, with the power setting of 60 W and 2.0 l/min of argon flow. The distal part of the EMS was removed and mechanical cleaning using balloon catheter was performed for remnant EMS. Without additional stent insertion, jaundice was relieved in a few days. No complication was recognized during the procedure and no recurrence of jaundice in the rest of his life

The Unmixing Problem: A Guide to Applying Single‐Cell RNA Sequencing to Bone

Bone is composed of a complex mixture of many dynamic cell types. Flow cytometry and in vivo lineage tracing have offered early progress toward deconvoluting this heterogeneous mixture of cells into functionally well‐defined populations suitable for further studies. Single‐cell sequencing is poised as a key complementary technique to better understand the cellular basis of bone metabolism and development. However, single‐cell sequencing approaches still have important limitations, including transcriptional effects of cell isolation and sparse sampling of the transcriptome, that must be considered during experimental design and analysis to harness the power of this approach. Accounting for these limitations requires a deep knowledge of the tissue under study. Therefore, with the emergence of accessible tools for conducting and analyzing single‐cell RNA sequencing (scRNA‐seq) experiments, bone biologists will be ideal leaders in the application of scRNA‐seq to the skeleton. Here we provide an overview of the steps involved with a single‐cell sequencing analysis of bone, focusing on practical considerations needed for a successful study. © 2019 American Society for Bone and Mineral Research.Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/150567/1/jbmr3802_am.pdfhttps://deepblue.lib.umich.edu/bitstream/2027.42/150567/2/jbmr3802.pd

Clinicopathological Features and Surgical Outcomes of Small Bowel Metastasis from Renal Cell Carcinoma

Small bowel metastasis from renal cell carcinoma (RCC) is rare, and its clinicopathological characteristics are unclear; thus, we revisited the concept of this tumor and reviewed its diagnostic and treatment modalities. We filtered MEDLINE searches of articles published in English between 1950 and 2019, and identified 100 patients who had undergone treatment, including 1 patient from our clinic. We extracted patient characteristics, treatment, and prognostic data, resulting in clinicopathological data on 100 patients (83 men, 17 women). Mean age was 63 years (range, 16-86 years). Tumor sites were duodenum, jejunum, ileum, and multiple sites in 30, 37, 25, and 7 patients, respectively. The 1-, 3-, and 5-year overall survival rates after diagnosis were 53.0%, 36.0%, and 36.0%. Curative resection patients showed 62.1% 5-year survival after surgery, vs. 27.5% in noncurative surgical management cases. Good prognoses can be expected if these tumors are identified early for complete removal. Surgery is the only curative option. To determine the best management strategy and improve prognostic accuracy, we continue to collect and analyze epidemiological and pathological data. Although this condition is rare, surgery should be considered if curative resection is expected. Prognosis after curative resection is not poor, but recurrence is not unlikely

A three‐dimensional analysis of primary failure of eruption in humans and mice

ObjectivesPrimary failure of eruption (PFE) is a genetic disorder exhibiting the cessation of tooth eruption. Loss‐of‐function mutations in parathyroid hormone (PTH)/parathyroid hormone‐related peptide (PTHrP) receptor (PTH/PTHrP receptor, PPR) were reported as the underlying cause of this disorder in humans. We showed in a PFE mouse model that PTHrP‐PPR signaling is responsible for normal dental follicle cell differentiation and tooth eruption. However, the mechanism underlying the eruption defect in PFE remains undefined. In this descriptive study, we aim to chronologically observe tooth eruption and root formation of mouse PFE molars through 3D microCT analyses.Setting and Sample PopulationTwo individuals with PFE were recruited at Showa University. A mouse PFE model was generated by deleting PPR specifically in PTHrP‐expressing dental follicle and divided into three groups, PPRfl/fl;R26RtdTomato/+(Control), PTHrP‐creER;PPRfl/+;R26RtdTomato/+(cHet), and PTHrP‐creER;PRRfl/fl;R26RtdTomato/+(cKO).Materials and MethodsImages from human PFE subjects were acquired by CBCT. All groups of mouse samples were studied at postnatal days 14, 25, 91, and 182 after a tamoxifen pulse at P3, and superimposition of 3D microCT images among three groups was rendered.ResultsMouse and human PFE molars exhibited a similar presentation in the 3D CT analyses. The quantitative analysis in mice demonstrated a statistically significant decrease in the eruption height of cKO first and second molars compared to other groups after postnatal day 25. Additionally, cKO molars demonstrated significantly shortened roots with dilacerations associated with the reduced interradicular bone height.ConclusionsMouse PFE molars erupt at a much slower rate compared to normal molars, associated with shortened and dilacerated roots and defective interradicular bones.Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/154523/1/odi13249_am.pdfhttps://deepblue.lib.umich.edu/bitstream/2027.42/154523/2/odi13249.pd