Proliferation and Osteogenic Differentiation of Mesenchymal Stem Cells Induced by a Short Isoform of NELL-1

Neural epidermal growth factor-like (NEL)-like protein 1 (NELL-1) has been identified as an osteoinductive differentiation factor that promotes mesenchymal stem cell (MSC) osteogenic differentiation. In addition to full-length NELL-1, there are several NELL-1-related transcripts reported. We used rapid amplification of cDNA ends to recover potential cDNA of NELL-1 isoforms. A NELL-1 isoform with the N-terminal 240 amino acid (aa) residues truncated was identified. While full-length NELL-1 that contains 810 aa residues (NELL-1810) plays an important role in embryologic skeletal development, the N-terminal-truncated NELL-1 isoform (NELL-1570) was expressed postnatally. Similar to NELL-1810, NELL-1570 induced MSC osteogenic differentiation. In addition, NELL-1570 significantly stimulated MSC proliferation in multiple MSC-like populations such as murine C3H10T1/2 MSC cell line, mouse primary MSCs, and perivascular stem cells, which is a type of stem cells proposed as the perivascular origin of MSCs. In contrast, NELL-1810 demonstrated only limited stimulation of MSC proliferation. Similar to NELL-1810, NELL-1570 was found to be secreted from host cells. Both NELL-1570 expression lentiviral vector and column-purified recombinant protein NELL-1570 demonstrated almost identical effects in MSC proliferation and osteogenic differentiation, suggesting that NELL-1570 may function as a pro-osteogenic growth factor. In vivo, NELL-1570 induced significant calvarial defect regeneration accompanied by increased cell proliferation. Thus, NELL-1570 has the potential to be used for cell-based or hormone-based therapy of bone regeneration. Stem Cells 2015;33:904-915 © 2014 AlphaMed Press

HSC-Independent Definitive Hematopoiesis Persists Into Adult Life

It is widely believed that hematopoiesis after birth is established by hematopoietic stem cells (HSCs) in the bone marrow and that HSC-independent hematopoiesis is limited only to primitive erythro-myeloid cells and tissue-resident innate immune cells arising in the embryo. Here, surprisingly, we find that significant percentages of lymphocytes are not derived from HSCs, even in 1-year-old mice. Instead, multiple waves of hematopoiesis occur from embryonic day 7.5 (E7.5) to E11.5 endothelial cells, which simultaneously produce HSCs and lymphoid progenitors that constitute many layers of adaptive T and B lymphocytes in adult mice. Additionally, HSC lineage tracing reveals that the contribution of fetal liver HSCs to peritoneal B-1a cells is minimal and that the majority of B-1a cells are HSC independent. Our discovery of extensive HSC-independent lymphocytes in adult mice attests to the complex blood developmental dynamics spanning the embryo-to-adult transition and challenges the paradigm of HSCs exclusively underpinning the postnatal immune system

HSC-Independent Definitive Hematopoiesis Persists Into Adult Life

It is widely believed that hematopoiesis after birth is established by hematopoietic stem cells (HSCs) in the bone marrow and that HSC-independent hematopoiesis is limited only to primitive erythro-myeloid cells and tissue-resident innate immune cells arising in the embryo. Here, surprisingly, we find that significant percentages of lymphocytes are not derived from HSCs, even in 1-year-old mice. Instead, multiple waves of hematopoiesis occur from embryonic day 7.5 (E7.5) to E11.5 endothelial cells, which simultaneously produce HSCs and lymphoid progenitors that constitute many layers of adaptive T and B lymphocytes in adult mice. Additionally, HSC lineage tracing reveals that the contribution of fetal liver HSCs to peritoneal B-1a cells is minimal and that the majority of B-1a cells are HSC independent. Our discovery of extensive HSC-independent lymphocytes in adult mice attests to the complex blood developmental dynamics spanning the embryo-to-adult transition and challenges the paradigm of HSCs exclusively underpinning the postnatal immune system

Genome-Wide Mapping of DNA Methylation in Chicken

Cytosine DNA methylation is an important epigenetic modification termed as the fifth base that functions in diverse processes. Till now, the genome-wide DNA methylation maps of many organisms has been reported, such as human, Arabidopsis, rice and silkworm, but the methylation pattern of bird remains rarely studied. Here we show the genome-wide DNA methylation map of bird, using the chicken as a model organism and an immunocapturing approach followed by high-throughput sequencing. In both of the red jungle fowl and the avian broiler, DNA methylation was described separately for the liver and muscle tissue. Generally, chicken displays analogous methylation pattern with that of animals and plants. DNA methylation is enriched in the gene body regions and the repetitive sequences, and depleted in the transcription start site (TSS) and the transcription termination site (TTS). Most of the CpG islands in the chicken genome are kept in unmethylated state. Promoter methylation is negatively correlated with the gene expression level, indicating its suppressive role in regulating gene transcription. This work contributes to our understanding of epigenetics in birds

A Hepatic Protein, Fetuin-A, Occupies a Protective Role in Lethal Systemic Inflammation

A liver-derived protein, fetuin-A, was first purified from calf fetal serum in 1944, but its potential role in lethal systemic inflammation was previously unknown. This study aims to delineate the molecular mechanisms underlying the regulation of hepatic fetuin-A expression during lethal systemic inflammation (LSI), and investigated whether alterations of fetuin-A levels affect animal survival, and influence systemic accumulation of a late mediator, HMGB1.LSI was induced by endotoxemia or cecal ligation and puncture (CLP) in fetuin-A knock-out or wild-type mice, and animal survival rates were compared. Murine peritoneal macrophages were challenged with exogenous (endotoxin) or endogenous (IFN-γ) stimuli in the absence or presence of fetuin-A, and HMGB1 expression and release was assessed. Circulating fetuin-A levels were decreased in a time-dependent manner, starting between 26 h, reaching a nadir around 24-48 h, and returning towards base-line approximately 72 h post onset of endotoxemia or sepsis. These dynamic changes were mirrored by an early cytokine IFN-γ-mediated inhibition (up to 50-70%) of hepatic fetuin-A expression. Disruption of fetuin-A expression rendered animals more susceptible to LSI, whereas supplementation of fetuin-A (20-100 mg/kg) dose-dependently increased animal survival rates. The protection was associated with a significant reduction in systemic HMGB1 accumulation in vivo, and parallel inhibition of IFN-γ- or LPS-induced HMGB1 release in vitro.These experimental data suggest that fetuin-A is protective against lethal systemic inflammation partly by inhibiting active HMGB1 release

Milk fat globule epidermal growth factor-factor 8-derived peptide attenuates organ injury and improves survival in sepsis

INTRODUCTION: Sepsis involves overwhelming inflammatory responses with subsequent immune-suppression that can lead to multiple organ dysfunction and ultimately death. Milk fat globule epidermal growth factor-factor 8 (MFG-E8) is a secretory protein found to have multiple biological activities against autoimmune and inflammatory diseases. MFG-E8 contains an Arg-Gly-Asp (RGD) motif involved in cell-cell and cell-matrix interactions. In sepsis, excessive neutrophils migration through endothelial cells and matrix to sites of inflammation results in organ damage. We hypothesized that MFG-E8-derived short peptides (MSP) flanking its RGD motif could provide protection against organ injury in sepsis. METHODS: The differentiated human neutrophil-like HL-60 cells (dHL60) were incubated with a series of peptides flanking the RGD motif of human MFG-E8 for a cell adhesion assay to fibronectin or human pulmonary artery endothelial cells (PAECs). For the induction of sepsis, male C57BL/6 mice (20–25 g) were subjected to cecal ligation and puncture (CLP). Peptide MSP68 (1 mg/kg body weight) or normal saline (vehicle) was injected intravenously at 2 h after CLP. Blood and tissue samples were collected at 20 h after CLP for various measurements. RESULTS: After screening, peptide MSP68 (VRGDV) had the highest inhibition of dHL-60 cell adhesion to fibronectin by 55.8 % and to PAEC by 67.7 %. MSP68 treatment significantly decreased plasma levels of organ injury marker AST by 37.1 % and the proinflammatory cytokines IL-6 and TNF-α by 61.9 % and 22.1 %, respectively after CLP. MSP68 improved the integrity of microscopic architectures, decreased IL-6 levels in the lungs by 85.1 %, and reduced apoptosis. MSP68 treatment also significantly reduced the total number of neutrophil infiltration by 61.9 % and 48.3 % as well as MPO activity by 40.8 % and 47.3 % in the lungs and liver, respectively, after CLP. Moreover, the number of bacteria translocated to mesenteric lymph nodes was decreased by 57 % with MSP68 treatment. Finally, the 10-day survival rate was increased from 26 % in the vehicle group to 58 % in the MSP68-treated group. CONCLUSIONS: MSP68 effectively inhibits excessive neutrophils infiltrating to organs, leading to moderate attenuation of organ injury and significantly improved survival in septic mice. Thus, MSP68 may be a potential therapeutic agent for treating sepsis