搜索结果: 'methocult media formulations for mouse hematopoietic cells serum containing'

In contrast to mammals,adult zebrafish achieve complete heart regeneration via proliferation of cardiomyocytes. Surprisingly,we found that regenerating cardiomyocytes experience DNA replication stress,which represents one reason for declining tissue regeneration during aging in mammals. Pharmacological inhibition of ATM and ATR kinases revealed that DNA damage response signaling is essential for zebrafish heart regeneration. Manipulation of Bone Morphogenetic Protein (BMP)-Smad signaling using transgenics and mutants showed that BMP signaling alleviates cardiomyocyte replication stress. BMP signaling also rescues neonatal mouse cardiomyocytes,human fibroblasts and human hematopoietic stem and progenitor cells (HSPCs) from replication stress. DNA fiber spreading assays indicate that BMP signaling facilitates re-start of replication forks after replication stress-induced stalling. Our results identify the ability to overcome replication stress as key factor for the elevated zebrafish heart regeneration capacity and reveal a conserved role for BMP signaling in promotion of stress-free DNA replication. Subject terms: Cardiac regeneration,DNA damage and repair,Ageing View Publication

Transforming growth factor-beta1 (TGF-beta1) is a pleiotropic cytokine with major in vitro effects on hematopoietic stem cells (HSCs) and lymphocyte development. Little is known about hematopoiesis from mice with constitutive TGF-beta1 inactivation largely because of important embryonic lethality and development of a lethal inflammatory disorder in TGF-beta1(-/-) pups,making these studies difficult. Here,we show that no sign of the inflammatory disorder was detectable in 8- to 10-day-old TGF-beta1(-/-) neonates as judged by both the number of T-activated and T-regulator cells in secondary lymphoid organs and the level of inflammatory cytokines in sera. After T-cell depletion,the inflammatory disease was not transplantable in recipient mice. Bone marrow cells from 8- to 10-day-old TGF-beta1(-/-) neonates showed strikingly impaired short- and long-term reconstitutive activity associated with a parallel decreased in vivo homing capacity of lineage negative (Lin(-)) cells. In addition an in vitro-reduced survival of immature progenitors (Lin(-) Kit(+) Sca(+)) was observed. Similar defects were found in liver cells from TGF-beta1(-/-) embryos on day 14 after vaginal plug. These data indicate that TGF-beta1 is a critical regulator for in vivo homeostasis of the HSCs,especially for their homing potential. View Publication

Cystic Fibrosis (CF) is a common genetic disease in the United States,resulting from mutations in the Cystic Fibrosis transmembrane conductance regulator (cftr) gene. CFTR modulators,particularly Elexacaftor/Tezacaftor/Ivacaftor (ETI),have significantly improved clinical outcomes for patients with CF. However,many CFTR mutations are not eligible for CFTR modulator therapy due to their rarity. In this study,we report that a patient carrying rare complex CFTR mutations,c.1680-877G>T and c.3067_3072delATAGTG,showed positive clinical outcomes after ETI treatment. We demonstrate that ETI was able to increase the expression of CFTR harboring c.3067_3072delATAGTG in a heterologous system. Importantly,patient-derived nasal epithelial cells in an air–liquid interface (ALI) culture showed improved CFTR function following ETI treatment. These findings supported the initiation of ETI with the patient. Retrospective studies have suggested that the patient has shown small but steady improvement over the past two years in several clinical metrics,including lung function,body mass index (BMI),and sweat chloride levels. Our studies suggest that ETI could be beneficial for patients carrying c.3067_3072delATAGTG. View Publication

Wnt/β-catenin signalling has a variety of roles in regulating stem cell fates. Its specific role in mouse epiblast stem cell self-renewal,however,remains poorly understood. Here we show that Wnt/β-catenin functions in both self-renewal and differentiation in mouse epiblast stem cells. Stabilization and nuclear translocation of β-catenin and its subsequent binding to T-cell factors induces differentiation. Conversely,retention of stabilized β-catenin in the cytoplasm maintains self-renewal. Cytoplasmic retention of β-catenin is effected by stabilization of Axin2,a downstream target of β-catenin,or by genetic modifications to β-catenin that prevent its nuclear translocation. We also find that human embryonic stem cell and mouse epiblast stem cell fates are regulated by β-catenin through similar mechanisms. Our results elucidate a new role for β-catenin in stem cell self-renewal that is independent of its transcriptional activity and will have broad implications in understanding the molecular regulation of stem cell fate. View Publication

Estrogens as well as some antiestrogens have been shown to prevent bone loss in postmenopausal women. These compounds seem to inhibit bone resorption,but their anabolic effects have been less explored. In this study,bone marrow cultures were used to compare the effect of 17beta-estradiol (E2),and two triphenylethylene derivatives,tamoxifen (TAM),and FC1271a,and a benzothiophene derivative raloxifene (RAL) on differentiation of osteoblasts. All enhanced osteoblastic differentiation of 21-day cultures as indicated by increased mineralization and bone nodule formation. All,except RAL,stimulated cell proliferation during the first 6 days of the culture. However,in the presence of RAL the content of total protein was increased in 13-day cultures. SDS-PAGE and autoradiography of [14C]-proline labeled proteins revealed elevated level of the newly synthesized collagen type I. The pure antiestrogen ICI 182,780 abolished the increase of the specific activity of alkaline phosphatase by E2,TAM,and FC1271a but not the effect of RAL on protein synthesis. Our results show that E2 as well as TAM,FC1271a,and RAL stimulate bone formation in vitro but the mechanism of the anabolic action of RAL in bone clearly differs from that of E2,TAM,and FC1271a. View Publication

Known molecular determinants of developmental plasticity are mainly transcription factors,while the extrinsic regulation of this process has been largely unexplored. Here we identify Cripto as one of the earliest epiblast markers and a key extracellular determinant of the naive and primed pluripotent states. We demonstrate that Cripto sustains mouse embryonic stem cell (ESC) self-renewal by modulating Wnt/β-catenin,whereas it maintains mouse epiblast stem cell (EpiSC) and human ESC pluripotency through Nodal/Smad2. Moreover,we provide unprecedented evidence that Cripto controls the metabolic reprogramming in ESCs to EpiSC transition. Remarkably,Cripto deficiency attenuates ESC lineage restriction in vitro and in vivo,and permits ESC transdifferentiation into trophectoderm lineage,suggesting that Cripto has earlier functions than previously recognized. All together,our studies provide novel insights into the current model of mammalian pluripotency and contribute to the understanding of the extrinsic regulation of the first cell lineage decision in the embryo. View Publication

Spinal muscular atrophy (SMA),a motor neuron disease (MND) and one of the most common genetic causes of infant mortality,currently has no cure. Patients with SMA exhibit muscle weakness and hypotonia. Stem cell transplantation is a potential therapeutic strategy for SMA and other MNDs. In this study,we isolated spinal cord neural stem cells (NSCs) from mice expressing green fluorescent protein only in motor neurons and assessed their therapeutic effects on the phenotype of SMA mice. Intrathecally grafted NSCs migrated into the parenchyma and generated a small proportion of motor neurons. Treated SMA mice exhibited improved neuromuscular function,increased life span,and improved motor unit pathology. Global gene expression analysis of laser-capture-microdissected motor neurons from treated mice showed that the major effect of NSC transplantation was modification of the SMA phenotype toward the wild-type pattern,including changes in RNA metabolism proteins,cell cycle proteins,and actin-binding proteins. NSC transplantation positively affected the SMA disease phenotype,indicating that transplantation of NSCs may be a possible treatment for SMA. View Publication

Oncogenic Kit mutations are found in somatic gastrointestinal (GI) stromal tumors (GISTs) and mastocytosis. A mouse model for the study of constitutive activation of Kit in oncogenesis has been produced by a knock-in strategy introducing a Kit exon 11-activating mutation into the mouse genome based on a mutation found in a case of human familial GIST syndrome. Heterozygous mutant KitV558Delta/+ mice develop symptoms of disease and eventually die from pathology in the GI tract. Patchy hyperplasia of Kit-positive cells is evident within the myenteric plexus of the entire GI tract. Neoplastic lesions indistinguishable from human GISTs were observed in the cecum of the mutant mice with high penetrance. In addition,mast cell numbers in the dorsal skin were increased. Therefore KitV558Delta/+ mice reproduce human familial GISTs,and they may be used as a model for the study of the role and mechanisms of Kit in neoplasia. Importantly,these results demonstrate that constitutive Kit signaling is critical and sufficient for induction of GIST and hyperplasia of interstitial cells of Cajal. View Publication

Ceramides are bioactive sphingolipids that have physiological effects on inflammation,apoptosis,and mitochondrial dysfunction. They may play a critical role in the harm of ischemia/reperfusion (IR). Ceramides and IR injury are not well-studied,and there is a lack of human data. Current studies aimed to investigate the role of ceramide buildup in cardiomyocytes (CMs) death using CMs derived from human induced pluripotent stem cell (hiPSC) as a model for simulating IR injury in vitro. In our model,serum- and glucose-free media was used to expose hiPSC-derived CMs to hypoxia (3% O 2 ) for 6 h (hrs),followed by reoxygenation (20% O 2 ) for 16 h. In contrast to normoxia (control) or hypoxia (ischemia),our data showed that following IR,there was an increase in the formation of mitochondrial superoxide and the mRNA levels of genes regulating ceramide synthesis,such as CerS2 and CerS4 in CMs. Further,there was a considerable rise in the levels of total ceramide,long-chain (C16:0,C18:0,and C18:1),and very long-chain (C22:0 and C24:1) ceramide species in CMs following reperfusion in comparison to control or ischemic CMs. Interestingly,compared to CMs exposed to IR without inhibitor,our data showed that inhibition of ceramide formation with fumonisin B1 (FB1) significantly lowered ceramide levels,reduced apoptosis,improved mitochondrial function,and enhanced survival of CMs exposed to IR. Furthermore,we used a transgenic mouse model,in which the CerS2 gene was overexpressed in the CMs of α-MHC-CerS2 mice,to validate the basic idea that ceramide contributes to heart disease in vivo. Our results showed that the heart tissues of α-MHC-CerS2 mice had significant levels of long-chain and very long-chain ceramides,which causes increased apoptosis,proinflammatory cytokines,interstitial inflammatory cell infiltration,and collagen deposition. Results from both in vitro and in vivo experiments show that ceramides have a significant role in either mediating or inducing damage to CMs. Additionally,in vitro findings show that ceramide reduction improves the outcome of IR injury by lowering intracellular Ca 2+ [Ca 2+ ] i concentration and improves mitochondrial function changes during IR. The online version contains supplementary material available at 10.1186/s13287-025-04340-3. View Publication

The aorta-gonad-mesonephros region plays an important role in hematopoietic stem cell (HSC) development during mouse embryogenesis. The vascular endothelial cadherin�?� CD45�?� (VE-cad�?�CD45�?�) population contains the major type of immature pre-HSCs capable of developing into long-term repopulating definitive HSCs. In this study,we developed a new coaggregation culture system,which supports maturation of a novel population of CD45-negative (VE-cad�?�CD45�?�CD41�?�) pre-HSCs into definitive HSCs. The appearance of these pre-HSCs precedes development of the VE-cad�?�CD45�?� pre-HSCs (termed here type I and type II pre-HSCs,respectively),thus establishing a hierarchical directionality in the developing HSC lineage. By labeling the luminal surface of the dorsal aorta,we show that both type I and type II pre-HSCs are distributed broadly within the endothelial and subendothelial aortic layers,in contrast to mature definitive HSCs which localize to the aortic endothelial layer. In agreement with expression of CD41 in pre-HSCs,in vivo CD41-Cre-mediated genetic tagging occurs in embryonic pre-HSCs and persists in all lymphomyeloid lineages of the adult animal. View Publication