Development Of Precursor Cells Research Articles

In vitro and in vivo evaluation of osteogenesis and antibacterial activity of Mg[sbnd]Ga alloys

MgGa alloys are considered highly potential biodegradable materials, owing to its good mechanical properties and appropriate corrosion resistance. However, it is still far from application due to the lack of biological evaluation. In the present study, biocompatibility, osteogenesis and antibacterial activity of extruded Mg–xGa (x = 1 and 5 wt%) alloys were investigated by in vitro cell culture experiments and in vivo implantation. The cell adhesion and proliferation of osteoblast precursor cells (MC3T3-E1) showed the excellent cytocompatibility of Mg–1Ga and poor cytocompatibility of Mg–5Ga. The osteogenic activity was evaluated and revealed that Ga3+ in the Mg–1Ga extract had the ability to enhance osteogenic differentiation through the facilitation of its early stages. In vivo studies in a rat femoral condyle model revealed that both Mg–1Ga and Mg–5Ga significantly promoted new bone formation without causing any adverse effects. Mg–5Ga exhibited a much higher corrosion rate in vivo than Mg–1Ga. Its osteogenic activity was better due to the rapid release of Mg2+ and Ga3+, but this caused premature structural integrity loss. Mg–1Ga and Mg–5Ga released Ga3+ to inhibit E. coli and S. aureus, with antibacterial rate increasing with Ga content. Our studies demonstrate that Mg–Ga alloys have the potential to be used as osteogenic and antibacterial implant materials. Statement of significanceThis study evaluates the biocompatibility, osteogenesis, and antibacterial activity of Mg–Ga alloys, which are promising biodegradable materials for medical applications. The study finds that Mg–1Ga exhibits excellent cytocompatibility and promotes osteogenic differentiation, facilitating the early stages of osteoblast precursor cell development. In vivo studies in a rat femoral condyle model reveal that Mg–1Ga and Mg–5Ga significantly promote new bone formation without causing any adverse effects. The antibacterial activity of both alloys is evaluated against E. coli and S. aureus, with the inhibition rate increasing with Ga content. These findings suggest that Mg–Ga alloys have the potential to serve as osteogenic and antibacterial implant materials, providing significant insights into the development of novel biomedical implants.

Synapses shape oligodendrocyte precursor cell development and predict myelination location.

Synapses shape oligodendrocyte precursor cell development and predict myelination location.

RIPK1 deficiency prevents thymic NK1.1 expression and subsequent iNKT cell development.

Receptor Interacting Protein Kinase 1 (RIPK1) and caspase-8 (Casp8) jointly orchestrate apoptosis, a key mechanism for eliminating developing T cells which have autoreactive or improperly arranged T cell receptors. Mutations in the scaffolding domain of Ripk1 gene have been identified in humans with autoinflammatory diseases like Cleavage Resistant RIPK1 Induced Autoinflammatory (CRIA) and Inflammatory Bowel Disease. RIPK1 protein also contributes to conventional T cell differentiation and peripheral T cell homeostasis through its scaffolding domain in a cell death independent context. Ripk1 deficient mice do not survive beyond birth, so we have studied the function of this kinase in vivo against a backdrop Ripk3 and Casp8 deficiency which allows the mice to survive to adulthood. These studies reveal a key role for RIPK1 in mediating NK1.1 expression, including on thymic iNKT cells, which is a key requirement for thymic stage 2 to stage 3 transition as well as iNKT cell precursor development. These results are consistent with RIPK1 mediating responses to TcR engagement, which influence NK1.1 expression and iNKT cell thymic development. We also used in vivo and in vitro stimulation assays to confirm a role for both Casp8 and RIPK1 in mediating iNKT cytokine effector responses. Finally, we also noted expanded and hyperactivated iNKT follicular helper (iNKTFH) cells in both DKO (Casp8-, Ripk3- deficient) and TKO mice (Ripk1-, Casp8-, Ripk3- deficient). Thus, while RIPK1 and Casp8 jointly facilitate iNKT effector function, RIPK1 uniquely influenced thymic iNKT cell development most likely by regulating molecular responses to T cell receptor engagement. iNKT developmental and functional aberrances were not evident in mice expressing a kinase-dead version of RIPK1 (RIPK1kd), indicating that the scaffolding function of this protein exerts the critical regulation of iNKT cells. Our findings suggest that small molecule inhibitors of RIPK1 could be used to regulate iNKT cell development and effector function to alleviate autoinflammatory conditions in humans.

Dynamic changes of oligodendrogenesis in neonatal rats with hypoxic-ischemic white matter injury

BackgroundWhite matter injury (WMI) is an important type of preterm brain injury, which may result in severe neurological sequelae and lack of effective treatments. It is ascertained that selective vulnerability of oligodendrocytes is closely related to the WMI in preterm infants. But the alteration of the endogenous oligodendrogenesis over long time after hypoxic-ischemic WMI is still not clearly elucidated. MethodsWe adopted an animal model of hypoxic-ischemic WMI in 3-day-old neonatal Sprague-Dawley rats. Immunofluorescence staining and western blotting were used to detect dynamic changes of oligodendrogenesis in the white matter region on postoperative day (POD) 1, 3, 7, 14, 28, 56 and 84. ResultsIn the sham group, the oligodendrocyte lineage in the white matter reached a developmental peak from POD 3 to 14. The proliferation and development of oligodendrocyte precursor cells (OPCs) occurred primarily within POD 14. The number of mature oligodendrocytes showed an upward trend and a dynamic change in proliferation over time. While in the WMI group, the oligodendrocyte lineage was upregulated on POD1 and 3 but downregulated on POD 7 and 14. The proliferation of OPCs increased on POD 1 and decreased on POD 3 and 7, with the total number of OPCs significantly reduced from POD 3 to 14. The number of mature oligodendrocytes decreased from POD 3 to 28, and return to the level of the sham group on POD 56 and 84, whereas the MBP expression was still significantly downregulated on POD 56 and 84. ConclusionsHypoxia-ischemia can have a long-term dynamic effect on the endogenous oligodendrogenesis of neonatal rat brain white matter. The proliferation of OPCs was promoted on POD 1 but inhibited from POD 3 to 14, which may be an early intervention target to improve oligodendrogenesis. The number of mature oligodendrocytes recover to the normal on POD 56 and 84 but the myelination is still blocked, which suggests it is essential to promote the maturation of oligodendrocyte and its function recovery at the same time within POD 28. Such efforts will provide the opportunity to test new interventions in pre-clinical studies for their promising clinical application.

Development of myelin in fetal and postnatal neocortex of the pig, the European wild boar Sus scrofa

Myelination of the neocortex of altricial species is mostly a postnatal event, and the appearance of myelin has been associated with the end of the critical period for ocular dominance plasticity in rodent visual cortex. Due to their precocality, ungulates may tell a different story. Here, we analyzed the development of PDGFRα positive oligodendrocyte precursor cells and expression of myelin proteins in the laminar compartments of fetal and postnatal porcine cortex from E45 onwards. Precursor cell density initially increased and then decreased but remained present at P90. MAG and MBP staining were detectable at E70 in subventricular zone and deep white matter, ascending into gyral white matter at E85, and into the gray matter and marginal zone at E100 (birth in pig at E114). Protein blots confirmed the declining expression of PDGFRα from E65 onwards, and the increase of MBP and MAG expression from E80 onwards. Somatosensory input elicited by spontaneous activity is considered important for the formation of the body representation. Indeed, PDGFRα, MBP and MAG expression started earlier in somatosensory than in visual cortex. Taken together, myelination proceeded in white and gray matter and marginal zone of pig cortex before birth with an areal-specific time course, and an almost mature pattern was present at P5 in visual cortex.

Impact of the Voltage-Gated Calcium Channel Antagonist Nimodipine on the Development of Oligodendrocyte Precursor Cells

Multiple sclerosis (MS) is an inflammatory demyelinating disease of the central nervous system (CNS). While most of the current treatment strategies focus on immune cell regulation, except for the drug siponimod, there is no therapeutic intervention that primarily aims at neuroprotection and remyelination. Recently, nimodipine showed a beneficial and remyelinating effect in experimental autoimmune encephalomyelitis (EAE), a mouse model of MS. Nimodipine also positively affected astrocytes, neurons, and mature oligodendrocytes. Here we investigated the effects of nimodipine, an L-type voltage-gated calcium channel antagonist, on the expression profile of myelin genes and proteins in the oligodendrocyte precursor cell (OPC) line Oli-Neu and in primary OPCs. Our data indicate that nimodipine does not have any effect on myelin-related gene and protein expression. Furthermore, nimodipine treatment did not result in any morphological changes in these cells. However, RNA sequencing and bioinformatic analyses identified potential micro (mi)RNA that could support myelination after nimodipine treatment compared to a dimethyl sulfoxide (DMSO) control. Additionally, we treated zebrafish with nimodipine and observed a significant increase in the number of mature oligodendrocytes (* p≤ 0.05). Taken together, nimodipine seems to have different positive effects on OPCs and mature oligodendrocytes.

Proper modulation of AHR signaling is necessary for establishing neural connectivity and oligodendrocyte precursor cell development in the embryonic zebrafish brain.

2,3,7,8-tetrachlorodibenzo-[p]-dioxin (TCDD) is a persistent global pollutant that exhibits a high affinity for the aryl hydrocarbon receptor (AHR), a ligand activated transcription factor. Epidemiological studies have associated AHR agonist exposure with multiple human neuropathologies. Consistent with the human data, research studies using laboratory models have linked pollutant-induced AHR activation to disruptions in learning and memory as well as motor impairments. Our understanding of endogenous AHR functions in brain development is limited and, correspondingly, scientists are still determining which cell types and brain regions are sensitive to AHR modulation. To identify novel phenotypes resulting from pollutant-induced AHR activation and ahr2 loss of function, we utilized the optically transparent zebrafish model. Early embryonic TCDD exposure impaired embryonic brain morphogenesis, resulted in ventriculomegaly, and disrupted neural connectivity in the optic tectum, habenula, cerebellum, and olfactory bulb. Altered neural network formation was accompanied by reduced expression of synaptic vesicle 2. Loss of ahr2 function also impaired nascent network development, but did not affect gross brain or ventricular morphology. To determine whether neural AHR activation was sufficient to disrupt connectivity, we used the Gal4/UAS system to express a constitutively active AHR specifically in differentiated neurons and observed disruptions only in the cerebellum; thus, suggesting that the phenotypes resulting from global AHR activation likely involve multiple cell types. Consistent with this hypothesis, we found that TCDD exposure reduced the number of oligodendrocyte precursor cells and their derivatives. Together, our findings indicate that proper modulation of AHR signaling is necessary for the growth and maturation of the embryonic zebrafish brain.

Modulation of Mesenchymal Stem Cells-Mediated Adaptive Immune Effectors' Repertoire in the Recovery of Systemic Lupus Erythematosus.

The breakdown of self-tolerance of the immune response can lead to autoimmune conditions in which chronic inflammation induces tissue damage. Systemic lupus erythematosus (SLE) is a debilitating multisystemic autoimmune disorder with a high prevalence in women of childbearing age; however, SLE incidence, prevalence, and severity are strongly influenced by ethnicity. Although the mystery of autoimmune diseases remains unsolved, disturbance in the proportion and function of B cell subsets has a major role in SLE's pathogenesis. Additionally, colocalizing hyperactive T helpercell subgroups within inflammatory niches are indispensable. Despite significant advances in standard treatments, nonspecific immunosuppression, the risk of serious infections, and resistance to conventional therapies in some cases have raised the urgent need for new treatment strategies. Without the need to suppress the immune system, mesenchymal stem cells (MSCs), as ''smart" immune modulators, are able to control cellular and humoral auto-aggression responses by participating in precursor cell development. In lupus, due to autologous MSCs disorder, the ability of allogenic engrafted MSCs in tissue regeneration and resetting immune homeostasis with the provision of a new immunocyte repertoire has been considered simultaneously. In Brief The bone marrow mesenchymal stem cells (BM-MSCs) lineage plays a critical role in maintaining the hematopoietic stem-cell microstructure and modulating immunocytes. The impairment of BM-MSCs and their niche partially contribute to the pathogenesis of SLE-like diseases. Allogenic MSC transplantation can reconstruct BM microstructure, possibly contributing to the recovery of immunocyte phenotype restoration of immune homeostasis. In terms of future prospects of MSCs, artificially gained by ex vivo isolation and culture adaptation, the wide variety of potential mediators and mechanisms might be linked to the promotion of the immunomodulatory function of MSCs.

GATA2 haploinsufficient patients lack innate lymphoid cells that arise after hematopoietic cell transplantation.

Innate lymphoid cells (ILC) are important barrier tissue immune regulators. They play a pivotal role in early non-specific protection against infiltrating pathogens, regulation of epithelial integrity, suppression of pro-inflammatory immune responses and shaping the intestinal microbiota. GATA2 haploinsufficiency causes an immune disorder that is characterized by bone marrow failure and (near) absence of monocytes, dendritic cells, B cells and natural killer (NK) cells. T cells develop normally, albeit at lower numbers. Here, we describe the absence of ILCs and their progenitors in blood and bone marrow of two patients with GATA2 haploinsufficiency and show that all subsets of ILCs appear after allogeneic hematopoietic stem cell transplantation, irrespective of the preparative conditioning regimen. Our data indicate that GATA2 is involved in the development of hematopoietic precursor cells (HPC) towards the ILC lineage.

A Role of the TEX101 Interactome in the Common Aetiology Behind Male Subfertility and Testicular Germ Cell Tumor.

Patients who develop testicular germ cell tumours (TGCT) are at higher risk to be subfertile than the general population. The conditions are believed to originate during foetal life, however, the mechanisms behind a common aetiology of TGCT and male subfertility remains unknown. Testis-expressed 101 (TEX101) is a glycoprotein that is related to male fertility, and downregulation of the TEX101 gene was shown in pre-diagnostic TGCT patients. In this review, we summarize the current knowledge of TEX101 and its interactome related to fertility and TGCT development. We searched literature and compilation of data from curated databases. There are studies from both human and animals showing that disruption of TEX101 result in abnormal semen parameters and sperm function. Members of the TEX101 interactome, like SPATA19, Ly6k, PICK1, and ODF genes are important for normal sperm function. We found only two studies of TEX101 related to TGCT, however, several genes in its interactome may be associated with TGCT development, such as PLAUR, PRSS21, CD109, and ALP1. Some of the interactome members are related to both fertility and cancer. Of special interest is the presence of the glycosylphosphatidylinositol anchored proteins TEX101 and PRSS21 in basophils that may be coupled to the immune response preventing further development of TGCT precursor cells. The findings of this review indicate that members of the TEX101 interactome could be a part of the link between TGCT and male subfertility.

Predicting signaling pathways regulating demyelination in a rat model of lithium-pilocarpine-induced acute epilepsy: A proteomics study

Demyelination is observed in animal models of intractable epilepsy (IE). Epileptogenesis damages the myelin sheath and dysregulates oligodendrocyte precursor cell (OPC) development. However, the molecular pathways regulating demyelination in epilepsy are unclear. Here, we predicted the molecular mechanisms regulating demyelination in a rat model of lithium-pilocarpine hydrochloride-induced epilepsy. We identified DGKA/Mboat2/Inpp5j and NOS/Keratin 28 as the main target molecules that regulate demyelination via glycerolipid and glycerophospholipid metabolism, phosphatidylinositol signaling, and estrogen signaling in demyelinated forebrain slice cultures (FSCs). In seizure-like FCSs, the actin cytoskeleton was regulated by Cnp and MBP via Pak4/Tmsb4x (also known as Tβ4) and Kif5c/Kntc1. Tβ4 possibly prevented OPC differentiation and maturation and inhibited MBP phosphorylation via the p38MAPK/ERK1/JNK1 pathway. The MAPK signaling pathway was more likely activated in seizure-like FCSs than in demyelinated FCSs. pMBP expression was decreased in the hippocampus of lithium-pilocarpine hydrochloride-induced acute epilepsy rats. The expression of remyelination-related factors was suppressed in the hippocampus and corpus callosum in lithium-pilocarpine hydrochloride-induced epilepsy rats. These findings suggest that the actin cytoskeleton, Tβ4, and MAPK signaling pathways regulate the decrease in pMBP in the hippocampus in a rat model of epilepsy. Our results indicate that regulating the actin cytoskeleton, Tβ4, and MAPK signaling pathways may facilitate the prevention of demyelination in IE.

Early cellular development induced by ecdysteroid in sex-specific wing degeneration of the wingless female winter moth.

The winter moth, Nyssiodes lefuarius, exhibits striking sexual dimorphism in wing form; males have functional wings of normal size, whereas females lack wings. We previously found that the steroid hormone 20-hydroxyecdysone (20E) triggered massive programmed cell death (PCD) only in the female pupal wing epithelium; however, when and how early sexual trait development of the pupal wings is initiated during pupal-adult metamorphosis remains obscure. To clarify the detailed morphological changes and mechanisms underlying early sexual trait development and cell death, we examined the effects of 20E on early ultrastructural and histological changes in the pupal wing epithelium of both sexes. Before the onset of adult differentiation, no morphological differences were observed in the epithelial cells of both sexes at an ultrastructural level. When 5.4µg of 20E was injected into pupae of both sexes at 15days after the onset of pupation, retraction of the wing epithelium from the pupal cuticle was initiated at day 2 after 20E injection in both sexes. Although overt degeneration of wing tissue was not still obvious, apoptotic body-like structures and auto-phagosomes were visible at day 3 after 20E injection in females, whereas development of scale precursor cells started on day 4 after injection in males. Our results suggest that (1) the injection of 20E induced sexually dimorphic changes in the pattern of organelle distribution in wing epithelial cells, and (2) abnormally shaped mitochondria in the cytoplasm of the female wing epithelium might be involved in the PCD that occurs during wing tissue degeneration.

Translational control in neurovascular brain development.

The human brain carries out complex tasks and higher functions and is crucial for organismal survival, as it senses both intrinsic and extrinsic environments. Proper brain development relies on the orchestrated development of different precursor cells, which will give rise to the plethora of mature brain cell-types. Within this process, neuronal cells develop closely to and in coordination with vascular cells (endothelial cells (ECs), pericytes) in a bilateral communication process that relies on neuronal activity, attractive or repulsive guidance cues for both cell types and on tight-regulation of gene expression. Translational control is a master regulator of the gene-expression pathway and in particular for neuronal and ECs, it can be localized in developmentally relevant (axon growth cone, endothelial tip cell) and mature compartments (synapses, axons). Herein, we will review mechanisms of translational control relevant to brain development in neurons and ECs in health and disease.

NSC1122002, a TRAF-binding domain inhibitor, inhibits collagen-induced arthritis in a mouse model.

Dysregulation of IL-12 and IL-23 is related to many autoimmune diseases including arthritis. The production of IL-12 and IL-23 were reported to be under the control of JMJD2D, whose activity and stability were promoted by the TRAF-binding domain (TRABID), a deubiquitinating enzyme that epigenetically modulated inflammatory gene expression. NSC1122002 is a novel inhibitor of TRABID, and this study aimed to examine the effects of NSC1122002 on the expression of IL-12 and IL-23 both in vitro and in vivo in the context of collagen-induced arthritis, consequently to evaluate its potential as a drug candidate for treating inflammatory disease. Bone marrow cells were isolated to detect the effect of NSC1122002 on the development of innate immune cells and other precursor cells. Primary macrophages and osteoclasts were used to examine the impact of NSC1122002 on cytokine expression. Collagen-induced arthritis was established to determine the function of NSC1122002 in vivo. NSC112200 did not affect the development of innate immune cells, primary osteoclast, and haematopoietic stem cells. NSC112200 specifically downregulated the expression of IL-12 and IL-23 through promoting degradation of JMJD2D by directly inhibited the deubiquitinating activity of TRABID. Besides, NSC112200 significantly suppressed the induction of CIA in mice. Our findings provided new insight into the pathological mechanism and intervention method for arthritis therapy and identified that NSC112200 could be a potential drug for treating autoimmune diseases.

Hepatocyte nuclear factor 1 alpha influences pancreatic cancer growth and metastasis

Hepatocyte nuclear factor 1 homeobox alpha (HNF1α) is a transcription factor involved in endodermal organogenesis and pancreatic precursor cell differentiation and development. Earlier studies have reported a role for HNF1α in pancreatic ductal adenocarcinoma (PDAC) but it is controversial. The mechanism by which it impacts PDAC is yet to be explored in depth. In this study, using the online databases we observed that HNF1α is upregulated in PDAC, which was also confirmed by our immunohistochemical analysis of PDAC tissue microarray. Silencing HNF1α reduced the proliferative, migratory, invasive and colony forming capabilities of pancreatic cancer cells. Key markers involved in these processes (pPI3K, pAKT, pERK, Bcl2, Zeb, Snail, Slug) were significantly changed in response to alterations in HNF1α expression. On the other hand, overexpression of HNF1α did not induce any significant change in the aggressiveness of pancreatic cancer cells. Our results demonstrate that reduced expression of HNF1α leads to inhibition of pancreatic cancer growth and progression, which indicates that it could be a potential oncogene and target for PDAC.

Maternal obesity causes fetal hypothalamic insulin resistance and disrupts development of hypothalamic feeding pathways

ObjectivePerinatal exposure to maternal obesity results in predisposition of offspring to develop obesity later in life. Increased weight gain in offspring exposed to maternal obesity is usually associated with hyperphagia, implicating altered central regulation of food intake as a cause. We aimed to define how maternal obesity impacts early development of the hypothalamus to program lasting dysfunction in feeding regulatory pathways. MethodsMice offspring of diet-induced obese mothers were compared to the offspring of lean control mothers. We analysed gene expression in the fetal hypothalamus, alongside neurosphere assays to investigate the effects of maternal obesity on neural progenitor cell proliferation in vitro. Western blotting was used to investigate the insulin signalling pathway in the fetal hypothalamus. Characterisation of cell type and neuropeptide profile in adulthood was linked with analyses of feeding behaviour. ResultsThere was a reduction in the expression of proliferative genes in the fetal hypothalamus of offspring exposed to maternal obesity. This reduction in proliferation was maintained in vitro when hypothalamic neural progenitor cells were grown as neurospheres. Hypothalamic fetal gene expression and neurosphere growth correlated with maternal body weight and insulin levels. Foetuses of obese mothers showed hypothalamic insulin resistance, which may be causative of reduced proliferation. Furthermore, maternal obesity activated the Notch signalling pathway in neonatal offspring hypothalamus, resulting in decreased neurogenesis. Adult offspring of obese mothers displayed an altered ratio of anorexigenic and orexigenic signals in the arcuate nucleus, associated with an inability to maintain energy homeostasis when metabolically challenged. ConclusionsThese findings show that maternal obesity alters the molecular signature in the developing hypothalamus, which is associated with disrupted growth and development of hypothalamic precursor cells and defective feeding regulation in adulthood. This is the first report of fetal hypothalamic insulin resistance in an obese pregnancy and suggests a mechanism by which maternal obesity causes permanent changes to hypothalamic structure and function.

Abstract P4-05-19: Genome wide cell specification of R-loops in BRCA1 mutation carriers without malignancy

Abstract An estimated 35,000 patients with breast cancer (BC) have pathogenic BRCA1/2 variants. Unfortunately, in the current state of medical practice, 50-80% of pathogenic mutation carriers remain undetected and undiagnosed. These are largely women with moderate penetrance mutations, including women with BRCA1 mutations, which may not have been identified. There is an unmet challenge to improve the identification and risks of patients who have an inherited increased lifetime risk of BC. BRCA1 mutation carriers (BRCA1-mc) consist ~8% of woman suffering from BCs. A transcriptional R-loop is a guanine-rich 3-stranded nucleic acid structure that plays key roles in (dis)regulation of transcription events, DNA damage, and genome (in)stability. R-loops can be involved in cancer cell precursor development in BRCA1-mc, but it is not clear how they affect luminal precursor differentiation to mature basal-like BCs associated with BRCA1-mc. The role of R-loops in basal-like cells in normal-like tissue of BRCA1-mc have not been studied, limiting the biological interpretation of previous observations. At a genome wide scale, accuracy and resolution of experimentally defined R-loop boundaries is still a challenge. Integrative statistical and genomic approaches have demonstrated that it is possible to computationally predict R-loop forming sequences (RLFSs) in the human genome at an accuracy of 84-92%. In these regards, integrative analysis of available datasets could be useful. We hypothesize that such analyses of RLFS-positive RNA:DNA hybrids/R-loops and gene expression profiles of BRCA1 wild-type and BRCA1 mutation non-cancer epithelial cells could distinguish normal individuals and pre-malignant risks in BRCA1-mc. To test this hypothesis, we used publicly available datasets and carried out a comprehensive genome-wide analysis for BRCA1-mc and non-carriers without BC malignancy. We identified differentially expressed genes (DEGs) for non-cancer breast epithelium cells from publicly available 10 non-cancer individuals with and 10 without BRCA1 mutations. Our QmRLFS finder, DRIP-seq dataset (GEO:GSE96672) and our R-loop database (http://rloop.bii.a-star.edu.sg/) were used to identify RLFS-positive R-loops genome wide. A majority of DEGs, 1813, in non-cancer epithelial cells of BRCA1-mc are downregulated; Gene Ontology terms include metabolism, immune system, cell signaling, cell adhesion, transcription, and stress response. But, 597 DEGs were upregulated; splicing events. 662 downregulated and 185 upregulated DEGs contained RLFS-positive R-loops. In BRCA1 mutation carrier and non-carrier genomes, RLFS and R-loops were commonly located upstream and downstream promoter regions of many genes regardless of breast cell types. Interestingly, RLFS-positive R-loops were observed most often in luminal cells, however they were also found in mature basal-like cells. In particular, RLFS-positive R-loops were found only in BRCA1 locus (in 1st intron) of mature basal-like cells of non-BRCA1 mutation breast tissue samples, but not in luminal cells. However, for BRCA1-mc, we observed R-loops in BRCA1 locus of luminal precursors and mature luminal cells, but not in basal-like cells. Also, in BRCA1 pathway BACH1 promoter region, the RLFS-positive R-loops were observed in all breast cell types for both BRCA1-mc and non-carriers. This study suggests strong association of RLFS-positive R-loop formation and its enrichment variations within mature basal-like breast cells. We highlight the potential clinical significance of early detection of RLFS-positive R-loops in non-cancer breast Citation Format: Andre Grageda, Debasree Sarkar, Vladimir A Kuznetsov. Genome wide cell specification of R-loops in BRCA1 mutation carriers without malignancy [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P4-05-19.

Expression of genes involved in neurogenesis, and neuronal precursor cell proliferation and development: Novel pathways of human ovarian granulosa cell differentiation and transdifferentiation capability invitro.

The process of neural tissue formation is associated primarily with the course of neurogenesis during embryonic life. The source of neural-like cells is stem cells, which, under the influence of appropriate differentiating factors, may differentiate/transdifferentiate towards a neural-like lineage. The present study suggested that, under long-term in vitro culture conditions, human ovarian granulosa cells (GCs), obtained from granulosa-rich follicular fluid, acquired new properties and expressed genes characteristic of the ontological groups ‘neurogenesis’ (GO:0022008), ‘neuronal precursor cell proliferation’ (GO:0061351) and ‘nervous system development’ (GO:0007399), which are closely related to the formation of neurons. The present study collected GCs from 20 women referred for the procedure of in vitro fertilization. Cells were maintained in long-term in vitro culture for 30 days, and RNA was isolated after 1, 7, 15 and 30 days of culture. The expression profile of individual genes was determined using the Affymetrix microarray method. The 131 genes with the highest expression change in relation to day 1 of culture were then selected; the 10 most affected genes found to be primarily involved in nerve cell formation processes were chosen for consideration in this study: CLDN11, OXTR, DFNA5, ATP8B1, ITGA3, CD9, FRY, NANOS1, CRIM1 and NTN4. The results of the present study revealed that these genes may be considered potential markers of the uninduced differentiation potential of GCs. In addition, it was suggested that GCs may be used to develop a cell line showing neuronal characteristics after 30 days of cultivation. In addition, due to their potential, these cells could possibly be used in the treatment of neurodegenerative diseases, not only in the form of ‘cultured neurons’ but also as producers of factors involved in the regeneration of the nervous system.

The transcriptional coactivator and histone acetyltransferase CBP regulates neural precursor cell development and migration

CREB (cyclic AMP response element binding protein) binding protein (CBP, CREBBP) is a ubiquitously expressed transcription coactivator with intrinsic histone acetyltransferase (KAT) activity. Germline mutations within the CBP gene are known to cause Rubinstein-Taybi syndrome (RSTS), a developmental disorder characterized by intellectual disability, specific facial features and physical anomalies. Here, we investigate mechanisms of CBP function during brain development in order to elucidate morphological and functional mechanisms underlying the development of RSTS. Due to the embryonic lethality of conventional CBP knockout mice, we employed a tissue specific knockout mouse model (hGFAP-cre::CBPFl/Fl, mutant mouse) to achieve a homozygous deletion of CBP in neural precursor cells of the central nervous system.Our findings suggest that CBP plays a central role in brain size regulation, correct neural cell differentiation and neural precursor cell migration. We provide evidence that CBP is both important for stem cell viability within the ventricular germinal zone during embryonic development and for unhindered establishment of adult neurogenesis. Prominent histological findings in adult animals include a significantly smaller hippocampus with fewer neural stem cells. In the subventricular zone, we observe large cell aggregations at the beginning of the rostral migratory stream due to a migration deficit caused by impaired attraction from the CBP-deficient olfactory bulb. The cerebral cortex of mutant mice is characterized by a shorter dendrite length, a diminished spine number, and a relatively decreased number of mature spines as well as a reduced number of synapses.In conclusion, we provide evidence that CBP is important for neurogenesis, shaping neuronal morphology, neural connectivity and that it is involved in neuronal cell migration. These findings may help to understand the molecular basis of intellectual disability in RSTS patients and may be employed to establish treatment options to improve patients’ quality of life.

Delineating Human B Cell Precursor Development With Genetically Identified PID Cases as a Model.

B-cell precursors (BCP) arise from hematopoietic stem cells in bone marrow (BM). Identification and characterization of the different BCP subsets has contributed to the understanding of normal B-cell development. BCP first rearrange their immunoglobulin (Ig) heavy chain (IGH) genes to form the pre-B-cell receptor (pre-BCR) complex together with surrogate light chains. Appropriate signaling via this pre-BCR complex is followed by rearrangement of the Ig light chain genes, resulting in the formation, and selection of functional BCR molecules. Consecutive production, expression, and functional selection of the pre-BCR and BCR complexes guide the BCP differentiation process that coincides with corresponding immunophenotypic changes. We studied BCP differentiation in human BM samples from healthy controls and patients with a known genetic defect in V(D)J recombination or pre-BCR signaling to unravel normal immunophenotypic changes and to determine the effect of differentiation blocks caused by the specific genetic defects. Accordingly, we designed a 10-color antibody panel to study human BCP development in BM by flow cytometry, which allows identification of classical preB-I, preB-II, and mature B-cells as defined via BCR-related markers with further characterization by additional markers. We observed heterogeneous phenotypes associated with more than one B-cell maturation pathway, particularly for the preB-I and preB-II stages in which V(D)J recombination takes place, with asynchronous marker expression patterns. Next Generation Sequencing of complete IGH gene rearrangements in sorted BCP subsets unraveled their rearrangement status, indicating that BCP differentiation does not follow a single linear pathway. In conclusion, B-cell development in human BM is not a linear process, but a rather complex network of parallel pathways dictated by V(D)J-recombination-driven checkpoints and pre-BCR/BCR mediated-signaling occurring during B-cell production and selection. It can also be described as asynchronous, because precursor B-cells do not differentiate as full population between the different stages, but rather transit as a continuum, which seems influenced (in part) by V-D-J recombination-driven checkpoints.