Progenitor Cell Markers Research Articles

The developmental toxicity of PM2.5 on the early stages of fetal lung with human lung bud tip progenitor organoids

Exposure to air pollution has been proven to be associated with impaired fetal lung development. However, due to the lack of reliable human source models, it is still challenging to deeply understand the human fetal lung development under PM2.5 exposure. Here, we utilized human embryonic stem cell (hESC) line H9 to generate lung bud tip progenitor organoids (LPOs), a process that mimics early stages of fetal lung development including definitive endoderm (DE) formation, anterior foregut endoderm (AFE) differentiation and lung progenitor cell specification, to evaluate potential pulmonary developmental toxicity of PM2.5. We demonstrated that PM2.5 exposure the entire LPOs induction from hESCs significantly affected cellular proliferation of LPOs, and altered the expression of lung progenitor cell markers NKX2.1, SOX2 and SOX9, which are canonically defined subsequently proximal–distal airways specification. To explore the dynamic influences of PM2.5 exposure at different stages of LPOs specification, we also found that PM2.5 exposure significantly affected the expression of several transcriptional factors that are important for the differentiation of DE and AFE. Mechanistically, we suggested PM2.5-induced developmental toxicity to LPOs was partially linked with the Wnt/β-catenin signaling pathway. Therefore, our findings further emphasize the substantial health risks in the development of respiratory system associated with prenatal exposure to PM2.5.

Microglia Cell Line SIM-A9 Features – New Data

SIM-A9 is a line of spontaneously immortalized mouse microglia cells obtained from newborn C57BL/6 mice’s cerebrum. The aim of this work is to characterize SIM-A9 line by the ratio of cells with the resting and activated microglia phenotype, to analyze the expression of stem/progenitor cell markers CD133 and nestin, growth factors receptors CSF-1R and EGFR, and the karyotype of this line. The light microscopy, immunocytochemistry, flow cytometry and RT/PCR were used to analyze the morphology, phenotype, and gene expression levels of pro-inflammatory cytokines, and the mFISH method was used to analyze the karyotype. It was shown for the first time that SIM-A9 cells express a high level of TSPO protein, CD68, CD11b and CD45 markers on the surface membrane of cells, which corresponds to the phenotype of activated microglia. Despite this, the cells of this line respond with additional activation to LPS stimulation, which leads to an increase in the pro-inflammatory cytokine genes IL-1β, TNFα, IL-6 expression and a high level of active oxygen and nitrogen metabolites formation. It was shown that SIM-A9 cells express stem and progenitor cells markers, CD133+ and nestin, which allows us to consider the cells of this line as early poorly differentiated progenitor cells, despite their phenotype corresponding to activated microglia. It was also found that SIM-A9 cells express receptors of two growth factors CSF-1 and EGF, CSF-1R and EGFR, which indicates the possibility of SIM-A9 cells proliferation stimulation by two alternative mechanisms under the action of the corresponding factors. SIM-A9 cells have a hypotetraploid karyotype with a large number of structural and quantitative chromosome anomalies.

Multi-scale systems genomics analysis predicts pathways, cell types, and drug targets involved in normative variation in peri-adolescent human cognition.

An open challenge in human genetics is to better understand the systems-level impact of genotype variation on developmental cognition. To characterize the genetic underpinnings of peri-adolescent cognition, we performed genotype-phenotype and systems analysis for binarized accuracy in nine cognitive tasks from the Philadelphia Neurodevelopmental Cohort (~2,200 individuals of European continental ancestry aged 8-21years). We report a region of genome-wide significance within the 3' end of the Fibulin-1 gene (P= 4.6 × 10-8), associated with accuracy in nonverbal reasoning, a heritable form of complex reasoning ability. Diffusion tensor imaging data from a subset of these participants identified a significant association of white matter fractional anisotropy with FBLN1 genotypes (P< 0.025); poor performers show an increase in the C and A allele for rs77601382 and rs5765534, respectively, which is associated with increased fractional anisotropy. Integration of published human brain-specific 'omic maps, including single-cell transcriptomes of the developing human brain, shows that FBLN1 demonstrates greatest expression in the fetal brain, as a marker of intermediate progenitor cells, demonstrates negligible expression in the adolescent and adult human brain, and demonstrates increased expression in the brain in schizophrenia. Collectively these findings warrant further study of this gene and genetic locus in cognition, neurodevelopment, and disease. Separately, genotype-pathway analysis identified an enrichment of variants associated with working memory accuracy in pathways related to development and to autonomic nervous system dysfunction. Top-ranking pathway genes include those genetically associated with diseases with working memory deficits, such as schizophrenia and Parkinson's disease. This work advances the "molecules-to-behavior" view of cognition and provides a framework for using systems-level organization of data for other biomedical domains.

Hyperbaric oxygen therapy efficacy on mandibular defect regeneration in rats with diabetes mellitus: an animal study

BackgroundThis study aimed to investigate the influence of hyperbaric oxygen therapy on mandibular critical-sized defect regeneration in rats with experimentally induced type I diabetes mellitus. Restoration of large osseous defects in an impaired osteogenic condition such as diabetes mellitus is a challenging task in clinical practice. Therefore, investigating adjunctive therapies to accelerate the regeneration of such defects is crucial.Materials and methodsSixteen albino rats were divided into two groups (n = 8/group). To induce diabetes mellitus, a single streptozotocin dosage was injected. Critical-sized defects were created in the right posterior mandibles and filled with beta-tricalcium phosphate graft. The study group was subjected to 90-min sessions of hyperbaric oxygen at 2.4 ATA, for 5 consecutive days per week. Euthanasia was carried out after 3 weeks of therapy. Bone regeneration was examined histologically and histomorphometrically. Angiogenesis was assessed by immunohistochemistry against vascular endothelial progenitor cell marker (CD34) and the microvessel density was calculated.ResultsExposure of diabetic animals to hyperbaric oxygen resulted in superior bone regeneration and increased endothelial cell proliferation, which were revealed histologically and immunohistochemically, respectively. These results were confirmed by histomorphometric analysis which disclosed a higher percentage of new bone surface area and microvessel density in the study group.ConclusionsHyperbaric oxygen has a beneficial effect on bone regenerative capacity, qualitatively and quantitively, as well as the ability to stimulate angiogenesis.

Intimal growth on the luminal surface of arteriovenous grafts in rats.

Endothelial cells are known to grow on the luminal surface of arteriovenous grafts (AVGs) used in hemodialysis. Although endothelial cells are important for preventing infection, a detailed growth of endothelial cells in AVGs is unknown. This study sought to create a simpler animal model of AVGs and to investigate how endothelial cells form on the luminal surface. Polyethylene grafts were placed between the cervical artery and vein of Wistar rats. The grafts were removed at 6h, 24h, 3days, or 7days after placement. The luminal surface was observed under optical and polarizing microscopy and stained with endothelial cell markers (LEL, CD31), the progenitor cell marker CD34, and the macrophage marker ED-1. Microscopy demonstrated many diffuse vascular endothelial cells on the luminal surface of AVGs after placement. While there was no difference in the number of LEL-positive cells between the arterial side (AS) and venous side (VS) at 6h or 7days, there were significantly more of these cells on the VS at both 24h and 3days (p < 0.05). Analysis at 24h showed some CD31-positive cells and few CD34-positive cells. This was the first study to use a simple rat model of AVG placement. Endothelial cell formation was initially more active on the VS than on the AS, but these cells subsequently increased in number across the luminal surface. Future clinical studies might contribute clinically by confirming whether AS versus VS puncture results in different infection rates.

Chondrocyte Isolation from Loose Bodies-An Option for Reducing Donor Site Morbidity for Autologous Chondrocyte Implantation.

Loose bodies (LBs) from patients with osteochondritis dissecans (OCD) are usually removed and discarded during surgical treatment of the defect. In this study, we address the question of whether these LBs contain sufficient viable and functional chondrocytes that could serve as a source for autologous chondrocyte implantation (ACI) and how the required prolonged in vitro expansion affects their phenotype. Chondrocytes were isolated from LBs of 18 patients and compared with control chondrocyte from non-weight-bearing joint regions (n = 7) and bone marrow mesenchymal stromal cells (BMSCs, n = 6) obtained during primary arthroplasty. No significant differences in the initial cell yield per isolation and the expression of the chondrocyte progenitor cell markers CD44 + /CD146+ were found between chondrocyte populations from LBs (LB-CH) and control patients (Ctrl-CH). During long-term expansion, LB-CH exhibited comparable viability and proliferation rates to control cells and no ultimate cell cycle arrest was observed within 12 passages respectively 15.3 ± 1.1 mean cumulative populations doublings (CPD). The chondrogenic differentiation potential was comparable between LB-CH and Ctrl-CH, but both groups showed a significantly higher ability to form a hyaline cartilage matrix in vitro than BMSC. Our data suggest that LBs are a promising cell source for obtaining qualitatively and quantitatively suitable chondrocytes for therapeutic applications, thereby circumventing donor site morbidity as a consequence of the biopsies required for the current ACI procedure.

Epithelial plasticity enhances regeneration of committed taste receptor cells following nerve injury

Taste receptor cells are taste bud epithelial cells that are dependent upon the innervating nerve for continuous renewal and are maintained by resident tissue stem/progenitor cells. Transection of the innervating nerve causes degeneration of taste buds and taste receptor cells. However, a subset of the taste receptor cells is maintained without nerve contact after glossopharyngeal nerve transection in the circumvallate papilla in adult mice. Here, we revealed that injury caused by glossopharyngeal nerve transection triggers the remaining differentiated K8-positive taste receptor cells to dedifferentiate and acquire transient progenitor cell-like states during regeneration. Dedifferentiated taste receptor cells proliferate, express progenitor cell markers (K14, Sox2, PCNA) and form organoids in vitro. These data indicate that differentiated taste receptor cells can enter the cell cycle, acquire stemness, and participate in taste bud regeneration. We propose that dedifferentiated taste receptor cells in combination with stem/progenitor cells enhance the regeneration of taste buds following nerve injury.

Expression of Indian hedgehog signaling in murine oviductal infundibulum and its relationship with epithelial homeostasis.

Homeostasis of the oviductal infundibulum epithelium is continuously regulated by signaling pathways under physiological and pathological conditions. Herein, we investigated the expression of hedgehog (Hh) signaling-related components in the murine oviductal infundibulum, which is known to maintain homeostasis in the adult epithelium. Additionally, using autoimmune disease-prone MRL/MpJ-Faslpr/lpr (MRL/lpr) mice showing abnormal morphofunction of the ciliated epithelium of the infundibulum related to the oviductal inflammation, we examined the relationship between Hh signaling and pathology of the infundibulum. The expression and localization of Pax8, a marker for progenitor cells in the oviductal epithelium, and Foxj1, a marker for ciliogenesis, were examined in the infundibulum. The results showed that Pax8 was downregulated and Foxj1 was upregulated with aging, suggesting that homeostasis of the infundibulum epithelium of MRL/lpr mice was disturbed at 6months of age. In all mice, the motile cilia of ciliated epithelial cells in the infundibulum harbored Hh signaling pathway-related molecules: patched (Ptch), smoothened (Smo), and epithelial cells harbor Gli. In contrast, Ptch, Smo, and Gli2 were significantly downregulated in the infundibulum of MRL/lpr mice at 6months of age. The expression levels of Pax8 and Foxj1 were significantly positively correlated with those of Ptch1, Smo, and Gli2. Hh signaling is thought to be involved in homeostasis of the ciliated epithelium in the infundibulum. In MRL/lpr mice, which show exacerbated severe systemic autoimmune abnormalities, molecular alterations in Hh signaling-related components are considered to interact with local inflammation in the infundibulum, leading to disturbances in epithelial homeostasis and reproductive function.

Bone Marrow-Derived Vasculogenic Mesenchymal Stem Cells Enhance In Vitro Angiogenic Sprouting of Human Umbilical Vein Endothelial Cells

Vasculogenic properties of bone marrow-derived mesenchymal stem cells (MSCs) have been reported, but it is still unclear whether the vasculogenic properties are restricted to some populations of MSCs or whether the entire population of MSCs has these properties. We cultured two different populations of MSCs in different culture media and their vasculogenic properties were evaluated using In vitro spheroid sprouting assay. Neither population of MSCs expressed markers of endothelial progenitor cells (EPCs), but they were different in the profiling of angiogenic factor expression as well as vasculogenic properties. One population of MSCs expressed basic fibroblast growth factor (bFGF) and another expressed hepatocyte growth factor (HGF). MSCs expressing HGF exhibited In vitro angiogenic sprouting capacity in response to bFGF derived from other MSCs as well as to their autocrine HGF. The vasculogenic mesenchymal stem cells (vMSCs) derived from the bone marrow also enhanced In vitro angiogenic sprouting capacity of human umbilical vein endothelial cells (HUVECs) in an HGF-dependent manner. These results suggest that MSCs exhibit different vasculogenic properties, and vMSCs that are different from EPCs may contribute to neovascularization and could be a promising cellular therapy for cardiovascular diseases.

Matrix protein Tenascin-C promotes kidney fibrosis via STAT3 activation in response to tubular injury

Accumulating evidence indicates that the extracellular matrix (ECM) is not only a consequence of fibrosis, but also contributes to the progression of fibrosis, by creating a profibrotic microenvironment. Tenascin-C (TNC) is an ECM glycoprotein that contains multiple functional domains. We showed that following kidney injury, TNC was markedly induced in fibrotic areas in the kidney from both mouse models and humans with kidney diseases. Genetically deletion of TNC in mice significantly attenuated unilateral ureteral obstruction-induced kidney fibrosis. Further studies showed that TNC promoted the proliferation of kidney interstitial cells via STAT3 activation. TNC-expressing cells in fibrotic kidney were activated fibroblast 2 (Act.Fib2) subpopulation, according to a previously generated single nucleus RNA-seq dataset profiling kidney of mouse UUO model at day 14. To identify and characterize TNC-expressing cells, we generated a TNC-promoter-driven CreER2-IRES-eGFP knock-in mouse line and found that the TNC reporter eGFP was markedly induced in cells around injured tubules that had lost epithelial markers, suggesting TNC was induced in response to epithelium injury. Most of the eGFP-positive cells were both NG2 and PDGFRβ positive. These cells did not carry markers of progenitor cells or macrophages. In conclusion, this study provides strong evidence that matrix protein TNC contributes to kidney fibrosis. TNC pathway may serve as a potential therapeutic target for interstitial fibrosis and the progression of chronic kidney disease.

Nestin as a diagnostic and prognostic marker for combined hepatocellular-cholangiocarcinoma.

Combined hepatocellular-cholangiocarcinoma (cHCC-CCA) is a rare primary liver cancer (PLC) associated with a poor prognosis. Given the challenges in its identification and its clinical implications, biomarkers are critically needed. We aimed to investigate the diagnostic and prognostic value of the immunohistochemical expression of Nestin, a progenitor cell marker, in a large multicentric series of PLCs. We collected 461 cHCC-CCA samples from 32 different clinical centers. Control cases included 368 hepatocellular carcinomas (HCCs) and 221 intrahepatic cholangiocarcinomas (iCCAs). Nestin immunohistochemistry was performed on whole tumor sections. Diagnostic and prognostic performances of Nestin expression were determined using receiver-operating characteristic curves and Cox regression modeling. Nestin was able to distinguish cHCC-CCA from HCC with AUCs of 0.85 and 0.86 on surgical and biopsy samples, respectively. Performance was lower for the distinction of cHCC-CCA from iCCA (AUCs of 0.59 and 0.60). Nestin, however, showed a high prognostic value, allowing identification of the subset of cHCC-CCA ("Nestin High", >30% neoplastic cells with positive staining) associated with the worst clinical outcome (shorter disease-free and overall survival) after surgical resection and liver transplantation, as well as when assessment was performed on biopsies. We show in different clinical settings that Nestin has diagnostic value and that it is a useful biomarker to identify the subset of cHCC-CCA associated with the worst clinical outcome. Nestin immunohistochemistry may be used to refine risk stratification and improve treatment allocation for patients with this highly aggressive malignancy. There are different types of primary liver cancers (i.e. cancers that originate in the liver). Accurately identifying a specific subtype of primary liver cancer (and determining its associated prognosis) is important as it can have a major impact on treatment allocation. Herein, we show that a protein called Nestin could be used to refine risk stratification and improve treatment allocation for patients with combined hepatocellular carcinoma, a rare but highly aggressive subtype of primary liver cancer.

Retinal pigment epithelium extracellular vesicles are potent inducers of age-related macular degeneration disease phenotype in the outer retina.

Age-related macular degeneration (AMD) is a leading cause of blindness. Vision loss is caused by the retinal pigment epithelium (RPE) and photoreceptors atrophy and/or retinal and choroidal angiogenesis. Here we use AMD patient-specific RPE cells with the Complement Factor H Y402H high-risk polymorphism to perform a comprehensive analysis of extracellular vesicles (EVs), their cargo and role in disease pathology. We show that AMD RPE is characterised by enhanced polarised EV secretion. Multi-omics analyses demonstrate that AMD RPE EVs carry RNA, proteins and lipids, which mediate key AMD features including oxidative stress, cytoskeletal dysfunction, angiogenesis and drusen accumulation. Moreover, AMD RPE EVs induce amyloid fibril formation, revealing their role in drusen formation. We demonstrate that exposure of control RPE to AMD RPE apical EVs leads to the acquisition of AMD features such as stress vacuoles, cytoskeletal destabilization and abnormalities in the morphology of the nucleus. Retinal organoid treatment with apical AMD RPE EVs leads to disrupted neuroepithelium and the appearance of cytoprotective alpha B crystallin immunopositive cells, with some co-expressing retinal progenitor cell markers Pax6/Vsx2, suggesting injury-induced regenerative pathways activation. These findings indicate that AMD RPE EVs are potent inducers of AMD phenotype in the neighbouring RPE and retinal cells.

A 3D-induced pluripotent stem cell-derived human neural culture model to study certain molecular and biochemical aspects of Alzheimer’s disease

PurposeAlzheimer’s disease (AD) early pathology needs better understanding and models. Here, we describe a human induced pluripotent stem cells (iPSCs)-derived 3D neural culture model to study certain aspects of AD biochemistry and pathology.MethodiPSCs derived from controls and AD patients with Presenilin1 mutations were cultured in a 3D platform with a similar microenvironment to the brain, to differentiate into neurons and astrocytes and self-organise into 3D structures by 3 weeks of differentiation in vitro.ResultsCells express astrocytic (GFAP), neuronal (β3-Tubulin, MAP2), glutamatergic (VGLUT1), GABAergic (GAD65/67), pre-synaptic (Synapsin1) markers and a low level of neural progenitor cell (Nestin) marker after 6 and 12 weeks of differentiation in 3D. The foetal 3R Tau isoforms and adult 4R Tau isoforms were detected at 6 weeks post differentiation, showing advanced neuronal maturity. In the 3D AD cells, total and insoluble Tau levels were higher than in 3D control cells.ConclusionOur data indicates that this model may recapitulate the early biochemical and pathological disease features and can be a relevant platform for studying early cellular and biochemical changes and the identification of drug targets.

SRSF10 is essential for progenitor spermatogonia expansion by regulating alternative splicing.

Alternative splicing expands the transcriptome and proteome complexity and plays essential roles in tissue development and human diseases. However, how alternative splicing regulates spermatogenesis remains largely unknown. Here, using a germ cell-specific knockout mouse model, we demonstrated that the splicing factor Srsf10 is essential for spermatogenesis and male fertility. In the absence of SRSF10, spermatogonial stem cells can be formed, but the expansion of Promyelocytic Leukemia Zinc Finger (PLZF)-positive undifferentiated progenitors was impaired, followed by the failure of spermatogonia differentiation (marked by KIT expression) and meiosis initiation. This was further evidenced by the decreased expression of progenitor cell markers in bulk RNA-seq, and much less progenitor and differentiating spermatogonia in single-cell RNA-seq data. Notably, SRSF10 directly binds thousands of genes in isolated THY+ spermatogonia, and Srsf10 depletion disturbed the alternative splicing of genes that are preferentially associated with germ cell development, cell cycle, and chromosome segregation, including Nasp, Bclaf1, Rif1, Dazl, Kit, Ret, and Sycp1. These data suggest that SRSF10 is critical for the expansion of undifferentiated progenitors by regulating alternative splicing, expanding our understanding of the mechanism underlying spermatogenesis.

Downregulation of p38 MAPK Signaling Pathway Ameliorates Tissue-Engineered Corneal Epithelium.

Tissue-engineered corneal epithelium transplantation is effective treatment for severe limbal stem cell deficiency (LSCD), while epithelial terminal differentiation, tans-differentiation, and insufficient stem cell during construction affect the quality of tissue-engineered corneal epithelium. In this study, we applied SB203580 in the culture medium to downregulate the p38 mitogen-activated protein kinase (MAPK) signaling pathway during construction of tissue-engineered corneal epithelium. With application of SB203580, tissue-engineered corneal epithelium showed enhanced strength and condensed structure. The expression of progenitor cell markers ATP-binding cassette sub-family G member 2, tumor protein p63, keratin 14, and Wnt family member 7A was increased, differentiation markers keratin 12, paired box 6, keratin 10, and keratin 13 and trans-differentiation markers actin alpha 2, smooth muscle and snail family transcriptional repressor 1 was decreased, while cell junction markers claudin 1 and cadherin 1 was increased in the tissue-engineered corneal epithelium. The Wnt/catenin beta 1 signaling pathway was upregulated in the epithelium after p38 MAPK inhibition. Transplantation of tissue-engineered corneal epithelium treated with SB203580 to rabbit LSCD model showed faster wound healing and improved epithelial quality. We conclude that downregulation of p38 MAPK signaling pathway helps maintain the stemness and prevent terminal differentiation and abnormal differentiation of corneal epithelial cells during epithelium construction process, and thus can improve the quality of tissue-engineered corneal epithelium. Impact statement Downregulation of p38 MAPK signaling pathway helps maintain the self-renewal of limbal stem cells and prevents terminal differentiation and abnormal differentiation of corneal epithelial cells. Small molecules modulating p38 MAPK signaling pathway ameliorate tissue-engineered corneal epithelium.

Myostatin is a negative regulator of adult neurogenesis after spinal cord injury in zebrafish.

Intrinsic and extrinsic inhibition of neuronal regeneration obstruct spinal cord (SC) repair in mammals. In contrast, adult zebrafish achieve functional recovery after complete SC transection. While studies of innate SC regeneration have focused on axon regrowth as a primary repair mechanism, how local adult neurogenesis affects functional recovery is unknown. Here, we uncover dynamic expression of zebrafish myostatin b (mstnb) in a niche of dorsal SC progenitors after injury. mstnb mutants show impaired functional recovery, normal glial and axonal bridging across the lesion, and an increase in the profiles of newborn neurons. Molecularly, neuron differentiation genes are upregulated, while the neural stem cell maintenance gene fgf1b is downregulated in mstnb mutants. Finally, we show that human fibroblast growth factor 1 (FGF1) treatment rescues the molecular and cellular phenotypes of mstnb mutants. These studies uncover unanticipated neurogenic functions for mstnb and establish the importance of local adult neurogenesis for innate SC repair.

OR08-1 PROP1 Is Not Essential For Pituitary Cells Terminal Differentiation

Abstract Introduction The pituitary gland controls several mechanisms as metabolism, growth, and reproduction, in response to hypothalamic stimuli. The adequate temporal/ spatial expression of transcription factors is mandatory for a normal pituitary development. PROP1 transcription factor is widely known as a key pituitary regulator. Pathogenic variants in the PROP1 gene are the main familial causes of hypopituitarism accounting for about 54% of the cases. Aim To characterize the expression of genes involved in pituitary cells determination and specification during pituitary high demand period such as sexual maturation compared to adulthood in the Ames mice. Methods Weight and naso-anal length were measured. Pituitary glands were collected from 5 wild type (WT) and 5 prop1 mutant (Mut) animals with 30 (P30), 40, (P40), and 60 (P60) days after birth. RT-qPCR was performed to analyze the pituitary transcription factors Sox2, Sox3, Hesx1, Otx2, and genes codifying the hormones GH, TSH, LH/ FSH, CGA, and PRL by SYBR® Green (QIAGEN, Valencia, CA). They were normalized by endogenous genes and performed in triplicate. The target genes relative quantification was performed using the mutant related to its age paired wild type as a calibrator and the results were expressed as fold change. Immunofluorescence of SOX2 (ab97959, Abcam, UK) (1: 1600) and pituitary hormones GH, LH, and FSH (1: 250) were done in pituitaries at P60 and its aged paired control, stained with propidium iodide (1: 1000) for nucleus and the secondary antibody Alexa FluorÒ488 (ab150077, Abcam, UK) (1: 1000). Results All mutant mice presented decreased weight and naso-anal length at the three analyzed periods. The expression of the pituitary stem/ progenitor cell marker Sox2 was increased at P30 and P60 and decreased at P40. Sox3 was increased at P30 and decreased at P40 and P60. Hesx1 was increased at P30 and P60 and decreased at P40. Otx2 was increased at all periods. At P30 the genes codifying pituitary hormones was decreased, except for Tsh and Fsh. At P40 it was observed increased expression of the genes codifying Gh, Lh, Fsh and Prl, while Tsh and Cga expression was reduced. At P60 all pituitary hormones codifying genes presented decreased expression. The immunofluorescence at P60 showed increased expression of SOX2, similar expression of FSH, and decreased expression of GH and LH related to their age paired WT. Conclusion Despite the absence of PROP1, Hesx1 is downregulated at P40. Pituitary cell subtype differentiation and hormonal production is observed at transcriptional and protein levels, showing that pituitary cells can differentiate and produce hormones in the absence of Prop1. The recovery of pituitary capacity to differentiate in mutant suggests the involvement of another factor in the pituitary cell differentiation pathway that could compensate the lack of Prop1. Presentation: Saturday, June 11, 2022 11:30 a.m. - 11:45 a.m.

Clonorchis sinensis infection contributes to hepatocellular carcinoma progression in rat.

Clonorchis sinensis (C. sinensis) infection is a risk factor for cholangiocarcinoma. Whether it also contributes to the development of hepatocellular carcinoma (HCC) is still unclear. This study explored the potential relationship between C. sinensis infection and HCC. A total of 110 Sprague-Dawley rats were divided into four treatment groups, the negative control group (NC) received intragastric (i.g.) administration of saline, while the clonorchiasis group (CS) received i.g. administration of 150 C. sinensis metacercariae. The diethylnitrosamine-induced group (DEN) received intraperitoneal (i.p.) administration of DEN. The clonorchiasis DEN-induced group (CSDEN) received i.g. administration of 150 C. sinensis metacercariae followed by i.p. administration of DEN. Hematoxylin and eosin staining, immunohistochemistry, and Masson's trichrome staining were performed for histopathological analysis of the isolated tissues. RNA-seq technology and RT-PCR were employed for gene expression. In the DEN group, 15 rats survived, of which 9 developed liver cirrhosis and 7 developed HCC. In the CSDEN group, all of the 17 surviving rats developed cirrhosis, and 15 showed development of HCC. The incidence of liver cirrhosis and HCC was significantly higher in the CSDEN group than in the DEN group. KEGG pathway analysis of the differentially expressed genes suggested significant upregulation in inflammation-associated pathways. Immunohistochemistry and RT-PCR results showed significant upregulation of hepatic progenitor cell markers (CK19, SOX9, EpCAM) in the CS group compared to the NC group, as well as in the CSDEN group compared to the DEN group. Our study suggests that C. sinensis infection increases risk of HCC in a rat model by stimulating proliferation of hepatic progenitor cells.

EARLY T-CELL PRECURSOR ACUTE LYMPHOBLASTIC LEUKEMIA IN CHILDHOOD

Early T-cell precursor (ETP) ALL accounts for 10% to 15% of T-ALL, which arises from an early T-cell lineage clone with aberrant expression of myeloid and/or early progenitor cell markers (1,2). ETPs are a subset of thymocytes representing recent immigrants from the bone marrow to the thymus, they retain multilineage differentiation potential, suggesting their direct derivation from hematopoietic stem cells (3). ETP-ALL, which was first reported by Coustan-Smith in 2009, largely overlaps with the pro-T subtype of the EGIL classification; its special diagnostic criteria in immunophenotypic screening are the absence of CD1a and CD8 expression, the absence or weak expression of CD5, and the presence of strong positive for at least one of CD34, CD117, HLADR, CD13, CD33, CD11b, and CD65 (2,4-6). In case of strong positivity of CD5, at least two of the latter must be strong positive (6). There is also novel evidence that the myeloid marker CD371 may be positive in ETP (6). The genetic features of ETP-ALL are similar to those of hematopoietic stem cells and myeloid progenitor cells. The genomic mutations of ETP-ALL are enriched in hematopoietic transcriptional regulators (such as BCL11B, ETV6, RUNX1, biallelic WT1, and GATA3), epigenetic factors (such as histone modification, including PHF6, CTCF, EED, EZH2, SUZ12, and SETD2), and signaling genes (such as activation JAK-STAT, IL-7R and RAS signaling pathway, including JAK1, JAK3, IL7R, SH2B3, NRAS, KRAS, FLT3, NF1, and PTPN11). ETP-ALL has a lower frequency of classical T-ALL genetic alterations such as NOTCH1/FBXW7/CDKN2A mutations and a higher prevalence of FLT3, NRAS/KRAS, DNMT3A, IDH1, IDH2, JAK3, and ETV6 mutations (1,2,5). ETP-ALL was initially thought to have a poor prognosis, but the opinions on it vary (2,3,7). ETP ALL is often corticosteroid resistant and a high percentage of ETP ALL patients have detectable MRD at day 29 including many induction failures (1,2,7,8). No difference in OS was observed in the COG AALL0434 study and UKALL 2003 trial between the patients with ETP-ALL and typical T-ALL (4,9). Therefore, it is important to continue with conventional therapy in ETP-ALL patients who have poor end-induction response; MRD based therapeutic approach is recommanded (7,10). Patients with ETP-ALL had high risk of hematological relapse treated at St Jude Children's Research Hospital (3). For relapsed and refractory patients, the use of acute myeloid leukemia-oriented therapies such as FLAG-IDA regimen or targeted agents may be of benefit for some patients, including FLT3 inhibitors, tyrosine kinase inhibitors, BCL-2 inhibitors such as venetoclax, or JAK/STAT inhibitors in patients with JAK mutations or fusions (1,2,4,5,7).

Prolonged activation of NMDA receptors induces dedifferentiation of islet β cells in mice.

The β-cell dedifferentiation is one of the critical mechanisms in diabetic β-cell loss. Long-term activation of N-methyl-D-aspartate (NMDA) receptors plays an essential role in the development of diabetes, but the underlying mechanisms have not been fully elucidated. This study aims to investigate the effect of prolonged activation of NMDA receptors on islet β-cell dedifferentiation. Male C57BL/6 mice were randomly divided into a normal control group (control group) and an NMDA group. The mice in the NMDA group were intraperitoneally injected with NMDA (8 mg/kg body weight) and those in the control group were injected with the same volume of saline every day for 6 months. At the end of the 6 th month, glucose tolerance and enzyme linked immunosorbent assay (ELISA) were used to detect the function of islets, and pancreatic tissues were taken for immunofluorescence staining to detect the expressions of insulin, glucagon, and proliferating cell nuclear antigen (PCNA). Real-time PCR was used to detect the mRNA expression of pancreatic β cells, α cells, and islet progenitor cell markers.The primary islets were treated with NMDA to observe the effect of NMDA on the dedifferentiation of β cells. The nuclear factor kappa-B (NF-κB) inhibitor BAY 11-7082 was used at the cellular level via detecting insulin secretion and the expression of endocrine cell markers. Compared with the control group, the mice in the NMDA group had higher blood glucose levels at each time point after glucose injection, and the area under the glucose tolerance curve was significantly increased ( P <0.05). The serum insulin content and insulin stimulatory index of the mice in the NMDA group were significantly lower than those in the control group at 30 min after glucose injection (both P <0.05). The double immunofluorescence staining for insulin and glucagon showed that the number of insulin-positive β cells in the pancreatic tissues of mice was significantly decreased after intraperitoneal injection of NMDA in mice for 6 months, while the number of glucagon-positive α cells was significantly increased. Real-time PCR results showed that β-cell markers ( Insulin , Pdx1 , Neurod1 , and Mafa ) were significantly down-regulated in mouse pancreatic tissues after intraperitoneal injection of NMDA for 6 months, while pancreatic progenitor cell markers ( Neurog3 , Gata6 , Hnf4a , Notch1, and Hes1 ) were significantly down-regulated; α-cell markers ( Glucagon , Arx , Irx2 , Mafb , Pou6f2 , Fev , Kcnj3, and Sv2b ) were significantly up-regulated. NMDA treatment of mouse primary islets for 48 h cause significant down-regulation of β-cell marker gene expression ( P <0.05 or P <0.01), accompanied by significant up-regulation of pancreatic progenitor cell markers and α-cell markers ( P <0.05, P <0.01 or P <0.001). The NF-κB inhibitor BAY 11-7082 significantly blocked the down-regulation of β-cell marker expression (all P <0.05) and the up-regulation of α-cell and pancreatic progenitor cell marker after NMDA treatment of islets for 48 h ( P <0.05 or P <0.01). Prolonged activation of NMDA receptors induces islet β-cell dedifferentiation via regulating the NF-κB pathway.