Progenitor Cell Markers Research Articles

7323 Prostatic Acid Phosphatase Is Negatively Regulated by Androgens and Persists in Castration-resistant Prostate Cancer

Abstract Disclosure: A. Kirschenbaum: None. P. Cheung: None. S. Yao: None. P. Cheung: None. Background: Prostate cancer (PCa) is the most common cancer and second leading cause of cancer death in American men. Most patients with metastatic PCa respond initially to androgen deprivation therapy (ADT) but the majority will progress to lethal, metastatic, castrate-resistant PCa (mCRPC). The identification of markers of mCRPC that are not regulated or negatively regulated by androgens can serve as markers and therapeutic targets in mCRPC. We hypothesized that prostatic acid phosphatase (PAP), a protein phosphatase and 5’ectonucleotidase that is expressed in both the cytoplasm and transmembrane (TM-PAP) of PCa cells, is negatively regulated by androgen signaling and can serve as a therapeutic target. Methods: PAP protein expression was assessed by immunohistochemistry (IHC) in PCa cell line spheroids (VCaP, 22Rv1, LNCaP, C42B) +/- androgen addition as well as in LNCaP s.c. xenografts +/- castration. Protein expression of PAP was assessed with western blotting in PCa cell lines after treatment with androgens (DHT) and anti-androgen (enzalutamide). Results: In spheroids derived from PCa cell lines, there is some IHC expression of PAP with the strongest staining in VCaP and 22Rv1 spheroids. DHT decreased PAP IHC expression in all spheroids. LNCaP and VCaP s.c. xenografts demonstrated that castration decreased tumor growth rates but that in castrate as well as control tumors PAP expression persisted. All forms of PAP including TM-PAP are expressed in VCaP cell line and were negatively regulated by androgen addition. Conclusions: The identification of markers of mCRPC that are not regulated or are negatively regulated by androgen could help identify recalcitrant tumor cells and mount targeted delivery of therapeutic agents. In several androgen sensitive PCa cell lines, DHT addition decreased PAP expression in spheroids. In vivo, PAP expression persists after castration in spite of decreases in tumor growth rates. Finally, DHT decreases and enzalutamide restores PAP expression in vitro. These data demonstrate that PAP is a more progenitor cell marker in PCa that persists after castration and may be targeted in castration-resistant disease. Presentation: 6/3/2024

561P PDGFRa as a reliable surface marker for fibro-adipogenic progenitor (FAP) cells

561P PDGFRa as a reliable surface marker for fibro-adipogenic progenitor (FAP) cells

Meflin/ISLR is a marker of adipose stem and progenitor cells in mice and humans that suppresses white adipose tissue remodeling and fibrosis.

Identifying specific markers of adipose stem and progenitor cells (ASPCs) in vivo is crucial for understanding the biology of white adipose tissues (WAT). PDGFRα-positive perivascular stromal cells represent the best candidates for ASPCs. This cell lineage differentiates into myofibroblasts that contribute to the impairment of WAT function. However, ASPC marker protein(s) that are functionally crucial for maintaining WAT homeostasis are unknown. We previously identified Meflin as a marker of mesenchymal stem cells (MSCs) in bone marrow and tissue-resident perivascular fibroblasts in various tissues. We also demonstrated that Meflin maintains the undifferentiated status of MSCs/fibroblasts. Here, we show that Meflin is expressed in WAT ASPCs. A lineage-tracing experiment showed that Meflin+ ASPCs proliferate in the WAT of obese mice induced by a high-fat diet (HFD), while some of them differentiate into myofibroblasts or mature adipocytes. Meflin knockout mice fed an HFD exhibited a significant fibrotic response as well as increases in adipocyte cell size and the number of crown-like structures in WAT, accompanied by impaired glucose tolerance. These data suggested that Meflin expressed by ASPCs may have a role in reducing disease progression associated with WAT dysfunction.

Garlic Extract Promotes Pancreatic Islet Neogenesis Through α-to-β-Cell Transdifferentiation and Normalizes Glucose Homeostasis in Diabetic Rats.

Garlic extract (GE) has been shown to ameliorate hyperglycemia in diabetic rats (DRs) by increasing insulin production. However, the mechanism through which it exerts its effects remains unclear. Here, it investigates the molecular process and the origin of regenerating β-cell in rats with streptozotocin (STZ)-induced diabetes in response to GE. In this study, quantitative RT-PCR (qRT-PCR), western blotting, and immunohistochemical analysis are carried out after pancreas isolation. These findings show that 1 week of GE treatment increases the expression of the endocrine progenitor cell markers Neurogenin3 (Neurog3), pancreatic and duodenal homeobox 1 (Pdx1), neurogenic differentiation factor 1 (Neurod1), paired box proteins (Pax)4, V-maf musculoaponeurotic fibrosarcoma oncogene homolog B(Mafb),and NK homeobox factors (Nkx)6-1 in STZ-induced DRs. Continuation with GE treatment for 8 weeks causes the expression of the mature β-cell markers insulin(Ins)2, urocortin3 (Ucn3), and glucose transporter 2 (Glut2) to peak. Comprehensive examination of the islet through immunohistochemical analysis reveals the presence of a heterogeneous cell population including INS+/GLUT2- and INS+/GLUT2+ β-cell subpopulations with few bihormonal INS+/GCG+ cells after 4 weeks. By week 8, islet architecture is reestablished, and glucose-stimulated insulin secretion was restored through the upregulation of Ucn3. GE induces β-cell neogenesis in DRs and restores islet architecture. The newly formed mature β-like cells could have originated through the differentiation of endocrine progenitor cells as well as α- to β-cell transdifferentiation.

Enhanced In Vitro Recapitulation of In Vivo Liver Regeneration by Co-Culturing Hepatocyte Organoids with Adipose-Derived Mesenchymal Stem Cells, Alleviating Steatosis and Apoptosis in Acute Alcoholic Liver Injury.

Hepatocyte organoids (HOs) have superior hepatic functions to cholangiocyte-derived organoids but suffer from shorter lifespans. To counteract this, we co-cultured pig HOs with adipose-derived mesenchymal stem cells (A-MSCs) and performed transcriptome analysis. The results revealed that A-MSCs enhanced the collagen synthesis pathways, which are crucial for maintaining the three-dimensional structure and extracellular matrix synthesis of the organoids. A-MSCs also increased the expression of liver progenitor cell markers (KRT7, SPP1, LGR5+, and TERT). To explore HOs as a liver disease model, we exposed them to alcohol to create an alcoholic liver injury (ALI) model. The co-culture of HOs with A-MSCs inhibited the apoptosis of hepatocytes and reduced lipid accumulation of HOs. Furthermore, varying ethanol concentrations (0-400 mM) and single-versus-daily exposure to HOs showed that daily exposure significantly increased the level of PLIN2, a lipid storage marker, while decreasing CYP2E1 and increasing CYP1A2 levels, suggesting that CYP1A2 may play a critical role in alcohol detoxification during short-term exposure. Moreover, daily alcohol exposure led to excessive lipid accumulation and nuclear fragmentation in HOs cultured alone. These findings indicate that HOs mimic in vivo liver regeneration, establishing them as a valuable model for studying liver diseases, such as ALI.

Role of HNF4alpha-cMyc interaction in liver regeneration after partial hepatectomy.

Hepatocyte nuclear factor 4 alpha (HNF4α) is the master regulator of hepatic differentiation. Recent studies have also revealed the role of HNF4α in hepatocyte proliferation via negatively regulating the expression of proto-mitogenic genes, including cMyc. Here, we aimed to study the interaction between HNF4α-cMyc during liver regeneration after partial hepatectomy (PHX). Wild-type (WT), hepatocyte-specific knockout of HNF4α (HNF4α-KO), cMyc (cMyc-KO), and HNF4α-cMyc double knockout (DKO) mice were subjected to PHX to induce liver regeneration. Blood and liver tissue samples were collected at 0h, 24h, 48h, 7D, and 14D after PHX for further analysis. WT, HNF4α-KO, cMyc-KO and DKO mice regained liver weight by 14 days after PHX. The deletion of cMyc did not affect liver regeneration, which was similar to the WT mice. WT and cMyc-KO mice started regaining liver weight as early as 24 hours after PHX, with a peak proliferation response at 48 hours after PHX. HNF4α- KO and DKO showed a delayed response with liver weight increase by day 7 after PHX. The overall hepatocyte proliferation response by DKO mice following PHX was lower than that of other genotypes. Interestingly, the surviving HNF4α-KO and DKO mice showed re-expression of HNF4α at mRNA and protein levels on day 14 after PHX. This was accompanied by a significant increase in the expression of Krt19 and Epcam, hepatic progenitor cell markers, in the DKO mice on day 14 after PHX. These data indicate that, in the absence of HNF4α, cMyc contributes to hepatocyte-driven proliferation to compensate for the lost tissue mass. Furthermore, in the absence of both HNF4α and cMyc, HPC-driven proliferation occurs to support liver regeneration.

The value of immunohistochemical expression of SOX9 and CD34 in alopecia areata

ABSTRACT Background Alopecia areata (AA), an immune-mediated disorder, is marked by temporary, nonscarring hair loss. The bulge area is protected from immune attacks by immune privilege; however, recent studies demonstrated immune cells infiltrating the bulge area. Objective This study aims to investigate the immunohistochemical expression of the sex-determining region Y-box 9 (SOX9) and cluster of differentiation 34 (CD34) in AA patients as markers of hair follicle stem cells (HFSCs) and progenitor cells, respectively. Methods Immunohistochemical staining of SOX9 and CD34 was applied on skin samples of 20 AA patients and 20 healthy controls. Results SOX9 and CD34 were significantly lower in lesional samples of cases compared to perilesional and control skin biopsies. Furthermore, SOX9 level was negatively correlated with the severity of alopecia tool score (SALT score) among the studied AA patients. Moreover, lowered SOX9 expression was present in patients with recurrent attacks. Conclusions The significant reduction of stem cell markers (SOX9 and CD34) in our studied AA cases signifies the pathological affection of HFSCs and their progeny in AA. This is thought to cause a loss of competence in generating new hair in some AA cases, which needs to be validated in further research. Limitations of the study This study has a small sample size.

Urine-derived renal epithelial cells isolated after kidney transplant are sensitive to neutrophil gelatinase-associated lipocalin exposure during in vitro culture

Urine-derived renal epithelial cells (URECs) are highly voided after kidney transplant and express typical kidney markers, including markers of kidney epithelial progenitor cells. Recently URECs have shown promising immunomodulatory properties when cultured with Peripheral Blood Mononuclear Cells (PBMCs), promoting an increase in the T regulatory cells. In vivo, kidney cells are highly exposed to damage associated molecules during both acute and chronic kidney injury. Neutrophil gelatinase-associated lipocalin (NGAL) is one of the most -known early marker of acute and chronic kidney damage. However, its role on the evolution of renal damage has not yet been fully described, nor has its impact on the characteristics of renal-derived cells during in vitro culture. The aim of this study is to investigate the effect of NGAL on the characteristics of URECs isolated after kidney transplant, by exposing these cells to the treatment with NGAL during in vitro culture and evaluating its effect on UREC viability, proliferation, and immunomodulatory potential. The exposure of URECs to NGAL reduced their viability and proliferative capacity, promoting the onset of apoptosis. The immunomodulatory properties of URECs were partially inhibited by NGAL, without affecting the increase of Treg cells observed during UREC-PBMCs coculture. These results suggest that the exposure to NGAL may compromise some features of kidney stem and specialized cell types, reducing their viability, increasing apoptosis, and partially altering their immunomodulatory properties. Thus, NGAL could represent a target for approaches acting on its inhibition or reduction to improve functional recovery.

The Effect of Bone Marrow Mesenchymal Stem Cells on Nestin and Sox-2 Gene Expression and Spatial Learning (Percent Alternation Y-Maze Test) against AlCl3-Induced Alzheimer's-like Pathology in a Rat Model.

Alzheimer's disease (AD) is a neurodegenerative condition characterized by gradual cognitive impairment, including loss of synapses and nerve cells involved in learning, memory, and habit formation processes. Bone Marrow Mesenchymal Stem Cells (BM-MSCs) are multipotent cells. Because of their self-renewable, differentiation, and immunomodulatory capabilities, they are commonly used to treat many disorders. Hence, the current study intends to examine the effect of BM-MSCs transplantation on Aluminum chloride (AlCl3)-induced cognitive problems, an experimental model resembling AD's hallmarks in rats. The study was conducted in 2022 at The Biomedical Laboratory Faculty of Medicine, Andalas University, Indonesia. Adult male Wistar rats (three groups: negative control; no intervention+treatment with PBS; positive control: AlCl3+treatment with aqua dest; AlCl3+BM-MSCs: AlCl3+treatment with BM-MSCs, n=5 each) were treated daily with AlCl3 orally for five days. Stem cells were intraperitoneally injected into rats at a dose of 1x106 cells/rat. The same quantity of phosphate-buffered saline was given to the control group. One month after stem cell injection, the rat brain tissue was removed and placed in the film bottles that had been created. The expression of neural progenitor cell markers, including nestin and sex-determining Y-box 2 (SOX-2), was analyzed using real-time polymerase chain reaction (RT-PCR). Rats' cognitive and functional memory were examined using Y-maze. Data were analyzed using SPSS software (version 26.0) with a one-way analysis of variance (ANOVA) test. The gene expression of nestin (29.74±0.42), SOX-2 (31.44±0.67), and percent alternation of Y-maze (67.04±2.28) increased in the AlCl3+BM-MSCs group compared to that in the positive control group. RT-PCR analysis indicated that nestin (P<0.001) and SOX-2 (P<0.001) were significantly enhanced in the AlCl3+BM-MSCs group compared to the positive control group. This group also indicated an increased percent alternation of Y-maze (P<0.001) in the AlCl3+BM-MSCs group compared to the positive control group. Due to its potential effects on cell therapy, BM-MSCs were found effective in a rat model of AD on the impairment of the rats' behavior and increased expression of neural progenitor cell markers.

Combined hepatocellular cholangiocarcinoma: A clinicopathological update.

Combined hepatocellular-cholangiocarcinoma (cHCC-CCA) is a rare primary liver cancer associated with an appalling prognosis. The diagnosis and management of this entity have been challenging to physicians, radiologists, surgeons, pathologists, and oncologists alike. The diagnostic and prognostic value of biomarkers such as the immunohistochemical expression of nestin, a progenitor cell marker, have been explored recently. With a better understanding of biology and the clinical course of cHCC-CCA, newer treatment modalities like immune checkpoint inhibitors are being tried to improve the survival of patients with this rare disease. In this review, we give an account of the recent developments in the pathology, diagnostic approach, and management of cHCC-CCA.

Collagen 17A1 in the Urothelium Regulates Epithelial Cell Integrity and Local Immunologic Responses in Obstructive Uropathy

Collagen 17A1 (COL17A1), an epidermal hemidesmosome component, is ectopically induced in the urothelium of mouse and human renal pelvis (RP) in parallel with urinary tract-associated lymphoid structure development. Here, COL17A1 was induced in obstructive uropathy-prone ureter of humans and cats. To ascertain its function, murine urinary organs with unilateral ureteral obstruction (UUO) were analyzed during 1 week after surgery. One day after UUO, COL17A1 expression increased in urothelial cells of RP and ureter, and was positively correlated with renal tubulointerstitial lesions. A portion of RP where the smooth muscle layer from the ureter was interrupted was sensitive to urothelium deciduation and COL17A1 induction, showing urine leaked from the RP lumen into the parenchyma. After urine stimulation, cultured immune cells expressed Cxcl2, also up-regulated in CD11b+ cells following COL17A1 stimulation. One day after UUO, CXCL2+ CD11b+ cells infiltrated the urothelium-disrupted area. However, these numbers were significantly lower in Col17a1-deficient mice. COL17A1+ urothelial cells partially co-expressed cytokeratin-14, a progenitor cell marker for urothelium, whereas Col17a1-deficient mice had lower numbers of cytokeratin-14+ cells. Gene Ontology analysis revealed that expression of epithelial- and immune-associated genes was up-regulated and down-regulated, respectively, in the ureter of Col17a1-deficient mice 4 days after UUO. Thus, COL17A1 maintains urothelium integrity by regulating urothelial cell adhesion, proliferation, and differentiation, and activates local immune responses during obstructive uropathy in mammals.

Evaluation of Vasculogenic Factors in the Developing Embryo at Weeks Five and Seven With a Special Focus on CD133 and TIE2 Markers.

Background Human embryo vasculogenesis (blood vessel development starting from endothelial precursors) includes the ability of mesenchymal cells and pluripotent stem cells to differentiate into endothelial cells. Quantification of endothelial progenitor cells is difficult to assess during the early steps of human embryo development due to several factors, especially due to the paucity of human embryo tissue which is usually discarded after early-stage pregnancy abortive methods. CD133 (Promimin-1) is a general marker of progenitor cells, but combined with other endothelial markers such as CD34, it may identify endothelial progenitor cells during embryonic development. CD34 immunohistochemistry was previously performed by our team to identify human embryo capillaries and comparatively assess microvessel density between different human embryonic tissues.TIE2 is an angiopoietin receptor strongly involved in the newly formed blood vessel maturation due to its expression in some mesenchymal precursors for future pericytes. CD34 assesses the presence of endothelial cells but its single use does not evaluate the endothelial progenitor state as CD133 may do nor vessel maturation as TIE2 may do. Data about the dynamics of CD133/TIE2 expression in the early stages of human embryo development are scarce. Hence, in this study, we aimed tocomparatively assess the dynamic of CD133+ endothelial precursors and TIE2 expression on five and seven-week-old human embryonic tissues with a special emphasis on their expression on embryonic vascular beds. Methodology CD133 and TIE2 immunohistochemistry was performed on five and seven-week-old human embryonic tissues followed by their quantification using the Qu Path digital image analysis (DIA) automated method. Results CD133 and TIE2 showed divergent patterns of expression during the initial phases of human embryonic development, specifically in the vascular endothelium of tiny capillaries. The expression of CD133 in endothelial cells lining the perfused lumen gradually decreased from five to seven-week-old embryos. It remained expressed with greater intensity in cells located at the tip of the vascular bud that emerged into pre-existing capillaries. TIE2 was much more specific than CD133, being restricted to the level of the vascular endothelium; therefore, it was easier to quantify using digital image analysis. The endothelium of the embryonic aorta was an exception to the divergent expression, as CD133 and TIE2 were consistently co-expressed in the seven-week-old embryo. The Qu Path DIA assessment increased the accuracy of CD133 and TIE2 evaluation, being the first time they were quantified by using automated software and not manually. Conclusions High heterogeneity of CD133 and TIE2 was observed between five and seven-week-old embryonic tissues as well as between different embryonic regions from the same gestational age. The unique finding of CD133/TIE2 co-expression persistenceinside aortic endothelium needs further studies to elucidate the role of this co-expression.

Mitigating Female Aging Kidney Damage from Ischemic Reperfusion Injury Using Renal Progenitor Cells

Acute kidney injury (AKI) is a sudden event that damages the kidney and disrupts normal function by various causes, such as ischemia, exposure to substances, or obstructions. With AKIs affecting 1 out of 5 hospitalized patients and their association with increased risk of chronic kidney disease (CKD), finding an effective treatment for AKIs is of vital importance. One possible treatment for AKIs could be found within the realm of progenitor cell therapy. Progenitor cells are currently used in the treatment of hematological diseases with the burgeoning information on their possible uses for other disorders. The purpose of this study was to determine whether there is a resident population of renal progenitor cells (RPCs) within the kidney and if these cells can be utilized as an effective treatment to mitigate aging AKI by decreasing inflammation through alteration of the M1:M2 macrophage ratio. This study utilized 75-78 weeks old C57BL/6J female mice to determine if RPCs can reduce the damage caused by ischemic reperfusion (IR)-induced AKI. Kidneys were harvested from young female (40 days old) mice and digested in collagenase type 1 to remove connective tissue. Cells were sorted by flow cytometry, positively selected for c-Kit using magnetic nanoparticles, and subsequently stained for Sca1 and Oct4. Aging female mice underwent renal IR injury (IRI) by clamping off the renal artery and vein for 27 minutes. Following reperfusion, the triple-positive cells were suspended in DMEM and injected into the kidney over the course of 8 minutes to reduce the possibility of pressure necrosis. A separate group of mice was injected with saline to serve as control. The mice were monitored for 3 days at which point kidney function was assessed and tissue harvested for biochemical analysis. Our results indicated an increase in inflammatory cytokines IL-17, NF-κB, and IL-6 in IRI mice which were mitigated with RPCs treatment along with an increase in anti-inflammatory marker IL-10. Furthermore, an increase in M1 macrophage markers was measured in IRI, and a trending increase in M2 macrophage markers in IRI animals treated with RPCs. The kidney function of IRI animals treated with RPC showed improvement compared to IRI control. In summary, we found a population of cells positive for three progenitor cell markers: c-Kit, Sca1, Oct4, and treating IRI aging female kidney with these cells reduced inflammatory markers along with an increase in kidney function, suggesting RPCs promote recovery from AKI by reducing inflammation. Grant Support: DK116591. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

A novel method for culturing enteric neurons generates neurospheres containing functional myenteric neuronal subtypes

BackgroundThe enteric nervous system (ENS) is comprised of neurons, glia, and neural progenitor cells that regulate essential gastrointestinal functions. Advances in high-efficiency enteric neuron culture would facilitate discoveries surrounding ENS regulatory processes, pathophysiology, and therapeutics. New methodDevelopment of a simple, robust, one-step method to culture murine enteric neurospheres in a 3D matrix that supports neural growth and differentiation. ResultsMyenteric plexus cells isolated from the entire length of adult murine small intestine formed ≥3000 neurospheres within 7 days. Matrigel-embedded neurospheres exhibited abundant neural stem and progenitor cells expressing Sox2, Sox10 and Msi1 by day 4. By day 5, neural progenitor cell marker Nestin appeared in the periphery of neurospheres prior to differentiation. Neurospheres produced extensive neurons and neurites, confirmed by Tubulin beta III, PGP9.5, HuD/C, and NeuN immunofluorescence, including neural subtypes Calretinin, ChAT, and nNOS following 8 days of differentiation. Individual neurons within and external to neurospheres generated depolarization induced action potentials which were inhibited in the presence of sodium channel blocker, Tetrodotoxin. Differentiated neurospheres also contained a limited number of glia and endothelial cells. Comparison with existing methodsThis novel one-step neurosphere growth and differentiation culture system, in 3D format (in the presence of GDNF, EGF, and FGF2), allows for ∼2-fold increase in neurosphere count in the derivation of enteric neurons with measurable action potentials. ConclusionOur method describes a novel, robust 3D culture of electrophysiologically active enteric neurons from adult myenteric neural stem and progenitor cells.

Vitamin A enhanced periosteal osteoclastogenesis is associated with increased number of tissue-derived macrophages/osteoclast progenitors

A deleterious effect of elevated levels of vitamin A on bone health has been reported in numerous clinical studies. Mechanistic studies in rodents have shown that numbers of periosteal osteoclasts are increased, while endocortical osteoclasts are simultaneously decreased by vitamin A treatment. These observations indicate that osteoclastogenesis on the endocortical and periosteal surfaces of bone is differentially controlled by vitamin A. The present study investigated the in vitro and in vivo effect of all-trans retinoic acid (ATRA), the active metabolite of vitamin A, on periosteal osteoclast progenitors. Mouse calvarial bone cells were cultured in media containing ATRA, with or without the osteoclastogenic cytokine RANKL, on plastic dishes or bone discs. Whereas ATRA did not stimulate osteoclast formation alone, the compound robustly potentiated the formation of RANKL-induced bone resorbing osteoclasts. This effect was due to stimulation by ATRA (EC50 ∼3nM) on the numbers of macrophages/osteoclast progenitors in the bone cell cultures, as assessed by mRNA and protein expression of several macrophage and osteoclast progenitor cell markers, such as M-CSF receptor, RANK, F4/80 and CD11b, as well as by FACS-analysis of CD11b+/F480+/Gr1- cells. The stimulation of macrophage numbers in the periosteal cell cultures was not mediated by increased M-CSF or IL-34. In contrast, ATRA did not enhance macrophages in bone marrow cell cultures. Importantly, ATRA treatment upregulated the mRNA expression of several macrophage-related genes also in the periosteum of tibia in adult mice. These observations demonstrate a novel mechanism by which vitamin A enhances osteoclast formation specifically on periosteal surfaces.

Clinical Utility of Cytokeratins for Accurate Diagnosis of Hepatocellular Carcinoma Among Hepatitis C Virus High-Risk Patients.

Hepatocellular carcinoma (HCC) is a primary malignancy of the liver and a global health problem. It is often diagnosed at advanced stage where hopeless for effective therapies. Identification of more reliable biomarkers for early detection of HCC is urgently needed. Cytokeratins are a marker of hepatic progenitor cells and act as a key player in tumor invasion. Herein, we sought to develop a novel score based on the combination of cytokeratin 18 (CK18) and cytokeratin 19 (CK19) with routine laboratory tests for accurate detection of HCC. Serum CK18, CK 19, α-fetoprotein, albumin and platelets count were assayed in HCC patients (75), liver cirrhosis patients (55) and healthy control (20). Areas under receiving operating curve (AUCs) were calculated and used for construction on novel score. A novel score named CK-HCC = CK 19 (ng/ml)×0.001+ CK18 (ng/ml)×0.004 + AFP (U/L)×5.4 - Platelets count (×109)/L×0.003 - Albumin (g/L)×0.27-36 was developed. CK-HCC score produces AUC of 0.919 for differentiating patients with HCC from those with liver cirrhosis with sensitivity and specificity of a cut-off 1.3 (i.e., less than 1.3 the case is considered cirrhotic, whereas above 1.3 it is considered HCC. CK-HCC score could replace AFP during screening of HCV patients and early detection of HCC.

TRIM27 Promotes Endothelial Progenitor Cell Apoptosis in Patients with In-Stent Restenosis by Ubiquitinating TBK1.

Approximately 2-10% in-stent restenosis (ISR) may occur following percutaneous coronary intervention (PCI) despite the use of modern drug-eluting stents (DES); thus, our study aimed to explore the effects of tripartite motif-containing (TRIM) 27 on ISR and the underlying mechanism. For this purpose, a total of 42 patients undergoing coronary angiography who had prior coronary angiography with DES implantation were recruited. Endothelial progenitor cells (EPCs) markers (defined as CD34 and vascular endothelial growth factoreceptor-2 (VEGFR-2)) in peripheral blood were measured to asses the circulating EPC level. The TRIM family-related gene expressions were detected by reverse transcription-quantitative polymerase chain reaction. Results suggested that ISR patients had reduced CD34+VEGFR-2+ and increased apoptosis rate of EPCs, along with upregulated TRIM27 and TRIM37 and downregulated TRIM28. TRIM27 promoted and TBK1 inhibited the apoptosis rate of EPCs. Mechanically, TRIM27 interacted with TBK1 to ubiquitinate TBK1 in in vitro study. In summary, TRIM27 promoted the progression of ISR in patients after PCI by ubiquitinating TBK1, which might provide novel ideas for the clinical treatment of ISR.

Abstract 5442: Terthighcells: key players in salivary gland homeostasis and regeneration after radiation therapy in adult mice

Abstract Salivary gland homeostasis and regeneration post-radiotherapy depend significantly on stem/progenitor cells. However, the lineage of SMG progenitor cells is not as well defined as in other normal organs, and better understanding of this population would be important for their future clinical application for tissue regeneration. Using a mouse strain expressing regulated CreERT2 recombinase from the endogenous Tert locus, we identify a distinct Terthigh cell population that are located in the ductal region throughout the submandibular gland (SMG), express ASCL3 (another progenitor cell marker) and can give rise to ductal cells during homeostasis. In contrast, Terthigh cells are found to repopulate both ductal and acinar cells at one year of tracing after radiotherapy. Terthigh cells from adult SMG retain self-renewal capacity when subjected in vitro culture, are resistant to radiation cell kill, and display enhanced proliferative activity post-radiation. Similarly, primary human SMG cells with high Tert expression display enhanced cell survival after radiotherapy. RNA sequencing reveals upregulation of the cell cycling and oxidative stress response pathways these cells after radiation. Mechanistically, Tert appears to modulate cell survival and ROS level in SMG spheres following radiation damage. Collectively, these data suggest that Terthigh cells constitute a novel subset of SMG progenitor cells located throughout the ductal region of the gland that can help to repopulate both ductal and acinar cells after RT damage. Citation Format: Li Guan, Vignesh Viswanathan, Sivakamasundari V, Hongbin Cao, Yuyan Jiang, Junfei Zhao, Deana R. Colburg, Patrick T. Neuhoefer, Yu Xu, Eyiwunmi E. Laseinde, Steven Artandi, Quynh-Thu Le. Terthighcells: key players in salivary gland homeostasis and regeneration after radiation therapy in adult mice [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5442.

Single nuclei transcriptomics of the in situ human limbal stem cell niche

The corneal epithelium acts as a barrier to pathogens entering the eye; corneal epithelial cells are continuously renewed by uni-potent, quiescent limbal stem cells (LSCs) located at the limbus, where the cornea transitions to conjunctiva. There has yet to be a consensus on LSC markers and their transcriptome profile is not fully understood, which may be due to using cadaveric tissue without an intact stem cell niche for transcriptomics. In this study, we addressed this problem by using single nuclei RNA sequencing (snRNAseq) on healthy human limbal tissue that was immediately snap-frozen after excision from patients undergoing cataract surgery. We identified the quiescent LSCs as a sub-population of corneal epithelial cells with a low level of total transcript counts. Moreover, TP63, KRT15, CXCL14, and ITGβ4 were found to be highly expressed in LSCs and transiently amplifying cells (TACs), which constitute the corneal epithelial progenitor populations at the limbus. The surface markers SLC6A6 and ITGβ4 could be used to enrich human corneal epithelial cell progenitors, which were also found to specifically express the putative limbal progenitor cell markers MMP10 and AC093496.1.

Fetal Leydig cells: What we know and what we don't.

During male fetal development, testosterone plays an essential role in the differentiation and maturation of the male reproductive system. Deficient fetal testosterone production can result in variations of sex differentiation that may cause infertility and even increased tumor incidence later in life. Fetal Leydig cells in the fetal testis are the major androgen source in mammals. Although fetal and adult Leydig cells are similar in their functions, they are two distinct cell types, and therefore, the knowledge of adult Leydig cells cannot be directly applied to understanding fetal Leydig cells. This review summarizes our current knowledge of fetal Leydig cells regarding their cell biology, developmental biology, and androgen production regulation in rodents and human. Fetal Leydig cells are present in basement membrane-enclosed clusters in between testis cords. They originate from the mesonephros mesenchyme and the coelomic epithelium and start to differentiate upon receiving a Desert Hedgehogsignal from Sertoli cells or being released from a NOTCH signal from endothelial cells. Mature fetal Leydig cells produce androgens. Human fetal Leydig cell steroidogenesis is LHCGR (Luteinizing Hormone Chronic Gonadotropin Receptor) dependent, while rodents are not, although other Gαs -protein coupled receptors might be involved in rodent steroidogenesis regulation. Fetal steroidogenesis ceases after sex differentiation is completed, and some fetal Leydig cells dedifferentiate to serve as stem cells for adult testicular cell types. Significant gaps are acknowledged: (1) Why are adult and fetal Leydig cells different? (2) What are bona fide progenitor and fetal Leydig cell markers? (3) Which signaling pathways and transcription factors regulate fetal Leydig cell steroidogenesis? It is critical to discover answers to these questions so that we can understand vulnerable targets in fetal Leydig cells and the mechanisms for androgen production that when disrupted, leads to variations in sex differentiation that range from subtle to complete sex reversal.