Reserve Cells Research Articles

Optimisation of cell fate determination for cultivated muscle differentiation

Production of cultivated meat requires defined medium formulations for the robust differentiation of myogenic cells into mature skeletal muscle fibres in vitro. Although these formulations can drive myogenic differentiation levels comparable to serum-starvation-based protocols, the resulting cultures are often heterogeneous, with a significant proportion of cells not participating in myofusion, limiting maturation of the muscle. To address this problem, we employed RNA sequencing to analyse heterogeneity in differentiating bovine satellite cells at single-nucleus resolution, identifying distinct cellular subpopulations including proliferative cells that fail to exit the cell cycle and quiescent ‘reserve cells’ that do not commit to myogenic differentiation. Our findings indicate that the MEK/ERK, NOTCH, and RXR pathways are active during the initial stages of myogenic cell fate determination, and by targeting these pathways, we can promote cell cycle exit while reducing reserve cell formation. This optimised medium formulation consistently yields fusion indices close to 100% in 2D culture. Furthermore, we demonstrate that these conditions enhance myotube formation and actomyosin accumulation in 3D bovine skeletal muscle constructs, providing proof of principle for the generation of highly differentiated cultivated muscle with excellent mimicry to traditional muscle.

Transformation zone at the vallate papillae: a significant source of papillomavirus infection at the base of the tongue?

ObjectiveThe aim of this study was to investigate whether the base of the tongue harbours a transformation zone (TZ), i.e., an initiation site for papillomavirus infection, analogous to that in the uterine cervix by examining the histological structure of Von Ebner’s gland ducts in the vallate papillae.MethodsImmunohistochemical staining and immunofluorescence techniques were used to detect markers associated with the uterine cervical TZ in the vallate papillae, and these results were compared with those in uterine cervical tissue. Additionally, tongue samples from mouse papillomavirus (MmuPV1)-infected mice were analysed to test our hypothesis.ResultsThe specific expression of CK17 in the squamous epithelium of the vallate papillae indicated the presence of immature squamous epithelium, arising from the transformation of reserve cells in this region. Moreover, a s determined using virus-infected mice, the TZ at the base of the tongue was a significant site for papilloma virus infection.ConclusionsThis is the first study to reveal the presence of a TZ in the vallate papillae, as determined by the presence of reserve cells and immature squamous epithelium, suggesting that the base of the tongue is a significant site for papillomavirus infection. This finding provides an entry point for the early prevention and diagnosis of HPV-associated lesions in the oropharynx.

Phthalates are detected in the follicular fluid of adolescents and oocyte donors with associated changes in the cumulus cell transcriptome

ObjectiveTo investigate follicular fluid (FF) phthalate levels in adolescents undergoing fertility preservation compared to oocyte donors and explore its association with ovarian reserve and cumulus cell gene expression. DesignRetrospective study, and molecular analysis of cumulus cells (CCs) and FF SubjectsAdolescents (n= 20, 16.7 ± 0.6 years old) undergoing fertility preservation and oocyte donors (n= 24, 26.2 ± 0.4 years old). Main Outcome MeasuresPatient demographics, ovarian stimulation, and oocyte retrieval outcomes were analyzed for each group. FF levels of 9 phthalate metabolites were assessed individually and as molar sums representative of common compounds (all phthalates: ƩPhthalates; di(2-ethylhexyl) phthalate (DEHP) associated phthalate metabolites: ƩDEHP), exposure sources (plastics: ƩPlastic; personal care products: ƩPCP), and modes of action (anti-androgenic: ƩAA) and compared between the two groups. The transcriptome of CC associated with mature oocytes was compared between adolescents and oocyte donors using bulk RNA-sequencing. ResultsFollicular fluid ƩPlastic and ƩPCP levels were significantly higher in adolescents compared to oocyte donors (p<0.05). Follicular fluid ƩDEHP, ƩPlastic, ƩPCP, ƩAA, and ƩPhthalates levels were positively associated with antral follicle count (AFC) (p<0.05) in oocyte donors when adjusted for age, BMI, and race/ethnicity. RNA-seq analysis revealed 248 differentially expressed genes (DEGs) in cumulus cells of adolescents within the top quartile (n=4) of FF ƩPhthalates levels compared to the adolescents within the bottom half (n=9). Genes enriched in pathways involved in cell motility and development were significantly downregulated. ConclusionAdolescents undergoing fertility preservation cycles demonstrate higher levels of phthalate metabolites in their follicular fluid compared to oocyte donors. Phthalate metabolite levels in FF are associated with higher AFC levels in oocyte donors.

Adenoid basal carcinoma cervix – A rare epithelial neoplasm

ABSTRACT Adenoid basal carcinoma of the uterine cervix is a rare epithelial neoplasm. Adenoid basal carcinoma constitutes less than 1% of all cervical adenocarcinomas and has a favorable prognosis. Adenoid basal carcinoma is assumed to arise from a multipotential basal or reserve cell layer in the cervical epithelium. We report a case of adenoid basal carcinoma of the uterine cervix with complaints of bleeding per vagina in a 40-year-old female. The carbohydrate antigen 125 level was elevated (100 U/ml), and the MRI abdomen and pelvis revealed increased signal intensity in the anterior lip of the cervix. The local examination revealed thickening of the anterior lip of the cervix and induration in the left fornix. The patient was diagnosed with carcinoma cervix stage IB2 (FIGO Stage). The patient was operated by a radical robotic hysterectomy. Microscopic examination revealed small basaloid tumor cell nests with peripheral cell palisading and microcyst formation. The tumor cells showed p63 immunoreactivity in basaloid tumor cells and were negative for CD117 and S-100P. This tumor should be differentiated from adenoid basal cell hyperplasia, which has a favorable outcome, and adenoid cystic carcinoma, with a dismal course.

Platelet-Rich Plasma Promotes the Expansion of Human Myoblasts and Favors the In Vitro Generation of Human Muscle Reserve Cells in a Deeper State of Quiescence

Stem cell therapy holds significant potential for skeletal muscle repair, with in vitro-generated human muscle reserve cells (MuRCs) emerging as a source of quiescent myogenic stem cells that can be injected to enhance muscle regeneration. However, the clinical translation of such therapies is hampered by the need for fetal bovine serum (FBS) during the in vitro generation of human MuRCs. This study aimed to determine whether fresh allogeneic human platelet-rich plasma (PRP) combined or not with hyaluronic acid (PRP-HA) could effectively replace xenogeneic FBS for the ex vivo expansion and differentiation of human primary myoblasts. Cells were cultured in media supplemented with either PRP or PRP-HA and their proliferation rate, cytotoxicity and myogenic differentiation potential were compared with those cultured in media supplemented with FBS. The results showed similar proliferation rates among human myoblasts cultured in PRP, PRP-HA or FBS supplemented media, with no cytotoxic effects. Human myoblasts cultured in PRP or PRP-HA showed reduced fusion ability upon differentiation. Nevertheless, we also observed that human MuRCs generated from PRP or PRP-HA myogenic cultures, exhibited increased Pax7 expression and delayed re-entry into the cell cycle upon reactivation, indicating a deeper quiescent state of human MuRCs. These results suggest that allogeneic human PRP effectively replaces FBS for the ex vivo expansion and differentiation of human myoblasts and favors the in vitro generation of Pax7High human MuRCs, with important implications for the advancement of stem cell-based muscle repair strategies.Graphical

Gastroduodenal injury and repair mechanisms.

Although the mucosal barrier serves as a primary interface between the environment and host, little is known about the repair of acute, superficial lesions or deeper, persistent lesions that if not healed, can be the site of increased permeability to luminal antigens, inflammation, and/or neoplasia development. Studies on acute superficial lesions have been sparse in the past year, with more focus given to novel mechanisms of mucosal protection, and the way in which mature epithelial cells or committed stem cells dedifferentiate, reprogram, proliferate, and then regenerate the gastroduodenal mucosa after injury. For this, adenoviral therapy showed organ specific targeting with mRNA and protein expression of effectors to protect against mucosal injury and ulceration. A large database of plant-based agents known to protect against injury and ulceration was published, along with studies using plant-based compounds delivered with alginates, polysaccharide/gel floating rafts, or incorporated into nanoparticles or green carbon dots to improve targeting and retention at the ulcerated lesion. With RNA technology developing rapidly, particularly single-cell RNA sequencing, important and novel data was forthcoming on mucosal regeneration. In particular, the role of interleukin-17 hub proteins in mucosal healing was highlighted. The presence and role of injury reserve cells was determined, as was the composition of ligand gradients for cell differentiation in both stomach and duodenum. The role of amphiregulin in parietal cell differentiation from lineage-restricted stem cells and the Yap1 gene signature in metaplasia vs. healing ulcers were of particular importance. Additionally, studies unveiled the important role of mesenchymal stromal cells in differentiation and repair mechanisms, in Muse cells as an exciting new therapy for mucosal repair after injury, and the role of sympathetic neurons in activating the immune system to regulate mucosal repair mechanisms. Recent studies highlight novel mechanisms that promote mucosal regeneration after injury of the gastroduodenal mucosa.

Trotter Procedure with Harmonic Scalpel : A Unique Approach to Mucoepidermoid Carcinoma of Tongue Base Revisited.

Mucoepidermoid carcinoma (MEC) is a malignant locally invasive tumour of salivary glands, presumed to arise from reserve cells of salivary gland ducts. It accounts approximately 35% of all malignancies of major and minor salivary glands [1]. MEC arising in minor salivary glands can be located in different areas such as palate, retromolar trigone, floor of mouth, buccal mucosa, lips and tongue. MEC of tongue base are not common but they constitute around 50% of malignant lesions of salivary glands in this region. It presents in fifth and sixth decades of life mostly [1]. We present a rare case of 29 year old woman with low grade mucoepidermoid carcinoma of base of tongue operated by the Trotter procedure with harmonic scalpel - median labio-mandibular glossotomy approach. It's an interesting approach first described by William Trotter in 1920s. It's used by head and neck surgeons for accessing tongue base lesions, pharyngeal wall and cervical spine.

Histopathological and Immunohistochemical Characterization of Sebaceous Adenoma and Epithelioma in Dogs.

Sebaceous gland tumors are neoplasms originating from the sebaceous gland and are the third most common type of skin tumor, accounting for 21-35% of all cutaneous neoplasms in dogs. According to their histopathological characteristics, sebaceous gland tumors can be classified into adenoma as a benign tumor and epithelioma as a malignant tumor. Sebaceous epithelioma is distinguished from sebaceous adenoma by containing 90% or more reserve cells. However, this simple numerical criterion is insufficient to histologically distinguish between epitheliomas and adenomas. In addition, sebaceoma in humans, a similar tumor to sebaceous epithelioma, is a term used for tumors with more than 50% of reserve cells, unlike epithelioma. Therefore, we aimed to compare and characterize the histological and immunohistochemical profiles of comprehensive sebaceous adenoma, epithelioma, and borderline tumors that have more than 50% but less than 90% of reserve cells. A total of 14 canine sebaceous tumors were diagnosed as seven adenomas, four borderline tumors, and three epitheliomas. Histologically, the sebaceous adenomas showed nodules consisting of mature sebocytes surrounded by monolayer basaloid cells. In contrast, the portion of the reserve cells was increased, the portion of lipidized cells was decreased, and the majority of lipidized cells were found to be immature in sebaceous epithelioma. In the sebaceous adenomas, necrosis was not observed and mitotic figures were rarely seen. However, necrosis and mitotic figures were highly frequent in both borderline tumor and sebaceous epithelioma. Immunohistochemistry revealed that borderline tumor and sebaceous epithelioma showed significantly higher expression against Ki-67 than sebaceous adenoma. We conclude that it is more accurate to employ the cut-off value of 50% reserve cells in humans rather than the current 90% reserve cells for classifying sebaceous gland tumors in dogs, thereby providing new insight into the characterization of the sebaceous gland tumors.

Determination of anti-Müllerian hormone levels in blood and urine in fertile cats

In recent years, blood anti-Müllerian hormone (AMH) levels have been investigated in female animals to diagnose many conditions, such as the presence of ovarian tissue, follicle reserve, and granulosa cell tumors. Since blood collection is an invasive method, diagnosis with a non-invasive method is important in terms of practicality and animal welfare. This study aimed to investigate the presence of AMH in cat urine and determine whether a correlation exists between blood and urine AMH levels. In addition, it was aimed at revealing whether there was a change in blood and urine AMH levels according to ovarian follicle distribution. Twenty-seven healthy, fertile female cats in the follicular phase were included. Following blood and urine sample collection, a routine ovariohysterectomy was performed. Histological analysis of the removed ovarian tissue was used to determine ovarian follicle types. While both AMH and estrogen levels were determined in blood samples, only AMH levels were investigated in urine samples. Blood AMH levels averaged 10.61 ± 0.75 ng/mL (range: 5–16 ng/mL), while urine AMH levels averaged 5.67 ± 0.91 ng/mL (range: 0.2–13 ng/mL). While urinary AMH level was <1 ng/mL in 7 cats, urinary AMH was >1 ng/mL in all remaining cats. While the study demonstrated AMH excretion in urine, no correlation was found between blood and urine AMH values. However, a significant positive correlation was observed between blood AMH levels and serum estrogen levels (P < 0.001). These findings suggest that urinary AMH may be a product of proteolytic degradation, potentially leading to inaccurate estimations of ovarian activity based solely on urine AMH levels.

Anti-apoptotic protein Bcl-2 contributes to the determination of reserve cells during myogenic differentiation of C2C12 cells.

Skeletal muscle's regenerative ability is vital for maintaining muscle function, but chronic diseases like Duchenne muscular dystrophy can deplete this capacity. Muscle satellite cells, quiescent in normal situations, are activated during muscle injury, expressing myogenic regulatory factors, and producing myogenic progenitor cells. It was reported that muscle stem cells in primary culture and reserve cells in C2C12 cells express anti-apoptotic protein Bcl-2. Although the role of Bcl-2 expressed in myogenic cells has been thought to be to enhance cell viability, we hypothesized that Bcl-2 may promote the formation of reserve cells. The expression pattern analysis showed the expression of Bcl-2 in undifferentiated mononucleated cells, emphasizing its usefulness as a reserve cell marker and reminding us that cells expressing Bcl-2 have low proliferative potential. Silencing of Bcl-2 by transfection with siRNA decreased cell viability and the number of reserve cells, while overexpression of Bcl-2 not only increases cell viability but also inhibits muscle differentiation and proliferation. These results emphasize dual roles of Bcl-2 in protecting cells from apoptosis and contributing to reserve cell formation by regulating myoblast proliferation and/or differentiation. Overall, the study sheds light on the multifaceted role of Bcl-2 in the maintenance of skeletal muscle regeneration.

In vivo PSC differentiation as a platform to identify factors for improving the engraftability of cultured muscle stem cells.

Producing an adequate number of muscle stem cells (MuSCs) with robust regenerative potential is essential for the successful cell therapy of muscle-wasting disorders. We have recently developed a method to produce skeletal myogenic cells with exceptional engraftability and expandability through an in vivo pluripotent stem cell (PSC) differentiation approach. We have subsequently mapped engraftment and gene expression and found that leukemia inhibitory factor receptor (Lifr) expression is positively correlated with engraftability. We therefore investigated the effect of LIF, the endogenous ligand of LIFR, on cultured MuSCs and examined their engraftment potential. We found that LIF-treated MuSCs exhibited elevated expression of PAX7, formed larger colonies from single cells, and favored the retention of PAX7+ "reserve cells" upon myogenic differentiation. This suggested that LIF promoted the maintenance of cultured MuSCs at a stem cell stage. Moreover, LIF enhanced the engraftment capability of MuSCs that had been expanded in vitro for 12 days by 5-fold and increased the number of MuSCs that repopulated the stem cell pool post-transplantation. These results thereby demonstrated the effectiveness of our in vivo PSC differentiation platform to identify positive regulators of the engraftability of cultured MuSCs.

Myosin heavy chain-perinatal regulates skeletal muscle differentiation, oxidative phenotype and regeneration.

Myosin heavy chain-perinatal (MyHC-perinatal) is one of two development-specific myosin heavy chains expressed exclusively during skeletal muscle development and regeneration. The specific functions of MyHC-perinatal are unclear, although mutations are known to lead to contracture syndromes such as Trismus-pseudocamptodactyly syndrome. Here, we characterize the functions of MyHC-perinatal during skeletal muscle differentiation and regeneration. Loss of MyHC-perinatal function leads to enhanced differentiation characterized by increased expression of myogenic regulatory factors and differentiation index as well as reduced reserve cell numbers in vitro. Proteomic analysis revealed that loss of MyHC-perinatal function results in a switch from oxidative to glycolytic metabolism in myofibers, suggesting a shift from slow type I to fast type IIb fiber type, also supported by reduced mitochondrial numbers. Paracrine signals mediate the effect of loss of MyHC-perinatal function on myogenic differentiation, possibly mediated by non-apoptotic caspase-3 signaling along with enhanced levels of the pro-survival apoptosis regulator Bcl2 and nuclear factor kappa-B (NF-κB). Knockdown of MyHC-perinatal during muscle regeneration in vivo results in increased expression of the differentiation marker myogenin (MyoG) and impaired differentiation, evidenced by smaller myofibers, elevated fibrosis and reduction in the number of satellite cells. Thus, we find that MyHC-perinatal is a crucial regulator of myogenic differentiation, myofiber oxidative phenotype and regeneration.

A269 SEX-DEPENDENT EFFECT OF PANCREASTATIN INHIBITION ON COLON MUCOSAL INTEGRITY IN HOMEOSTATIC CONDITION

Abstract Background Sex bias in inflammatory bowel disease (IBD)-associated colorectal cancer (CRC) epidemiology is well known. Pancreastatin (PST), an epithelial cells-derived peptide overexpressed in IBD patients, regulates the endoplasmic reticulum (ER) and survival signalling involved in CRC. Pancreastatin inhibitor 8 (PSTi8), an inhibitor of PST, showed promising anti-inflammatory effects in IBD male mice model. However, no data exists regarding PST’s role in colon mucosal regeneration and epithelium maintenance. Aims Here, we investigated the impact of PSTi8 treatment on colonic epithelial cells in male and female mice under homeostatic conditions. Methods Male and female C57BL/6 mice were treated with PSTi8 (2.5mg/mL/kg, i.r.) or PBS for six days. Epithelial cells’ proliferation (Ki67) and differentiation (cytokeratin-20) were measured by immunofluorescence. Genetic markers for differentiated epithelial cells (goblet Muc2 and Tuft Dclk1), stem cells (fast-cycling Lgr5, reserve Hopx and fetal-like Ly6a), stem cell regulator (BMP4), epithelial cells lineage commitment (Atoh1, Hes1, Gfl1), ER activation (ATF6), and apoptosis (CHOP) were tested by qRT-PCR. Results PSTi8 treatment decreased epithelial cell proliferation in both sexes. The inhibition of PST lowered the differentiation of epithelial cells and mucus production and increased tuft cell gene expression in females but not in male mice. In females, PSTi8 treatment increased fast-cycling stem cells but lower reserve and fetal-like stem cells in both sexes. In addition, PST inhibition significantly downregulated BMP4, which regulates epithelial cell differentiation, in females while not altering its expression in males. In contrast, PSTi8 treatment significantly increased transcription factors regulating epithelial cells' self-renewal and lineage commitments Hes1 and GFI1 in females but decreased Atoh1 in both sexes. PST inhibition enhanced gene expression of ER stress molecules ATF6 and CHOP in female and ATF6 in male mice. CHOP remained unaltered by PST inhibition in males. Conclusions PSTi8 alters colonic epithelial regeneration, differentiation, and thus maintenance in a sex-dependent manner. Funding Agencies CIHRChildren’s Hospital Research Institute of Manitoba (CHRIM)/Research Manitoba

Динамика гиперхромных и MyoD-позитивных ядер регенерирующих скелетных мышц при инфракрасной фотомодуляции

Introduction. Currently, there are no techniques that allow to fully restore skeletal muscle tissue after injury. Infrared laser exposure is a universal physiotherapeutic method used to activate proliferation, increase enzyme activity, and reduce inflammation and edema. At the same time, the influence of the exposure time of infrared laser irradiation on the activation of myosatellite cells has been poorly studied. The paper aimed to analyze the effect of the duration of infrared laser irradiation on the state of cambial reserve cells in regenerating skeletal muscle tissue. Materials and methods. In the experiment (128 male Wistar rats), 4 groups were formed: 0 – intact group; I (dynamic control) – incised wound m. gastrocnemius; II – laser irradiation of the wound with a 60- second exposure; and III – laser irradiation of the wound with a 180-second exposure.The laser was used in a continuous mode, the power being 1.0 W. We performed a morphological study of the area of hyperchromatic muscle nuclei and the density of MyoD+ nuclei at different periods of the experiment. Results. In group III, the area of hyperchromatic nuclei decreased on day 1, and on day 7, it was 30% more than in group I. The density of the arrangement of MyoD+ nuclei in groups II and III increased compared to that in group I for a period of 3 days, regardless of the exposure time. However, on day 7, the number of nuclei was greater in group II. Conclusion. After the use of an infrared laser with a 60-second exposure, the density of MyoD+ nuclei increases, whereas the effect of a 180-second laser exposure has a more dramatic effect on the increase in the area of hyperchromic nuclei of muscle tissue.

Organ Regeneration Without Relying on Regeneration-Dedicated Stem Cells.

The classic conception of tissue regeneration assumed the existence of tissue-proper regeneration stem cells that are set aside during normal tissue development and reserved as stem cells for regeneration. However, modern studies using cell tracing and other approaches have ruled out the presence of regeneration-proper stem cells in most cases in vertebrate tissue regeneration. The only experimentally validated regeneration-dedicated reserve cells are the satellite cells in skeletal muscle (e.g., Michele 2022) (see Sect. 5.2.3 ). Here, we will first discuss examples of large-scale tissue regeneration, liver regeneration in mammals, and lens and limb regeneration in newts. Then, attempts to widen the tissue regeneration capacity in mammals with exogenous transcription factor genes will be reviewed.

Inhibition of cytosine 5-hydroxymethylation during progression of cancer precursor lesions in the uterine cervix.

Methylation and hydroxymethylation of cytosine moieties in CpG islands of specific genes are epigenetic processes shown to be involved in the development of cervical (pre)neoplastic lesions. We studied global (hydroxy)methylation during the subsequent steps in the carcinogenic process of the uterine cervix by using immunohistochemical protocols for the detection of 5-methylcytosine (5-mC) and 5-hydroxymethylcytosine (5-hmC) in paraffin-embedded tissues of the normal epithelia and (pre)malignant lesions. This approach allowed obtaining spatially resolved information of (epi)genetic alterations for individual cell populations in morphologically heterogeneous tissue samples. The normal ectocervical squamous epithelium showed a high degree of heterogeneity for both modifications, with a major positivity for 5-mC in the basal and parabasal layers in the ectocervical region, while 5-hmC immunostaining was even more restricted to the cells in the basal layer. Immature squamous metaplasia, characterized by expression of SOX17, surprisingly showed a decrease of 5-hmC in the basal compartments and an increase in the more superficial layers of the epithelium. The normal endocervical glandular epithelium showed a strong immunostaining reactivity for both modifications. At the squamocolumnar junctions, a specific 5-hmC pattern was observed in the squamous epithelium, resembling that of metaplasia, with the typical weak to negative reaction for 5-hmC in the basal cell compartment. The reserve cells underlying the glandular epithelium were also largely negative for 5-hmC but showed immunostaining for 5-mC. While the overall methylation status remained relatively constant, about 20% of the high-grade squamous lesions showed a very low immunostaining reactivity for 5-hmC. The (pre)malignant glandular lesions, including adenocarcinoma in situ (AIS) and adenocarcinoma showed a progressive decrease of hydroxymethylation with advancement of the lesion, resulting in cases with regions that were negative for 5-hmC immunostaining. These data indicate that inhibition of demethylation, which normally follows cytosine hydroxymethylation, is an important epigenetic switch in the development of cervical cancer.

In Vitro and In Vivo Evidence towards Fibronectin's Protective Effects against Prion Infection.

A distinctive signature of the prion diseases is the accumulation of the pathogenic isoform of the prion protein, PrPSc, in the central nervous system of prion-affected humans and animals. PrPSc is also found in peripheral tissues, raising concerns about the potential transmission of pathogenic prions through human food supplies and posing a significant risk to public health. Although muscle tissues are considered to contain levels of low prion infectivity, it has been shown that myotubes in culture efficiently propagate PrPSc. Given the high consumption of muscle tissue, it is important to understand what factors could influence the establishment of a prion infection in muscle tissue. Here we used in vitro myotube cultures, differentiated from the C2C12 myoblast cell line (dC2C12), to identify factors affecting prion replication. A range of experimental conditions revealed that PrPSc is tightly associated with proteins found in the systemic extracellular matrix, mostly fibronectin (FN). The interaction of PrPSc with FN decreased prion infectivity, as determined by standard scrapie cell assay. Interestingly, the prion-resistant reserve cells in dC2C12 cultures displayed a FN-rich extracellular matrix while the prion-susceptible myotubes expressed FN at a low level. In agreement with the in vitro results, immunohistopathological analyses of tissues from sheep infected with natural scrapie demonstrated a prion susceptibility phenotype linked to an extracellular matrix with undetectable levels of FN. Conversely, PrPSc deposits were not observed in tissues expressing FN. These data indicate that extracellular FN may act as a natural barrier against prion replication and that the extracellular matrix composition may be a crucial feature determining prion tropism in different tissues.

Elevated stress response marks deeply quiescent reserve cells of gastric chief cells

Gastrointestinal tract organs harbor reserve cells, which are endowed with cellular plasticity and regenerate functional units in response to tissue damage. However, whether the reserve cells in gastrointestinal tract exist as long-term quiescent cells remain incompletely understood. In the present study, we systematically examine H2b-GFP label-retaining cells and identify a long-term slow-cycling population in the gastric corpus but not in other gastrointestinal organs. The label-retaining cells, which reside near the basal layers of the corpus, comprise a subpopulation of chief cells. The identified quiescent cells exhibit induction of Atf4 and its target genes including Atf3, a marker of paligenosis, and activation of the unfolded protein response, but do not show elevated expression of Troy, Lgr5, or Mist. External damage to the gastric mucosa induced by indomethacin treatment triggers proliferation of the quiescent Atf4+ population, indicating that the gastric corpus harbors a specific cell population that is primed to facilitate stomach regeneration.

Invasive stratified mucin-producing carcinoma (ISMC) of the cervix: a clinicopathological and molecular analysis of 59 cases with special emphasis on histogenesis and potential therapeutic targets.

This study aimed to better characterize the clinical and molecular features in invasive stratified mucin-producing carcinoma (ISMC), an uncommon aggressive subtype of endocervical adenocarcinoma (EAC). We recruited 59 ISMC for clinicopathological analysis, immunohistochemistry (n = 56), and targeted next-generation sequencing (n = 17). Our cases contained 29 pure and 30 mixed-type ISMC.Five patients developed local recurrence at 6-32 months (median: 13 months), and died of disease at 16-55 months (median: 16 months). Pure and mixed-type ISMC showed no significant difference in overall survival and tumour relapse (P > 0.05) except larger tumour size in the pure-type (P = 0.009). Compared to the usual-type EAC (n = 217), ISMsC were more frequently associated with large tumour size (P = 0.003), advanced FIGO stage (P = 0.017), lymph node metastasis (P = 0.022), Silva pattern C (P < 0.001), and poor overall survival and short tumour recurrence. SOX2 expression was observed in 82.1% (46/56) ISMC, substantially higher than p63 expression (P < 0.001), while positive SOX17 was present in 3.6%(2/56) cases. PD-L1 was positive in 41/56 ISMC (73.21%) (combined positive score: range: 1-92, median: 22). Three ISMC patients (17.65%) had PIK3CA mutations, while one each (5.88%) patient harboured an ERBB2, TP53, STK11, and PTEN mutation, respectively. We conclude that ISMC is clinically more aggressive than the usual-type EAC. ISMC may originate from cervical reserve cells with bidirectional differentiation. PD-L1 overexpression and the molecular profiles raise the possibility that a subset of ISMC patients may benefit from anti-PD-L1 immunotherapy and other targeted therapy, such as mTOR inhibitor and T-DM1.

In vitro-generated human muscle reserve cells are heterogeneous for Pax7 with distinct molecular states and metabolic profiles

BackgroundThe capacity of skeletal muscles to regenerate relies on Pax7+ muscle stem cells (MuSC). While in vitro-amplified MuSC are activated and lose part of their regenerative capacity, in vitro-generated human muscle reserve cells (MuRC) are very similar to quiescent MuSC with properties required for their use in cell-based therapies.MethodsIn the present study, we investigated the heterogeneity of human MuRC and characterized their molecular signature and metabolic profile.ResultsWe observed that Notch signaling is active and essential for the generation of quiescent human Pax7+ MuRC in vitro. We also revealed, by immunofluorescence and flow cytometry, two distinct subpopulations of MuRC distinguished by their relative Pax7 expression. After 48 h in differentiation medium (DM), the Pax7High subpopulation represented 35% of the total MuRC pool and this percentage increased to 61% after 96 h in DM. Transcriptomic analysis revealed that Pax7High MuRC were less primed for myogenic differentiation as compared to Pax7Low MuRC and displayed a metabolic shift from glycolysis toward fatty acid oxidation. The bioenergetic profile of human MuRC displayed a 1.5-fold decrease in glycolysis, basal respiration and ATP-linked respiration as compared to myoblasts. We also observed that AMPKα1 expression was significantly upregulated in human MuRC that correlated with an increased phosphorylation of acetyl-CoA carboxylase (ACC). Finally, we showed that fatty acid uptake was increased in MuRC as compared to myoblasts, whereas no changes were observed for glucose uptake.ConclusionsOverall, these data reveal that the quiescent MuRC pool is heterogeneous for Pax7 with a Pax7High subpopulation being in a deeper quiescent state, less committed to differentiation and displaying a reduced metabolic activity. Altogether, our data suggest that human Pax7High MuRC may constitute an appropriate stem cell source for potential therapeutic applications in skeletal muscle diseases.