Role of Hedgehog Signaling Pathways in Multipotent Mesenchymal Stem Cells Differentiation.

Multipotent mesenchymal stem cells (MSCs) have high self-renewal and multi-lineage differentiation potentials and low immunogenicity, so they have attracted much attention in the field of regenerative medicine and have a promising clinical application. MSCs originate from the mesoderm and can differentiate not only into osteoblasts, cartilage, adipocytes, and muscle cells but also into ectodermal and endodermal cell lineages across embryonic layers. To design cell therapy for replacement of damaged tissues, it is essential to understand the signaling pathways, which have a major impact on MSC differentiation, as this will help to integrate the signaling inputs to initiate a specific lineage. Hedgehog (Hh) signaling plays a vital role in the development of various tissues and organs in the embryo. As a morphogen, Hh not only regulates the survival and proliferation of tissue progenitor and stem populations but also is a critical moderator of MSC differentiation, involving tri-lineage and across embryonic layer differentiation of MSCs. This review summarizes the role of Hh signaling pathway in the differentiation of MSCs to mesodermal, endodermal, and ectodermal cells.

The effect of diabetes mellitus on differentiation of mesenchymal stem cells into insulin-producing cells.

BACKGROUND: Diabetes mellitus (DM) is a global epidemic with increasing incidences. DM is a metabolic disease associated with chronic hyperglycemia. Aside from conventional treatments, there is no clinically approved cure for DM up till now. Differentiating mesenchymal stem cells (MSCs) into insulin-producing cells (IPCs) is a promising approach for curing DM. Our study was conducted to investigate the effect of DM on MSCs differentiation into IPCs in vivo and in vitro. METHODS: We isolated adipose-derived mesenchymal stem cells (Ad-MSCs) from the epididymal fat of normal and STZ-induced diabetic Sprague-Dawley male rats. Afterwards, the in vitro differentiation of normal-Ad-MSCs (N-Ad-MSCs) and diabetic-Ad-MSCs (DM-Ad-MSCs) into IPCs was compared morphologically then through determining the gene expression of ß-cell markers including neurogenin-3 (Ngn-3), homeobox protein (Nkx6.1), musculoaponeurotic fibrosarcoma oncogene homolog A (MafA), and insulin-1 (Ins-1) and eventually, through performing glucose-stimulated insulin secretion test (GSIS). Finally, the therapeutic potential of N-Ad-MSCs and DM-Ad-MSCs transplantation was compared in vivo in STZ-induced diabetic animals. RESULTS: Our results showed no significant difference in the characteristics of N-Ad-MSCs and DM-Ad-MSCs. However, we demonstrated a significant difference in their abilities to differentiate into IPCs in vitro morphologically in addition to ß-cell markers expression, and functional assessment via GSIS test. Furthermore, the abilities of both Ad-MSCs to control hyperglycemia in diabetic rats in vivo was assessed through measuring fasting blood glucose (FBGs), body weight (BW), histopathological examination of both pancreas and liver and immunoexpression of insulin in pancreata of study groups. CONCLUSION: Our findings reveal the effectiveness of N-Ad-MSCs in differentiating into IPCs in vitro and controlling the hyperglycemia of STZ-induced diabetic rats in vivo compared to DM-Ad-MSCs.

Lats1 and Lats2 regulate YAP and TAZ activity to control the development of mouse Sertoli cells.

Recent reports suggest that the Hippo signaling pathway regulates testis development, though its exact roles in Sertoli cell differentiation remain unknown. Here, we examined the functions of the main Hippo pathway kinases, large tumor suppressor homolog kinases 1 and 2 (Lats1 and Lats2) in developing mouse Sertoli cells. Conditional inactivation of Lats1/2 in Sertoli cells resulted in the disorganization and overgrowth of the testis cords, the induction of a testicular inflammatory response and germ cell apoptosis. Stimulated by retinoic acid 8 (STRA8) expression in germ cells additionally suggested that germ cells may have been preparing to enter meiosis prior to their loss. Gene expression analyses of the developing testes of conditional knockout animals further suggested impaired Sertoli cell differentiation, epithelial-to-mesenchymal transition, and the induction of a specific set of genes associated with Yes-associated protein (YAP) and transcriptional coactivator with PDZ-binding motif (TAZ)-mediated integrin signaling. Finally, the involvement of YAP/TAZ in Sertoli cell differentiation was confirmed by concomitantly inactivating Yap/Taz in Lats1/2 conditional knockout model, which resulted in a partial rescue of the testicular phenotypic changes. Taken together, these results identify Hippo signaling as a crucial pathway for Sertoli cell development and provide novel insight into Sertoli cell fate maintenance.

Neuron-derived neurotrophic factor promotes the differentiation of intramuscular and subcutaneous adipocytes in goat.

Adipocyte play an important role in human health and meat quality by influencing the tenderness, flavor, and juiciness of mutton It has been shown that neuron-derived neurotrophic factor (NENF) is closely related to energy metabolism and adipocyte differentiation in bovine. However, the role of NENF in the goats remains unclear. The aim of this study was to detect the expression of NENF in goat subcutaneous and intramuscular adipocytes, temporal expression profiles of the NENF, and overexpressed NENF on the differentiation of different adipocytes. In this study, PCR amplification successfully cloned the goat NENF gene with a fragment length of 521 bp. In addition, the time point of highest expression of NENF differed between these two adipocytes differentiation processes. Overexpression of NENF in intramuscular and subcutaneous adipocytes promoted the expression levels of differentiation markers CEBPß and SREBP, which in turn promoted the differentiation of intramuscular and subcutaneous adipocytes. This study will provide basic data for further study of the role of goats in goat adipocyte differentiation and for the final elucidation of its molecular mechanisms in regulating goat adipocyte deposition.

Safety of GMP-compliant iPSC lines generated by Sendai virus transduction is dependent upon clone identity and sex of the donor.

Human induced pluripotent stem cells (hiPSCs) are a potential source of somatic cells for cell therapies due to their ability to self-renew and differentiate into various cells of the body. To date, the clinical application of hiPSCs has been limited due to safety issues. The present study aims to standardize the safety procedure of the derivation of GMP-compliant induced pluripotent stem cell (iPSC) lines from human fibroblasts. The hiPSC lines were generated using the nonintegrative Sendai virus method to incorporate Yamanaka reprogramming factors (OCT3/4, SOX2, KLF4 and c-MYC) into cells. A constant temperature was maintained during the cell culture, including all stages of the culture after transduction with Sendai virus. Pluripotency was proved in six independently generated hiPSC lines from adult female (47 years old) and male (57 years old) donors' derived fibroblasts via alkaline phosphatase live (ALP) staining, qPCR, and immunocytochemistry. The hiPSC lines showed a gradual decrease in the presence of the virus with each subsequent passage, and this reduction was specific to the hiPSC line. The frequency and probability of chromosomal aberrations in hiPSCs were dependent on both the iPSC clone identity and sex of the donor. In summary, the generation of hiPSC for clinical applications requires safety standards application (biosafety protocol, quality control of hiPSC lines, viral and genetic integrity screening) from the first stages of the clonal selection of hiPSC from the same donor.

[Cell technologies as a basis for the development of regenerative principles for the treatment of lacrimal gland diseases]. / Kletochnye tekhnologii kak osnova razrabotki regeneratornykh printsipov lecheniya zabolevanii sleznoi zhelezy.

The lacrimal gland (LG) is a tubuloacinar exocrine gland composed of acinar, ductal, and myoepithelial cells. Three-dimensional distribution of acinar lobules, ducts, and myoepithelial cells is necessary for the effective functioning of the organ. LG is the main organ of immune surveillance of the ocular surface system. The embryogenesis of the gland is regulated by the interaction of genetic mechanisms, internal epigenetic (enzyme systems, hormones) and exogenous factors. There is no doubt that there is a clear genetic program for the implementation of the complex process of embryonic development. The mechanisms regulating LG organogenesis initiate the work of a huge number of structural oncogenes, transcription and growth factors, etc. Studying the expression and selective activity of regulatory genes during organ development, their participation in the differentiation of different cell types is a current trend at the nexus of clinical genetics, molecular biology, embryology and immunocytochemistry. Due to its relatively simple structure and accessibility, human LG is a suitable object for potential application in regenerative medicine. Development of a universal protocol for obtaining functional differentiated secretory epithelium of LG capable of expressing tissue-specific markers is an urgent task. Determining the nature and origin of stem cells and progenitor cells will allow the isolation and multiplication of these cells in culture. After obtaining a functionally active culture of LG cells, it is possible to create a model of autoimmune diseases.

Iron deficiency in astrocytes alters cellular status and impacts on oligodendrocyte differentiation.

Iron deficiency (ID) has been shown to affect central nervous system (CNS) development and induce hypomyelination. Previous work from our laboratory in a gestational ID model showed that both oligodendrocyte (OLG) and astrocyte (AST) maturation was impaired. To explore the contribution of AST iron to the myelination process, we generated an in vitro ID model by silencing divalent metal transporter 1 (DMT1) in AST (siDMT1 AST) or treating AST with Fe3+ chelator deferoxamine (DFX; DFX AST). siDMT1 AST showed no changes in proliferation but remained immature. Co-cultures of oligodendrocyte precursors cells (OPC) with siDMT1 AST and OPC cultures incubated with siDMT1 AST-conditioned media (ACM) rendered a reduction in OPC maturation. These findings correlated with a decrease in the expression of AST-secreted factors IGF-1, NRG-1, and LIF, known to promote OPC differentiation. siDMT1 AST also displayed increased mitochondrial number and reduced mitochondrial size as compared to control cells. DFX AST also remained immature and DFX AST-conditioned media also hampered OPC maturation in culture, in keeping with a decrease in the expression of AST-secreted growth factors IGF-1, NRG-1, LIF, and CNTF. DFX AST mitochondrial morphology and number showed results similar to those observed in siDMT1 AST. In sum, our results show that ID, induced through two different methods, impacts AST maturation and mitochondrial functioning, which in turn hampers OPC differentiation.

Mesenchymal cells regulate enteric neural crest cell migration via RET-GFRA1b trans-signaling.

During early development, the enteric nervous system forms from the migration of enteric neural crest cells (ENCCs) from the foregut to the hindgut, where they undergo proliferation and differentiation facilitated by interactions with enteric mesenchymal cells (EMCs). This study investigates the impact on ENCC migration of EMC-ENCC communication mediated by GFRA1b expressed in EMCs. GFRA1-expressing cells in day 11-12 (E11-12) mouse embryos differentiated into smooth muscle cells from E12 onwards. Observations at E12-13.5 revealed high levels of GFRA1 expression on the anti-mesenteric side of the hindgut, correlating with enhanced ENCC migration. This indicates that GFRA1 in EMCs plays a role in ENCC migration during development. Examining GFRA1 isoforms, we found high levels of GFRA1b, which lacks amino acids 140-144, in EMCs. To assess the impact of GFRA1 isoforms on EMC-ENCC communication, we conducted neurosphere drop assays. This revealed that GFRA1b-expressing cells promoted GDNF-dependent extension and increased neurite density in ENCC neurospheres. Co-culture of ENCC mimetic cells expressing RET and GFRA1a with EMC mimetic cells expressing GFRA1a, GFRA1b, or vector alone showed that only GFRA1b-expressing co-cultured cells sustained RET phosphorylation in ENCC-mimetic cells for over 120 min upon GDNF stimulation. Our study provides evidence that GFRA1b-mediated cell-to-cell communication plays a critical role in ENCC motility in enteric nervous system development. These findings contribute to understanding the cellular interactions and signaling mechanisms that underlie enteric nervous system formation and highlight potential therapeutic targets for gastrointestinal motility disorders.

Comparison of techniques for corneal epithelium cell culture for the collection of conditioned medium.

PURPOSES: To determine the best protocol in obtaining the higher yield of conditioned culture medium to be used for the bone marrow mesenchymal stem cell differentiation into corneal epithelial cells, five techniques for the primary culture of human corneal epithelial cells were evaluated. METHODS: The studied culture techniques of corneal epithelial cells were: explants in culture flasks with and without hydrophilic surface treatment, on amniotic membrane, with enzymatic digestion, and by corneal scraping. The conditioned culture medium collected from these cultures was used to differentiate human bone marrow mesenchymal stem cells into corneal epithelial cells, which were characterized using flow cytometry with pan-cytokeratin and the corneal-specific markers, cytokeratin 3 and cytokeratin 12. RESULTS: The culture technique using flasks with hydrophilic surface treatment resulted in the highest yield of conditioned culture medium. Flasks without surface treatment resulted to a very low success rate. Enzymatic digestion and corneal scraping showed contamination with corneal fibroblasts. The culture on amniotic membranes only allowed the collection of culture medium during the 1st cell confluence. The effectiveness of cell differentiation was confirmed by cytometry analysis using the collected conditioned culture medium, as demonstrated by the expressions of cytokeratin 3 (95.3%), cytokeratin 12 (93.4%), and pan-cytokeratin (95.3%). CONCLUSION: The culture of corneal epithelial cell explants in flasks with hydrophilic surface treatment is the best technique for collecting a higher yield of conditioned culture medium to be used to differentiate mesenchymal stem cells.

Current perspectives on the multiple roles of osteoclasts: Mechanisms of osteoclast-osteoblast communication and potential clinical implications.

Bone remodeling is a complex process involving the coordinated actions of osteoblasts and osteoclasts to maintain bone homeostasis. While the influence of osteoblasts on osteoclast differentiation is well established, the reciprocal regulation of osteoblasts by osteoclasts has long remained enigmatic. In the past few years, a fascinating new role for osteoclasts has been unveiled in promoting bone formation and facilitating osteoblast migration to the remodeling sites through a number of different mechanisms, including the release of factors from the bone matrix following bone resorption and direct cell-cell interactions. Additionally, considerable evidence has shown that osteoclasts can secrete coupling factors known as clastokines, emphasizing the crucial role of these cells in maintaining bone homeostasis. Due to their osteoprotective function, clastokines hold great promise as potential therapeutic targets for bone diseases. However, despite long-standing work to uncover new clastokines and their effect in vivo, more substantial efforts are still required to decipher the mechanisms and pathways behind their activity in order to translate them into therapies. This comprehensive review provides insights into our evolving understanding of the osteoclast function, highlights the significance of clastokines in bone remodeling, and explores their potential as treatments for bone diseases suggesting future directions for the field.

An improved method for generating human spinal cord neural stem cells.

Neural stem cells have exhibited efficacy in pre-clinical models of spinal cord injury (SCI) and are on a translational path to human testing. We recently reported that neural stem cells must be driven to a spinal cord fate to optimize host axonal regeneration into sites of implantation in the injured spinal cord, where they subsequently form neural relays across the lesion that support significant functional improvement. We also reported methods of deriving and culturing human spinal cord neural stem cells derived from embryonic stem cells that can be sustained over serial high passage numbers in vitro, providing a potentially optimized cell source for human clinical trials. We now report further optimization of methods for deriving and sustaining cultures of human spinal cord neural stem cell lines that result in improved karyotypic stability while retaining anatomical efficacy in vivo. This development improves prospects for safe human translation.

In vitro platform to model the function of ionocytes in the human airway epithelium.

BACKGROUND: Pulmonary ionocytes have been identified in the airway epithelium as a small population of ion transporting cells expressing high levels of CFTR (cystic fibrosis transmembrane conductance regulator), the gene mutated in cystic fibrosis. By providing an infinite source of airway epithelial cells (AECs), the use of human induced pluripotent stem cells (hiPSCs) could overcome some challenges of studying ionocytes. However, the production of AEC epithelia containing ionocytes from hiPSCs has proven difficult. Here, we present a platform to produce hiPSC-derived AECs (hiPSC-AECs) including ionocytes and investigate their role in the airway epithelium. METHODS: hiPSCs were differentiated into lung progenitors, which were expanded as 3D organoids and matured by air-liquid interface culture as polarised hiPSC-AEC epithelia. Using CRISPR/Cas9 technology, we generated a hiPSCs knockout (KO) for FOXI1, a transcription factor that is essential for ionocyte specification. Differences between FOXI1 KO hiPSC-AECs and their wild-type (WT) isogenic controls were investigated by assessing gene and protein expression, epithelial composition, cilia coverage and motility, pH and transepithelial barrier properties. RESULTS: Mature hiPSC-AEC epithelia contained basal cells, secretory cells, ciliated cells with motile cilia, pulmonary neuroendocrine cells (PNECs) and ionocytes. There was no difference between FOXI1 WT and KO hiPSCs in terms of their capacity to differentiate into airway progenitors. However, FOXI1 KO led to mature hiPSC-AEC epithelia without ionocytes with reduced capacity to produce ciliated cells. CONCLUSION: Our results suggest that ionocytes could have role beyond transepithelial ion transport by regulating epithelial properties and homeostasis in the airway epithelium.

Dynamic Roles of Signaling Pathways in Maintaining Pluripotency of Mouse and Human Embryonic Stem Cells.

Culturing of mouse and human embryonic stem cells (ESCs) in vitro was a major breakthrough in the field of stem cell biology. These models gained popularity very soon mainly due to their pluripotency. Evidently, the ESCs of mouse and human origin share typical phenotypic responses due to their pluripotent nature, such as self-renewal capacity and potency. The conserved network of core transcription factors regulates these responses. However, significantly different signaling pathways and upstream transcriptional networks regulate expression and activity of these core pluripotency factors in ESCs of both the species. In fact, ample evidence shows that a pathway, which maintains pluripotency in mouse ESCs, promotes differentiation in human ESCs. In this review, we discuss the role of canonical signaling pathways implicated in regulation of pluripotency and differentiation particularly in mouse and human ESCs. We believe that understanding these distinct and at times-opposite mechanisms-is critical for the progress in the field of stem cell biology and regenerative medicine.

Isolation and Culture of Mouse Primary Microglia and Oligodendrocyte Precursor Cells.

Microglia and oligodendrocyte precursor cells (OPCs) are critical glia subsets in the central nervous system (CNS) and are actively engaged in a body of diseases, such as stroke, Alzheimer's disease, multiple sclerosis, etc. Microglia and OPC serve as compelling tools for the study of CNS diseases as well as the repair and damage of myelin sheath in vitro. In this protocol, we summarized a method which is capable of using the same batch of new-born mice to isolate and culture microglia and OPCs. It integrates the characteristics of practicality, convenience, and efficiency, providing a convenient, easy, and reliable technique for research.

Fluid shear force and hydrostatic pressure jointly promote osteogenic differentiation of BMSCs by activating YAP1 and NFAT2.

Natural bone tissue features a complex mechanical environment, with cells responding to diverse mechanical stimuli, including fluid shear stress (FSS) and hydrostatic pressure (HP). However, current in vitro experiments commonly employ a singular mechanical stimulus to simulate the mechanical environment in vivo. The understanding of the combined effects and mechanisms of multiple mechanical stimuli remains limited. Hence, this study constructed a mechanical stimulation device capable of simultaneously applying FSS and HP to cells. This study investigated the impact of FSS and HP on the osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs) and examined the distinctions and interactions between the two mechanisms. The results demonstrated that both FSS and HP individually enhanced the osteogenic differentiation of BMSCs, with a more pronounced effect observed through their combined application. BMSCs responded to external FSS and HP stimulation through the integrin-cytoskeleton and Piezo1 ion channel respectively. This led to the activation of downstream biochemical signals, resulting in the dephosphorylation and nuclear translocation of the intracellular transcription factors Yes Associated Protein 1 (YAP1) and nuclear factor of activated T cells 2 (NFAT2). Activated YAP1 could bind to NFAT2 to enhance transcriptional activity, thereby promoting osteogenic differentiation of BMSCs more effectively. This study highlights the significance of composite mechanical stimulation in BMSCs' osteogenic differentiation, offering guidance for establishing a complex mechanical environment for in vitro functional bone tissue construction.

How to differentiate induced pluripotent stem cells into sensory neurons for disease modelling: a functional assessment.

BACKGROUND: Human induced pluripotent stem cell (iPSC)-derived peripheral sensory neurons present a valuable tool to model human diseases and are a source for applications in drug discovery and regenerative medicine. Clinically, peripheral sensory neuropathies can result in maladies ranging from a complete loss of pain to severe painful neuropathic disorders. Sensory neurons are located in the dorsal root ganglion and are comprised of functionally diverse neuronal types. Low efficiency, reproducibility concerns, variations arising due to genetic factors and time needed to generate functionally mature neuronal populations from iPSCs remain key challenges to study human nociception in vitro. Here, we report a detailed functional characterization of iPSC-derived sensory neurons with an accelerated differentiation protocol ("Anatomic" protocol) compared to the most commonly used small molecule approach ("Chambers" protocol). Anatomic's commercially available RealDRG™ were further characterized for both functional and expression phenotyping of key nociceptor markers. METHODS: Multiple iPSC clones derived from different reprogramming methods, genetics, age, and somatic cell sources were used to generate sensory neurons. Manual patch clamp was used to functionally characterize both control and patient-derived neurons. High throughput techniques were further used to demonstrate that RealDRGs™ derived from the Anatomic protocol are amenable to high throughput technologies for disease modelling. RESULTS: The Anatomic protocol rendered a purer culture without the use of mitomycin C to suppress non-neuronal outgrowth, while Chambers differentiations yielded a mix of cell types. Chambers protocol results in predominantly tonic firing when compared to Anatomic protocol. Patient-derived nociceptors displayed higher frequency firing compared to control subject with both, Chambers and Anatomic differentiation approaches, underlining their potential use for clinical phenotyping as a disease-in-a-dish model. RealDRG™ sensory neurons show heterogeneity of nociceptive markers indicating that the cells may be useful as a humanized model system for translational studies. CONCLUSIONS: We validated the efficiency of two differentiation protocols and their potential application for functional assessment and thus understanding the disease mechanisms from patients suffering from pain disorders. We propose that both differentiation methods can be further exploited for understanding mechanisms and development of novel treatments in pain disorders.

RIO-kinase 2 is essential for hematopoiesis.

Regulation of protein synthesis is a key factor in hematopoietic stem cell maintenance and differentiation. Rio-kinase 2 (RIOK2) is a ribosome biogenesis factor that has recently been described an important regulator of human blood cell development. Additionally, we have previously identified RIOK2 as a regulator of protein synthesis and a potential target for the treatment of acute myeloid leukemia (AML). However, its functional relevance in several organ systems, including normal hematopoiesis, is not well understood. Here, we investigate the consequences of RIOK2 loss on normal hematopoiesis using two different conditional knockout mouse models. Using competitive and non-competitive bone marrow transplantations, we demonstrate that RIOK2 is essential for the differentiation of hematopoietic stem and progenitor cells (HSPCs) as well as for the maintenance of fully differentiated blood cells in vivo as well as in vitro. Loss of RIOK2 leads to rapid death in full-body knockout mice as well as mice with RIOK2 loss specific to the hematopoietic system. Taken together, our results indicate that regulation of protein synthesis and ribosome biogenesis by RIOK2 is essential for the function of the hematopoietic system.

[Mitochondrial metabolism and erythroid differentiation].

The transcription factor GATA-1 is essential for erythroid differentiation. Recently, FAM210B, which encodes a mitochondrial inner membrane protein, has been identified as a novel target of GATA-1. To clarify the role of FAM210B, we depleted endogenous FAM210B in human iPS-derived erythroid progenitor (HiDEP-1) cells, and found that erythroid differentiation was more pronounced in the FAM210B depleted cells. Comprehensive metabolite analysis revealed a decline in mitochondrial function accompanied by increased lactate production, indicative of anaerobic glycolysis. Mass spectrometry revealed that FAM210B could interact with multiple subunits of mitochondrial ATP synthases, such as subunit alpha (ATP5A) and beta (ATP5B). Our results suggested that FAM210B contributes prominently to erythroid differentiation by regulating mitochondrial energy metabolism. This review will discuss the potential association between mitochondrial metabolism and erythropoiesis.

IL-33-ST2 signaling in fibro-adipogenic progenitors alleviates immobilization-induced muscle atrophy in mice.

BACKGROUND: The regenerative and adaptive capacity of skeletal muscles reduces with age, leading to severe disability and frailty in the elderly. Therefore, development of effective therapeutic interventions for muscle wasting is important both medically and socioeconomically. In the present study, we aimed to elucidate the potential contribution of fibro-adipogenic progenitors (FAPs), which are mesenchymal stem cells in skeletal muscles, to immobilization-induced muscle atrophy. METHODS: Young (2-3 months), adult (12-14 months), and aged (20-22 months) mice were used for analysis. Muscle atrophy was induced by immobilizing the hind limbs with a steel wire. FAPs were isolated from the hind limbs on days 0, 3, and 14 after immobilization for transcriptome analysis. The expression of ST2 and IL-33 in FAPs was evaluated by flow cytometry and immunostaining, respectively. To examine the role of IL-33-ST2 signaling in vivo, we intraperitoneally administered recombinant IL-33 or soluble ST2 (sST2) twice a week throughout the 2-week immobilization period. After 2-week immobilization, the tibialis anterior muscles were harvested and the cross-sectional area of muscle fibers was evaluated. RESULTS: The number of FAPs increased with the progression of muscle atrophy after immobilization in all age-groups. Transcriptome analysis of FAPs collected before and after immobilization revealed that Il33 and Il1rl1 transcripts, which encode the IL-33 receptor ST2, were transiently induced in young mice and, to a lesser extent, in aged mice. The number of FAPs positive for ST2 increased after immobilization in young mice. The number of ST2-positive FAPs also increased after immobilization in aged mice, but the difference from the baseline was not statistically significant. Immunostaining for IL-33 in the muscle sections revealed a significant increase in the number of FAPs expressing IL-33 after immobilization. Administration of recombinant IL-33 suppressed immobilization-induced muscle atrophy in aged mice but not in young mice. CONCLUSIONS: Our data reveal a previously unknown protective role of IL-33-ST2 signaling against immobilization-induced muscle atrophy in FAPs and suggest that IL-33-ST2 signaling is a potential new therapeutic target for alleviating disuse muscle atrophy, particularly in older adults.

Autophagy and Exocytosis of Lipofuscin Into the Basolateral Extracellular Space of Human Retinal Pigment Epithelium From Fetal Development to Adolescence.

Purpose: To undertake the first ultrastructural characterization of human retinal pigment epithelial (RPE) differentiation from fetal development to adolescence. Methods: Ten fetal eyes and three eyes aged six, nine, and 17 years were examined in the temporal retina adjacent to the optic nerve head by transmission electron microscopy. The area, number, and distribution of RPE organelles were quantified and interpreted within the context of adjacent photoreceptors, Bruch's membrane, and choriocapillaris maturation. Results: Between eight to 12 weeks' gestation (WG), pseudostratified columnar epithelia with apical tight junctions differentiate to a simple cuboidal epithelium with random distribution of melanosomes and mitochondria. Between 12 to 26 WG, cells enlarge and show long apical microvilli and apicolateral junctional complexes. Coinciding with eye opening at 26 WG, melanosomes migrate apically whereas mitochondria distribute to perinuclear regions, with the first appearance of phagosomes, complex granules, and basolateral extracellular space (BES) formation. Significantly, autophagy and heterophagy, as evidenced by organelle recycling, and the gold standard of ultrastructural evidence for autophagy of double-membrane autophagosomes and mitophagosomes were evident from 32 WG, followed by basal infoldings of RPE cell membrane at 36 WG. Lipofuscin formation and deposition into the BES evident at six years increased at 17 years. Conclusions: We provide compelling ultrastructural evidence that heterophagy and autophagy begins in the third trimester of human fetal development and that deposition of cellular byproducts into the extracellular space of RPE takes place via exocytosis. Transplanted RPE cells must also demonstrate the capacity to subserve autophagic and heterophagic functions for effective disease mitigation.