Efficacy of injection of autologous adipose tissue in the treatment of patients with complex and recurrent fistula-in-ano of cryptoglandular origin.

BACKGROUND: Adipose tissue injections, a rich source of mesenchymal stem cells, have been successfully used to promote anal fistula healing. This study aimed to investigate the efficacy of adipose tissue injection in treating patients with complex and recurrent fistulas of cryptoglandular origin. METHODS: We conducted a prospective, single-center, open-label, non-randomized, interventional clinical trial from January 2020 to December 2022. We enrolled nine patients, who were evaluated after at least 12 months of follow-up. All patients had seton removal, fistula tract excision or curettage, and a mucosal flap if possible or, alternatively, an internal opening suture. We used a commercially available system to collect and process adipose tissue prior to injection. This system allowed the collection, microfragmentation, and filtration of tissue. RESULTS: Selected cases included six men and three women with a median age of 42 (range 31-55) years. All patients had an extended disease course period, ranging from 3 to 13 (mean 6.6) years, and a history of multiple previous surgeries, including two to eight interventions (a mean of 4.4 per case). All fistulas were high transsphincteric, four cases horseshoe and two cases with secondary suprasphincteric or peri-elevator tract fistulas. Six cases (66%) achieved complete fistula healing at a mean follow-up of 18 (range 12-36) months. Three cases (33.3%) experienced reduced secretion and decreased anal discomfort. CONCLUSIONS: In patients with complex and recurrent fistulas, such as the ones described, many from palliative treatments with setons, the adjuvant injection of adipose tissue might help achieve complete healing or improvement in a significant percentage of cases. CLINICALTRIALS: The study protocol was prospectively registered on ClinicalTrials.gov (NCT04750499).

Platelet-rich fibrin (PRF) modified nano-hydroxyapatite/chitosan/gelatin/alginate scaffolds increase adhesion and viability of human dental pulp stem cells (DPSC) and osteoblasts derived from DPSC.

Bone tissue regeneration strategies have incorporated the use of natural polymers, such as hydroxyapatite (nHA), chitosan (CH), gelatin (GEL), or alginate (ALG). Additionally, platelet concentrates, such as platelet-rich fibrin (PRF) have been suggested to improve scaffold biocompatibility. This study aimed to develop scaffolds composed of nHA, GEL, and CH, with or without ALG and lyophilized PRF, to evaluate the scaffold's properties, growth factor release, and dental pulp stem cells (DPSC), and osteoblast (OB) derived from DPSC viability. Four scaffold variations were synthesized and lyophilized. Then, degradation, swelling profiles, and morphological analysis were performed. Furthermore, PDGF-BB and FGF-B growth factors release were quantified by ELISA, and cytotoxicity and cell viability were evaluated. The swelling and degradation profiles were similar in all scaffolds, with pore sizes ranging between 100 and 250 µm. FGF-B and PDGF-BB release was evidenced after 24 h of scaffold immersion in cell culture medium. DPSC and OB-DPSC viability was notably increased in PRF-supplemented scaffolds. The nHA-CH-GEL-PRF scaffold demonstrated optimal physical-biological characteristics for stimulating DPSC and OB-DPSC cell viability. These results suggest lyophilized PRF improves scaffold biocompatibility for bone tissue regeneration purposes.

HPV8-induced STAT3 activation led keratinocyte stem cell expansion in human actinic keratoses.

Despite epidermal turnover, the skin is host to a complex array of microbes including viruses, such as the human papillomavirus (HPV), which must infect and manipulate skin keratinocyte stem cells (KSC) to survive. This crosstalk between the virome and KSC populations remains largely unknown. Here, we investigated the effect of HPV8 on KSCs using various mouse models. We observed that the HPV8 early region gene E6 specifically caused Lrig1+ hair follicle junctional zone KSC proliferation and expansion, which would facilitate viral transmission. Within Lrig1+ KSCs specifically, HPV8 E6 bound intracellular p300 to phosphorylate the STAT3 transcriptional regulatory node. This induces ΔNp63 expression, resulting in KSC expansion into the overlying epidermis. HPV8 was associated with 70% of human actinic keratoses (AK). Together these results define the "hit and run" mechanism for HPV8 in human actinic keratosis as an expansion of KSCs, which lacks melanosome protection and is thus susceptible to sun-light-induced malignant transformation.

Molecular cascade reveals sequential milestones underlying hippocampal neural stem cell development into an adult state.

Quiescent adult neural stem cells (NSCs) in the mammalian brain arise from proliferating NSCs during development. Beyond acquisition of quiescence, an adult NSC hallmark, little is known about the process, milestones, and mechanisms underlying the transition of developmental NSCs to an adult NSC state. Here, we performed targeted single-cell RNA-seq analysis to reveal the molecular cascade underlying NSC development in the early postnatal mouse dentate gyrus. We identified two sequential steps, first a transition to quiescence followed by further maturation, each of which involved distinct changes in metabolic gene expression. Direct metabolic analysis uncovered distinct milestones, including an autophagy burst before NSC quiescence acquisition and cellular reactive oxygen species level elevation along NSC maturation. Functionally, autophagy is important for the NSC transition to quiescence during early postnatal development. Together, our study reveals a multi-step process with defined milestones underlying establishment of the adult NSC pool in the mammalian brain.

Isthmus progenitor cells contribute to homeostatic cellular turnover and support regeneration following intestinal injury.

The currently accepted intestinal epithelial cell organization model proposes that Lgr5+ crypt-base columnar (CBC) cells represent the sole intestinal stem cell (ISC) compartment. However, previous studies have indicated that Lgr5+ cells are dispensable for intestinal regeneration, leading to two major hypotheses: one favoring the presence of a quiescent reserve ISC and the other calling for differentiated cell plasticity. To investigate these possibilities, we studied crypt epithelial cells in an unbiased fashion via high-resolution single-cell profiling. These studies, combined with in vivo lineage tracing, show that Lgr5 is not a specific ISC marker and that stemness potential exists beyond the crypt base and resides in the isthmus region, where undifferentiated cells participate in intestinal homeostasis and regeneration following irradiation (IR) injury. Our results provide an alternative model of intestinal epithelial cell organization, suggesting that stemness potential is not restricted to CBC cells, and neither de-differentiation nor reserve ISC are drivers of intestinal regeneration.

Human Papillomavirus Carcinogenicity and the Need of New Perspectives: Thoughts from a Retrospective Analysis on Human Papillomavirus Outcomes Conducted at the Hospital University of Bari, Apulia, Italy, between 2011 and 2022.

BACKGROUND: The current manuscript's aim was to determine the human papillomavirus (HPV) genotype-specific prevalence and distribution among individuals, males, and females, of different ages in the region of Apulia, Italy, highlighting the possible variables involved in the carcinogenicity mechanism. In addition, we proposed two hypothetical models of HPV's molecular dynamics, intending to clarify the impact of prevention and therapeutic strategies, explicitly modeled by recent survey data. METHODS: We presented clinical data from 9647 participants tested for either high-risk (HR) or low-risk (LR) HPV at the affiliated Bari Policlinic University Hospital of Bari from 2011 to 2022. HPV DNA detection was performed using nested-polymerase chain reaction (PCR) and multiplex real-time PCR assay. Statistical analysis showed significant associations for all genders and ages and both HR- and LR-HPV types. A major number of significant pairwise associations were detected for the higher-risk types and females and lower-risk types and males. RESULTS: The overall prevalence of HPV was 50.5% (n-4.869) vs. 49.5% (n-4.778) of the study population, of which 74.4% (n-3621) were found to be HPV high-risk (HR-HPV) genotypes and 57.7% (n-2.807) low-risk HPV (LR-HPV) genotypes, of which males were 58% and females 49%; the three most prevalent HR-HPV genotypes were HPV 53 (n707-15%), 16 (n704-14%), and 31 (n589-12%), and for LR-HPV, they were 42 (19%), 6 (16%), and 54 (13%); 56% of patients screened for HPV were ≤ 30 years old, 53% were between 31 and 40 years old, 46% were 41-50 and 51-60 years old, and finally, 44% of subjects were >60 years old. CONCLUSIONS: Our study provided comprehensive epidemiological data on HPV prevalence and genotype distribution among 9647 participants, which could serve as a significant reference for clinical practice, and it implied the necessity for more effective screening methods for HPV carcinogenesis covering the use of more specific molecular investigations. Although this is a predominantly descriptive and epidemiological study, the data obtained offer not only a fairly unique trend compared to other studies of different realities and latitudes but also lead us to focus on the HPV infection within two groups of young people and adults and hypothesize the possible involvement of dysbiosis, stem cells, and the retrotransposition mechanism.

Differential Colonization and Mucus Ultrastructure Visualization in Bovine Ileal and Rectal Organoid-Derived Monolayers Exposed to Enterohemorrhagic Escherichia coli.

Enterohemorrhagic Escherichia coli (EHEC) is a critical public health concern due to its role in severe gastrointestinal illnesses in humans, including hemorrhagic colitis and the life-threatening hemolytic uremic syndrome. While highly pathogenic to humans, cattle, the main reservoir for EHEC, often remain asymptomatic carriers, complicating efforts to control its spread. Our study introduces a novel method to investigate EHEC using organoid-derived monolayers from adult bovine ileum and rectum. These polarized epithelial monolayers were exposed to EHEC for four hours, allowing us to perform comparative analyses between the ileal and rectal tissues. Our findings mirrored in vivo observations, showing a higher colonization rate in the rectum compared with the ileum (44.0% vs. 16.5%, p < 0.05). Both tissues exhibited an inflammatory response with increased expression levels of TNF-a (p < 0.05) and a more pronounced increase of IL-8 in the rectum (p < 0.01). Additionally, the impact of EHEC on the mucus barrier varied across these gastrointestinal regions. Innovative visualization techniques helped us study the ultrastructure of mucus, revealing a net-like mucin glycoprotein organization. While further cellular differentiation could enhance model accuracy, our research significantly deepens understanding of EHEC pathogenesis in cattle and informs strategies for the preventative measures and therapeutic interventions.

Pathogen-epithelium interactions and inflammatory responses in Salmonella Dublin infections using ileal monolayer models derived from adult bovine organoids.

Salmonella enterica serovar Dublin (S. Dublin) is an important enteric pathogen affecting cattle and poses increasing public health risks. Understanding the pathophysiology and host-pathogen interactions of S. Dublin infection are critical for developing effective control strategies, yet studies are hindered by the lack of physiologically relevant in vitro models. This study aimed to generate a robust ileal monolayer derived from adult bovine organoids, validate its feasibility as an in vitro infection model with S. Dublin, and evaluate the epithelial response to infection. A stable, confluent monolayer with a functional epithelial barrier was established under optimized culture conditions. The model's applicability for studying S. Dublin infection was confirmed by documenting intracellular bacterial invasion and replication, impacts on epithelial integrity, and a specific inflammatory response, providing insights into the pathogen-epithelium interactions. The study underscores the utility of organoid-derived monolayers in advancing our understanding of enteric infections in livestock and highlights implications for therapeutic strategy development and preventive measures, with potential applications extending to both veterinary and human medicine. The established bovine ileal monolayer offers a novel and physiologically relevant in vitro platform for investigating enteric pathogen-host interactions, particularly for pathogens like S. Dublin.

Generation, Expansion, and Biobanking of Gastrointestinal Patient-Derived Organoids from Tumor and Normal Tissues.

Patient-derived organoids (PDOs) generated from adult stem cells present in tissues are invaluable tools for translational cancer research (Drost, Clevers, Nat Rev Cancer 18(7):407-418, 2018). The generation of this 3D cultures is not trivial and requires dedicated procedures. Despite the rapid increase in the use of organoids in cancer research, it is noteworthy that published procedures regarding their generation often lack critical information and standardized protocols remain elusive. Addressing these limitations, the protocol described in this chapter offers an in-depth and comprehensive guide to establishing, expanding, and freezing gastrointestinal PDOs obtained from normal and tumor tissue biopsies. Notably, it also provides valuable insights in the form of tips and tricks to guide and overcome potential challenges that may arise during the procedure.

Phase separation of SHP2E76K promotes malignant transformation of mesenchymal stem cells by activating mitochondrial complexes.

Mesenchymal stem cells (MSCs), suffering from diverse gene hits, undergo malignant transformation and aberrant osteochondral differentiation. Src homology region 2-containing protein tyrosine phosphatase 2 (SHP2), a nonreceptor protein tyrosine phosphatase, regulates multicellular differentiation, proliferation, and transformation. However, the role of SHP2 in MSC fate determination remains unclear. Here, we showed that MSCs bearing the activating SHP2E76K mutation underwent malignant transformation into sarcoma stem-like cells. We revealed that the SHP2E76K mutation in mouse MSCs led to hyperactive mitochondrial metabolism by activating mitochondrial complexes I and III. Inhibition of complexes I and III prevented hyperactive mitochondrial metabolism and malignant transformation of SHP2E76K MSCs. Mechanistically, we verified that SHP2 underwent liquid-liquid phase separation (LLPS) in SHP2E76K MSCs. SHP2 LLPS led to its dissociation from complexes I and III, causing their hyperactivation. Blockade of SHP2 LLPS by LLPS-defective mutations or allosteric inhibitors suppressed complex I and III hyperactivation as well as malignant transformation of SHP2E76K MSCs. These findings reveal that complex I and III hyperactivation driven by SHP2 LLPS promotes malignant transformation of SHP2E76K MSCs and suggest that inhibition of SHP2 LLPS could be a potential therapeutic target for the treatment of activated SHP2-associated cancers.

Neuropeptide substance P alters stem cell fate to aid wound healing and promote epidermal stratification through asymmetric stem cell divisions.

Loss of sensory innervation delays wound healing and administration of the neuropeptide substance P improves re-epithelialization. Keratinocyte hyperproliferation post-wounding may result from symmetric stem cell (SC) self-renewal, asymmetric SC self-renewal, committed progenitor divisions, or a combination of these. However, the effects of sensory denervation and of neuropeptides on SC proliferation are not known. Here we show that early after wounding both asymmetric and symmetric SC self-renewal increase, without significant committed progenitor (CP) activation. Decreased sensory innervation is associated with a decrease in both SC and CP proliferation. Based on previous work showing that substance P is decreased in capsaicin-treated mice and improves wound healing in normal skin, we examined the effects of substance P on SC and CP proliferation during wound healing. Substance P restored asymmetric SC proliferation in skin with decreased sensory innervation, both at baseline and following wounding. Epidermis with decreased sensory innervation was severely thinned. Consistent with this, substance P-induced asymmetric SC proliferation resulted in increased stratification in skin with both normal and decreased innervation. Lapatinib prevented the substance P-induced increase in asymmetric SC divisions in murine epidermis, as well as the increase in epidermal stratification, suggesting that asymmetric SC divisions are required for epidermal stratification.

The nuclei of human adult stem cells can move within the cell and generate cellular protrusions to contact other cells.

BACKGROUND: The neuronal transdifferentiation of adult bone marrow cells (BMCs) is still considered an artifact based on an alternative explanation of experimental results supporting this phenomenon obtained over decades. However, recent studies have shown that following neural induction, BMCs enter an intermediate cellular state before adopting neural-like morphologies by active neurite extension and that binucleated BMCs can be formed independent of any cell fusion events. These findings provide evidence to reject the idea that BMC neural transdifferentiation is merely an experimental artifact. Therefore, understanding the intermediate states that cells pass through during transdifferentiation is crucial given their potential application in regenerative medicine and disease modelling. METHODS: In this study, we examined the functional significance of the variety of morphologies and positioning that cell nuclei of human bone marrow-derived mesenchymal stem cells (hBM-MSCs) can adopt during neural-like differentiation using live-cell nuclear fluorescence labelling, time-lapse microscopy, and confocal microscopy analysis. RESULTS: Here, we showed that after neural induction, hBM-MSCs enter an intermediate cellular state in which the nuclei are able to move within the cells, switching shapes and positioning and even generating cellular protrusions as they attempt to contact the cells around them. These findings suggest that changes in nuclear positioning occur because human cell nuclei somehow sense their environment. In addition, we showed the process of direct interactions between cell nuclei, which opens the possibility of a new level of intercellular interaction. CONCLUSIONS: The present study advances the understanding of the intermediate stage through which hBM-MSCs pass during neural transdifferentiation, which may be crucial to understanding the mechanisms of these cell conversion processes and eventually harness them for use in regenerative medicine. Importantly, our study provides for the first time evidence that the nuclei of hBM-MSC-derived intermediate cells somehow sense their environment, generating cellular protrusions to contact other cells. In summary, human mesenchymal stromal cells could not only help to increase our understanding of the mechanisms underlying cellular plasticity but also facilitate the exact significance of nuclear positioning in cellular function and in tissue physiology.

Clinical Evaluation of Safety and Efficacy of a Central Current Good Manufacturing Practices Laboratory Produced Autologous Adipose-Derived Stromal Vascular Fraction Cell Therapy Product for the Treatment of Knee Osteoarthritis.

Knee osteoarthritis (KOA) is a prevalent condition characterized by the progressive deterioration of the entire joint and has emerged as a prominent contributor to disability on a global scale. The nature of the disease and its impact on joint function significantly limit mobility and daily activities, highlighting its substantial influence on patients' overall well-being. Stromal vascular fraction (SVF) is a heterogenous, autologous cell product, containing mesenchymal stem cells, derived from the patient's subcutaneous adipose tissue with demonstrated safety and efficacy in the treatment of KOA patients. We conducted a single-arm, open-label, multisite, FDA approved clinical study in Kellgren-Lawrence severity grade 2-4 KOA patients. The cellular product was manufactured from patient-specific lipoaspirate in a centrally located FDA-compliant manufacturing facility. Twenty-nine subjects were treated with a quality tested single intra-articular injection of GMP manufactured SVF. Adverse events, laboratory values, vital signs, and physical examination findings were monitored during the study period. Robust tolerability, without any substantial safety issues, was demonstrated. Knee pain and function, assessed through the Knee Injury and Osteoarthritis Outcome Score (KOOS), demonstrated notable improvements. These positive benefits persisted for up to 12 months, and the majority of participants expressed satisfaction. SVF from each patient was stored in a liquid nitrogen freezer for future clinical treatments. Unique to this study of autologous cells is the shipment of lipoaspirate from the clinic to a central FDA-compliant manufacturing facility for cleanroom-controlled manufacturing. The cell product characterization data demonstrate that this method produces an equivalent product in terms of cell count and viability with the added benefit of further quality assurance testing, including sterility, endotoxin, and flow cytometry, before patient administration. Clinical Trial Registration Number: NCT04043819.

Connexin43 in mesenchymal lineage cells regulates body adiposity and energy metabolism in mice.

Connexin43 (Cx43) is the most abundant gap junction protein present in the mesenchymal lineage. In mature adipocytes, Cx43 mediates white adipose tissue (WAT) beiging in response to cold exposure and maintains the mitochondrial integrity of brown adipose tissue (BAT). We found that genetic deletion of Gja1 (Cx43 gene) in cells that give rise to chondro-osteogenic and adipogenic precursors driven by the Dermo1/Twist2 promoter led to lower body adiposity and partial protection against the weight gain and metabolic syndrome induced by a high-fat diet (HFD) in both sexes. These protective effects were related to increased locomotion, fuel utilization, energy expenditure, nonshivering thermogenesis, and better glucose tolerance in conditionally Gja1-ablated mice. Accordingly, Gja1-mutant mice exhibited reduced adipocyte hypertrophy, partially preserved insulin sensitivity, increased BAT lipolysis, and decreased whitening under HFD. This metabolic phenotype was not reproduced with more restricted Gja1 ablation in differentiated adipocytes, suggesting that Cx43 in adipocyte progenitors or other targeted cells restrains energy expenditures and promotes fat accumulation. These results reveal what we believe is a hitherto unknown action of Cx43 in adiposity, and offer a promising new pharmacologic target for improving metabolic balance in diabetes and obesity.

Seizure-induced LIN28A disrupts pattern separation via aberrant hippocampal neurogenesis.

Prolonged seizures can disrupt stem cell behavior in the adult hippocampus, an important brain structure for spatial memory. Here, using a mouse model of pilocarpine-induced status epilepticus (SE), we characterized spatiotemporal expression of Lin28a mRNA and proteins after SE. Unlike Lin28a transcripts, induction of LIN28A protein after SE was detected mainly in the subgranular zone, where immunoreactivity was found in progenitors, neuroblasts, and immature and mature granule neurons. To investigate roles of LIN28A in epilepsy, we generated Nestin-Cre:Lin28aloxP/loxP (conditional KO [cKO]) and Nestin-Cre:Lin28a+/+ (WT) mice to block LIN28A upregulation in all neuronal lineages after acute seizure. Adult-generated neuron- and hippocampus-associated cognitive impairments were absent in epileptic LIN28A-cKO mice, as evaluated by pattern separation and contextual fear conditioning tests, respectively, while sham-manipulated WT and cKO animals showed comparable memory function. Moreover, numbers of hilar PROX1-expressing ectopic granule cells (EGCs), together with PROX1+/NEUN+ mature EGCs, were significantly reduced in epileptic cKO mice. Transcriptomics analysis and IHC validation at 3 days after pilocarpine administration provided potential LIN28A downstream targets such as serotonin receptor 4. Collectively, our findings indicate that LIN28A is a potentially novel target for regulation of newborn neuron-associated memory dysfunction in epilepsy by modulating seizure-induced aberrant neurogenesis.

In vivo confocal microscopy evaluation of infiltrated immune cells in corneal stroma treated with cell therapy in advanced keratoconus.

PURPOSE: This study investigates immune cell (ICs) infiltration in advanced keratoconus patients undergoing autologous adipose-derived adult stem cell (ADASC) therapy with recellularized human donor corneal laminas (CL). METHODS: A prospective clinical trial included fourteen patients divided into three groups: G-1, ADASCs; G-2, decellularized CL (dCL); and G-3, dCL recellularized with ADASCs (ADASCs-rCL). Infiltrated ICs were assessed using in vivo confocal microscopy (IVCM) at 1,3,6, and12 months post-transplant. RESULTS: Infiltrated ICs, encompassing granulocytes and agranulocytes, were observed across all groups, categorized by luminosity, structure, and area. Stromal ICs infiltration ranged from 1.19% to 6.62%, with a consistent increase in group-related cell density (F = 10.68, P < .0001), independent of post-op time (F = 0.77, P = 0.511); the most substantial variations were observed in G-3 at 6 and 12 months (2.0 and 1.87-fold, respectively). Similarly, significant size increases were more group-dependent (F = 5.76, P < .005) rather than time-dependent (F = 2.84, P < .05); G-3 exhibited significant increases at 6 and 12 months (3.70-fold and 2.52-fold, respectively). A lamina-induced shift in IC size occurred (F = 110.23, P < .0001), primarily with 50-100 µm2 sizes and up to larger cells > 300µm2, presumably macrophages, notably in G-3, indicating a potential role in tissue repair and remodeling, explaining reductions in cells remnants < 50µm2. CONCLUSIONS: ADASCs-rCL therapy may lead to increased IC infiltration compared to ADASCs alone, impacting cell distribution and size due to the presence of the lamina. The findings reveal intricate immune patterns shaped by the corneal microenvironment and highlight the importance of understanding immune responses for the development of future therapeutic strategies.

Three-Dimensional Cell Culture of Epididymal Basal Cells and Organoids: A Novel Tool for Toxicology.

Spermatozoa are formed in the testis but must transit through the epididymis to acquire motility and the ability to fertilize. The epididymis is a single convoluted tubule comprising several anatomically and physiologically distinct regions. The pseudostratified epithelium consists of multiple cell types, including principal cells, clear cells, narrow cells, and apical cells, that line the lumen of the epididymis. Basal cells are present at the base of the epithelium, and halo cells, which includes macrophages/monocytes, mononuclear phagocytes, and T lymphocytes, are also present in the epithelium. Several aspects of this complex spermatozoan maturation process are well established, but a great deal remains poorly understood. Given that dysfunction of the epididymis has been associated with male infertility, in vitro tools to study epididymal function and epididymal sperm maturation are required. Our lab and others have previously developed human, rat, and mouse epithelial principal cell lines, which have been used to address certain questions, such as about the regulation of junctional proteins in the epididymis, as well as the toxicity of nonylphenols. Given that the epididymal epithelium comprises multiple cell types, however, a 3D in vitro model provides a more comprehensive and realistic tool that can be used to study and elucidate the multiple aspects of epididymal function. The purpose of this article is to provide detailed information regarding the preparation, maintenance, passaging, and immunofluorescent staining of rat epididymal organoids derived from adult basal cells, which we have demonstrated to be a type of adult stem cell in the rat epididymis. © 2024 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Isolation of epididymal cells Basic Protocol 2: Magnetic activated cell sorting and isolation of basal cells Basic Protocol 3: Preparation and culture of epididymal basal cell organoids Basic Protocol 4: Passage of epididymal basal cell organoids Basic Protocol 5: Freezing and thawing of epididymal basal cell organoids Basic Protocol 6: Immunofluorescent staining of epididymal basal cell organoids.

Adult stem cells in the eye: Identification, characterisation, and therapeutic application in ocular regeneration - A review.

Adult stem cells, present in various parts of the human body, are undifferentiated cells that can proliferate and differentiate to replace dying cells within tissues. Stem cells have specifically been identified in the cornea, trabecular meshwork, crystalline lens, iris, ciliary body, retina, choroid, sclera, conjunctiva, eyelid, lacrimal gland, and orbital fat. The identification of ocular stem cells broadens the potential therapeutic strategies for untreatable eye diseases. Currently, stem cell transplantation for corneal and conjunctival diseases remains the most common stem cell-based therapy in ocular clinical management. Lens epithelial stem cells have been applied in the treatment of paediatric cataracts. Several early-phase clinical trials for corneal and retinal regeneration using ocular stem cells are also underway. Extensive preclinical studies using ocular stem cells have been conducted, showing encouraging outcomes. Ocular stem cells currently demonstrate great promise in potential treatments of eye diseases. In this review, we focus on the identification, characterisation, and therapeutic application of adult stem cells in the eye.

Bioengineering and vascularization strategies for islet organoids: advancing toward diabetes therapy.

Diabetes presents a pressing healthcare crisis, necessitating innovative solutions. Organoid technologies have rapidly advanced, leading to the emergence of bioengineering islet organoids as an unlimited source of insulin-producing cells for treating insulin-dependent diabetes. This advancement surpasses the need for cadaveric islet transplantation. However, clinical translation of this approach faces two major limitations: immature endocrine function and the absence of a perfusable vasculature compared to primary human islets. In this review, we summarize the latest developments in bioengineering functional islet organoids in vitro and promoting vascularization of organoid grafts before and after transplantation. We highlight the crucial roles of the vasculature in ensuring long-term survival, maturation, and functionality of islet organoids. Additionally, we discuss key considerations that must be addressed before clinical translation of islet organoid-based therapy, including functional immaturity, undesired heterogeneity, and potential tumorigenic risks.

Hallmarks of stemness in mammalian tissues.

All adult tissues experience wear and tear. Most tissues can compensate for cell loss through the activity of resident stem cells. Although the cellular maintenance strategies vary greatly between different adult (read: postnatal) tissues, the function of stem cells is best defined by their capacity to replace lost tissue through division. We discuss a set of six complementary hallmarks that are key enabling features of this basic function. These include longevity and self-renewal, multipotency, transplantability, plasticity, dependence on niche signals, and maintenance of genome integrity. We discuss these hallmarks in the context of some of the best-understood adult stem cell niches.