Stem Cell Activity Research Articles

Locally released H2 and O2 from an artificial periosteum modulate the survival and reparative activities of stem cells derived from the aged human nasal mucosa after transplantation

Stem cell therapies are limited by poor cell survival and engraftment. Elderly diabetic patients (EDPs) have a greater risk of bone graft failure. Herein, a g-C3N4/polydopamine (PDA@g-C3N4) composite nanosheet was successfully fabricated via in situ polymerization of PDA on a g-C3N4 surface. Then, the PDA/g-C3N4 composite was crosslinked with a fibrin hydrogel to prepare an artificial periosteum for EDPs. With the help of NIR, we showed that the synthetic artificial periosteum released H2 and O2 and enhanced the survival and osteogenic differentiation of aged human nasal mucosa-derived ectodermal stem cells (hEMSCs) in microenvironments characterized by oxidative stress and hypoxia. The artificial periosteum had pleiotropic effects on aged hEMSC reparative activities, modulating cytokine secretion and the protein expression of effectors associated with vascularization, and bone formation in vivo. Our findings imply that the developed artificial periosteum loaded with hEMSCs has great promise for the repair of bone defects in EDPs.

Tracking of Nascent Matrix Deposition during Muscle Stem Cell Activation across Lifespan Using Engineered Hydrogels.

Adult stem cells occupy a niche that contributes to their function, but how stem cells rebuild their microenvironment after injury remains an open-ended question. Herein, biomaterial-based systems and metabolic labeling are utilized to evaluate how skeletal muscle stem cells deposit extracellular matrix. Muscle stem cells and committed myoblasts are observed to generate less nascent matrix than muscle resident fibro-adipogenic progenitors. When cultured on substrates that matched the stiffness of physiological uninjured and injured muscles, muscle stem cells increased nascent matrix deposition with activation kinetics. Reducing the ability to deposit nascent matrix by an inhibitor of vesicle trafficking (Exo-1) attenuated muscle stem cell function and mimicked impairments observed from muscle stem cells isolated from old muscles. Old muscle stem cells are observed to deposit less nascent matrix than young muscle stem cells, which is rescued with therapeutic supplementation of insulin-like growth factors. These results highlight the role of nascent matrix production with muscle stem cell activation.

Viscoelastic hydrogels regulate adipose-derived mesenchymal stem cells for nucleus pulposus regeneration

Low back pain is a leading cause of disability worldwide, often attributed to intervertebral disc (IVD) degeneration with loss of the functional nucleus pulposus (NP). Regenerative strategies utilizing biomaterials and stem cells are promising for NP repair. Human NP tissue is highly viscoelastic, relaxing stress rapidly under deformation. However, the impact of tissue-specific viscoelasticity on the activities of adipose-derived stem cells (ASC) remains largely unexplored. Here, we investigated the role of matrix viscoelasticity in regulating ASC differentiation for IVD regeneration. Viscoelastic alginate hydrogels with stress relaxation time scales ranging from 100 s to 1000s were developed and used to culture human ASCs for 21 days. Our results demonstrated that the fast-relaxing hydrogel significantly enhanced ASCs long-term cell survival and NP-like extracellular matrix secretion of aggrecan and type-II collagen. Moreover, gene expression analysis revealed a substantial upregulation of the mechanosensitive ion channel marker TRPV4 and NP-specific markers such as SOX9, HIF-1α, KRT18, CDH2 and CD24 in ASCs cultured within the fast-relaxing hydrogel, compared to slower-relaxing hydrogels. These findings highlight the critical role of matrix viscoelasticity in regulating ASC behavior and suggest that viscoelasticity is a key parameter for novel biomaterials design to improve the efficacy of stem cell therapy for IVD regeneration. Statement of significanceSystematically characterized the influence of tissue-mimetic viscoelasticity on ASC. NP-mimetic hydrogels with tunable viscoelasticity and tissue-matched stiffness. Long-term survival and metabolic activity of ASCs are substantially improved in the fast-relaxing hydrogel. The fast-relaxing hydrogel allows higher rate of cell protrusions formation and matrix remodeling. ASC differentiation towards an NP-like cell phenotype is promoted in the fast-relaxing hydrogel, with more CD24 positive expression indicating NP committed cell fate. The expression of TRPV4, a molecular sensor of matrix viscoelasticity, is significantly enhanced in the fast-relaxing hydrogel, indicating ASC sensing matrix viscoelasticity during cell development. The NP-specific ECM secretion of ASC is considerably influenced by matrix viscoelasticity, where the deposition of aggrecan and type-II collagen are significantly enhanced in the fast-relaxing hydrogel.

Two‐Way Regulation of MgFe‐LDH on ESC Cell Fate via Temporal Selective Activation of Different Cell Membrane Receptors

AbstractStem cell fate regulation by biomaterials has considerable therapeutic potential; however, the mechanisms whereby biomaterials instruct stem cells for accurate fate regulation still need to be elucidated. Layered double hydroxide (LDH) nanoparticles are widely used for tissue regeneration because of their excellent physicochemical properties and biocompatibility. This study reveals that MgFe‐LDH has bidirectional regulatory effects on the differentiation of mouse embryonic stem cells into neural progenitor cells, essentially suppressing cell differentiation on the 1st day and promoting cell differentiation on the 3rd day. Single‐cell transcriptome sequencing and simulated computation of structural analysis demonstrate that MgFe‐LDH interacts with the membrane receptors leukemia inhibitory factor receptor (LIFR, 15 nm above the cell membrane) and pached1 (PTCH1, 6 nm above the cell membrane) on the 1st and 3rd day of differentiation, respectively. LIFR has more contact with MgFe‐LDH than PTCH1 and relatively weaker electrostatic interaction energies (−11010.0 and −9829.3 kJ mol−1 for PTCH1 and LIFR, respectively). These factors determine receptor binding and activation priority, followed by cell fate regulation. The results indicate that MgFe‐LDH regulate the activity and fate of stem cells dually, thus creating possibilities for personalized therapy in neurodevelopment and regenerative medicine through an in‐depth study of cell membrane receptors.

Classification of Neural Stem Cell Activation State In Vitro using Autofluorescence.

Neural stem cells (NSCs) divide and produce newborn neurons in the adult brain through a process called adult neurogenesis. Adult NSCs are primarily quiescent, a reversible cell state where they have exited the cell cycle (G0) yet remain responsive to the environment. In the first step of adult neurogenesis, quiescent NSCs (qNSCs) receive a signal and activate, exiting quiescence and re-entering the cell cycle. Thus, understanding the regulators of NSC quiescence and quiescence exit is critical for future strategies targeting adult neurogenesis. However, our understanding of NSC quiescence is limited by technical constraints in identifying quiescent NSCs (qNSCs) and activated NSCs (aNSCs). This protocol describes a new approach to identify and enrich qNSCs and aNSCs generated in in vitro cultures by imaging NSC autofluorescence. First, this protocol describes how to use a confocal microscope to identify autofluorescent markers of qNSCs and aNSCs to classify NSC activation state using autofluorescence intensity. Second, this protocol describes how to use a fluorescent activated cell sorter (FACS) to classify NSC activation state and enrich samples for qNSCs or aNSCs using autofluorescence intensity. Third, this protocol describes how to use a multiphoton microscope to perform fluorescence lifetime imaging (FLIM) at single-cell resolution, classify NSC activation state, and track the dynamics of quiescent exit using both autofluorescence intensitiesand fluorescence lifetimes. Thus, this protocol provides a live-cell, label-free, single-cell resolution toolkit for studying NSC quiescence and quiescence exit.

Loss of the stress sensor GADD45A promotes stem cell activity and ferroptosis resistance in LGR4/HOXA9-dependent AML

AbstractThe overall prognosis of acute myeloid leukemia (AML) remains dismal, largely because of the inability of current therapies to kill leukemia stem cells (LSCs) with intrinsic resistance. Loss of the stress sensor growth arrest and DNA damage-inducible 45 alpha (GADD45A) is implicated in poor clinical outcomes, but its role in LSCs and AML pathogenesis is unknown. Here, we define GADD45A as a key downstream target of G protein-coupled receptor (LGR)4 pathway and discover a regulatory role for GADD45A loss in promoting leukemia-initiating activity and oxidative resistance in LGR4/HOXA9-dependent AML, a poor prognosis subset of leukemia. Knockout of GADD45A enhances AML progression in murine and patient-derived xenograft (PDX) mouse models. Deletion of GADD45A induces substantial mutations, increases LSC self-renewal and stemness in vivo, and reduces levels of reactive oxygen species (ROS), accompanied by a decreased response to ROS-associated genotoxic agents (eg, ferroptosis inducer RSL3) and acquisition of an increasingly aggressive phenotype on serial transplantation in mice. Our single-cell cellular indexing of transcriptomes and epitopes by sequencing analysis on patient-derived LSCs in PDX mice and subsequent functional studies in murine LSCs and primary AML patient cells show that loss of GADD45A is associated with resistance to ferroptosis (an iron-dependent oxidative cell death caused by ROS accumulation) through aberrant activation of antioxidant pathways related to iron and ROS detoxification, such as FTH1 and PRDX1, upregulation of which correlates with unfavorable outcomes in patients with AML. These results reveal a therapy resistance mechanism contributing to poor prognosis and support a role for GADD45A loss as a critical step for leukemia-initiating activity and as a target to overcome resistance in aggressive leukemia.

Treatment of Syringomyelia Characterized by Focal Dilatation of the Central Canal Using Mesenchymal Stem Cells and Neural Stem Cells.

Syringomyelia is a progressive chronic disease that leads to nerve pain, sensory dissociation, and dyskinesia. Symptoms often do not improve after surgery. Stem cells have been widely explored for the treatment of nervous system diseases due to their immunoregulatory and neural replacement abilities. In this study, we used a rat model of syringomyelia characterized by focal dilatation of the central canal to explore an effective transplantation scheme and evaluate the effect of mesenchymal stem cells and induced neural stem cells for the treatment of syringomyelia. The results showed that cell transplantation could not only promote syrinx shrinkage but also stimulate the proliferation of ependymal cells, and the effect of this result was related to the transplantation location. These reactions appeared only when the cells were transplanted into the cavity. Additionally, we discovered that cell transplantation transformed activated microglia into the M2 phenotype. IGF1-expressing M2 microglia may play a significant role in the repair of nerve pain. Cell transplantation can promote cavity shrinkage and regulate the local inflammatory environment. Moreover, the proliferation of ependymal cells may indicate the activation of endogenous stem cells, which is important for the regeneration and repair of spinal cord injury.

Hypoxic preconditioning accelerates the healing of ischemic intestinal injury by activating HIF-1α/PPARα pathway-mediated fatty acid oxidation

Hypoxic preconditioning (HPC) has been shown to improve organ tolerance to subsequent severe hypoxia or ischemia. However, its impact on intestinal ischemic injury has not been well studied. In this study, we evaluated the effects of HPC on intestinal ischemia in rats. Intestinal rehabilitation, levels of fatty acid oxidation (FAO) by-products, intestinal stem cells (ISCs), levels of hypoxia-inducible factor 1 subunit α (HIF-1α) and its downstream genes such as peroxisome proliferator-activated receptor α (PPARα), and carnitine palmitoyltransferase 1a (CPT1A) were assessed at distinct time intervals following intestinal ischemia with or without the interference of HIF-1α. Our data showed that HPC facilitates the restoration of the intestinal structure and enhances the FAO, by boosting intestinal stem cells. Additionally, HIF-1α, PPARα, and CPT1A mRNA and their protein levels were generally up-regulated in the small intestine of HPC rats as compared to the control group. Our vitro experiment also shows low-oxygen induces highly levels of HIF-1α and its downstream genes, with a concurrent increase in FAO products in IEC-6 cells. Furthermore, the above phenomenon could be reversed by silencing HIF-1α. In conclusion, we hypothesize that HPC can stimulate the activation of intestinal stem cells via HIF-1α/PPARα pathway-mediated FAO, thereby accelerating the healing process post ischemic intestinal injury.

Response of Male Reproductive System Against Ionizing Radiation and Available Radio-protective Agents: Cellular and Molecular Insight.

The reproductive organ, housing spermatogonial stem cells (SSCs), undergoes ongoing division impacted by the irradiation dosage and exposure duration. Within the male reproductive organ, germ stem cells (spermatogonia) and somatic cells (Sertoli and Leydig cells) are present. Lower doses of ionizing (>4-6 Gy) and non-ionizing radiation (radiofrequency and microwave range 900 MHz - 2.45 GHz) may cause sperm-related issues, while higher doses (15 Gy) may affect Leydig cells and testosterone production. Response to radiation varies with age and pubescence. Spermatogonial stem cells, crucial for regenerating the spermatogenic lineage, express molecular markers like Estrogen receptor, FSH (Follicular Stimulating Hormone) receptor, TLR-4 (Toll-like Receptor-4), TLR-5 (Toll-like Receptor-5), FGF2 (Fibroblast Growth Factor-2), KIT (Receptor Tyrosine Kinase), AT-1 (Angiotensin II Type-1 Receptor), LXRs-γ (Liver X Receptor-γ), TNF-β (Tumor Necrosis Factor-β), and PCNA (Proliferating Cell Nuclear Antigen), influencing stem cell activity in testes. This study aimed to review the various available radioprotective agents and their efficacy in targeting the male reproductive system from the available literature. Various radioprotective herbal/synthetic/microbial/metallic extracts/formulations/ drugs [Septilin, Silymarin, Organic Turmeric, Oestrogen, Melatonin, Febuxostat, SQGD (Semiquinone glucoside derivative), Rapamycin, Entolimod, Zinc, Selenium, etc.] have been investigated up to exposure, but owing to effectiveness issues, they are unable to fulfil the aim to the fullest of restoring male fertility and normal testosterone levels during such eventuality. Further study is needed to optimize these tactics and fill knowledge gaps. Also, the effective components of herbal, synthetic drugs, etc., should be isolated and tested up to clinical levels, paving the way for successful radioprotection and radiomitigation strategies in the male reproductive system.

Toxicity and cytokine release from human dental pulp stem cells after exposure to universal dental adhesives cured by single peak and polywave LEDs

Objectivesto assess the impact of universal adhesives, cured with single-peak and polywave LEDs, on the metabolic activity and cytokine release of human dental pulp stem cells (hDPSCs). In addition, analyze the degree of conversion (DC) of the adhesives cured with the different LEDs. MethodsDiscs (5 mm diameter, 1 mm thick) were prepared using three universal adhesives: Single Bond Universal (SBU, 3 M ESPE), Optibond Universal (OBU, Kerr), and Zipbond Universal (ZBU, SDI). These discs were cured for 40 s using a single-peak (DeepCure, 3 M ESPE) or a polywave light-emmiting diode (LED) curing unit (Valo Grand, Ultradent). After 24 h, the specimens were placed in 24-well culture plates, each containing 1 mL of culture medium for 24 h. hDPSCs (1.8 ×104) were seeded in 96-well plates and allowed to grow for 24 h. Subsequently, the cells were exposed to the extracts (culture medium containing eluates from the adhesive discs) for an additional 24 h. Cells not exposed to the extracts were used as a control group. The mitochondrial metabolism was assessed using the MTT assay and the cytokine release evaluated through MAGPIX. The degree of conversion of the adhesives was analyzed using FTIR (n = 5). The results were analyzed by ANOVA two-way and Tukey’s test. ResultsOBU and ZBU eluates caused a statistically significant reduction in mitochondrial metabolism, regardless of the LED used, indicating their cytotoxicity. In contrast, SBU did not significantly affect the MTT results, resembling the control group. A higher release of cytokines IL-1, IL-6, IL-10, and TNF-α were found in association to ZBU. SBU, on the other hand, increased the release of IL-8. OBU did not influenced the cytokine release. SBU presented the higher DC, while OBU and ZBU had similar DC, lower than SBU. SignificanceIn conclusion, universal adhesives exhibit toxicity towards hDPSCs, but the extent of toxicity varies depending on the adhesive material. ZBU was associated with increased cytokine release, particularly pro-inflammatory mediators, from hDPSCs. The different LEDs did not influenced the cytotoxicity of the evaluated adhesives.

A core scientific problem in the treatment of central nervous system diseases: newborn neurons.

It has long been asserted that failure to recover from central nervous system diseases is due to the system's intricate structure and the regenerative incapacity of adult neurons. Yet over recent decades, numerous studies have established that endogenous neurogenesis occurs in the adult central nervous system, including humans'. This has challenged the long-held scientific consensus that the number of adult neurons remains constant, and that new central nervous system neurons cannot be created or renewed. Herein, we present a comprehensive overview of the alterations and regulatory mechanisms of endogenous neurogenesis following central nervous system injury, and describe novel treatment strategies that target endogenous neurogenesis and newborn neurons in the treatment of central nervous system injury. Central nervous system injury frequently results in alterations of endogenous neurogenesis, encompassing the activation, proliferation, ectopic migration, differentiation, and functional integration of endogenous neural stem cells. Because of the unfavorable local microenvironment, most activated neural stem cells differentiate into glial cells rather than neurons. Consequently, the injury-induced endogenous neurogenesis response is inadequate for repairing impaired neural function. Scientists have attempted to enhance endogenous neurogenesis using various strategies, including using neurotrophic factors, bioactive materials, and cell reprogramming techniques. Used alone or in combination, these therapeutic strategies can promote targeted migration of neural stem cells to an injured area, ensure their survival and differentiation into mature functional neurons, and facilitate their integration into the neural circuit. Thus can integration replenish lost neurons after central nervous system injury, by improving the local microenvironment. By regulating each phase of endogenous neurogenesis, endogenous neural stem cells can be harnessed to promote effective regeneration of newborn neurons. This offers a novel approach for treating central nervous system injury.

Facile synthesis of mechanically robust and injectable tetra-polyethylene glycol/methacrylate chitosan double-network hydrogel cartilage repair

The development of advanced hydrogel scaffolds with good biocompatibility, mechanical strength and bioactive property is highly desirable in cell growth, proliferation, and differentiation for the cartilage tissue regeneration. Herein, we reported a simple, inexpensive, feasible, and environmentally benign strategy to synthesize a biocompatible polyethylene glycol-based methacrylate-modified chitosan (PCSMA) double network (DN) hydrogel via the reversible Schiff base reaction and successive photopolymerization between the tetra-poly (ethylene glycol) aldehyde (Tetra-PEG-CHO) and methacrylate-modified chitosan (CSMA) in mild conditions. The four-terminated CHO groups were hypothesized to not only construct the whole architectural network through the dynamic pH-responsive Schiff base but also contribute to the injectable behavior and good tissue adhesion, which may furnish the surgeon with the availability of injectable and shapeless ability to fill in the gaps. Compared to the single PCS hydrogel with the main similar components of the tetra-PEG-CHO and pure CS moieties, the post-fabrication of PCSMA DN hydrogel possessed slower degradation time, denser network, and stronger mechanical stability, which could facilitate the bone marrow mesenchymal stem cell (BMSCs) activity and promote the chondrogenic differentiation for facilitating cartilage regeneration. Our study highlights the positive effects on chondrogenic performance and supports the PCSMA hydrogel as a promising tissue-engineered scaffold for the cartilage defect repair.

EXPERIENCE OF USING STEM CELLS IN PATIENTS WITH ATOPIC DERMATITIS

Recent research in this area has shown that in many tissues, including skin, stem cells become dormant as they mature, slowing down their rate of division and not responding as effectively to stimulation [1]. A decrease in the activity of stem cells leads to the fact that the rate of skin renewal slows down and defects begin to accumulate in it; the skin ages [2]. Recently, the attention of scientists has been attracted by plant stem cells, which produce a complex of molecular regulators that are universal. These regulators are capable of changing the functioning of human skin stem cells, partially returning them the energy of youth [3]. One of the most popular ingredients today is Swiss apple stem cells. Each adult apple stem cell can independently generate new cells and, in a concentration of just 0.1%, stimulate the division of human stem cells by 80% [4]. Phytostem (plant) cells were discovered relatively recently - only in the second half of the 20th century [5]. And the most effective in influencing human cells are considered to be plant stem cells, green apple phytostem cells. Scientists have proven that they are active biostimulators of human cells [6]. Plant cell preparations are absolutely hypoallergenic, unlike those obtained from animal or human tissues.

Multifunctional Sr, Mg, Ag-substituted octacalcium phosphate/carboxymethyl chitosan scaffolds: Antibacterial activity and osteogenic differentiation of human mesenchymal stem cells

Numerous studies indicate that octacalcium phosphate (OCP) possesses promising bone regenerative properties. To further improve the regenerative properties of OCP, several ionic substitutions have been proposed. However, knowledge regarding the synergic effect of multiple ions substituted in OCP crystals and fabricated scaffolds is lacking. In this work, plate-like OCP crystals substituted with 1 and 2.5 mol.% of Mg2+, Sr2+ and Ag+ were prepared by the wet precipitation method. Rietveld refinement analysis indicated a successful substitution of Ca2+ by Mg2+, Sr2+and Ag+ ions. The prepared substituted OCP powders showed a high antibacterial effect towards Gram-negative and Gram-positive bacteria. Further, composite scaffolds based on substituted OCP and carboxymethyl chitosan were prepared by the freeze-crosslinking method. The scaffolds had a highly porous structure, with interconnected pores and homogeneously dispersed OCP within the polymer matrix, and high stability in simulated physiological conditions during 28 days. Good viability and proliferation of human bone marrow-derived mesenchymal stem/stromal cells were confirmed by Live/Dead and CyQUANT assays. Additionally, successful osteogenic differentiation was confirmed by quantification of alkaline phosphatase activity and immunocytochemical staining for type I collagen. Results indicate that ionic substitutions with 1.0 mol.% have the most beneficial effect on stem cell differentiation towards osteoblast phenotype.

Strontium and Zinc Co-Doped Mesoporous Bioactive Glass Nanoparticles for Potential Use in Bone Tissue Engineering Applications.

Mesoporous bioactive glass nanoparticles (MBGNs) have attracted significant attention as multifunctional nanocarriers for various applications in both hard and soft tissue engineering. In this study, multifunctional strontium (Sr)- and zinc (Zn)-containing MBGNs were successfully synthesized via the microemulsion-assisted sol-gel method combined with a cationic surfactant (cetyltrimethylammonium bromide, CTAB). Sr-MBGNs, Zn-MBGNs, and Sr-Zn-MBGNs exhibited spherical shapes in the nanoscale range of 100 ± 20 nm with a mesoporous structure. Sr and Zn were co-substituted in MBGNs (60SiO2-40CaO) to induce osteogenic potential and antibacterial properties without altering their size, morphology, negative surface charge, amorphous nature, mesoporous structure, and pore size. The synthesized MBGNs facilitated bioactivity by promoting the formation of an apatite-like layer on the surface of the particles after immersion in Simulated Body Fluid (SBF). The effect of the particles on the metabolic activity of human mesenchymal stem cells was concentration-dependent. The hMSCs exposed to Sr-MBGNs, Zn-MBGNs, and Sr-Zn-MBGNs at 200 μg/mL enhanced calcium deposition and osteogenic differentiation without osteogenic supplements. Moreover, the cellular uptake and internalization of Sr-MBGNs, Zn-MBGNs, and Sr-Zn-MBGNs in hMSCs were observed. These novel particles, which exhibited multiple functionalities, including promoting bone regeneration, delivering therapeutic ions intracellularly, and inhibiting the growth of Staphylococcus aureus and Escherichia coli, are potential nanocarriers for bone regeneration applications.

Bioactivity of human dental pulp-derived stem cells with boron-controlled S-PRG filler eluate by anion exchange.

Surface pre-reacted glass-ionomer (S-PRG) filler is a bioactive glass filler capable of releasing various ions. A culture medium to which was added an S-PRG filler eluate rich in boron was reported to enhance alkaline phosphatase (ALP) activity in human dental pulp-derived stem cells (hDPSC). To clarify the role of boron eluted from S-PRG fillers, the modified S-PRG filler eluate with different boron concentrations was prepared by using an anion exchange material. Therefore, elemental mapping analysis of anion exchange material, adsorption ratio, hDPSCs proliferation and ALP activity were evaluated. For statistical analysis, Kruskal-Wallis test was used, with statistical significance determined at p<0.05. ALP activity enhancement was not observed in hDPSC cultured in the medium that contained the S-PRG filler eluate from which boron had been removed. The result suggested the possibility that an S-PRG filler eluate with controlled boron release could be useful for the development of novel dental materials.

Abstract 1473: Exposure of arsenic promotes malignant stemness and induces bladder malignancy in preclinical models

Abstract Numerous epidemiological and scientific studies have consistently demonstrated that populations exposed to arsenic (As) concentrations ranging from &amp;gt;10 ppb to 2500 ppb in their drinking water face an elevated risk of urogenital cancers, with a particular emphasis on bladder cancer (BCa). Hence, the goal of the study is to investigate the underlying mechanisms that trigger malignant transformation in the bladder using physiologically relevant arsenic concentrations observed in BCa patients. In our results, we observed significantly elevated levels of arsenic in the urine of BCa patients, ranging from 20 to 50 mg/L, in stark contrast to the levels observed in healthy controls (&amp;gt;10 mg/L) and the recommended guidelines set by the Environmental Protection Agency (EPA) and Centers for Disease Control and Prevention (CDC). We used a median physiological concentration of arsenic (30 PPM) in drinking water-induced bladder hyperplasia in male and female mice over six months. Likewise, chronic exposure of healthy bladder epithelial cells (TRT-HU1) to similar arsenic concentrations (250nM:30 PPM) for 12 months resulted in malignant transformation, and arsenic-transformed cells (AsT) were capable of inducing tumors in xenografted and orthotopic models. Nonetheless, the molecular mechanism responsible for this malignancy remains to be elucidated. Our molecular analysis has pointed towards the activation of stem cells, notably ALDH1A1 (with a 50,000-fold increase), as a pivotal factor driving this transformation. Furthermore, our research has demonstrated that the activation of ALDH1A1 is contingent on the induction of Metal Response Element-Binding Transcription Factor-1 (MTF1). Consequently, silencing MTF1 inhibited the expression of ALDH1A1 and its associated malignant properties in As-transformed cells, strongly suggesting that MTF1 functions as the master regulator in As-induced bladder carcinogenesis. We have also observed similar molecular signatures (MTF1 and ALDH1A1 expression) in arsenic-exposed versus non-exposed mice and bladder cancer patients. In conclusion, our studies suggest that activation of stem cells is a critical step in arsenic-induced bladder carcinogenesis. Citation Format: Bhawna Tyagi, Mohit Vashishta, Neha Tyagi, Balaji Chandrasekaran, Vaibhav Shukla, Ashish Tyagi, Chendil Damodaran. Exposure of arsenic promotes malignant stemness and induces bladder malignancy in preclinical models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1473.

Abstract 5967: Blockade of Wnt5A/ROR1 signaling as cancer stem cell targeting therapy for metastatic prostate cancer

Abstract BACKGROUND– Wnt5A and its non-canonical Wnt receptor, ROR1, have emerged as a promising new signaling pathway target for lethal, metastatic prostate cancer. Wnt5A has emerged as a significant marker of poor prognosis in circulating tumor cells (CTCs) of metastatic castration resistant prostate cancer (mCRPC) patients. ROR1 is expressed in lethal types of mCRPC, especially neuroendocrine prostate cancer (NEPC). A therapeutic ROR1 antibody, Zilovertamab, has been clinically tested in chronic lymphocytic leukemia (CLL) and metastatic breast cancer and shown to be safe. We sought to investigate Zilovertamab-based therapies for metastatic prostate cancer in pre-clinical model systems. HYPOTHESIS – Wnt5A/ROR1 signaling may mediate a cancer stem cell program which makes cells resistant to therapies which target the cell cycle and proliferation. Blocking ROR1 may reveal vulnerabilities which sensitize cancer cells to chemotherapies like docetaxel. METHODS– ROR1 expression was determined using RNASeq, qRT-PCR, FACS, IHC and Western blotting. ROR1 signaling was blocked using the anti-ROR1 therapeutic antibody, Zilovertamab, or CRISPRCas9 ROR1 knock out. Cell growth was measured using an Incucyte real time imaging system. Stem cell activity was assessed in 3D organoids. Cell cycle analysis was performed in live cells in 2D cultures and 3D organoids using the Fucci2BL bicistronic Fluorescent, Ubiquitination-based Cell Cycle Indicator reporter system. PDX PCSD13 tumor growth was measured via calipers and IVIS. RNASeq was performed on tumors. RESULTS– We showed that ROR1 was expressed at high levels in mCRPC cell lines: PC3, DU145, and in the bone metastatic prostate cancer PDX: PCSD13. CRISPR/Cas9 Knock out of ROR1 in PC3 and DU145 cells showed increased sensitivity to docetaxel inhibition of proliferation in vitro in 2D real time Incucyte proliferation assays and in 3D organoids. Organoid formation was significantly reduced in ROR1 KO cells. Cells expressing the FUCCI live cell cycle tracker showed docetaxel led to G2 arrest and ROR1 signaling inhibition increased efficacy of docetaxel induced cell cycle arrest. Treatment of PCSD13 PDX in vivo with Zilovertamab plus docetaxel synergistically increased tumor growth inhibition in vivo and modulated cancer stem cell and cell cycle expression profiles. CONCLUSIONS– Cancer stem cells represent the fundamental precursors from which all diverse tumor subpopulations evolve. Thus, therapeutic targeting of these tumor initiating stem/progenitor cells may prevent the evolutionary diversification of a tumor and overcome a critical clinical barrier to cancer treatment. We showed synergistic response in our PDX and PCs cell line models to Zilovertamab plus docetaxel. We are now conducting a phase 1b clinical trial with zilovertamab plus docetaxel in patients with metastatic CRPC (CirmD, NCT05156905, PI R Mckay). Citation Format: Christina A.M. Jamieson, Jamillah Murtadha, Christopher S. Oh, Michelle T. Muldong, Niloofar Etemadfard, Evodie Koutouan, Young E. Yoon, Sanghee Lee, Oh Kwon, Jong J. Oh, Min S. Kim, Karl Willert, Nicholas A. Cacalano, Catriona H.M. Jamieson, Terry Gaasterland, Rana R. Mckay, Christopher J. Kane, Anna A. Kulidjian, Charles E. Prussak. Blockade of Wnt5A/ROR1 signaling as cancer stem cell targeting therapy for metastatic prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5967.

Hierarchically Biomimetic Scaffolds with Anisotropic Micropores and Nanotopological Patterns to Promote Bone Regeneration via Geometric Modulation.

Structural engineering is an appealing means to modulate osteogenesis without the intervention of exogenous cells or therapeutic agents. In this work, a novel 3D scaffold with anisotropic micropores and nanotopographical patterns is developed. Scaffolds with oriented pores are fabricated via the selective extraction of water-soluble polyethylene oxide from its poly(ε-caprolactone) co-continuous mixture and uniaxial stretching. The plate apatite-like lamellae are subsequently hatched on the pore walls through surface-induced epitaxial crystallization. Such a unique geometric architecture yields a synergistic effect on the osteogenic capability. The prepared scaffold leads to a 19.2% and 128.0% increase in the alkaline phosphatase activity of rat bone mesenchymal stem cells compared to that of the scaffolds with only oriented pores and only nanotopographical patterns, respectively. It also induces the greatest upregulation of osteogenic-related gene expression in vitro. The cranial defect repair results demonstrate that the prepared scaffold effectively promotes new bone regeneration, as indicated by a 350% increase in collagen I expression in vivo compared to the isotropic porous scaffold without surface nanotopology after implantation for 14weeks. Overall, this work provides geometric motifs for the transduction of biophysical cues in 3D porous scaffolds, which is a promising option for tissue engineering applications.

Autofluorescence is a biomarker of neural stem cell activation state

Neural stem cells (NSCs) must exit quiescence to produce neurons; however, our understanding of this process remains constrained by the technical limitations of current technologies. Fluorescence lifetime imaging (FLIM) of autofluorescent metabolic cofactors has been used in other cell types to study shifts in cell states driven by metabolic remodeling that change the optical properties of these endogenous fluorophores. Using this non-destructive, live-cell, and label-free strategy, we found that quiescent NSCs (qNSCs) and activated NSCs (aNSCs) have unique autofluorescence profiles. Specifically, qNSCs display an enrichment of autofluorescence localizing to a subset of lysosomes, which can be used as a graded marker of NSC quiescence to predict cell behavior at single-cell resolution. Coupling autofluorescence imaging with single-cell RNA sequencing, we provide resources revealing transcriptional features linked to deep quiescence and rapid NSC activation. Together, we describe an approach for tracking mouse NSC activation state and expand our understanding of adult neurogenesis.